Metabolic engineering of Selenocysteine Biosynthesis and Insertion Pathway in Lactococcus lactis

Metabolic engineering of Selenocysteine Biosynthesis and Insertion Pathway in Lactococcus lactis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Metabolic engineering of Selenocysteine Biosynthesis and Insertion Pathway in Lactococcus lactis Jing-Jing Peng, Yao Qin, Liang-Hua Lu, Shi-Yang Yue, Ping Shi, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5428752/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Se-enriched lactic acid bacteria (LAB) exist unclear metabolic flow, unstable composition of selenium spectrum and low selenoprotein content such prominent problems caused by complex metabolic pathway and non-specific incorporation of selenium currently. Accordingly, this study reports how to introduce the firstly proposed Selenocysteine Biosynthesis and Insertion Pathway (SBIP) into Lactococcus lactis ( L. lactis ) and specifically guide selenium metabolic flow to direct synthesis of specific selenoprotein with employed multi-level metabolic engineering strategies. In result, the integration of these key factors turned out to facilitate the establishment of SBIP in NZ9000: SelA, SelB, SelC, SelD, GshF and FDH from NZ9000/SBIP up-regulated 8.01, 19.03, 925982.32, 34.51, 31879.16 and 28367.04 multiples compared with NZ9000/p-p; FI/OD 600 of NZ9000/SBIP-sfGFP was 362.25 ± 0.43; FDH enzyme activity of NZ9000/SBIP reached 28.11 ± 0.12 mU/mg, and GshF 219.47 ± 0.79 mU/mg under the optimal expression. This first successful implementation of directed synthesis of selenoprotein FDH would indicate a whole new direction to supply Sec-contained proteins through biosynthesis in LAB factory. Selenocysteine Lactococcus lactis Formate dehydrogenase Bifunctional glutathione synthetase selenium enrichment Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Selenium (Se) is a trace element rich in biological functions[1]. Commercial selenium supplement is mainly through the intake of selenium-rich food or products, inorganic selenium and organic selenium[2]. In dietary selenium and selenium-rich yeast, selenium mainly exists as free SeMet, or is randomly inserted into proteins instead of Met and intaked by body[3]. However, the complex metabolic pathway and non-specific incorporation of selenium will lead to some common problems such as unclear metabolic flow, unstable composition of selenium spectrum and low selenoprotein content. Due to the wider medicinal value of organic selenoprotein, there have been peptide synthesis, chemical mutagenesis, cysteine nutrition-deficient biosynthesis and such preparation methods[4, 5]. However, these methods are deficient in high cost, low yield and lack of directivity, which limit their sustainable and economically feasible application[6]. Therefore, to develop a new biological selenium enrichment, specifically guiding selenium metabolic flow to direct synthesis of specific selenoprotein is urgently needed. In 1995, Calomme et al.[7] firstly demonstrated that Lactobacillus could transform inorganic selenium into organic selenium whose main species was Sec, different from those commercial selenium-rich products with SeMet as the main species, indicating a feasible new direction for selenium production and supplement. Current studies on Se-enriched LAB and their selenoproteins mainly focus on strain screening, cultivation optimization, antioxidant effects and morphology identification, while there are no studies on LAB Sec and selenoprotein metabolic pathways and directed synthesis. And LAB don’t contain selenoprotein coding genes or regulatory factors after query from NCBI (National Center of Biotechnology Information) and bSECIS (bacterial selenocysteine insertion sequence). Therefore, as Fig. 1 illustrated, this study desired to establish a microbial cell factory[8] for directed and efficient synthesis of novel biomass specific selenoproteins by constructing SBIP (Selenocysteine Biosynthesis and Insertion Pathway)[9] equipped with one key codon (UGA), one cis-acting element (SECIS) and four regulatory factors SelA (Selenocysteine synthase), SelB (Selenocysteine-specific elongation factor), SelC (tRNA [Ser]sec ) and SelD (Seleno-phosphate synthase) to specifically guide the selenium metabolism flow in LAB. Although SBIP of E.coli has been clarified[13, 14], it does not belong to the food grade safe strain, which limits its application. Comparatively, L. lactis , the most representative strain of LAB, is authorized as GRAS microorganism by FDA[15] and commonly used for genetic engineering[16]. Since the only commercial expression system in LAB[17] is the nisin controlled expression (NICE) proposed by Kuipers et al.[18], and L. lactis NZ9000 [20] is the most applied model strain among all the derivatives of L. lactis MG1363[19], NZ9000 with NICE inducible system was preferentially used for the early SBIP construction of free expression system[20, 21]. Besides, constitutive expression system[22] which is also suitable for large-scale production in industry can continuously express target protein without spatiotemporal specificity or adding inducers, this study would combine these two systems. At present, P1, P2, P3, P5, P8 [23] and P21, P23, P32, P44, P59[24] have been applied for the expression of foreign genes in L. lactis . Considering that the transcriptional activity of P8, P5, P3, PnisA, P2, P45, P1, P6, P32 decreased, and high expression of SelA may inhibit the expression of selenoprotein[25], strong promoter P8 and P5, moderately strong promoter PnisA and weak promoters P1 and P32 were selected by high-throughput screening for optimal co-expression of the target genes in SBIP. Meanwhile, promoter P23[26] and P59[27, 28] with good application effect were also tried to verify the fused FDH-sfGFP expression. Many attempts have been made to overexpress the SBIP regulatory factors homologically by Escherichia coli ( E. coli ) so far[29–32], and it has been used to express heterologous selenoprotein[11, 33, 34], but no metabolic pathway application or modification of SBIP has been reported in other prokaryotic host bacteria, neither in LAB. In this study, the SBIP would be introduced into L. lactis NZ9000 by two major shutter plasmids: pNZ8148[35] and pTRKH2[28, 36]. The former one was equipped with four regulatory factors to be expressed in a mixed form due to the combination of PnisA with P1, PnisA or P5 (Fig. 1 -a). While the latter one was simple constructive expression by ligating GshF (SA) with P8 or P32 to enhance the selenium donor required for Sec synthesis (Fig. 1 -b), and ligating fused FDH-sfGFP with P23 or P59 to introduce SECIS to translate UGA (Fig. 1 -c). Finally, the transfer of mechanism from E. coli to L. lactis was turned out to be a triumph when FDH and other target genes were verified, providing a reference for the engineering production of other artificial selenoproteins, an in-depth understanding for specific selenoprotein synthesis and regulation, and sustainable development of biological resources. 2. Material and methods 2.1 Chemicals, enzymes and media Chemicals and enzymes: T4 DNA ligase and pEASY®-Basic Seamless Cloning and Assembly Kit were purchased from TransGen Biotech (Beijing, China); PrimeSTAR® HS DNA Polymerase, PrimeScript™ II 1st Strand cDNA Synthesis Kit, TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) were purchased from TaKaRa Biotech (Kyoto, Japan)༛Plasmid Mini Kit I, DNA Gel Extraction Kit, Bacterial RNA Kit and DNase I Set were purchased from Omega Bio-Tek (New York, USA); Reduced glutathione (GSH) test kit (microplate method) and FDH test kit (microplate method) were purchased from Grace Biotech (Suzhou, China); BCA protein concentration determination kit was purchased from Beyotime Biotech (Shanghai, China); All fast digest restriction enzymes were purchased from Thermo Fisher Scientific Inc. (Massachusetts, USA). Media: LB medium (pH 7.0 ± 0.1) contained 10g /L tryptone, 5 g/L yeast extract and 10 g/L NaCl; M17 medium contained 2.5 g/L bacterial peptone, 2.5 g/L bacterial casein peptone, 5 g/L soya peptone, 5 g/L beef extract, 2.5 g/L yeast extract, 0.5 g/L sodium ascorbate, 19 g/L β-sodium glycerophosphate, and 0.25 g/L MgSO4; GM17 medium was M17 supplemented with 0.5% glucose; GGC-GM17 medium was GM17 supplemented with 10 mM L-Glutamic acid (Glu), L-Glycine (Gly) and L-Cysteine (Cys), respectively[37, 38]; G-SGM17B medium was GM17 supplemented with 1.5% glycine, 0.5 M sucrose, 2 mM CaCl 2 and 20 mM MgCl 2 . The pHs of M17 and the mediums derived from it were adjusted by 1% NaOH and 1% HCl to 7.0 ~ 7.4. All above were provided by Solarbio (Beijing, China) and Macklin (Shanghai, China). 2.2 Strains, plasmids and cultivation conditions All bacterial strains and plasmids used in this study were listed in Table S1 . E. coli MC1061 and E. coli TOP10 were used as competent cell for the chemical transformation of pNZ8148 and its recombinant plasmids, while E. coli JM110 and E. coli DH5α were used for the transformation of pTRKH2 and its recombinant plasmids. E. coli was cultured in LB broth at 37℃ and 220 r/min. L. lactis NZ9000 was a host to verify the expression of several key factors. NZ9000 strains were cultured in GM17 broth at 30℃ without agitation. The standard of supplemented antibiotics for screening recombinants and transformants were as follows: 200 µg/mL erythromycin and 25 µg/mL chloramphenicol was added into LB broth respectively when pTRKH2 or its recombinant plasmids and pNZ8148 or its recombinant plasmids existed in E. coli strains, while only 12.5 µg/mL erythromycin and 12.5 µg/mL chloramphenicol was added into GM17 broth when pTRKH2 or its recombinant plasmids and pNZ8148 or its recombinant plasmids existed in NZ9000 strains, and only add 8 µg/mL erythromycin and chloramphenicol each when pTRKH2 and pNZ8148 or two of their recombinant plasmids both existed in NZ9000 strains. 2.3 DNA manipulation and strain construction All plasmids listed in Table S1 were prepared with Omega plasmid kit, while an additional incubation with 1 mg/mL lysozyme (Solarbio) in 50 mM PBS buffer at 37℃ for 30 min was called for those from NZ9000. All primers listed in Table S2 were provided by BGI•Tech (Shenzhen, China). PCR amplifications were conducted with TaKaRa DNA Polymerase and the target linearized DNA fragments were purified with Omega gel extraction kit. GshF (GenBank: MN020375.1) was synthesized by Jinsirui Biotech (Jiangsu, China) and inserted onto pTRKH2 through SacI and SalI sites to generate pTRKH2-GshF by Jinsirui Biotech. SelC (Gene ID: 948167) derived from pBAD18-SelABC2-GPX-GW was inserted into pNZ8148 through PstI and KpnI sites to generate pNZ8148-SelC using T4 DNA ligase. For the rest target genes, SelA-SelB with nested expressed genes derived from pBAD18-SelABC2-GPX-GW was amplified by PCR using primer pairs “SelA-SelB-F/SelA-SelB-R”, while SelD and FDH derived from E. coli MG1655 genomic DNA were amplified by PCR using primer pairs “SelD-F/SelD-R” and “fdhF-F/fdhF-R” respectively. And fluorescent protein gene sfGFP derived from pRHU3-sfGFP was amplified by PCR using primer pairs “sfGFP -F/sfGFP -R”. However, the schematic overview for the construction of regulatory plasmid and verifiable plasmid were shown by Fig. 2 . Firstly, SelA-SelB and SelD purified fragments were assembled together onto linearized pNZ8148-SelC which has been already digested with XbaI and HindIII to generate pCABD (pNZ8148-SelC-SelA-SelB-SelD) using In-Fusion assembly kit. Likewise, FDH and sfGFP purified fragments were assembled together onto linearized pTRKH2-GshF which has been already digested with ApaLI and KpnI to generate pGFS (pTRKH2-GshF-FDH-sfGFP). In order to initiate the transcription and expression of the seven target genes (“SelA ~ SelD” on pNZ8148 and “GshF, FDH and sfGFP” on pTRKH2) in this work, above two main plasmids were inserted various promoters from pUC6P, which was chemically synthesized by Jinsirui Biotech. To preliminarily verify the expression of GshF in NZ9000, the strong promoter P8 was inserted before the GshF on pTRKH2-GshF with primers “P8-F/ P8-R” by In-Fusion assembly. According to Fig. 3 and Table S3, these promoters were installed independently or in combination with the primer pairs listed in Table S2 by In-Fusion assembly. In this study, the TGA codon[39] and the 11nt downstream SECIS[40] on fdhF[41], as well as the termination codon at the end were key point of penetration. On one hand, due to the need of codon readability and fusion expression of FDH-sfGFP, the TGA at 418–420 site of FDH was replaced by TGC, and the TAA at the end was replaced by AAA to generate pGFS1 (pTRKH2-GshF-FDH-sfGFP1). The bases were replaced successively by long-distance inverse-PCR[42] with primer pairs “(TGA→TGC)-F/(TGA→TGC)-R” and “(TAA→AAA)-F/(TAA→AAA)-R”. On the other hand, due to the need of independent expression of FDH, TGC at site 418–420 of FDH should be replaced back by TGA, and AAA at the end of FDH should be replaced back by TAA to generate p8G23FS2 (pTRKH2-P8-GshF-P23-FDH-sfGFP2). The bases were replaced successively by long-distance inverse PCR with primer pairs “(TGC→TGA)-F/(TGC→TGA)-R” and “(AAA→TAA)-F/(AAA→TAA)-R”. All recombinant plasmids were screened first by LB agar plates and then by GM17 agar plates according to the standard of supplemented antibiotics in 2.2. Finally, the recombinant plasmids were electro-transformed into NZ9000 with 2mm electroporation cuvettes[43] following the BTX ECM® 830 (Harvard Apparatus, Massachusetts, USA) protocol and under the condition of 2 kv, 150 µs of PL, 30 of MP, 100 ms of PI. So NZ9000/SBIP-sfGFP was constructed by transforming two optimal vectors after the screening and integration of promoters for regulatory factors and verifiable factors. Then NZ9000/SBIP was established after the base substitution of two different sites on one plasmid with dual constructive promoters. 2.4 Inducible and constitutive expression and crude enzyme solution preparation 2.4.1 Inducible and constitutive expression When NZ9000 transformants containing pNZ8148 or its recombinant plasmids harboring both PnisA and P1, such as NZ9000/pC1ABD (NZ9000/pNZ8148-SelC-P1-SelA-SelB-SelD), NZ9000/SBIP-sfGFP (NZ9000/(pC1ABD + p8G23FS1)) and NZ9000/SBIP (NZ9000/(pC1ABD + p8G23FS2)), the precultured cells were 3% inoculated into 30 mL fresh medium and expanded until OD 600 which was measured by MAPADA UV-1800PC spectrophotometer (Shanghai, China) reached 0.5 around 3 h later, then 30 ng/mL Nisin with or without 8 µg/mL Na 2 SeO 3 were added to induce the expression of regulatory gene for another 15 h[44], while the controls were added nothing for constitutive expression. And when NZ9000 transformants containing pTRKH2 or its recombinant plasmids only harboring P8, P32, P23, P59 or their combination, such as NZ9000/p8G (NZ9000/pTRKH2-P8-GshF), NZ9000/pG23FS1 (NZ9000/pTRKH2-GshF-P23-FDH-sfGFP1), NZ9000/pG59FS1 (NZ9000/pTRKH2-GshF-P59-FDH-sfGFP1), NZ9000/p8G23FS1 (NZ9000/pTRKH2-P8-GshF-P23-FDH-sfGFP1) and NZ9000/p32G23FS1 (NZ9000/pTRKH2-P32-GshF-P23-FDH-sfGFP1), the precultured cells were 3% inoculated into 30 mL fresh medium and expanded overnight till 18 h for constitutive expression. 2.4.2 Preparation of crude enzyme solution Each 30 mL NZ9000 recombinant bacteria solution was firstly centrifugated at 5000 xg, 25℃ for 10 min, then cell precipitation was harvested and washed 2 ~ 3 times with PBS (50 mM, pH 7.4), and finally suspended into 1.0 mL PBS with 1 mg/mL lysozyme. After incubation at 37℃ for 60 min, add 100 mg SiO 2 powder and 15 particles of Coolaber stainless steel beads (Beijing, China) to complete the cells grinding by Sceintz-48L cryogenic high-throughput tissue grinder (Jiangsu, China) under the following conditions: precooling at -30℃, 25 Hz, 600 rpm, 600 seconds. Lastly, the grinding blend was centrifuged in a high-speed refrigerated centrifuge at 12000 xg, 4℃ for 15 min, and the supernatant harvested was the crude enzyme solution. 2.5 Preliminary analysis for the target gene elements I. Regulatory genes: The function of SelA ~ SelD were achieved in L. lactis through the construction of pCABD and the subsequent screening from pC1ABD, pCPABD (pNZ8148-SelC-PnisA-SelA-SelB-SelD) and pC5ABD (pNZ8148-SelC-P5-SelA-SelB-SelD) one by one for the optimal. After the successful verification by endonuclease digestion, AGE (agarose gel electrophoresis) and DNA sequencing, the optimal strain would be cultured according to 2.4.1 and performed with SDS-PAGE. II. Verifiable genes: The expression of GshF and fused FDH-sfGFP were fulfilled in L. lactis through the construction of p8G and subsequent screening from “pG23FS1 and pG59FS1” and “p8G23FS1 and p32G23FS1”. Likewise, after the successful verification by endonuclease digestion, AGE and DNA sequencing, GshF, FDH and FDH-sfGFP would be verified with SDS-PAGE and enzyme activity assay, while the reporter gene sfGFP would be verified by the fluorescence intensity (FI) determination by TECAN Infinite M200 PRO microplate reader (Männedorf, Switzerland). 2.6 Confirmatory analysis for SBIP(-sfGFP) in NZ9000 After verification by endonuclease digestion, AGE and DNA sequencing of the double plasmids in NZ9000/SBIP(-sfGFP), the co-expressed proteins bands of SelA, SelB, SelD, GshF and FDH or FDH-sfGFP would be wholly tested by SDS-PAGE. To further analyze whether these inserted factors in the pathway really worked, RT-qPCR analysis, enzyme activity assay, as well as FI/OD 600 determination were demanded as described below. 2.7 Analytical methods 2.7.1 Endonuclease digestion and electrophoresis 20 µL digestion system for single plasmid was as follows: 1 µg plasmid, 1 µL fast digest endonuclease, 2 µL 10×fast digest buffer, and finally add sterilized ultra-pure water up to 20 µL. And 20 µL digestion system for double plasmid was as follows: 2 µg plasmid, 2 µL fast digest endonuclease, 2 µL 10×fast digest buffer, and finally add sterilized ultra-pure water up to 20 µL. While parameters for AGE were as follows: 1% agarose in 100 mL 1×TAE solution with 10 µL gel red nucleic acid dye (TransGen); 3 µL Thermo Scientific GeneRuler 1 kb Plus DNA Ladder Marker (Massachusetts, USA); 120 V, 108 mA, and 40 min by DYY-8C Nucleic acid electrophoresis apparatus (Beijing, China) and images taken by Bio-Rad Molecular Imager with ChemiDoc™ XRS + System (California, USA). 2.7.2 SDS-PAGE Parameters for polyacrylamide gel electrophoresis were as follows: the lower resolving gel was set as 10% and the upper spacer gel was 5%; 3 µL Solarbio broad-spectrum protein marker (Beijing, China); the loading sample was 3 µL 4×loading buffer with 9 µL diluted crude enzyme solution by 2 ~ 8 times; 80 V for 20 min, and up to 120 V for 40 min by Bio-Rad PowerPac Basic protein electrophoresis apparatus (California, USA); Dyed with Coomassie bright blue buffer (100 mg dye powder in 25 mL isopropyl alcohol, 10 mL glacial acetic acid and 65 mL distilled water) for 30 min at 45 r/min and decolorized with eluent (10 mL acetic acid, 5 mL anhydrous ethanol and 85 mL distilled water) for 3 h at 90 r/min. Finally, the protein glue immersed in distilled water was taken images by Bio-Rad Molecular Imager with ChemiDoc™ XRS + System. 2.7.3 Relative fluorescence intensity(FI/OD 600 ) determination[26, 45] The recombinant plasmids harboring the read-through FDH-sfGFP were electroporated into L. lactis NZ9000 competent cells, and then single colonies were screened and inoculated into 5 mL GM17 broth (+ Em༆Cr / +Em) with stand culture at 30℃ to logarithmic stage. Finally, cells precipitation was harvested after centrifugation at 6000 xg for 8 min, washed twice and resuspended in 1 mL 50 mM PBS, from which 200 µL cell resuspension solution with 3 parallels were transferred into a 96 well plate and performed by OD 600 detection and FI measurement with excitation at 485 nm and emission at 525 nm by TECAN Infinite M200 PRO microplate reader, while PBS buffer was as the blank control. 2.7.4 Establishment of growth curves In this study, NZ9000/pNZ8148, NZ9000/pTRKH2, and NZ9000/p-p(NZ9000/(pNZ8148 + pTRKH2)) were all constructed in advance for later comparison. Besides the target strain NZ9000/SBIP and the slow growing NZ9000/p8G in this study, NZ9000, NZ9000/pNZ8148, NZ9000/pTRKH2 and NZ9000/p-p such four strains were all set as controls to compare the growth process, providing references for optimizing the cultivation of the recombinant strains afterwards. During the monitoring, each first generation of these bacteria solution was 3% inoculated into 100 mL GM17 broth, and 200 µL with 3 parallels was taken every 2 h within 24 h to measure OD 600 against fresh medium by TECAN microplate reader. 2.7.5 Assay for GSH and the enzyme activity of GshF and FDH 1 mL GSH synthesis reaction system[46]: 20 mM Glu, 20 mM Gly, 20 mM Cys, 10 mM ATP, 30 mM MgCl 2 and 100 mM Tris-HCl buffer (pH 8.0). Each component was dissolved in Tris-HCl buffer. An appropriate 100 µL of supernatant crude enzyme solution was added to 1 mL reaction system, incubated at 37℃ for 20 min by Guowang DTH-100 dry bath (Jiangsu, China), and terminated by water boiling bath for 3 min. After chill for 30 min on ice, it was centrifuged at 12000 xg 4℃ for 10 min, and the supernatant was harvested to determine the yield of GSH by Grace kit. And the unit of GshF activity (U) was calculated by the amount of GshF enzyme (U/mg or U/mL) required to catalyze the formation of 1 µmol GSH per minute at 37℃. Similarly, 1mL FDH enzyme reaction system was conducted by NADH colorimetric method[47, 48]: 1 mM NAD +, 6 mM sodium formate, 100 mM PBS buffer (pH 7.5). Each component was dissolved in PBS buffer. And the unit of FDH activity (U) was calculated by the amount of FDH enzyme (U/mg or U/mL) required to catalyze the formation of 1 nmol NADH per minute at 35℃. 2.7.6 RT-qPCR NZ9000/SBIP was the strain containing double plasmids with optimal promoters combination screened by GM17 (+ Em + Cr) agar plates, and first generation bacterial solution was 3% inoculated into 10 mL fresh GM17 medium for about 3 h, after when 13 h Nisin induction culturing was performed. Then 3 mL bacterial solution with 3 parallels would be harvested to extract total RNA with genomic DNA erased by Omega kits. And a few RNA was taken to complete AGE. When three obvious bands occurred on the imaging system, the extracted RNA was immediately reverse-transcribed into cDNA by TaKaRa Kit. In this study, NZ9000/p-p was treated as control and 16S rRNA[49] was decided as reference gene. The final RT-qPCR for target genes with designed specific primers in Table S4 would be performed following the instruction of TaKaRa TB Green kit, and each 20 µL reaction solution was transferred into 100 µL white eight-connected tubes, after which they were detected by Roche LightCycler® 96 (Basel, Switzerland) following two-step RT-qPCR procedure in Table S5. Based on the output data from Roche software, the differential expression multiples of target genes between samples and controls were calculated and analyzed by the method of comparative Cq value and F value showed in the following formulas. (1)△△Cq=△Cq sample− △Cq control ; ༈2༉F = 2 −△△Cq Note F equaled multiple change of target gene expression between tested samples and control ones. When F > 1, it meant that the expression of the tested sample was up-regulated by F times compared with the control one; When 1 > F > 0, it meant that the expression level of the tested sample was down-regulated by 1/F times compared with the control one. 2.7.7 Transcriptome analysis for SBIP element genes Both 50 mL the sample NZ9000/SBIP and the control NZ9000/p-p with 3 parallels were cultured as 2.7.6 said. After 16 h, bacteria solution was firstly centrifugated at 5000 xg, 4℃ for 15 min, then cell precipitation was harvested and washed with 50 mM PBS. Finally, the six samples would be sent to Majorbio Bio-pharm Tech (Shanghai, China) to accomplish the transcriptome sequencing and analysis after frozen at -80℃ one night. I. Differential expression analysis of genes between NZ9000/SBIP and NZ9000/p-p was conducted to identify differential expression genes between samples with volcano plot and statistics bar. II. Gene set analysis of similar and special genes was performed by Go and KEGG such annotations classification or enrichment analysis. 2.7.8 Statistical analysis All the measured data in this study were expressed in the form of “Mean ± SD”. SPSS 20.0 was firstly applied to conduct one-way ANOVA with t-test and Duncan multiple range test for samples differences within and among groups (P > 0.05 means no significant difference while P < 0.05 means significant difference and P < 0.01 extremely significant difference), then the contrast difference significance was presented directly through the bar chart by Graph Pad Prism 6. 3. Results 3.1 The construction and growth comparison of NZ9000/SBIP and other strains As Fig. S1 clearly shown, the AGE results of NZ9000/SBIP and most other strains were in line with expectations after endonuclease digestion, and the DNA sequencing results from BGI further confirmed the accuracy. However, in Fig. S2: pC5ABD and pCPABD transformants both lost about 3000 bp, while pG59FS1 transformant also lost 96 bp, and thus they failed to be screened out. Among the generated different recombinant L. lactis strains in Fig. 3 , several typical representatives were monitored within 24 h to find their growth trends. As following Fig. 4 showed, NZ9000/p8G adapted to grow more slowly than others before the first 4 h and the growth increased significantly after 6 h; The introduction of pNZ8148 into NZ9000 had little effect on the growth trend, and their logarithmic phase were both from 2 to 8 h, during which the growth increased significantly after 4 h; Compared with NZ9000 and NZ9000/pNZ8148, the growth force became weaker after the introduction of pTRKH2 into NZ9000 or double plasmids including it. Consequently, the growth rate of NZ9000/p-p was between NZ9000/pNZ8148 and NZ9000/pTRKH2 though NZ9000/SBIP carrying optimal combination of promoters presented better growth activity than NZ9000/p-p, which was similar to that of Tiange Ma’s study[50]. Unlike NZ9000/p-p, it was known that the growth curve of NZ9000/SBIP carrying protein genes and promoters was similar to that of NZ9000/pNZ8148. 3.2 Preliminary verification of expression of the target gene elements 3.2.1 SDS-PAGE analysis of regulatory genes by L. lactis NZ9000/pC1ABD The expression of the regulatory factors in the optimal NZ9000/pC1ABD was analyzed by SDS-PAGE. As shown in Fig. S3, compared with NZ9000/pNZ8148 under non-induced and Nisin induced culturing (lane 1 and 2), both NZ9000/pC1ABD without and with Nisin induction (lane 3 and 4) showed obvious overexpressed bands, corresponding well with the calculated theoretical molecular weights of SelA, SelB and SelD, which are 50.62 kDa (about 51kDa), 68.88 kDa (about 69kDa) and 36.69 kDa (about 37kDa), respectively. However, SelA showed much thicker band than SelB and SelD, probably due to that P1 was ligated in front of SelA and far away from SelB and SelD. To sum up, SelA, SelB and SelD on the regulatory plasmid was preliminarily confirmed to be expressed in NZ9000, but whether they promoted to assist UGA translated into Sec would be furtherly verified with the optimal verifiable plasmid. 3.2.2 Expression of GshF (SA) by L. lactis NZ9000/p8G According to the practically weak growth force of NZ9000/p8G in Fig. 4 , this strain was harvested at 6 h, 12 h, 18 h, 24 h and 30 h, and verified uniformly compared with constitutive expression after cultured for 24 h in medium GGC-GM17[37, 38]. Consequently, no obvious protein production was found when NZ9000/p8G only cultured 6h during prelogarithmic phase in Fig. S4-a, but the protein production was obvious at 12 h, and stable at 18 h ~ 30 h. Through comparing lane 7 with lane 5, we found that GshF band was thicker in medium GGC-GM17, indicating that the addition of substrate such as Glu with a final concentration of 10 mM was conducive to the expression of GshF[51]. Then GSH concentration and GshF enzyme activity of NZ9000/p8G cultured by GM17 at five different time was measured. And the yield of GSH was found to be positively correlated with the enzyme activity of GshF in Fig. S5. the expression of GshF (SA) by NZ9000/p8G cultured for 24 h were similar to that of E. coli BL21/pET28a(+)-gshFSA[46], from which GshF (SA) was assayed as 1.242 U/mL or 0.1275 U/mg. 3.2.3 The fusion expression of FDH-sfGFP by L. lactis recombinant strains The expression of fusion protein FDH-sfGFP to light up reporter gene sfGFP were mainly decided by ligating promoter P23[26] or P59[27] in front of FDH on pGFS1. Since they have been both verified with good application effect, the optimal fluorescent strain and optimal time for FI determination would be analyzed at five different time from 0 h ~ 30 h. As can be seen from Fig. S6, the FI of NZ9000/pG23FS1 was significantly stronger than that of NZ9000/pG59FS1, stating that P23 should be the preferred promoter for the expression of FDH-sfGFP. Moreover, the FI kept increasing before 18 h and remained at a stable level after 18 h, which would be picked to harvest the strains and conduct the measurement as recorded in Table 1 . To further verify NZ9000/pG23FS1, the SDS-PAGE analysis of fused FDH-sfGFP (106 kDa) at five different time could be seen from Fig. S4-b. Those recombinant L. lactis strains were found to have a better expression of GshF and fused FDH-sfGFP after cultured for 24 h from Fig. S4-a and Fig. S4-b. Then on the backbone of the preferred pG23FS1, P8 and P32 would be ligated to screen a better one for the combination with P23. As shown in Table 1 , the FI of the recombinant NZ9000/p8G23FS1 was superior to NZ9000/p32G23FS1, but much lower than NZ9000/pG23FS1. Besides, superior expression of GshF and fused FDH-sfGFP by P8 + P23 to P32 + P23 could be seen by SDS-PAGE analysis in Fig. S4-c and from the results of GshF enzyme activity assay: the former 0.1990 U/mg was higher than the later 0.1038 U/mg, but lower than NZ9000/p8G. Table 1 FI analysis of recombinant L. lactis strains in this study Recombinant L. lactis NZ9000 with sfGFP FI/OD 600 NZ9000/pG23FS1 2431.46 ± 2.19 NZ9000/pG59FS1 274.53 ± 0.70 NZ9000/p8G23FS1 381.68 ± 0.54 NZ9000/p32G23FS1 344.49 ± 0.72 NZ9000/ SBIP-sfGFP 362.25 ± 0.43 3.3 Verification analysis of differences after optimized expression for NZ9000 SBIP 3.3.1 Expression optimization and SDS-PAGE analysis for SBIP in L. lactis Based on the screened results from 3.1 and 3.2.3 and the afterwards optimal recombinant plasmids combination, NZ9000/SBIP-sfGFP was NZ9000/(pC1ABD + p8G23FS1) and NZ9000/SBIP was NZ9000/(pC1ABD + p8G23FS2). According to the growth trend of NZ9000/SBIP in Fig. 4 , its expression optimization would depend on differed induction condition and medium composition as Fig. S7-a represented. In this work, the four cultivation methods were compared simultaneously and were all analyzed by SDS-PAGE. As expected, the target protein bands of 37 kDa, 51 kDa, 69 kDa, 79 kDa and 86 kDa could be obviously seen in lane 2 ~ 5 of Fig. 5 , indicating that SelD, SelA, SelB, FDH and GshF were all successfully expressed in NZ9000/SBIP. Moreover, the target bands of lane 3 were more obvious than others, indicating NZ9000/SBIP cultured in GGC-GM17 with Nisin induction and Na 2 SeO 3 addition[1] was optimal. 3.3.2 Differential analysis of enzyme activity In this work, NZ9000/SBIP-sfGFP was set as one control to find out the enzyme activity difference between the fused FDH-sfGFP from it and FDH from L. lactis NZ9000/SBIP under the existing of SelA ~ SelD. Meanwhile, NZ9000/NoABCD (i.e. NZ9000/p8G23FS2) was set as another control to analyze whether FDH could produce enzyme activity without SelA ~ SelD. According to Fig. 6 , the enzyme activity of FDH could both be detected out by NZ9000/SBIP and NZ9000/SBIP-sfGFP under different culture modes. As Table 2 stated, the effect of independent expression of FDH was two orders of magnitude higher than that of FDH-sfGFP, while the negative control NZ9000/NoABCD showed trace enzyme activity in the absence of SelA ~ SelD, indicating that there might be other mechanisms in L. lactis background to support the random and slight insertion of Sec in FDH. Among the four culturing modes as Fig. S7-a showed, FDH of NZ9000/SBIP 4 had the highest enzyme activity (28.11 mU/mg), but was significantly different from that of Rémi Thomé (0.32 U/mg)[52] and MJ Axley (1.4 U/mg)[53]; As one comparison, the enzyme activity decreased by 4 times (about 6.90 mU/mg) without induction. And the addition of Na 2 SeO 3 could slightly weaken the enzyme activity of FDH since that of SBIP 3 was a bit lower than SBIP 2’s, indicating that selenium would affect the level of enzyme activity[54, 55]. Moreover, when NZ9000 strain existed only one p8G23FS1 or p8G23FS2 and regardless of whether pC1ABD existed or not, the enzyme activity of GshF in these 8 groups were all distributed around 200 mU/mg and had no significant difference, while the addition of Na 2 SeO 3 also had a slight negative effect on it. In conclusion, the SBIP could enable Sec with TGA codon at 418–420 site of selenoprotein FDH to be expressed in L. lactis with directional insertion, and the expression effect of NZ9000/SBIP was the best when cultured by GGC-GM17 with Nisin induction and Na 2 SeO 3 addition. Table 2 Assay of FDH and GshF from crude enzyme extract of NZ9000/SBIP and its controls NZ9000 Samples Enzyme activity (Mean ± SD, mU/mL) Protein (Mean ± SD, mg/mL) Specific activity ( Mean ± SD, mU/mg ) FDH GshF Crude enzyme extract FDH GshF SBIP 1 18.6610 ± 0.0538 d 514.6075 ± 1.5820 d 2.7031 ± 0.2703 6.9036 ± 0.0056 d 190.3790 ± 0.5882 f SBIP 2 56.9473 ± 0.2329 b 668.5793 ± 2.3449 a 2.8023 ± 0.1460 20.3218 ± 0.0994 b 208.7811 ± 0.6815 b SBIP 3 54.2308 ± 0.3696 c 550.4916 ± 1.1651 c 3.2023 ± 0.1920 16.9349 ± 0.0829 c 196.4446 ± 0.5425 e SBIP 4 84.2076 ± 0.4114 a 657.3410 ± 3.2301 b 2.9952 ± 0.1677 28.1146 ± 0.1249 a 219.4677 ± 0.7930 a SBIP-sfGFP 1 0.4041 ± 0.0051 e 456.4654 ± 2.5675 h 2.3028 ± 0.3108 0.1755 ± 0.0018 e 191.7217 ± 0.7792 f SBIP-sfGFP 2 0.6859 ± 0.0062 e 480.8223 ± 2.4187 e 2.5079 ± 0.3972 0.2735 ± 0.0021 e 198.2211 ± 0.5523 d NoABCD 1 0.1242 ± 0.0034 f 470.1924 ± 2.7635 f 2.4067 ± 0.1225 0.0516 ± 0.0014 f 195.3682 ± 1.1719 e NoABCD 2 0.1240 ± 0.0033 f 460.7237 ± 2.7150 e 2.2999 ± 0.4188 0.0539 ± 0.0014 f 200.3270 ± 1.1160 c Note: “SBIP 1” represented NZ9000/SBIP cultured without induction; “SBIP 2” represented NZ9000/SBIP cultured with Nisin induction; “SBIP 3” represented NZ9000/SBIP cultured with Nisin induction and Na 2 SeO 3 addition; “SBIP 4” represented NZ9000/SBIP cultured by GGC-GM17 with Nisin induction and Na 2 SeO 3 addition; “SBIP-sfGFP 1” represented NZ9000/SBIP-sfGFP cultured without induction; “SBIP-sfGFP 2” represented NZ9000/SBIP-sfGFP cultured with Nisin induction; “NoABCD 1” represented NZ9000/p8G23FS2 cultured without induction; “NoABCD 2” represented NZ9000/p8G23FS2 cultured with Nisin induction. The last four groups serve as controls to illustrate the FDH expression problem. Differences among groups were compared using Duncan multiple range test of one-way ANOVA: "a” ~ “f” represented a diminishing significant difference. 3.3.3 RT-qPCR analysis for SBIP in L. lactis Because SelA ~ SelD couldn’t be assayed like the other two enzymes, RT-qPCR would be an suitably applicable method to further verify the feasibility to produce site-inserted Sec by constructing SBIP in L. lactis . As Fig. S8 showed, the 23S (1450 bp), 16S (770 bp) and 5S (120 bp) bands were clear, bright and complete for both the sample NZ9000/SBIP and the control NZ9000/p-p, and the OD 260 /OD 280 was all between the ideal 1.8 ~ 2.0, proving that total RNA had high purity. Secondly, the shape and trend of RT-qPCR amplification curves displayed by Fig. S9 of the six target protein genes were consistent, indicating that their amplification efficiency was similar. According to F values calculated with the formulas in 2.7.6, the target genes of SelA, SelB, SelC, SelD, GshF and FDH from NZ9000/SBIP was up-regulated by 8.01, 19.03, 925982.32, 34.51, 31879.16 and 28367.04 multiples compared with the control NZ9000/p-p, indicating that all these inserted factors were all proved to play roles along the route of SBIP in L. lactis . 3.4 Transcriptome analysis of NZ9000/SBIP and NZ9000/p-p 3.4.1 Statistical analysis of differential genes between NZ9000/SBIP and NZ9000/p-p Differential genes between NZ9000/SBIP and NZ9000/p-p would be calculated by statistical expression amount. As shown in Fig. S10, compared with NZ9000/p-p, NZ9000/SBIP had a total of 358 differential genes, among which 211 up-regulated genes, including the six target genes SelA, SelB, SelC, SelD, GshF and FDH. The up-regulation of respective 23.2, 19.86, 8.19, 22.80, 18.0 and 21.60 fold compared with NZ9000/p-p, consisted well with the results of RT-qPCR analysis in 3.3.3. All in all, NZ9000/SBIP was successfully constructed through the whole work in this study, indicating that selenium metabolic flow was successfully guided to synthesize Sec-containing selenoprotein by introducing SBIP into LAB. 3.4.2 GO analysis of similar genes compared with NZ9000 reference genome GO (Gene Ontology) annotation of similar genes shared by NZ9000/SBIP, NZ9000/p-p and NZ9000 reference genomes was shown in Fig. S11. Firstly, according to BP (Biological Process) statistics, genes involved in peptide and protein transport accounted for 16.38%, while phosphorylation 15.55%. Secondly, according to MF (Molecular Function) statistics, genes involved in ATP binding, DNA binding and nucleic acid binding in target bacteria accounted for 23.75%, 21.61% and 4.55%, respectively. And based on the statistical information of transcriptome differential genes, it was found that PLP (pyridoxal phosphate) dependent aminotransferase involved in phosphorylation was also an up-regulated gene, which should have a synergistic effect with SelA, a member of the folded type I superprotein family of PLP dependent enzymes[56, 57]; ATP binding gene functions are mainly involved in phosphorylase, peptide transporter binding protein, amino acid transporter binding protein, etc. Although SelD is also an ATP binding protein, and there have also been reports proposing that some LAB (e.g. Enterococcus faecalis , Leuconostoc citreum , Lactobacillus futsaii ) contained SelA and SelD genes[58, 59], they were not yet found in the reference genome of the background bacterium NZ9000[60] (GenBank: CP002094.1). 3.4.3 KEGG analysis of differential genes and SBIP route created in LAB KEGG (Kyoto Encyclopedia of Genes and Genomes) annotations analysis was performed on the differential genes set between NZ9000/SBIP and NZ9000/p-p. The distribution of the major biochemical metabolic pathways and signal transduction pathways in which these differential genes participated was shown in Fig. S12. Among them, most genes involved in amino acid metabolism, accounting for 12.33%. However, the results of the KEGG pathway enrichment analysis for differential genes between NZ9000/SBIP and NZ9000/p-p were shown as Fig. 7 a: The introduction of SBIP into NZ9000 had a great effect on the intracellular amino acid metabolism of L. lactis , and there were significant differences in the amino acid metabolism of valine, leucine, isoleucine, arginine and others. Meanwhile, it also had a great impact on selenocompound metabolism. Based on the transcriptome differential gene statistics, there were 5 selenocompounds having significant differences, including SelA, SelB, SelC, SelD and FDH. In summary, those key gene tools non-existed in NZ9000 genome originally were all working along the metabolic pathway after introduced. Moreover, the amino acid synthesis and metabolic pathway were significantly restructed after the introduction of SBIP, which was consistent with the directed synthesis of selenoprotein. At last, the heterologous SBIP route created in LAB was shown by Fig. 7 b. 4. Discussion 4.1 Differences between fusion expression and independent expression As well-known, green fluorescent protein (GFP) has been generally used as a marker and a fusion tag, but the folding rate of wild-type GFP could be affected by temperature, and the fusion partner would reduce its folding efficiency and FI[61]. Therefore, a more stable GFP mutant sfGFP (super-folder GFP) was used in this study. The FI was reported to be proportional to the total expression, and would not be affected by misfolding of the fusion partner, and was independent of the solubility of the fusion protein[62, 63]. According to Fig. 6 in this study, the enzyme activity of FDH could both be detected out by NZ9000/SBIP and NZ9000/SBIP-sfGFP under different culture modes, but the latter one was much lower than that of the former one. This result was inconsistent with the case by KV Solovyov et al.[64], and was presumed to be affected by the promoters used, because strong promoters do not always lead to higher expression levels due to the burden and toxicity of some target proteins on host cells[65], and the same promoter may even behave differently for different proteins[66]. Moreover, the expression of introduced gene protein might also impose an adverse effect on the NICE system in L. lactis [67]. 4.2 The addition of amino acids and the red selenium phenomenon As a rule, the optimization of protein expression generally starts from four aspects: induction time, concentration of inducer, culture temperature, and composition of medium (such as metal ions, amino acids, ATP, special nitrogen sources or carbon sources)[46, 68]. Accordingly, a new phenomenon was found when expression optimization of NZ9000/SBIP was conducted by four cultivation methods. As Fig. S7-a showed, the red selenium phenomenon of sample ④ (Nisin induction and Na 2 SeO 3 addition with GGC-GM17 medium) was more significant than that of ③ (Nisin induction and Na 2 SeO 3 addition with GM17 medium). In order to figure out which amino acid was responsible for the deepest Se-enriched color in this strain, another three cultivation methods shown in Fig. S7-b were used to illustrate it. And it turned out that the red color of cells solution gradually lightened when GM17 medium was added with Glu, Gly and Cys, respectively. Se(Ⅳ) might cause oxidation damage to these AAs, while Se(Ⅳ) was reduced to red nano-selenium which would bound to the cell membrane of the bacteria[69, 70]. 4.3 Gene loss during construction of recombinant L. lactis strains To weaken the cell wall of gram-positive bacteria and improve the transformation efficiency, lysozyme, mutanolysin, penicillin or glycine would be added to the culture-medium[43, 71]. The previous issue about electroporation was mainly experimental conditions, while the reasons for partial gene fragment loss when heterologous plasmids were electroporated into LAB have been few reported. Although the plasmid pIL253 transformant (4.8 kilobases) from L. lactis was found to miss a multiple cloning site which equaled 0.8 kilobase[71], it was hard to be explained. Unlike the accurately expected Fig. S1 , the similar issues occurred in this study in Fig. S2: pC5ABD and pCPABD transformants was presumed that SelA-SelB (3233bp) or SelB-SelD (2889 bp) might be deleted during the electroporation, while pG59FS1 transformant also lost 96 bp on the 3’ end of P59. However, Andreas Schӓfer et al.[72] proposed that gene disruption and allelic exchange would be facilitated by homologous recombination, while plasmids equipped with a genetically modified sacB gene would not be lost from Gram − to Gram + , which offers valuable suggestions for solving these problems in the future. 4.4 The codon selection for Sec insertion and selenoprotein production Up to now, two types of codons have been reported for encoding Sec: (1) Stop codon (UAG or UGA): First, site-specific insertion of Sec into selenoproteins by heterozygous tRNA is mainly dependent on UAG encoding[25, 73]. The stop codon UAG has encoded many UAAs (unnatural amino acids) with different functions and structures, and achieved their site-specific insertion in proteins[74, 75]. However, a single UAG cannot meet the requirements of inserting multiple UAAs into a protein at the same time when research continues to deepen, and the number of stop codons determines that the reuse of it is limited[76]. Second, the stop codon UGA can be recognized as a meaningful codon to encode Sec under certain conditions although its read-through efficiency is less than 5% in prokaryotes and less than 3% in eukaryotes[77]. (2) Sense codon: Markus J. BrÖcker[78] found that E.coli could decode 55 sense codons into Sec via the prokaryotic read-through mechanism and Sec encoding didn’t strictly depend on specific codons. Therefore, considering the + 1 frameshift[79] exists, Yan Qi[80] tried to express human GPx1 to realize the in vitro encoding of Sec with quadruplet codons. 5. Conclusion In this study, a multistage metabolic engineering strategy to construct SBIP in L. lactis NZ9000 for site-directed biosynthesis of Sec-containing selenoprotein was proposed for the first time. By the means of DNA manipulation, the building of recombinant plasmids equipped with the regulatory factors and verifiable factors, and the optimal recombinant plasmids combination to achieve the successful construction of SBIP by L. lactis , which would be verified by multidimensional determination and analysis. The result turned out that: SelA, SelB, SelC, SelD, GshF and FDH from NZ9000/SBIP up-regulated 8.01, 19.03, 925982.32, 34.51, 31879.16 and 28367.04 multiples compared with NZ9000/p-p; FI/OD 600 of NZ9000/ SBIP-sfGFP was 362.25 ± 0.43; FDH enzyme activity of NZ9000/SBIP reached 28.11 ± 0.12 mU/mg, and GshF 219.47 ± 0.79 mU/mg under the optimal expression.. The mechanism of selenoprotein synthesis by LAB was analyzed: NZ9000/NoABCD with SBIP unpresent detected a very small amount of FDH enzyme activity, indicating a few random incorporation of Sec. However, the enzyme activity and transcription level of Sec-containing FDH were significantly improved after pathway inserted and optimized culturing, and the amino acid anabolic pathway was the most significant, indicating the increased incorporation rate of Sec in LAB selenoprotein by directed synthesis. This was such a pioneered case to offer an innovative reference for the engineering transformation and production of specific selenoprotein in the food, fodder or drug supply field. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Funding This work was financially supported by the National Natural Science Foundation of China General Project (31972050), which should be sincerely acknowledged. Author Contribution J.P., Y.Q., L.L., S.Y.,P.S., Y.F.,X.P. and F.Y.searched and collected the literatures; J.P., Y.Q., L.L, S.Y., P.S., L.W., C.L. and T.G. completed investigation, analysis and experiment; J.P. made the tables and figures and wrote the original manuscript; C.W. , X.L and X.H. supervised the project and reviewed the manuscript; C.W. got the project from government and edited the draft. All authors read and approved the manuscript. Data availability Data will be made available on request. References Liang X, Xue Z, Zheng Y, Li S, Zhou L, Cao L, Zou Y: Selenium supplementation enhanced the expression of selenoproteins in hippocampus and played a neuroprotective role in LPS-induced neuroinflammation. International Journal of Biological Macromolecules: Structure, Function and Interactions 2023. Schrauzer GN: Nutritional selenium supplements:product types, quality and safety. Journal of the American College of Nutrition 2001, 20: 1-4. Infante HG, Hearn R, Catterick T: Current mass spectrometry strategies for selenium speciation in dietary sources of high-selenium. Analytical and bioanalytical chemistry 2005, 382: 957-967. Stadtman TC: Selenocysteine. Annual Review of Biochemistry 1996, 65: 83-100. Wang C: Generation of Selenoprotein (Selenopeptide) and its Antioxidant Ability Jilin University2014. Renga W, Dong L, Qihang C, Zhengshan L, Jingwen Z, Jian M: Optimization of vanillin biosynthesis in Escherichia coli K12 MG1655 through metabolic engineering. Bioresource Technology 2024, 411 . Calomme MR, Branden KVD, Berghe DAV: Selenium and lactobacillus species. Journal of Applied Microbiology 1995, 79: 331-340. Zhang C, Li Y, Zhu F, Li Z, Chen N: Metabolic engineering of an auto-regulated Corynebacterium glutamicum chassis for biosynthesis of 5-aminolevulinic acid. Bioresource Technology 2020 : 124064. Jingjing P, Shiyang Y, Yuhui F, Xiaoling L, Chenghua W: Mechanisms affecting the biosynthesis and incorporation rate of selenocysteine. Molecules 2021, 26: 7120. Zhang J, Fu RY, Hugenholtz J, Li Y, Chen J: Glutathione protects Lactococcus lactis against acid stress. Applied and Environmental Microbiology 2007, 73: 5268-5275. Arnér E, Sarioglu H, Lottspeich F, Holmgren A, Böck A: High-level expression in Escherichia coli of selenocysteine-containing rat thioredoxin reductase utilizing gene fusions with engineered bacterial-type SECIS elements and co-expression with the selA, selB and selC genes. Journal of Molecular Biology 1999, 292: 1003-1016. Li M, Meng X, Sun Z, Zhu C, Ji H: Effects of NADH Availability on 3-Phenyllactic Acid Production by Lactobacillus plantarum Expressing Formate Dehydrogenase. Current Microbiology 2019, 76: 706-712. Heider J, Baron C, Böck A: Coding from a distance: dissection of the mRNA determinants required for the incorporation of selenocysteine into protein. The EMBO Journal 1992, 11: 3759-3766. Zhang M, Jiang L, Zhang Y: Advance on Bioinformatic Analysis of Selenium Metabolic Network and Selenoproteomes. Current Biotechnology 2017, 7: 537-543. Mao R, Wu D, Wang Y: Surface display on lactic acid bacteria without genetic modification: strategies and applications. Applied Microbiology and Biotechnology 2016, 100: 9407-9421. Bolotin A, Wincker P, Mauger S, Jaillon O, Malarme K, Weissenbach J, Ehrlich SD, Sorokin A: The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp. lactis IL1403. Genome Research 2001, 11: 731-753. Xiong ZQ, Wei YY, Kong LH, Song X, Ai LZ: Short communication: An inducible CRISPR/dCas9 gene repression system in Lactococcus lactis. Journal of Dairy Science 2019, 103: 161-165. Kuipers OP, Beerthuyzen MM, Ruyter PD, Luesink EJ, Vos WD: Autoregulation of Nisin Biosynthesis in Lactococcus lactis by Signal Transduction. Journal of Biological Chemistry 1995, 270: 27299-27304. Gasson MJ: Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing. Journal of Bacteriology 1983, 154: 1-9. Hui W, Xiao L, Linlin H, Yicheng F, Zhiqiang X, Lianzhong A, Xin S: Progress in Research on Lactococcus lactis Expression Systems and Their Promoter Regulatory Elements. Journal of Food Science 2022, 43: 330-336. Soumya MP, Parameswaran R, Nampoothiri KM: Nisin controlled homologous Over-expression of an exopolysaccharide biosynthetic glycosyltransferase gene for enhanced EPS production in Lactobacillus plantarum BR2. Bioresource Technology 2023, 385: 129387. Song AAL, Lionel LAI, Lim SHE, Rahim RA: A review on Lactococcus lactis: from food to factory. Microbial Cell Factories 2017, 16: 1-15. Zhu D, Liu F, Xu H, Bai Y, Zhang X, Saris PEJ, Qiao M: Isolation of strong constitutive promoters from Lactococcus lactis subsp. lactis N8. FEMS Microbiology Letters 2015, 362: fnv107. Vossen JVD, Lelie DVD, Venema G: Isolation and characterization of Streptococcus cremoris Wg2-specific promoters. Applied and Environmental Microbiology 1987, 53: 2452-2457. Mukai T, Sevostyanova A, Suzuki T, Fu X, Söll D: A Facile Method for Producing Selenocysteine-Containing Proteins. Angewandte Chemie-International Edition 2018, 57: 7215-7219. Lu L, Lianzhong A, Yongjun X, Zhiqiang X, Tong G, Xin S: A reporter system with the enhanced green fluorescent protein in Lactococcus lactis NZ9000. Food and Fermentation Industries 2020, 46: 6. Martinez-Jaramillo E, Garza-Morales R, Loera-Arias M, Saucedo-Cardenas O, Montes-de-Oca-Luna R, McNally L, Gomez-Gutierrez J: Development of Lactococcus lactis encoding fluorescent proteins, GFP, mCherry and iRFP regulated by the nisin-controlled gene expression system. Biotechnic & Histochemistry 2017, 92: 167-174. Gaeng S, Scherer S, Neve H, Loessner MJ: Gene cloning and expression and secretion of Listeria monocytogenes bacteriophage-lytic enzymes in Lactococcus lactis. Applied and Environmental Microbiology 2000, 66: 2951-2958. Manzine LR, Cassago A, da Silva MTA, Thiemann OH: An efficient protocol for the production of tRNA-free recombinant Selenocysteine Synthase (SELA) from Escherichia coli and its biophysical characterization. Protein expression and purification 2013, 88: 80-84. Haruna K-i, Alkazemi MH, Liu Y, Söll D, Englert M: Engineering the elongation factor Tu for efficient selenoprotein synthesis. Nucleic acids research 2014, 42: 9976-9983. Mansell JB, Guévremont D, Poole ES, Tate WP: A dynamic competition between release factor 2 and the tRNASec decoding UGA at the recoding site of Escherichia coli formate dehydrogenase H. The EMBO journal 2001. Noinaj, Nicholas: Structural Insights into the Catalytic Mechanism of Escherichia coli Selenophosphate Synthetase. Journal of Bacteriology 2012. Gursinsky T, Grobe D, Schierhorn A, Jager J, Andreesen JR, Sohling B: Factors and Selenocysteine Insertion Sequence Requirements for the Synthesis of Selenoproteins from a Gram-Positive Anaerobe in Escherichia coli. Applied & Environmental Microbiology 2008, 74: 1385-1393. Xiuxiu F, Jingyan W: Efficient Expression of Glutathione Peroxidase with Chimeric tRNA in Amber-less Escherichia coli. ACS Synthetic Biology 2018, 7 . Mierau I, Kleerebezem M: 10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis. Applied Microbiology and Biotechnology 2005, 68: 705-717. O'Sullivan DJ, Klaenhammer TR: High-and low-copy-number Lactococcus shuttle cloning vectors with features for clone screening. Gene 1993, 137: 227-231. Xu C, Shi Z, Shao J, Yu C, Xu Z: Metabolic engineering of Lactococcus lactis for high level accumulation of glutathione and S-adenosyl-L-methionine. World Journal of Microbiology and Biotechnology 2019, 35: 185. Rui-yan F, Jian C, Yin L: Production of glutathione by recombinant Lactococcus lactis. Chinese Journal of Bioprocess Engineering 2004, 2: 30-35. Berry MJ, Banu L, Chen Y, Mandel SJ, Kieffer JD, Harney JW, Larsen PR: Recognition of UGA as a selenocysteine codon in type I deiodinase requires sequences in the 3′ untranslated region. Nature 1991, 353: 273-276. Fourmy D, Guittet E, Yoshizawa S: Structure of prokaryotic SECIS mRNA hairpin and its interaction with elongation factor SelB. Journal of Molecular Biology 2002, 324: 137-150. Pecher A, Zinoni F, Bck A: The seleno-polypeptide of formic dehydrogenase (formate hydrogen-lyase linked) from Escherichia coli: genetic analysis. Archives of Microbiology 1985, 141: 359-363. Willis T, Jadayel D, Coignet L, Abdul-Rauf M, Treleaven J, Catovsky D, Dyer M: Rapid molecular cloning of rearrangements of the IGHJ locus using long-distance inverse polymerase chain reaction. Blood, The Journal of the American Society of Hematology 1997, 90: 2456-2464. Dayong R, Chang L, Yan-qing Q, Shou-wen D, Huan-huan G, Hong-feng L, Yang L, Ning-yi J: Optimization of High-Efficiency Electrotransformation Conditions for Lactococcus lactis. Chinese Journal of Veterinary Medicine 2012, 46: 5-9. Pieniz S, Okeke BC, Andreazza R, Brandelli A: Evaluation of selenite bioremoval from liquid culture by Enterococcus species. Microbiological Research 2011, 166: 176-185. Xiao F, Wang H, Shi Z, Huang Q, Xu Z: Multi-level metabolic engineering of Pseudomonas mutabilis ATCC31014 for efficient production of biotin. Metabolic Engineering 2019, 61: 406-415. Liu X: Directed evolution of glutathione bifunctional svnthase and its application in L-Glutathione synthesis. Zhejiang University2018. Slusarczyk H, Felber S, Kula MR, Pohl M: Stabilization of NAD‐dependent formate dehydrogenase from Candida boidinii by site‐directed mutagenesis of cysteine residues. European Journal of Biochemistry 2000, 267: 1280-1289. Zhenhua Z, Yuli X, Tiejun W, Hong Z, Cunduo T, Yunchao K, Lunguang Y: Directed evolution of catalytic activity of formate dehydrogenase and its efficient expression. Chinese Journal of Applied Chemistry 2021, 38: 704. Zhu Z: Efficient identification of anti-acid components in Lactococcus lactis and its function analysis. Jiangnan University2019. Ma T, Lu J, Zhu J, Li X, Gu H, Montalbán-López M, Wu X, Luo S, Zhao Y, Jiang S: The Secretion of Streptomyces monbaraensis Transglutaminase From Lactococcus lactis and Immobilization on porous magnetic nanoparticles. Frontiers in Microbiology 2019, 10: 1675. Binbin C: Cloning, expression, characteristics andapplication of bifunctional glutathione synthetase. Zhejiang University, College Of Chemical&Biological Engineering; 2015. Thomé R, Gust A, Toci R, Mendel R, Bittner F, Magalon A, Walburger A: A sulfurtransferase is essential for activity of formate dehydrogenases in Escherichia coli. Journal of Biological Chemistry 2012, 287: 4671-4678. Axley M, Grahame DA, Stadtman TC: Escherichia coli formate-hydrogen lyase. Purification and properties of the selenium-dependent formate dehydrogenase component. Journal of Biological Chemistry 1990, 265: 18213-18218. Ashrafi H, Sadeghi AA, Chamani M: Effect of Organic Selenium Supplementation on the Antioxidant Status, Immune Response, and the Relative Expression of IL-2 and IFN-γ Genes in Ewes During the Hot Season. Biological Trace Element Research 2024, 202: 2052-2061. Caiyi S, Yuting L, Xiaomin L, Yihua W, Guangzhi F, Yu S: Effect of Selenium on Extracellular Enzyme Activities of Pleurotus ostreatus. Journal of Anhui Agricultural Sciences 2023, 51: 42-44. Araiso Y, Palioura S, Ishitani R, Sherrer RL, O’Donoghue P, Yuan J, Oshikane H, Domae N, DeFranco J, Söll D: Structural insights into RNA-dependent eukaryal and archaeal selenocysteine formation. Nucleic Acids Research 2008, 36: 1187-1199. Ganichkin OM, Xu XM, Carlson BA, Mix H, Hatfield DL, Gladyshev VN, Wahl MC: Structure and catalytic mechanism of eukaryotic selenocysteine synthase. Journal of Biological Chemistry 2008, 283: 5849-5865. Yan Z, Romero H, Salinas G, Gladyshev VN: Dynamic evolution of selenocysteine utilization in bacteria: a balance between selenoprotein loss and evolution of selenocysteine from redox active cysteine residues. Genome Biology 2006, 7: R94. Morales AE, González L, Contreras EL, Serrano GR: SelA and SelD genes involved in selenium absorption metabolism in lactic acid bacteria isolated from Mexican cheeses. International Dairy Journal 2019, 103: 104629. Linares DM, Kok J, Poolman B: Genome Sequences of Lactococcus lactis MG1363 (Revised) and NZ9000 and Comparative Physiological Studies. Journal of Bacteriology 2010, 192: 5806-5812. Huang C, Cheng Y, Lixin MA, Yan H: The expression of methyl parathion hydrolase fusion with gfp in E. coli. Journal of Hubei University(Natural Science) 2019. Zimmer M: Green fluorescent protein (GFP): applications, structure, and related photophysical behavior. Cheminform 2002, 102: 759-781. Schaefer SK, Heidemann J, Puchert A, Koelbel K: Crystal structure of a domain-swapped photoactivatable sfGFP variant provides evidence for GFP folding pathway. FEBS Journal 2019, 286: 2329-2340. Solovyov KV, Kern AM, Grudinina NA, Aleynikova TD: Genetic Structures and Conditions of their Expression, which Allow Receiving Native Recombinant Proteins with High Output. International Journal of Biomedicine 2012, 2: 78-81. Hannig G, Makrides SC: Strategies for optimizing heterologous protein expression in Escherichia coli. Trends in Biotechnology 1998, 16: 54-60. Mutalik VK, Guimaraes JC, Cambray G, Mai QA, Christoffersen MJ, Martin L, Yu A, Lam C, Rodriguez C, Bennett G: Quantitative estimation of activity and quality for collections of functional genetic elements. Nature Methods 2013, 10: 347. Liang X, Sun Z, Zhong J, Zhang Q, Huan L: Adverse effect of nisin resistance protein on nisin-induced expression system in Lactococcus lactis. Microbiological Research 2010, 165: 458-465. JiaHui L: Expression and Optimization of the Novel Bifunctional.Glutathione Synthetase in Pichia pastoris and Escherichia coli. Zhejiang University2019. Fang Y, Zhi H, Zhenjiang G, Wenjie Z: Oxidation of phycocyanin molecules by Se(IV) and formation of nano-Se(0). Marine Sciences 2010, 34: 55-65. Sun Y, Wang H, Zhou L, Chang M, Yue T, Yuan Y, Shi Y: Distribution characteristics of organic selenium in Se-enriched Lactobacillus (Lactobacillus paracasei). LWT Food Science and Technology 2022, 165: 113699. Holo H, Nes IF: High-Frequency Transformation, by Electroporation, of Lactococcus lactis subsp. cremoris Grown with Glycine in Osmotically Stabilized Media. Applied and Environmental Microbiology 1990, 55: 3119-3123. Schaefer A, Tauch A, Jaeger W, Kalinowski J: Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 1994, 145: 69-73. Hoffman KS, Chung CZ, Sll TMK-KJBOD: Recoding UAG to selenocysteine in Saccharomyces cerevisiae. RNA 2023, 29: 1400-1410. Krahn N, Tharp JM, Crnković A, Söll D: Engineering aminoacyl-tRNA synthetases for use in synthetic biology. The Enzymes 2020. Aldag C, Bröcker MJ, Hohn MJ, Prat L, Hammond G, Plummer A, Söll D: Rewiring Translation for Elongation Factor Tu‐Dependent Selenocysteine Incorporation. Angewandte Chemie 2013, 52: 1441-1445. Hankore ED, Zhang L, Chen Y, Liu K, Niu W, Guo J: Genetic Incorporation of Noncanonical Amino Acids Using Two Mutually Orthogonal Quadruplet Codons. ACS Synthetic Biology 2019. Suppmann S, Persson BC, Bck A: Dynamics and efficiency in vivo of UGA-directed selenocysteine insertion at the ribosome. The EMBO Journal 1999, 18: 2284-2293. Bröcker DMJ, Ho JML, Church PGM, Söll PD, O'Donoghue PP: Recoding the Genetic Code with Selenocysteine. Angewandte Chemie International Edition 2014. Curran J, Yarus M: Reading frame selection and transfer RNA anticodon loop stacking. Science 1987, 238: 1545-1550. Qi Y: Expression and characterization of novel bifunctional antioxidant enzymes and exploration of a new encoding method for Sec in vitro. Jilin University2024. Additional Declarations No competing interests reported. Supplementary Files Supplementarydata11.11.doc Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5428752","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":384852456,"identity":"be97b4c4-9c57-4dc1-8536-0d2db00a02b7","order_by":0,"name":"Jing-Jing Peng","email":"","orcid":"","institution":"Guangxi Health Science College","correspondingAuthor":false,"prefix":"","firstName":"Jing-Jing","middleName":"","lastName":"Peng","suffix":""},{"id":384852457,"identity":"cd1885eb-29bb-49d4-952c-bba5bcbb609a","order_by":1,"name":"Yao Qin","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"prefix":"","firstName":"Yao","middleName":"","lastName":"Qin","suffix":""},{"id":384852458,"identity":"e792cbc8-fbd6-4a7a-a001-e0972d45fcec","order_by":2,"name":"Liang-Hua Lu","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"prefix":"","firstName":"Liang-Hua","middleName":"","lastName":"Lu","suffix":""},{"id":384852459,"identity":"dd70a297-1677-4353-b14c-394768ffd059","order_by":3,"name":"Shi-Yang Yue","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"prefix":"","firstName":"Shi-Yang","middleName":"","lastName":"Yue","suffix":""},{"id":384852460,"identity":"41ae6af2-6ff5-44c7-87ad-9a992a1ab3f8","order_by":4,"name":"Ping Shi","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"prefix":"","firstName":"Ping","middleName":"","lastName":"Shi","suffix":""},{"id":384852461,"identity":"000c1048-3d93-4d3d-b91e-dcc502fdbab7","order_by":5,"name":"Ling-Yu Wang","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"prefix":"","firstName":"Ling-Yu","middleName":"","lastName":"Wang","suffix":""},{"id":384852462,"identity":"b4711930-484e-42df-a1f0-90a7b4d8124d","order_by":6,"name":"Chen-Xing Liu","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"prefix":"","firstName":"Chen-Xing","middleName":"","lastName":"Liu","suffix":""},{"id":384852463,"identity":"6ad8c09d-45f5-4131-a546-1cf438baee0f","order_by":7,"name":"Yu-Hui Fang","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"prefix":"","firstName":"Yu-Hui","middleName":"","lastName":"Fang","suffix":""},{"id":384852465,"identity":"f01bcd1f-629c-4e09-a287-e454dbf8beca","order_by":8,"name":"Tian-Tian Gao","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"prefix":"","firstName":"Tian-Tian","middleName":"","lastName":"Gao","suffix":""},{"id":384852466,"identity":"7983889b-9af9-4544-860c-17147cddac8f","order_by":9,"name":"Xiao-Dong Pei","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"prefix":"","firstName":"Xiao-Dong","middleName":"","lastName":"Pei","suffix":""},{"id":384852467,"identity":"f32c5982-bbf6-43ee-9920-1245bfc105f7","order_by":10,"name":"Fu-Tian Yu","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"prefix":"","firstName":"Fu-Tian","middleName":"","lastName":"Yu","suffix":""},{"id":384852471,"identity":"274e0d29-b5b4-4ea7-9064-160f2bdb5cbe","order_by":11,"name":"Xin-bi Huang","email":"","orcid":"","institution":"Guangxi Health Science College","correspondingAuthor":false,"prefix":"","firstName":"Xin-bi","middleName":"","lastName":"Huang","suffix":""},{"id":384852476,"identity":"01ffae65-e80f-4884-b14a-41d1cf774c09","order_by":12,"name":"Xiao-Ling Liu","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"prefix":"","firstName":"Xiao-Ling","middleName":"","lastName":"Liu","suffix":""},{"id":384852477,"identity":"db672ab6-aa7d-40f5-aa68-7229db488dfa","order_by":13,"name":"Cheng-Hua Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYDACCTBpA+HwkKAljXQth0nQwj+7x/Bzwa/zdhtuJDA+eNvGIG9O0JI7Z4ylZ/bdTp45I4HZcG4bg+HOBgJaDCRyN0jz9txO5pdIYJPmbWNIMDhAWMvm37w955LZJBLYfxOrZZs0z48DdiBbmInSInEj/5s1b0NygmTPw2bJOeckDDcQ0sI/Iy35Ns8fO3uD48kHP7wps5EnaAsYMLYxJDYwMDYwwKKJCPCHwZ5YpaNgFIyCUTACAQC1dz1QMTJoXwAAAABJRU5ErkJggg==","orcid":"","institution":"Guangxi University","correspondingAuthor":true,"prefix":"","firstName":"Cheng-Hua","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-11-11 04:23:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5428752/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5428752/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":71323957,"identity":"0a1f60ce-8563-4052-b0b2-d06f1c003e2b","added_by":"auto","created_at":"2024-12-13 10:39:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":111200,"visible":true,"origin":"","legend":"\u003cp\u003eOverall scheme for engineering construction of SBIP into \u003cem\u003eL. lactis\u003c/em\u003e. a. The necessary regulatory factors and their respective roles. b. The increased source of selenium donor H\u003csub\u003e2\u003c/sub\u003eSe by promoting GSH synthesis[10] after inserting GshF. c. The incorporation of the key UGA codon and SECIS for SBIP by introducing FDH[11, 12].\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5428752/v1/c0ed5aafc120dcea1653c580.png"},{"id":71324451,"identity":"311ee17a-3200-4162-b557-cba64b0496a1","added_by":"auto","created_at":"2024-12-13 10:47:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":292576,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic description of the construction of two main plasmids. a. Construction of regulatory vector pCABD by ligating main genes SelA, SelB, SelC and SelD onto the shutter vector pNZ8148 backbone; b. Construction of verifiable vector pGFS by ligating main genes GshF, FDH and sfGFP onto another shuttle vector pTRKH2 backbone. The ligation process for the two main plasmids combined the traditional T4 ligation and the new Infusion seamless cloning assembly.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5428752/v1/1de87dae79f196787c620b44.png"},{"id":71323956,"identity":"a4657e08-67c5-414d-ab11-49e6df415f20","added_by":"auto","created_at":"2024-12-13 10:39:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":415330,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of promoter screening engineering (a) and optimal promoter combination (b) for SBIP construction in \u003cem\u003eL. lactis\u003c/em\u003e NZ9000. I. Promoter optimization for transcription and expression of SelA ~ SelD by inserting P1, PnisA or P5 with progressively enhanced transcriptional activity in front of SelA on pCABD. II. Promoter optimization for expression of GshF and FDH-sfGFP by inserting P8 or P32 in front of gshF (SA), and P23 or P59 in front of FDH on pGFS1, and darker green meant brighter fluorescence. III. The screened two plasmids with the optimal promoter combination were electroporated into \u003cem\u003eL. lactis \u003c/em\u003eNZ9000 to construct SBIP. Note: The abbreviations or descriptions of all strains and plasmids were shown in Table S1.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5428752/v1/0dce69e2c16fdc3d9b9eb517.png"},{"id":71323959,"identity":"7e9ce1d2-ea2d-4f30-b157-285eb8e69a57","added_by":"auto","created_at":"2024-12-13 10:39:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":129333,"visible":true,"origin":"","legend":"\u003cp\u003eThe growth comparison of NZ9000/SBIP and its controls at 30℃. Each dot represented every monitoring time point. The rising curves of different colors represented the growth trend of different strains.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5428752/v1/d6288790c159000ab92826ac.png"},{"id":71323961,"identity":"4aa5b012-09e8-419b-8bc8-22d7a4e4dc89","added_by":"auto","created_at":"2024-12-13 10:39:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":108488,"visible":true,"origin":"","legend":"\u003cp\u003eSDS-PAGE verification of the target gene proteins expressed by NZ9000/SBIP. Lane M represented broad-spectrum protein marker; Lane 1 represented crude enzyme solution from \u003cem\u003eL. lactis\u003c/em\u003e NZ9000/p-p; Lane 2 represented crude enzyme solution from NZ9000/SBIP with Nisin induction and Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e3\u003c/sub\u003e addition; Lane 3 represented crude enzyme solution from NZ9000/SBIP cultured in GGC-GM17 with Nisin induction and Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e3\u003c/sub\u003e addition; Lane 4 represented crude enzyme solution from NZ9000/SBIP with Nisin induction; Lane 5 represented crude enzyme solution from NZ9000/SBIP without induction.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5428752/v1/f08d6dfc9041de436114439f.png"},{"id":71323958,"identity":"62b24aa1-b009-4af9-9afe-bf958b59c91c","added_by":"auto","created_at":"2024-12-13 10:39:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":134253,"visible":true,"origin":"","legend":"\u003cp\u003eSignificance comparison of FDH and GshF from crude enzyme solution of NZ9000/SBIP and its controls. Differences between groups were compared using t-test of one-way ANOVA: “\u003csub\u003e*\u003c/sub\u003e” stands for P\u0026lt;0.05, “\u003csub\u003e**\u003c/sub\u003e” stands for P\u0026lt;0.01, “\u003csub\u003e***\u003c/sub\u003e” stands for P\u0026lt;0.001, and “ns” stands for non-significant.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5428752/v1/2ef9a07f8e5db6f0c04c4072.png"},{"id":71323960,"identity":"d0271972-8416-4d45-8fc0-b5c15cf06d71","added_by":"auto","created_at":"2024-12-13 10:39:06","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":347280,"visible":true,"origin":"","legend":"\u003cp\u003eKEGG enrichment analysis (a) when NZ9000/SBIP vs NZ9000/p-p and SBIP route created in LAB (b). Note: Rich factor represented the ratio of the number of differential genes to that of annotated genes. The greater the Rich factor, the greater the degree of enrichment. FDR was Qvalue, and the value range was [0,1]. The closer it was to zero, the more significant the enrichment.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5428752/v1/dd37012d5bfd7c8431ef61c6.png"},{"id":74005411,"identity":"af348031-6089-4ad4-843e-4b7e4f59bf06","added_by":"auto","created_at":"2025-01-17 00:31:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6535119,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5428752/v1/1667a57f-ca92-4c24-b11e-d344175663ec.pdf"},{"id":71323964,"identity":"6f45a7b0-95a5-466b-a9eb-d3522a75fbe5","added_by":"auto","created_at":"2024-12-13 10:39:07","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4964864,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata11.11.doc","url":"https://assets-eu.researchsquare.com/files/rs-5428752/v1/baddc962ea8cabf44d771291.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"Metabolic engineering of Selenocysteine Biosynthesis and Insertion Pathway in Lactococcus lactis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSelenium (Se) is a trace element rich in biological functions[1]. Commercial selenium supplement is mainly through the intake of selenium-rich food or products, inorganic selenium and organic selenium[2]. In dietary selenium and selenium-rich yeast, selenium mainly exists as free SeMet, or is randomly inserted into proteins instead of Met and intaked by body[3]. However, the complex metabolic pathway and non-specific incorporation of selenium will lead to some common problems such as unclear metabolic flow, unstable composition of selenium spectrum and low selenoprotein content. Due to the wider medicinal value of organic selenoprotein, there have been peptide synthesis, chemical mutagenesis, cysteine nutrition-deficient biosynthesis and such preparation methods[4, 5]. However, these methods are deficient in high cost, low yield and lack of directivity, which limit their sustainable and economically feasible application[6]. Therefore, to develop a new biological selenium enrichment, specifically guiding selenium metabolic flow to direct synthesis of specific selenoprotein is urgently needed.\u003c/p\u003e \u003cp\u003eIn 1995, Calomme et al.[7] firstly demonstrated that \u003cem\u003eLactobacillus\u003c/em\u003e could transform inorganic selenium into organic selenium whose main species was Sec, different from those commercial selenium-rich products with SeMet as the main species, indicating a feasible new direction for selenium production and supplement. Current studies on Se-enriched LAB and their selenoproteins mainly focus on strain screening, cultivation optimization, antioxidant effects and morphology identification, while there are no studies on LAB Sec and selenoprotein metabolic pathways and directed synthesis. And LAB don\u0026rsquo;t contain selenoprotein coding genes or regulatory factors after query from NCBI (National Center of Biotechnology Information) and bSECIS (bacterial selenocysteine insertion sequence). Therefore, as Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e illustrated, this study desired to establish a microbial cell factory[8] for directed and efficient synthesis of novel biomass specific selenoproteins by constructing SBIP (Selenocysteine Biosynthesis and Insertion Pathway)[9] equipped with one key codon (UGA), one cis-acting element (SECIS) and four regulatory factors SelA (Selenocysteine synthase), SelB (Selenocysteine-specific elongation factor), SelC (tRNA\u003csup\u003e[Ser]sec\u003c/sup\u003e) and SelD (Seleno-phosphate synthase) to specifically guide the selenium metabolism flow in LAB.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlthough SBIP of \u003cem\u003eE.coli\u003c/em\u003e has been clarified[13, 14], it does not belong to the food grade safe strain, which limits its application. Comparatively, \u003cem\u003eL. lactis\u003c/em\u003e, the most representative strain of LAB, is authorized as GRAS microorganism by FDA[15] and commonly used for genetic engineering[16]. Since the only commercial expression system in LAB[17] is the nisin controlled expression (NICE) proposed by Kuipers et al.[18], and \u003cem\u003eL. lactis\u003c/em\u003e NZ9000\u003csup\u003e[20]\u003c/sup\u003e is the most applied model strain among all the derivatives of \u003cem\u003eL. lactis\u003c/em\u003e MG1363[19], NZ9000 with NICE inducible system was preferentially used for the early SBIP construction of free expression system[20, 21]. Besides, constitutive expression system[22] which is also suitable for large-scale production in industry can continuously express target protein without spatiotemporal specificity or adding inducers, this study would combine these two systems. At present, P1, P2, P3, P5, P8 [23] and P21, P23, P32, P44, P59[24] have been applied for the expression of foreign genes in \u003cem\u003eL. lactis\u003c/em\u003e. Considering that the transcriptional activity of P8, P5, P3, PnisA, P2, P45, P1, P6, P32 decreased, and high expression of SelA may inhibit the expression of selenoprotein[25], strong promoter P8 and P5, moderately strong promoter PnisA and weak promoters P1 and P32 were selected by high-throughput screening for optimal co-expression of the target genes in SBIP. Meanwhile, promoter P23[26] and P59[27, 28] with good application effect were also tried to verify the fused FDH-sfGFP expression.\u003c/p\u003e \u003cp\u003eMany attempts have been made to overexpress the SBIP regulatory factors homologically by \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e) so far[29\u0026ndash;32], and it has been used to express heterologous selenoprotein[11, 33, 34], but no metabolic pathway application or modification of SBIP has been reported in other prokaryotic host bacteria, neither in LAB. In this study, the SBIP would be introduced into \u003cem\u003eL. lactis\u003c/em\u003e NZ9000 by two major shutter plasmids: pNZ8148[35] and pTRKH2[28, 36]. The former one was equipped with four regulatory factors to be expressed in a mixed form due to the combination of PnisA with P1, PnisA or P5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e-a). While the latter one was simple constructive expression by ligating GshF (SA) with P8 or P32 to enhance the selenium donor required for Sec synthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e-b), and ligating fused FDH-sfGFP with P23 or P59 to introduce SECIS to translate UGA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e-c). Finally, the transfer of mechanism from \u003cem\u003eE. coli\u003c/em\u003e to \u003cem\u003eL. lactis\u003c/em\u003e was turned out to be a triumph when FDH and other target genes were verified, providing a reference for the engineering production of other artificial selenoproteins, an in-depth understanding for specific selenoprotein synthesis and regulation, and sustainable development of biological resources.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Chemicals, enzymes and media\u003c/h2\u003e \u003cp\u003eChemicals and enzymes: T4 DNA ligase and pEASY\u0026reg;-Basic Seamless Cloning and Assembly Kit were purchased from TransGen Biotech (Beijing, China); PrimeSTAR\u0026reg; HS DNA Polymerase, PrimeScript\u0026trade; II 1st Strand cDNA Synthesis Kit, TB Green\u0026reg; Premix Ex Taq\u0026trade; II (Tli RNaseH Plus) were purchased from TaKaRa Biotech (Kyoto, Japan)༛Plasmid Mini Kit I, DNA Gel Extraction Kit, Bacterial RNA Kit and DNase I Set were purchased from Omega Bio-Tek (New York, USA); Reduced glutathione (GSH) test kit (microplate method) and FDH test kit (microplate method) were purchased from Grace Biotech (Suzhou, China); BCA protein concentration determination kit was purchased from Beyotime Biotech (Shanghai, China); All fast digest restriction enzymes were purchased from Thermo Fisher Scientific Inc. (Massachusetts, USA).\u003c/p\u003e \u003cp\u003eMedia: LB medium (pH 7.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1) contained 10g /L tryptone, 5 g/L yeast extract and 10 g/L NaCl; M17 medium contained 2.5 g/L bacterial peptone, 2.5 g/L bacterial casein peptone, 5 g/L soya peptone, 5 g/L beef extract, 2.5 g/L yeast extract, 0.5 g/L sodium ascorbate, 19 g/L β-sodium glycerophosphate, and 0.25 g/L MgSO4; GM17 medium was M17 supplemented with 0.5% glucose; GGC-GM17 medium was GM17 supplemented with 10 mM L-Glutamic acid (Glu), L-Glycine (Gly) and L-Cysteine (Cys), respectively[37, 38]; G-SGM17B medium was GM17 supplemented with 1.5% glycine, 0.5 M sucrose, 2 mM CaCl\u003csub\u003e2\u003c/sub\u003e and 20 mM MgCl\u003csub\u003e2\u003c/sub\u003e. The pHs of M17 and the mediums derived from it were adjusted by 1% NaOH and 1% HCl to 7.0\u0026thinsp;~\u0026thinsp;7.4. All above were provided by Solarbio (Beijing, China) and Macklin (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Strains, plasmids and cultivation conditions\u003c/h2\u003e \u003cp\u003eAll bacterial strains and plasmids used in this study were listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. \u003cem\u003eE. coli\u003c/em\u003e MC1061 and \u003cem\u003eE. coli\u003c/em\u003e TOP10 were used as competent cell for the chemical transformation of pNZ8148 and its recombinant plasmids, while \u003cem\u003eE. coli\u003c/em\u003e JM110 and \u003cem\u003eE. coli\u003c/em\u003e DH5α were used for the transformation of pTRKH2 and its recombinant plasmids. \u003cem\u003eE. coli was\u003c/em\u003e cultured in LB broth at 37℃ and 220 r/min. \u003cem\u003eL. lactis\u003c/em\u003e NZ9000 was a host to verify the expression of several key factors. NZ9000 strains were cultured in GM17 broth at 30℃ without agitation. The standard of supplemented antibiotics for screening recombinants and transformants were as follows: 200 \u0026micro;g/mL erythromycin and 25 \u0026micro;g/mL chloramphenicol was added into LB broth respectively when pTRKH2 or its recombinant plasmids and pNZ8148 or its recombinant plasmids existed in \u003cem\u003eE. coli\u003c/em\u003e strains, while only 12.5 \u0026micro;g/mL erythromycin and 12.5 \u0026micro;g/mL chloramphenicol was added into GM17 broth when pTRKH2 or its recombinant plasmids and pNZ8148 or its recombinant plasmids existed in NZ9000 strains, and only add 8 \u0026micro;g/mL erythromycin and chloramphenicol each when pTRKH2 and pNZ8148 or two of their recombinant plasmids both existed in NZ9000 strains.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 DNA manipulation and strain construction\u003c/h2\u003e \u003cp\u003eAll plasmids listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e were prepared with Omega plasmid kit, while an additional incubation with 1 mg/mL lysozyme (Solarbio) in 50 mM PBS buffer at 37℃ for 30 min was called for those from NZ9000. All primers listed in Table S2 were provided by BGI\u0026bull;Tech (Shenzhen, China). PCR amplifications were conducted with TaKaRa DNA Polymerase and the target linearized DNA fragments were purified with Omega gel extraction kit. GshF (GenBank: MN020375.1) was synthesized by Jinsirui Biotech (Jiangsu, China) and inserted onto pTRKH2 through SacI and SalI sites to generate pTRKH2-GshF by Jinsirui Biotech. SelC (Gene ID: 948167) derived from pBAD18-SelABC2-GPX-GW was inserted into pNZ8148 through PstI and KpnI sites to generate pNZ8148-SelC using T4 DNA ligase. For the rest target genes, SelA-SelB with nested expressed genes derived from pBAD18-SelABC2-GPX-GW was amplified by PCR using primer pairs \u0026ldquo;SelA-SelB-F/SelA-SelB-R\u0026rdquo;, while SelD and FDH derived from \u003cem\u003eE. coli\u003c/em\u003e MG1655 genomic DNA were amplified by PCR using primer pairs \u0026ldquo;SelD-F/SelD-R\u0026rdquo; and \u0026ldquo;fdhF-F/fdhF-R\u0026rdquo; respectively. And fluorescent protein gene sfGFP derived from pRHU3-sfGFP was amplified by PCR using primer pairs \u0026ldquo;sfGFP -F/sfGFP -R\u0026rdquo;.\u003c/p\u003e \u003cp\u003eHowever, the schematic overview for the construction of regulatory plasmid and verifiable plasmid were shown by Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Firstly, SelA-SelB and SelD purified fragments were assembled together onto linearized pNZ8148-SelC which has been already digested with XbaI and HindIII to generate pCABD (pNZ8148-SelC-SelA-SelB-SelD) using In-Fusion assembly kit. Likewise, FDH and sfGFP purified fragments were assembled together onto linearized pTRKH2-GshF which has been already digested with ApaLI and KpnI to generate pGFS (pTRKH2-GshF-FDH-sfGFP). In order to initiate the transcription and expression of the seven target genes (\u0026ldquo;SelA\u0026thinsp;~\u0026thinsp;SelD\u0026rdquo; on pNZ8148 and \u0026ldquo;GshF, FDH and sfGFP\u0026rdquo; on pTRKH2) in this work, above two main plasmids were inserted various promoters from pUC6P, which was chemically synthesized by Jinsirui Biotech. To preliminarily verify the expression of GshF in NZ9000, the strong promoter P8 was inserted before the GshF on pTRKH2-GshF with primers \u0026ldquo;P8-F/ P8-R\u0026rdquo; by In-Fusion assembly. According to Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Table S3, these promoters were installed independently or in combination with the primer pairs listed in Table S2 by In-Fusion assembly.\u003c/p\u003e \u003cp\u003eIn this study, the TGA codon[39] and the 11nt downstream SECIS[40] on fdhF[41], as well as the termination codon at the end were key point of penetration. On one hand, due to the need of codon readability and fusion expression of FDH-sfGFP, the TGA at 418\u0026ndash;420 site of FDH was replaced by TGC, and the TAA at the end was replaced by AAA to generate pGFS1 (pTRKH2-GshF-FDH-sfGFP1). The bases were replaced successively by long-distance inverse-PCR[42] with primer pairs \u0026ldquo;(TGA\u0026rarr;TGC)-F/(TGA\u0026rarr;TGC)-R\u0026rdquo; and \u0026ldquo;(TAA\u0026rarr;AAA)-F/(TAA\u0026rarr;AAA)-R\u0026rdquo;. On the other hand, due to the need of independent expression of FDH, TGC at site 418\u0026ndash;420 of FDH should be replaced back by TGA, and AAA at the end of FDH should be replaced back by TAA to generate p8G23FS2 (pTRKH2-P8-GshF-P23-FDH-sfGFP2). The bases were replaced successively by long-distance inverse PCR with primer pairs \u0026ldquo;(TGC\u0026rarr;TGA)-F/(TGC\u0026rarr;TGA)-R\u0026rdquo; and \u0026ldquo;(AAA\u0026rarr;TAA)-F/(AAA\u0026rarr;TAA)-R\u0026rdquo;.\u003c/p\u003e \u003cp\u003eAll recombinant plasmids were screened first by LB agar plates and then by GM17 agar plates according to the standard of supplemented antibiotics in 2.2. Finally, the recombinant plasmids were electro-transformed into NZ9000 with 2mm electroporation cuvettes[43] following the BTX ECM\u0026reg; 830 (Harvard Apparatus, Massachusetts, USA) protocol and under the condition of 2 kv, 150 \u0026micro;s of PL, 30 of MP, 100 ms of PI. So NZ9000/SBIP-sfGFP was constructed by transforming two optimal vectors after the screening and integration of promoters for regulatory factors and verifiable factors. Then NZ9000/SBIP was established after the base substitution of two different sites on one plasmid with dual constructive promoters.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Inducible and constitutive expression and crude enzyme solution preparation\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 Inducible and constitutive expression\u003c/h2\u003e \u003cp\u003eWhen NZ9000 transformants containing pNZ8148 or its recombinant plasmids harboring both PnisA and P1, such as NZ9000/pC1ABD (NZ9000/pNZ8148-SelC-P1-SelA-SelB-SelD), NZ9000/SBIP-sfGFP (NZ9000/(pC1ABD\u0026thinsp;+\u0026thinsp;p8G23FS1)) and NZ9000/SBIP (NZ9000/(pC1ABD\u0026thinsp;+\u0026thinsp;p8G23FS2)), the precultured cells were 3% inoculated into 30 mL fresh medium and expanded until OD\u003csub\u003e600\u003c/sub\u003e which was measured by MAPADA UV-1800PC spectrophotometer (Shanghai, China) reached 0.5 around 3 h later, then 30 ng/mL Nisin with or without 8 \u0026micro;g/mL Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e3\u003c/sub\u003e were added to induce the expression of regulatory gene for another 15 h[44], while the controls were added nothing for constitutive expression. And when NZ9000 transformants containing pTRKH2 or its recombinant plasmids only harboring P8, P32, P23, P59 or their combination, such as NZ9000/p8G (NZ9000/pTRKH2-P8-GshF), NZ9000/pG23FS1 (NZ9000/pTRKH2-GshF-P23-FDH-sfGFP1), NZ9000/pG59FS1 (NZ9000/pTRKH2-GshF-P59-FDH-sfGFP1), NZ9000/p8G23FS1 (NZ9000/pTRKH2-P8-GshF-P23-FDH-sfGFP1) and NZ9000/p32G23FS1 (NZ9000/pTRKH2-P32-GshF-P23-FDH-sfGFP1), the precultured cells were 3% inoculated into 30 mL fresh medium and expanded overnight till 18 h for constitutive expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 Preparation of crude enzyme solution\u003c/h2\u003e \u003cp\u003eEach 30 mL NZ9000 recombinant bacteria solution was firstly centrifugated at 5000 xg, 25℃ for 10 min, then cell precipitation was harvested and washed 2\u0026thinsp;~\u0026thinsp;3 times with PBS (50 mM, pH 7.4), and finally suspended into 1.0 mL PBS with 1 mg/mL lysozyme. After incubation at 37℃ for 60 min, add 100 mg SiO\u003csub\u003e2\u003c/sub\u003e powder and 15 particles of Coolaber stainless steel beads (Beijing, China) to complete the cells grinding by Sceintz-48L cryogenic high-throughput tissue grinder (Jiangsu, China) under the following conditions: precooling at -30℃, 25 Hz, 600 rpm, 600 seconds. Lastly, the grinding blend was centrifuged in a high-speed refrigerated centrifuge at 12000 xg, 4℃ for 15 min, and the supernatant harvested was the crude enzyme solution.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Preliminary analysis for the target gene elements\u003c/h2\u003e \u003cp\u003eI. Regulatory genes:\u003c/p\u003e \u003cp\u003eThe function of SelA\u0026thinsp;~\u0026thinsp;SelD were achieved in \u003cem\u003eL. lactis\u003c/em\u003e through the construction of pCABD and the subsequent screening from pC1ABD, pCPABD (pNZ8148-SelC-PnisA-SelA-SelB-SelD) and pC5ABD (pNZ8148-SelC-P5-SelA-SelB-SelD) one by one for the optimal. After the successful verification by endonuclease digestion, AGE (agarose gel electrophoresis) and DNA sequencing, the optimal strain would be cultured according to 2.4.1 and performed with SDS-PAGE.\u003c/p\u003e \u003cp\u003eII. Verifiable genes:\u003c/p\u003e \u003cp\u003eThe expression of GshF and fused FDH-sfGFP were fulfilled in \u003cem\u003eL. lactis\u003c/em\u003e through the construction of p8G and subsequent screening from \u0026ldquo;pG23FS1 and pG59FS1\u0026rdquo; and \u0026ldquo;p8G23FS1 and p32G23FS1\u0026rdquo;. Likewise, after the successful verification by endonuclease digestion, AGE and DNA sequencing, GshF, FDH and FDH-sfGFP would be verified with SDS-PAGE and enzyme activity assay, while the reporter gene sfGFP would be verified by the fluorescence intensity (FI) determination by TECAN Infinite M200 PRO microplate reader (M\u0026auml;nnedorf, Switzerland).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Confirmatory analysis for SBIP(-sfGFP) in NZ9000\u003c/h2\u003e \u003cp\u003eAfter verification by endonuclease digestion, AGE and DNA sequencing of the double plasmids in NZ9000/SBIP(-sfGFP), the co-expressed proteins bands of SelA, SelB, SelD, GshF and FDH or FDH-sfGFP would be wholly tested by SDS-PAGE. To further analyze whether these inserted factors in the pathway really worked, RT-qPCR analysis, enzyme activity assay, as well as FI/OD\u003csub\u003e600\u003c/sub\u003e determination were demanded as described below.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Analytical methods\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.7.1 Endonuclease digestion and electrophoresis\u003c/h2\u003e \u003cp\u003e20 \u0026micro;L digestion system for single plasmid was as follows: 1 \u0026micro;g plasmid, 1 \u0026micro;L fast digest endonuclease, 2 \u0026micro;L 10\u0026times;fast digest buffer, and finally add sterilized ultra-pure water up to 20 \u0026micro;L. And 20 \u0026micro;L digestion system for double plasmid was as follows: 2 \u0026micro;g plasmid, 2 \u0026micro;L fast digest endonuclease, 2 \u0026micro;L 10\u0026times;fast digest buffer, and finally add sterilized ultra-pure water up to 20 \u0026micro;L. While parameters for AGE were as follows: 1% agarose in 100 mL 1\u0026times;TAE solution with 10 \u0026micro;L gel red nucleic acid dye (TransGen); 3 \u0026micro;L Thermo Scientific GeneRuler 1 kb Plus DNA Ladder Marker (Massachusetts, USA); 120 V, 108 mA, and 40 min by DYY-8C Nucleic acid electrophoresis apparatus (Beijing, China) and images taken by Bio-Rad Molecular Imager with ChemiDoc\u0026trade; XRS\u0026thinsp;+\u0026thinsp;System (California, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.7.2 SDS-PAGE\u003c/h2\u003e \u003cp\u003eParameters for polyacrylamide gel electrophoresis were as follows: the lower resolving gel was set as 10% and the upper spacer gel was 5%; 3 \u0026micro;L Solarbio broad-spectrum protein marker (Beijing, China); the loading sample was 3 \u0026micro;L 4\u0026times;loading buffer with 9 \u0026micro;L diluted crude enzyme solution by 2\u0026thinsp;~\u0026thinsp;8 times; 80 V for 20 min, and up to 120 V for 40 min by Bio-Rad PowerPac Basic protein electrophoresis apparatus (California, USA); Dyed with Coomassie bright blue buffer (100 mg dye powder in 25 mL isopropyl alcohol, 10 mL glacial acetic acid and 65 mL distilled water) for 30 min at 45 r/min and decolorized with eluent (10 mL acetic acid, 5 mL anhydrous ethanol and 85 mL distilled water) for 3 h at 90 r/min. Finally, the protein glue immersed in distilled water was taken images by Bio-Rad Molecular Imager with ChemiDoc\u0026trade; XRS\u0026thinsp;+\u0026thinsp;System.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.7.3 Relative fluorescence intensity(FI/OD\u003csub\u003e600\u003c/sub\u003e) determination[26, 45]\u003c/h2\u003e \u003cp\u003eThe recombinant plasmids harboring the read-through FDH-sfGFP were electroporated into \u003cem\u003eL. lactis\u003c/em\u003e NZ9000 competent cells, and then single colonies were screened and inoculated into 5 mL GM17 broth (+\u0026thinsp;Em༆Cr / +Em) with stand culture at 30℃ to logarithmic stage. Finally, cells precipitation was harvested after centrifugation at 6000 xg for 8 min, washed twice and resuspended in 1 mL 50 mM PBS, from which 200 \u0026micro;L cell resuspension solution with 3 parallels were transferred into a 96 well plate and performed by OD\u003csub\u003e600\u003c/sub\u003e detection and FI measurement with excitation at 485 nm and emission at 525 nm by TECAN Infinite M200 PRO microplate reader, while PBS buffer was as the blank control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.7.4 Establishment of growth curves\u003c/h2\u003e \u003cp\u003eIn this study, NZ9000/pNZ8148, NZ9000/pTRKH2, and NZ9000/p-p(NZ9000/(pNZ8148\u0026thinsp;+\u0026thinsp;pTRKH2)) were all constructed in advance for later comparison. Besides the target strain NZ9000/SBIP and the slow growing NZ9000/p8G in this study, NZ9000, NZ9000/pNZ8148, NZ9000/pTRKH2 and NZ9000/p-p such four strains were all set as controls to compare the growth process, providing references for optimizing the cultivation of the recombinant strains afterwards. During the monitoring, each first generation of these bacteria solution was 3% inoculated into 100 mL GM17 broth, and 200 \u0026micro;L with 3 parallels was taken every 2 h within 24 h to measure OD\u003csub\u003e600\u003c/sub\u003e against fresh medium by TECAN microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.7.5 Assay for GSH and the enzyme activity of GshF and FDH\u003c/h2\u003e \u003cp\u003e1 mL GSH synthesis reaction system[46]: 20 mM Glu, 20 mM Gly, 20 mM Cys, 10 mM ATP, 30 mM MgCl\u003csub\u003e2\u003c/sub\u003e and 100 mM Tris-HCl buffer (pH 8.0). Each component was dissolved in Tris-HCl buffer. An appropriate 100 \u0026micro;L of supernatant crude enzyme solution was added to 1 mL reaction system, incubated at 37℃ for 20 min by Guowang DTH-100 dry bath (Jiangsu, China), and terminated by water boiling bath for 3 min. After chill for 30 min on ice, it was centrifuged at 12000 xg 4℃ for 10 min, and the supernatant was harvested to determine the yield of GSH by Grace kit. And the unit of GshF activity (U) was calculated by the amount of GshF enzyme (U/mg or U/mL) required to catalyze the formation of 1 \u0026micro;mol GSH per minute at 37℃. Similarly, 1mL FDH enzyme reaction system was conducted by NADH colorimetric method[47, 48]: 1 mM NAD +, 6 mM sodium formate, 100 mM PBS buffer (pH 7.5). Each component was dissolved in PBS buffer. And the unit of FDH activity (U) was calculated by the amount of FDH enzyme (U/mg or U/mL) required to catalyze the formation of 1 nmol NADH per minute at 35℃.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e2.7.6 RT-qPCR\u003c/h2\u003e \u003cp\u003eNZ9000/SBIP was the strain containing double plasmids with optimal promoters combination screened by GM17 (+\u0026thinsp;Em\u0026thinsp;+\u0026thinsp;Cr) agar plates, and first generation bacterial solution was 3% inoculated into 10 mL fresh GM17 medium for about 3 h, after when 13 h Nisin induction culturing was performed. Then 3 mL bacterial solution with 3 parallels would be harvested to extract total RNA with genomic DNA erased by Omega kits. And a few RNA was taken to complete AGE. When three obvious bands occurred on the imaging system, the extracted RNA was immediately reverse-transcribed into cDNA by TaKaRa Kit. In this study, NZ9000/p-p was treated as control and 16S rRNA[49] was decided as reference gene. The final RT-qPCR for target genes with designed specific primers in Table S4 would be performed following the instruction of TaKaRa TB Green kit, and each 20 \u0026micro;L reaction solution was transferred into 100 \u0026micro;L white eight-connected tubes, after which they were detected by Roche LightCycler\u0026reg; 96 (Basel, Switzerland) following two-step RT-qPCR procedure in Table S5.\u003c/p\u003e \u003cp\u003eBased on the output data from Roche software, the differential expression multiples of target genes between samples and controls were calculated and analyzed by the method of comparative Cq value and F value showed in the following formulas.\u003c/p\u003e \u003cp\u003e(1)△△Cq=△Cq \u003csub\u003esample\u0026minus;\u003c/sub\u003e△Cq \u003csub\u003econtrol\u003c/sub\u003e; ༈2༉F\u0026thinsp;=\u0026thinsp;2\u003csup\u003e\u0026minus;△△Cq\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eNote\u003c/strong\u003e \u003cp\u003eF equaled multiple change of target gene expression between tested samples and control ones. When F\u0026thinsp;\u0026gt;\u0026thinsp;1, it meant that the expression of the tested sample was up-regulated by F times compared with the control one; When 1\u0026thinsp;\u0026gt;\u0026thinsp;F\u0026thinsp;\u0026gt;\u0026thinsp;0, it meant that the expression level of the tested sample was down-regulated by 1/F times compared with the control one.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e2.7.7 Transcriptome analysis for SBIP element genes\u003c/h2\u003e \u003cp\u003eBoth 50 mL the sample NZ9000/SBIP and the control NZ9000/p-p with 3 parallels were cultured as 2.7.6 said. After 16 h, bacteria solution was firstly centrifugated at 5000 xg, 4℃ for 15 min, then cell precipitation was harvested and washed with 50 mM PBS. Finally, the six samples would be sent to Majorbio Bio-pharm Tech (Shanghai, China) to accomplish the transcriptome sequencing and analysis after frozen at -80℃ one night. I. Differential expression analysis of genes between NZ9000/SBIP and NZ9000/p-p was conducted to identify differential expression genes between samples with volcano plot and statistics bar. II. Gene set analysis of similar and special genes was performed by Go and KEGG such annotations classification or enrichment analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e2.7.8 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll the measured data in this study were expressed in the form of \u0026ldquo;Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u0026rdquo;. SPSS 20.0 was firstly applied to conduct one-way ANOVA with t-test and Duncan multiple range test for samples differences within and among groups (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05 means no significant difference while P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 means significant difference and P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 extremely significant difference), then the contrast difference significance was presented directly through the bar chart by Graph Pad Prism 6.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.1 The construction and growth comparison of NZ9000/SBIP and other strains\u003c/h2\u003e \u003cp\u003eAs Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e clearly shown, the AGE results of NZ9000/SBIP and most other strains were in line with expectations after endonuclease digestion, and the DNA sequencing results from BGI further confirmed the accuracy. However, in Fig. S2: pC5ABD and pCPABD transformants both lost about 3000 bp, while pG59FS1 transformant also lost 96 bp, and thus they failed to be screened out. Among the generated different recombinant \u003cem\u003eL. lactis\u003c/em\u003e strains in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, several typical representatives were monitored within 24 h to find their growth trends. As following Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e showed, NZ9000/p8G adapted to grow more slowly than others before the first 4 h and the growth increased significantly after 6 h; The introduction of pNZ8148 into NZ9000 had little effect on the growth trend, and their logarithmic phase were both from 2 to 8 h, during which the growth increased significantly after 4 h; Compared with NZ9000 and NZ9000/pNZ8148, the growth force became weaker after the introduction of pTRKH2 into NZ9000 or double plasmids including it. Consequently, the growth rate of NZ9000/p-p was between NZ9000/pNZ8148 and NZ9000/pTRKH2 though NZ9000/SBIP carrying optimal combination of promoters presented better growth activity than NZ9000/p-p, which was similar to that of Tiange Ma\u0026rsquo;s study[50]. Unlike NZ9000/p-p, it was known that the growth curve of NZ9000/SBIP carrying protein genes and promoters was similar to that of NZ9000/pNZ8148.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Preliminary verification of expression of the target gene elements\u003c/h2\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1 SDS-PAGE analysis of regulatory genes by \u003cem\u003eL. lactis\u003c/em\u003e NZ9000/pC1ABD\u003c/h2\u003e \u003cp\u003eThe expression of the regulatory factors in the optimal NZ9000/pC1ABD was analyzed by SDS-PAGE. As shown in Fig. S3, compared with NZ9000/pNZ8148 under non-induced and Nisin induced culturing (lane 1 and 2), both NZ9000/pC1ABD without and with Nisin induction (lane 3 and 4) showed obvious overexpressed bands, corresponding well with the calculated theoretical molecular weights of SelA, SelB and SelD, which are 50.62 kDa (about 51kDa), 68.88 kDa (about 69kDa) and 36.69 kDa (about 37kDa), respectively. However, SelA showed much thicker band than SelB and SelD, probably due to that P1 was ligated in front of SelA and far away from SelB and SelD. To sum up, SelA, SelB and SelD on the regulatory plasmid was preliminarily confirmed to be expressed in NZ9000, but whether they promoted to assist UGA translated into Sec would be furtherly verified with the optimal verifiable plasmid.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2 Expression of GshF (SA) by \u003cem\u003eL. lactis\u003c/em\u003e NZ9000/p8G\u003c/h2\u003e \u003cp\u003eAccording to the practically weak growth force of NZ9000/p8G in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, this strain was harvested at 6 h, 12 h, 18 h, 24 h and 30 h, and verified uniformly compared with constitutive expression after cultured for 24 h in medium GGC-GM17[37, 38]. Consequently, no obvious protein production was found when NZ9000/p8G only cultured 6h during prelogarithmic phase in Fig. S4-a, but the protein production was obvious at 12 h, and stable at 18 h\u0026thinsp;~\u0026thinsp;30 h. Through comparing lane 7 with lane 5, we found that GshF band was thicker in medium GGC-GM17, indicating that the addition of substrate such as Glu with a final concentration of 10 mM was conducive to the expression of GshF[51]. Then GSH concentration and GshF enzyme activity of NZ9000/p8G cultured by GM17 at five different time was measured. And the yield of GSH was found to be positively correlated with the enzyme activity of GshF in Fig. S5. the expression of GshF (SA) by NZ9000/p8G cultured for 24 h were similar to that of \u003cem\u003eE. coli\u003c/em\u003e BL21/pET28a(+)-gshFSA[46], from which GshF (SA) was assayed as 1.242 U/mL or 0.1275 U/mg.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e3.2.3 The fusion expression of FDH-sfGFP by \u003cem\u003eL. lactis\u003c/em\u003e recombinant strains\u003c/h2\u003e \u003cp\u003eThe expression of fusion protein FDH-sfGFP to light up reporter gene sfGFP were mainly decided by ligating promoter P23[26] or P59[27] in front of FDH on pGFS1. Since they have been both verified with good application effect, the optimal fluorescent strain and optimal time for FI determination would be analyzed at five different time from 0 h\u0026thinsp;~\u0026thinsp;30 h. As can be seen from Fig. S6, the FI of NZ9000/pG23FS1 was significantly stronger than that of NZ9000/pG59FS1, stating that P23 should be the preferred promoter for the expression of FDH-sfGFP. Moreover, the FI kept increasing before 18 h and remained at a stable level after 18 h, which would be picked to harvest the strains and conduct the measurement as recorded in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further verify NZ9000/pG23FS1, the SDS-PAGE analysis of fused FDH-sfGFP (106 kDa) at five different time could be seen from Fig. S4-b. Those recombinant \u003cem\u003eL. lactis\u003c/em\u003e strains were found to have a better expression of GshF and fused FDH-sfGFP after cultured for 24 h from Fig. S4-a and Fig. S4-b. Then on the backbone of the preferred pG23FS1, P8 and P32 would be ligated to screen a better one for the combination with P23. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the FI of the recombinant NZ9000/p8G23FS1 was superior to NZ9000/p32G23FS1, but much lower than NZ9000/pG23FS1. Besides, superior expression of GshF and fused FDH-sfGFP by P8\u0026thinsp;+\u0026thinsp;P23 to P32\u0026thinsp;+\u0026thinsp;P23 could be seen by SDS-PAGE analysis in Fig. S4-c and from the results of GshF enzyme activity assay: the former 0.1990 U/mg was higher than the later 0.1038 U/mg, but lower than NZ9000/p8G.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFI analysis of recombinant \u003cem\u003eL. lactis\u003c/em\u003e strains in this study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRecombinant \u003cem\u003eL. lactis\u003c/em\u003e NZ9000 with sfGFP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFI/OD\u003csub\u003e600\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNZ9000/pG23FS1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2431.46\u0026thinsp;\u0026plusmn;\u0026thinsp;2.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNZ9000/pG59FS1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e274.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNZ9000/p8G23FS1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e381.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNZ9000/p32G23FS1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e344.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNZ9000/ SBIP-sfGFP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e362.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Verification analysis of differences after optimized expression for NZ9000 SBIP\u003c/h2\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1 Expression optimization and SDS-PAGE analysis for SBIP in \u003cem\u003eL. lactis\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eBased on the screened results from 3.1 and 3.2.3 and the afterwards optimal recombinant plasmids combination, NZ9000/SBIP-sfGFP was NZ9000/(pC1ABD\u0026thinsp;+\u0026thinsp;p8G23FS1) and NZ9000/SBIP was NZ9000/(pC1ABD\u0026thinsp;+\u0026thinsp;p8G23FS2). According to the growth trend of NZ9000/SBIP in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, its expression optimization would depend on differed induction condition and medium composition as Fig. S7-a represented. In this work, the four cultivation methods were compared simultaneously and were all analyzed by SDS-PAGE. As expected, the target protein bands of 37 kDa, 51 kDa, 69 kDa, 79 kDa and 86 kDa could be obviously seen in lane 2 ~ 5 of Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003e, indicating that SelD, SelA, SelB, FDH and GshF were all successfully expressed in NZ9000/SBIP. Moreover, the target bands of lane 3 were more obvious than others, indicating NZ9000/SBIP cultured in GGC-GM17 with Nisin induction and Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e3\u003c/sub\u003e addition[1] was optimal.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2 Differential analysis of enzyme activity\u003c/h2\u003e \u003cp\u003eIn this work, NZ9000/SBIP-sfGFP was set as one control to find out the enzyme activity difference between the fused FDH-sfGFP from it and FDH from \u003cem\u003eL. lactis\u003c/em\u003e NZ9000/SBIP under the existing of SelA\u0026thinsp;~\u0026thinsp;SelD. Meanwhile, NZ9000/NoABCD (i.e. NZ9000/p8G23FS2) was set as another control to analyze whether FDH could produce enzyme activity without SelA\u0026thinsp;~\u0026thinsp;SelD. According to Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003e, the enzyme activity of FDH could both be detected out by NZ9000/SBIP and NZ9000/SBIP-sfGFP under different culture modes. As Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e stated, the effect of independent expression of FDH was two orders of magnitude higher than that of FDH-sfGFP, while the negative control NZ9000/NoABCD showed trace enzyme activity in the absence of SelA\u0026thinsp;~\u0026thinsp;SelD, indicating that there might be other mechanisms in \u003cem\u003eL. lactis\u003c/em\u003e background to support the random and slight insertion of Sec in FDH.\u003c/p\u003e \u003cp\u003eAmong the four culturing modes as Fig. S7-a showed, FDH of NZ9000/SBIP 4 had the highest enzyme activity (28.11 mU/mg), but was significantly different from that of R\u0026eacute;mi Thom\u0026eacute; (0.32 U/mg)[52] and MJ Axley (1.4 U/mg)[53]; As one comparison, the enzyme activity decreased by 4 times (about 6.90 mU/mg) without induction. And the addition of Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e3\u003c/sub\u003e could slightly weaken the enzyme activity of FDH since that of SBIP 3 was a bit lower than SBIP 2\u0026rsquo;s, indicating that selenium would affect the level of enzyme activity[54, 55]. Moreover, when NZ9000 strain existed only one p8G23FS1 or p8G23FS2 and regardless of whether pC1ABD existed or not, the enzyme activity of GshF in these 8 groups were all distributed around 200 mU/mg and had no significant difference, while the addition of Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e3\u003c/sub\u003e also had a slight negative effect on it.\u003c/p\u003e \u003cp\u003eIn conclusion, the SBIP could enable Sec with TGA codon at 418\u0026ndash;420 site of selenoprotein FDH to be expressed in \u003cem\u003eL. lactis\u003c/em\u003e with directional insertion, and the expression effect of NZ9000/SBIP was the best when cultured by GGC-GM17 with Nisin induction and Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e3\u003c/sub\u003e addition.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAssay of FDH and GshF from crude enzyme extract of NZ9000/SBIP and its controls\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNZ9000 Samples\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eEnzyme activity\u003c/p\u003e \u003cp\u003e(Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, mU/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProtein\u003c/p\u003e \u003cp\u003e(Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, mg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eSpecific activity\u003c/p\u003e \u003cp\u003e( Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, mU/mg )\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFDH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGshF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCrude enzyme extract\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFDH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGshF\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSBIP 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18.6610\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0538\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e514.6075\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5820\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.7031\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2703\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.9036\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0056\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e190.3790\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5882\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSBIP 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e56.9473\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2329\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e668.5793\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3449\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.8023\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1460\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20.3218\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0994\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e208.7811\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6815\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSBIP 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e54.2308\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3696\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e550.4916\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1651\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3.2023\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1920\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16.9349\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0829\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e196.4446\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5425\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSBIP 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e84.2076\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4114\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e657.3410\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2301\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.9952\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1677\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e28.1146\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1249\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e219.4677\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7930\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSBIP-sfGFP 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.4041\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0051\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e456.4654\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5675\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.3028\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3108\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1755\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0018\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e191.7217\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7792\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSBIP-sfGFP 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.6859\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0062\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e480.8223\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4187\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.5079\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3972\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.2735\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0021\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e198.2211\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5523\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNoABCD 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.1242\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0034\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e470.1924\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7635\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.4067\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1225\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0516\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0014\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e195.3682\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1719\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNoABCD 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.1240\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0033\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e460.7237\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7150\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.2999\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4188\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0539\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0014\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e200.3270\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1160\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eNote: \u0026ldquo;SBIP 1\u0026rdquo; represented NZ9000/SBIP cultured without induction; \u0026ldquo;SBIP 2\u0026rdquo; represented NZ9000/SBIP cultured with Nisin induction; \u0026ldquo;SBIP 3\u0026rdquo; represented NZ9000/SBIP cultured with Nisin induction and Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e3\u003c/sub\u003e addition; \u0026ldquo;SBIP 4\u0026rdquo; represented NZ9000/SBIP cultured by GGC-GM17 with Nisin induction and Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e3\u003c/sub\u003e addition; \u0026ldquo;SBIP-sfGFP 1\u0026rdquo; represented NZ9000/SBIP-sfGFP cultured without induction; \u0026ldquo;SBIP-sfGFP 2\u0026rdquo; represented NZ9000/SBIP-sfGFP cultured with Nisin induction; \u0026ldquo;NoABCD 1\u0026rdquo; represented NZ9000/p8G23FS2 cultured without induction; \u0026ldquo;NoABCD 2\u0026rdquo; represented NZ9000/p8G23FS2 cultured with Nisin induction. The last four groups serve as controls to illustrate the FDH expression problem. Differences among groups were compared using Duncan multiple range test of one-way ANOVA: \"a\u0026rdquo; ~ \u0026ldquo;f\u0026rdquo; represented a diminishing significant difference.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section3\"\u003e \u003ch2\u003e3.3.3 RT-qPCR analysis for SBIP in \u003cem\u003eL. lactis\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eBecause SelA ~ SelD couldn\u0026rsquo;t be assayed like the other two enzymes, RT-qPCR would be an suitably applicable method to further verify the feasibility to produce site-inserted Sec by constructing SBIP in \u003cem\u003eL. lactis\u003c/em\u003e. As Fig. S8 showed, the 23S (1450 bp), 16S (770 bp) and 5S (120 bp) bands were clear, bright and complete for both the sample NZ9000/SBIP and the control NZ9000/p-p, and the OD\u003csub\u003e260\u003c/sub\u003e/OD\u003csub\u003e280\u003c/sub\u003e was all between the ideal 1.8\u0026thinsp;~\u0026thinsp;2.0, proving that total RNA had high purity. Secondly, the shape and trend of RT-qPCR amplification curves displayed by Fig. S9 of the six target protein genes were consistent, indicating that their amplification efficiency was similar. According to F values calculated with the formulas in 2.7.6, the target genes of SelA, SelB, SelC, SelD, GshF and FDH from NZ9000/SBIP was up-regulated by 8.01, 19.03, 925982.32, 34.51, 31879.16 and 28367.04 multiples compared with the control NZ9000/p-p, indicating that all these inserted factors were all proved to play roles along the route of SBIP in \u003cem\u003eL. lactis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Transcriptome analysis of NZ9000/SBIP and NZ9000/p-p\u003c/h2\u003e \u003cdiv id=\"Sec31\" class=\"Section3\"\u003e \u003ch2\u003e3.4.1 Statistical analysis of differential genes between NZ9000/SBIP and NZ9000/p-p\u003c/h2\u003e \u003cp\u003eDifferential genes between NZ9000/SBIP and NZ9000/p-p would be calculated by statistical expression amount. As shown in Fig. S10, compared with NZ9000/p-p, NZ9000/SBIP had a total of 358 differential genes, among which 211 up-regulated genes, including the six target genes SelA, SelB, SelC, SelD, GshF and FDH. The up-regulation of respective 23.2, 19.86, 8.19, 22.80, 18.0 and 21.60 fold compared with NZ9000/p-p, consisted well with the results of RT-qPCR analysis in 3.3.3. All in all, NZ9000/SBIP was successfully constructed through the whole work in this study, indicating that selenium metabolic flow was successfully guided to synthesize Sec-containing selenoprotein by introducing SBIP into LAB.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section3\"\u003e \u003ch2\u003e3.4.2 GO analysis of similar genes compared with NZ9000 reference genome\u003c/h2\u003e \u003cp\u003eGO (Gene Ontology) annotation of similar genes shared by NZ9000/SBIP, NZ9000/p-p and NZ9000 reference genomes was shown in Fig. S11. Firstly, according to BP (Biological Process) statistics, genes involved in peptide and protein transport accounted for 16.38%, while phosphorylation 15.55%. Secondly, according to MF (Molecular Function) statistics, genes involved in ATP binding, DNA binding and nucleic acid binding in target bacteria accounted for 23.75%, 21.61% and 4.55%, respectively. And based on the statistical information of transcriptome differential genes, it was found that PLP (pyridoxal phosphate) dependent aminotransferase involved in phosphorylation was also an up-regulated gene, which should have a synergistic effect with SelA, a member of the folded type I superprotein family of PLP dependent enzymes[56, 57]; ATP binding gene functions are mainly involved in phosphorylase, peptide transporter binding protein, amino acid transporter binding protein, etc. Although SelD is also an ATP binding protein, and there have also been reports proposing that some LAB (e.g. \u003cem\u003eEnterococcus faecalis\u003c/em\u003e, \u003cem\u003eLeuconostoc citreum\u003c/em\u003e, \u003cem\u003eLactobacillus futsaii\u003c/em\u003e) contained SelA and SelD genes[58, 59], they were not yet found in the reference genome of the background bacterium NZ9000[60] (GenBank: CP002094.1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003e3.4.3 KEGG analysis of differential genes and SBIP route created in LAB\u003c/h2\u003e \u003cp\u003eKEGG (Kyoto Encyclopedia of Genes and Genomes) annotations analysis was performed on the differential genes set between NZ9000/SBIP and NZ9000/p-p. The distribution of the major biochemical metabolic pathways and signal transduction pathways in which these differential genes participated was shown in Fig. S12. Among them, most genes involved in amino acid metabolism, accounting for 12.33%. However, the results of the KEGG pathway enrichment analysis for differential genes between NZ9000/SBIP and NZ9000/p-p were shown as Fig.\u0026nbsp;\u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e7\u003c/span\u003ea: The introduction of SBIP into NZ9000 had a great effect on the intracellular amino acid metabolism of \u003cem\u003eL. lactis\u003c/em\u003e, and there were significant differences in the amino acid metabolism of valine, leucine, isoleucine, arginine and others. Meanwhile, it also had a great impact on selenocompound metabolism. Based on the transcriptome differential gene statistics, there were 5 selenocompounds having significant differences, including SelA, SelB, SelC, SelD and FDH. In summary, those key gene tools non-existed in NZ9000 genome originally were all working along the metabolic pathway after introduced. Moreover, the amino acid synthesis and metabolic pathway were significantly restructed after the introduction of SBIP, which was consistent with the directed synthesis of selenoprotein. At last, the heterologous SBIP route created in LAB was shown by Fig.\u0026nbsp;\u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e7\u003c/span\u003eb.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec35\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Differences between fusion expression and independent expression\u003c/h2\u003e \u003cp\u003eAs well-known, green fluorescent protein (GFP) has been generally used as a marker and a fusion tag, but the folding rate of wild-type GFP could be affected by temperature, and the fusion partner would reduce its folding efficiency and FI[61]. Therefore, a more stable GFP mutant sfGFP (super-folder GFP) was used in this study. The FI was reported to be proportional to the total expression, and would not be affected by misfolding of the fusion partner, and was independent of the solubility of the fusion protein[62, 63]. According to Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003e in this study, the enzyme activity of FDH could both be detected out by NZ9000/SBIP and NZ9000/SBIP-sfGFP under different culture modes, but the latter one was much lower than that of the former one. This result was inconsistent with the case by KV Solovyov et al.[64], and was presumed to be affected by the promoters used, because strong promoters do not always lead to higher expression levels due to the burden and toxicity of some target proteins on host cells[65], and the same promoter may even behave differently for different proteins[66]. Moreover, the expression of introduced gene protein might also impose an adverse effect on the NICE system in \u003cem\u003eL. lactis\u003c/em\u003e[67].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec36\" class=\"Section2\"\u003e \u003ch2\u003e4.2 The addition of amino acids and the red selenium phenomenon\u003c/h2\u003e \u003cp\u003eAs a rule, the optimization of protein expression generally starts from four aspects: induction time, concentration of inducer, culture temperature, and composition of medium (such as metal ions, amino acids, ATP, special nitrogen sources or carbon sources)[46, 68]. Accordingly, a new phenomenon was found when expression optimization of NZ9000/SBIP was conducted by four cultivation methods. As Fig. S7-a showed, the red selenium phenomenon of sample ④ (Nisin induction and Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e3\u003c/sub\u003e addition with GGC-GM17 medium) was more significant than that of ③ (Nisin induction and Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e3\u003c/sub\u003e addition with GM17 medium). In order to figure out which amino acid was responsible for the deepest Se-enriched color in this strain, another three cultivation methods shown in Fig. S7-b were used to illustrate it. And it turned out that the red color of cells solution gradually lightened when GM17 medium was added with Glu, Gly and Cys, respectively. Se(Ⅳ) might cause oxidation damage to these AAs, while Se(Ⅳ) was reduced to red nano-selenium which would bound to the cell membrane of the bacteria[69, 70].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec37\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Gene loss during construction of recombinant \u003cem\u003eL. lactis\u003c/em\u003e strains\u003c/h2\u003e \u003cp\u003eTo weaken the cell wall of gram-positive bacteria and improve the transformation efficiency, lysozyme, mutanolysin, penicillin or glycine would be added to the culture-medium[43, 71]. The previous issue about electroporation was mainly experimental conditions, while the reasons for partial gene fragment loss when heterologous plasmids were electroporated into LAB have been few reported. Although the plasmid pIL253 transformant (4.8 kilobases) from \u003cem\u003eL. lactis\u003c/em\u003e was found to miss a multiple cloning site which equaled 0.8 kilobase[71], it was hard to be explained. Unlike the accurately expected Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, the similar issues occurred in this study in Fig. S2: pC5ABD and pCPABD transformants was presumed that SelA-SelB (3233bp) or SelB-SelD (2889 bp) might be deleted during the electroporation, while pG59FS1 transformant also lost 96 bp on the 3\u0026rsquo; end of P59. However, Andreas Schӓfer et al.[72] proposed that gene disruption and allelic exchange would be facilitated by homologous recombination, while plasmids equipped with a genetically modified sacB gene would not be lost from Gram\u003csup\u003e\u0026minus;\u003c/sup\u003e to Gram\u003csup\u003e+\u003c/sup\u003e, which offers valuable suggestions for solving these problems in the future.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec38\" class=\"Section2\"\u003e \u003ch2\u003e4.4 The codon selection for Sec insertion and selenoprotein production\u003c/h2\u003e \u003cp\u003eUp to now, two types of codons have been reported for encoding Sec: (1) Stop codon (UAG or UGA): First, site-specific insertion of Sec into selenoproteins by heterozygous tRNA is mainly dependent on UAG encoding[25, 73]. The stop codon UAG has encoded many UAAs (unnatural amino acids) with different functions and structures, and achieved their site-specific insertion in proteins[74, 75]. However, a single UAG cannot meet the requirements of inserting multiple UAAs into a protein at the same time when research continues to deepen, and the number of stop codons determines that the reuse of it is limited[76]. Second, the stop codon UGA can be recognized as a meaningful codon to encode Sec under certain conditions although its read-through efficiency is less than 5% in prokaryotes and less than 3% in eukaryotes[77]. (2) Sense codon: Markus J. Br\u0026Ouml;cker[78] found that \u003cem\u003eE.coli\u003c/em\u003e could decode 55 sense codons into Sec via the prokaryotic read-through mechanism and Sec encoding didn\u0026rsquo;t strictly depend on specific codons. Therefore, considering the +\u0026thinsp;1 frameshift[79] exists, Yan Qi[80] tried to express human GPx1 to realize the in vitro encoding of Sec with quadruplet codons.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn this study, a multistage metabolic engineering strategy to construct SBIP in \u003cem\u003eL. lactis\u003c/em\u003e NZ9000 for site-directed biosynthesis of Sec-containing selenoprotein was proposed for the first time. By the means of DNA manipulation, the building of recombinant plasmids equipped with the regulatory factors and verifiable factors, and the optimal recombinant plasmids combination to achieve the successful construction of SBIP by \u003cem\u003eL. lactis\u003c/em\u003e, which would be verified by multidimensional determination and analysis. The result turned out that: SelA, SelB, SelC, SelD, GshF and FDH from NZ9000/SBIP up-regulated 8.01, 19.03, 925982.32, 34.51, 31879.16 and 28367.04 multiples compared with NZ9000/p-p; FI/OD\u003csub\u003e600\u003c/sub\u003e of NZ9000/ SBIP-sfGFP was 362.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43; FDH enzyme activity of NZ9000/SBIP reached 28.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 mU/mg, and GshF 219.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79 mU/mg under the optimal expression.. The mechanism of selenoprotein synthesis by LAB was analyzed: NZ9000/NoABCD with SBIP unpresent detected a very small amount of FDH enzyme activity, indicating a few random incorporation of Sec. However, the enzyme activity and transcription level of Sec-containing FDH were significantly improved after pathway inserted and optimized culturing, and the amino acid anabolic pathway was the most significant, indicating the increased incorporation rate of Sec in LAB selenoprotein by directed synthesis. This was such a pioneered case to offer an innovative reference for the engineering transformation and production of specific selenoprotein in the food, fodder or drug supply field.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was financially supported by the National Natural Science Foundation of China General Project (31972050), which should be sincerely acknowledged.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJ.P., Y.Q., L.L., S.Y.,P.S., Y.F.,X.P. and F.Y.searched and collected the literatures; J.P., Y.Q., L.L, S.Y., P.S., L.W., C.L. and T.G. completed investigation, analysis and experiment; J.P. made the tables and figures and wrote the original manuscript; C.W. , X.L and X.H. supervised the project and reviewed the manuscript; C.W. got the project from government and edited the draft. All authors read and approved the manuscript.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLiang X, Xue Z, Zheng Y, Li S, Zhou L, Cao L, Zou Y: \u003cstrong\u003eSelenium supplementation enhanced the expression of selenoproteins in hippocampus and played a neuroprotective role in LPS-induced neuroinflammation.\u003c/strong\u003e \u003cem\u003eInternational Journal of Biological Macromolecules: Structure, Function and Interactions \u003c/em\u003e2023.\u003c/li\u003e\n\u003cli\u003eSchrauzer GN: \u003cstrong\u003eNutritional selenium supplements:product types, quality and safety.\u003c/strong\u003e \u003cem\u003eJournal of the American College of Nutrition \u003c/em\u003e2001, \u003cstrong\u003e20:\u003c/strong\u003e1-4.\u003c/li\u003e\n\u003cli\u003eInfante HG, Hearn R, Catterick T: \u003cstrong\u003eCurrent mass spectrometry strategies for selenium speciation in dietary sources of high-selenium.\u003c/strong\u003e \u003cem\u003eAnalytical and bioanalytical chemistry \u003c/em\u003e2005, \u003cstrong\u003e382:\u003c/strong\u003e957-967.\u003c/li\u003e\n\u003cli\u003eStadtman TC: \u003cstrong\u003eSelenocysteine.\u003c/strong\u003e \u003cem\u003eAnnual Review of Biochemistry \u003c/em\u003e1996, \u003cstrong\u003e65:\u003c/strong\u003e83-100.\u003c/li\u003e\n\u003cli\u003eWang C: \u003cstrong\u003eGeneration of Selenoprotein (Selenopeptide) and its Antioxidant Ability \u003c/strong\u003eJilin University2014.\u003c/li\u003e\n\u003cli\u003eRenga W, Dong L, Qihang C, Zhengshan L, Jingwen Z, Jian M: \u003cstrong\u003eOptimization of vanillin biosynthesis in Escherichia coli K12 MG1655 through metabolic engineering.\u003c/strong\u003e \u003cem\u003eBioresource Technology \u003c/em\u003e2024, \u003cstrong\u003e411\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eCalomme MR, Branden KVD, Berghe DAV: \u003cstrong\u003eSelenium and lactobacillus species.\u003c/strong\u003e \u003cem\u003eJournal of Applied Microbiology \u003c/em\u003e1995, \u003cstrong\u003e79:\u003c/strong\u003e331-340.\u003c/li\u003e\n\u003cli\u003eZhang C, Li Y, Zhu F, Li Z, Chen N: \u003cstrong\u003eMetabolic engineering of an auto-regulated Corynebacterium glutamicum chassis for biosynthesis of 5-aminolevulinic acid.\u003c/strong\u003e \u003cem\u003eBioresource Technology \u003c/em\u003e2020\u003cstrong\u003e:\u003c/strong\u003e124064.\u003c/li\u003e\n\u003cli\u003eJingjing P, Shiyang Y, Yuhui F, Xiaoling L, Chenghua W: \u003cstrong\u003eMechanisms affecting the biosynthesis and incorporation rate of selenocysteine.\u003c/strong\u003e \u003cem\u003eMolecules \u003c/em\u003e2021, \u003cstrong\u003e26:\u003c/strong\u003e7120.\u003c/li\u003e\n\u003cli\u003eZhang J, Fu RY, Hugenholtz J, Li Y, Chen J: \u003cstrong\u003eGlutathione protects Lactococcus lactis against acid stress.\u003c/strong\u003e \u003cem\u003eApplied and Environmental Microbiology \u003c/em\u003e2007, \u003cstrong\u003e73:\u003c/strong\u003e5268-5275.\u003c/li\u003e\n\u003cli\u003eArn\u0026eacute;r E, Sarioglu H, Lottspeich F, Holmgren A, B\u0026ouml;ck A: \u003cstrong\u003eHigh-level expression in Escherichia coli of selenocysteine-containing rat thioredoxin reductase utilizing gene fusions with engineered bacterial-type SECIS elements and co-expression with the selA, selB and selC genes.\u003c/strong\u003e \u003cem\u003eJournal of Molecular Biology \u003c/em\u003e1999, \u003cstrong\u003e292:\u003c/strong\u003e1003-1016.\u003c/li\u003e\n\u003cli\u003eLi M, Meng X, Sun Z, Zhu C, Ji H: \u003cstrong\u003eEffects of NADH Availability on 3-Phenyllactic Acid Production by Lactobacillus plantarum Expressing Formate Dehydrogenase.\u003c/strong\u003e \u003cem\u003eCurrent Microbiology \u003c/em\u003e2019, \u003cstrong\u003e76:\u003c/strong\u003e706-712.\u003c/li\u003e\n\u003cli\u003eHeider J, Baron C, B\u0026ouml;ck A: \u003cstrong\u003eCoding from a distance: dissection of the mRNA determinants required for the incorporation of selenocysteine into protein.\u003c/strong\u003e \u003cem\u003eThe EMBO Journal \u003c/em\u003e1992, \u003cstrong\u003e11:\u003c/strong\u003e3759-3766.\u003c/li\u003e\n\u003cli\u003eZhang M, Jiang L, Zhang Y: \u003cstrong\u003eAdvance on Bioinformatic Analysis of Selenium Metabolic Network and Selenoproteomes.\u003c/strong\u003e \u003cem\u003eCurrent Biotechnology \u003c/em\u003e2017, \u003cstrong\u003e7:\u003c/strong\u003e537-543.\u003c/li\u003e\n\u003cli\u003eMao R, Wu D, Wang Y: \u003cstrong\u003eSurface display on lactic acid bacteria without genetic modification: strategies and applications.\u003c/strong\u003e \u003cem\u003eApplied Microbiology and Biotechnology \u003c/em\u003e2016, \u003cstrong\u003e100:\u003c/strong\u003e9407-9421.\u003c/li\u003e\n\u003cli\u003eBolotin A, Wincker P, Mauger S, Jaillon O, Malarme K, Weissenbach J, Ehrlich SD, Sorokin A: \u003cstrong\u003eThe complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp. lactis IL1403.\u003c/strong\u003e \u003cem\u003eGenome Research \u003c/em\u003e2001, \u003cstrong\u003e11:\u003c/strong\u003e731-753.\u003c/li\u003e\n\u003cli\u003eXiong ZQ, Wei YY, Kong LH, Song X, Ai LZ: \u003cstrong\u003eShort communication: An inducible CRISPR/dCas9 gene repression system in Lactococcus lactis.\u003c/strong\u003e \u003cem\u003eJournal of Dairy Science \u003c/em\u003e2019, \u003cstrong\u003e103:\u003c/strong\u003e161-165.\u003c/li\u003e\n\u003cli\u003eKuipers OP, Beerthuyzen MM, Ruyter PD, Luesink EJ, Vos WD: \u003cstrong\u003eAutoregulation of Nisin Biosynthesis in Lactococcus lactis by Signal Transduction.\u003c/strong\u003e \u003cem\u003eJournal of Biological Chemistry \u003c/em\u003e1995, \u003cstrong\u003e270:\u003c/strong\u003e27299-27304.\u003c/li\u003e\n\u003cli\u003eGasson MJ: \u003cstrong\u003ePlasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing.\u003c/strong\u003e \u003cem\u003eJournal of Bacteriology \u003c/em\u003e1983, \u003cstrong\u003e154:\u003c/strong\u003e1-9.\u003c/li\u003e\n\u003cli\u003eHui W, Xiao L, Linlin H, Yicheng F, Zhiqiang X, Lianzhong A, Xin S: \u003cstrong\u003eProgress in Research on Lactococcus lactis Expression Systems and Their Promoter Regulatory Elements.\u003c/strong\u003e \u003cem\u003eJournal of Food Science \u003c/em\u003e2022, \u003cstrong\u003e43:\u003c/strong\u003e330-336.\u003c/li\u003e\n\u003cli\u003eSoumya MP, Parameswaran R, Nampoothiri KM: \u003cstrong\u003eNisin controlled homologous Over-expression of an exopolysaccharide biosynthetic glycosyltransferase gene for enhanced EPS production in Lactobacillus plantarum BR2.\u003c/strong\u003e \u003cem\u003eBioresource Technology \u003c/em\u003e2023, \u003cstrong\u003e385:\u003c/strong\u003e129387.\u003c/li\u003e\n\u003cli\u003eSong AAL, Lionel LAI, Lim SHE, Rahim RA: \u003cstrong\u003eA review on Lactococcus lactis: from food to factory.\u003c/strong\u003e \u003cem\u003eMicrobial Cell Factories \u003c/em\u003e2017, \u003cstrong\u003e16:\u003c/strong\u003e1-15.\u003c/li\u003e\n\u003cli\u003eZhu D, Liu F, Xu H, Bai Y, Zhang X, Saris PEJ, Qiao M: \u003cstrong\u003eIsolation of strong constitutive promoters from Lactococcus lactis subsp. lactis N8.\u003c/strong\u003e \u003cem\u003eFEMS Microbiology Letters \u003c/em\u003e2015, \u003cstrong\u003e362:\u003c/strong\u003efnv107.\u003c/li\u003e\n\u003cli\u003eVossen JVD, Lelie DVD, Venema G: \u003cstrong\u003eIsolation and characterization of Streptococcus cremoris Wg2-specific promoters.\u003c/strong\u003e \u003cem\u003eApplied and Environmental Microbiology \u003c/em\u003e1987, \u003cstrong\u003e53:\u003c/strong\u003e2452-2457.\u003c/li\u003e\n\u003cli\u003eMukai T, Sevostyanova A, Suzuki T, Fu X, S\u0026ouml;ll D: \u003cstrong\u003eA Facile Method for Producing Selenocysteine-Containing Proteins.\u003c/strong\u003e \u003cem\u003eAngewandte Chemie-International Edition \u003c/em\u003e2018, \u003cstrong\u003e57:\u003c/strong\u003e7215-7219.\u003c/li\u003e\n\u003cli\u003eLu L, Lianzhong A, Yongjun X, Zhiqiang X, Tong G, Xin S: \u003cstrong\u003eA reporter system with the enhanced green fluorescent protein in Lactococcus lactis NZ9000.\u003c/strong\u003e \u003cem\u003eFood and Fermentation Industries \u003c/em\u003e2020, \u003cstrong\u003e46:\u003c/strong\u003e6.\u003c/li\u003e\n\u003cli\u003eMartinez-Jaramillo E, Garza-Morales R, Loera-Arias M, Saucedo-Cardenas O, Montes-de-Oca-Luna R, McNally L, Gomez-Gutierrez J: \u003cstrong\u003eDevelopment of Lactococcus lactis encoding fluorescent proteins, GFP, mCherry and iRFP regulated by the nisin-controlled gene expression system.\u003c/strong\u003e \u003cem\u003eBiotechnic \u0026amp; Histochemistry \u003c/em\u003e2017, \u003cstrong\u003e92:\u003c/strong\u003e167-174.\u003c/li\u003e\n\u003cli\u003eGaeng S, Scherer S, Neve H, Loessner MJ: \u003cstrong\u003eGene cloning and expression and secretion of Listeria monocytogenes bacteriophage-lytic enzymes in Lactococcus lactis.\u003c/strong\u003e \u003cem\u003eApplied and Environmental Microbiology \u003c/em\u003e2000, \u003cstrong\u003e66:\u003c/strong\u003e2951-2958.\u003c/li\u003e\n\u003cli\u003eManzine LR, Cassago A, da Silva MTA, Thiemann OH: \u003cstrong\u003eAn efficient protocol for the production of tRNA-free recombinant Selenocysteine Synthase (SELA) from Escherichia coli and its biophysical characterization.\u003c/strong\u003e \u003cem\u003eProtein expression and purification \u003c/em\u003e2013, \u003cstrong\u003e88:\u003c/strong\u003e80-84.\u003c/li\u003e\n\u003cli\u003eHaruna K-i, Alkazemi MH, Liu Y, S\u0026ouml;ll D, Englert M: \u003cstrong\u003eEngineering the elongation factor Tu for efficient selenoprotein synthesis.\u003c/strong\u003e \u003cem\u003eNucleic acids research \u003c/em\u003e2014, \u003cstrong\u003e42:\u003c/strong\u003e9976-9983.\u003c/li\u003e\n\u003cli\u003eMansell JB, Gu\u0026eacute;vremont D, Poole ES, Tate WP: \u003cstrong\u003eA dynamic competition between release factor 2 and the tRNASec decoding UGA at the recoding site of Escherichia coli formate dehydrogenase H.\u003c/strong\u003e \u003cem\u003eThe EMBO journal \u003c/em\u003e2001.\u003c/li\u003e\n\u003cli\u003eNoinaj, Nicholas: \u003cstrong\u003eStructural Insights into the Catalytic Mechanism of Escherichia coli Selenophosphate Synthetase.\u003c/strong\u003e \u003cem\u003eJournal of Bacteriology \u003c/em\u003e2012.\u003c/li\u003e\n\u003cli\u003eGursinsky T, Grobe D, Schierhorn A, Jager J, Andreesen JR, Sohling B: \u003cstrong\u003eFactors and Selenocysteine Insertion Sequence Requirements for the Synthesis of Selenoproteins from a Gram-Positive Anaerobe in Escherichia coli.\u003c/strong\u003e \u003cem\u003eApplied \u0026amp; Environmental Microbiology \u003c/em\u003e2008, \u003cstrong\u003e74:\u003c/strong\u003e1385-1393.\u003c/li\u003e\n\u003cli\u003eXiuxiu F, Jingyan W: \u003cstrong\u003eEfficient Expression of Glutathione Peroxidase with Chimeric tRNA in Amber-less Escherichia coli.\u003c/strong\u003e \u003cem\u003eACS Synthetic Biology \u003c/em\u003e2018, \u003cstrong\u003e7\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eMierau I, Kleerebezem M: \u003cstrong\u003e10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis.\u003c/strong\u003e \u003cem\u003eApplied Microbiology and Biotechnology \u003c/em\u003e2005, \u003cstrong\u003e68:\u003c/strong\u003e705-717.\u003c/li\u003e\n\u003cli\u003eO\u0026apos;Sullivan DJ, Klaenhammer TR: \u003cstrong\u003eHigh-and low-copy-number Lactococcus shuttle cloning vectors with features for clone screening.\u003c/strong\u003e \u003cem\u003eGene \u003c/em\u003e1993, \u003cstrong\u003e137:\u003c/strong\u003e227-231.\u003c/li\u003e\n\u003cli\u003eXu C, Shi Z, Shao J, Yu C, Xu Z: \u003cstrong\u003eMetabolic engineering of Lactococcus lactis for high level accumulation of glutathione and S-adenosyl-L-methionine.\u003c/strong\u003e \u003cem\u003eWorld Journal of Microbiology and Biotechnology \u003c/em\u003e2019, \u003cstrong\u003e35:\u003c/strong\u003e185.\u003c/li\u003e\n\u003cli\u003eRui-yan F, Jian C, Yin L: \u003cstrong\u003eProduction of glutathione by recombinant Lactococcus lactis.\u003c/strong\u003e \u003cem\u003eChinese Journal of Bioprocess Engineering \u003c/em\u003e2004, \u003cstrong\u003e2:\u003c/strong\u003e30-35.\u003c/li\u003e\n\u003cli\u003eBerry MJ, Banu L, Chen Y, Mandel SJ, Kieffer JD, Harney JW, Larsen PR: \u003cstrong\u003eRecognition of UGA as a selenocysteine codon in type I deiodinase requires sequences in the 3\u0026prime; untranslated region.\u003c/strong\u003e \u003cem\u003eNature \u003c/em\u003e1991, \u003cstrong\u003e353:\u003c/strong\u003e273-276.\u003c/li\u003e\n\u003cli\u003eFourmy D, Guittet E, Yoshizawa S: \u003cstrong\u003eStructure of prokaryotic SECIS mRNA hairpin and its interaction with elongation factor SelB.\u003c/strong\u003e \u003cem\u003eJournal of Molecular Biology \u003c/em\u003e2002, \u003cstrong\u003e324:\u003c/strong\u003e137-150.\u003c/li\u003e\n\u003cli\u003ePecher A, Zinoni F, Bck A: \u003cstrong\u003eThe seleno-polypeptide of formic dehydrogenase (formate hydrogen-lyase linked) from Escherichia coli: genetic analysis.\u003c/strong\u003e \u003cem\u003eArchives of Microbiology \u003c/em\u003e1985, \u003cstrong\u003e141:\u003c/strong\u003e359-363.\u003c/li\u003e\n\u003cli\u003eWillis T, Jadayel D, Coignet L, Abdul-Rauf M, Treleaven J, Catovsky D, Dyer M: \u003cstrong\u003eRapid molecular cloning of rearrangements of the IGHJ locus using long-distance inverse polymerase chain reaction.\u003c/strong\u003e \u003cem\u003eBlood, The Journal of the American Society of Hematology \u003c/em\u003e1997, \u003cstrong\u003e90:\u003c/strong\u003e2456-2464.\u003c/li\u003e\n\u003cli\u003eDayong R, Chang L, Yan-qing Q, Shou-wen D, Huan-huan G, Hong-feng L, Yang L, Ning-yi J: \u003cstrong\u003eOptimization of High-Efficiency Electrotransformation Conditions for Lactococcus lactis.\u003c/strong\u003e \u003cem\u003eChinese Journal of Veterinary Medicine \u003c/em\u003e2012, \u003cstrong\u003e46:\u003c/strong\u003e5-9.\u003c/li\u003e\n\u003cli\u003ePieniz S, Okeke BC, Andreazza R, Brandelli A: \u003cstrong\u003eEvaluation of selenite bioremoval from liquid culture by Enterococcus species.\u003c/strong\u003e \u003cem\u003eMicrobiological Research \u003c/em\u003e2011, \u003cstrong\u003e166:\u003c/strong\u003e176-185.\u003c/li\u003e\n\u003cli\u003eXiao F, Wang H, Shi Z, Huang Q, Xu Z: \u003cstrong\u003eMulti-level metabolic engineering of Pseudomonas mutabilis ATCC31014 for efficient production of biotin.\u003c/strong\u003e \u003cem\u003eMetabolic Engineering \u003c/em\u003e2019, \u003cstrong\u003e61:\u003c/strong\u003e406-415.\u003c/li\u003e\n\u003cli\u003eLiu X: \u003cstrong\u003eDirected evolution of glutathione bifunctional svnthase and its application in L-Glutathione synthesis.\u003c/strong\u003e Zhejiang University2018.\u003c/li\u003e\n\u003cli\u003eSlusarczyk H, Felber S, Kula MR, Pohl M: \u003cstrong\u003eStabilization of NAD‐dependent formate dehydrogenase from Candida boidinii by site‐directed mutagenesis of cysteine residues.\u003c/strong\u003e \u003cem\u003eEuropean Journal of Biochemistry \u003c/em\u003e2000, \u003cstrong\u003e267:\u003c/strong\u003e1280-1289.\u003c/li\u003e\n\u003cli\u003eZhenhua Z, Yuli X, Tiejun W, Hong Z, Cunduo T, Yunchao K, Lunguang Y: \u003cstrong\u003eDirected evolution of catalytic activity of formate dehydrogenase and its efficient expression.\u003c/strong\u003e \u003cem\u003eChinese Journal of Applied Chemistry \u003c/em\u003e2021, \u003cstrong\u003e38:\u003c/strong\u003e704.\u003c/li\u003e\n\u003cli\u003eZhu Z: \u003cstrong\u003eEfficient identification of anti-acid components in Lactococcus lactis and its function analysis.\u003c/strong\u003e Jiangnan University2019.\u003c/li\u003e\n\u003cli\u003eMa T, Lu J, Zhu J, Li X, Gu H, Montalb\u0026aacute;n-L\u0026oacute;pez M, Wu X, Luo S, Zhao Y, Jiang S: \u003cstrong\u003eThe Secretion of Streptomyces monbaraensis Transglutaminase From Lactococcus lactis and Immobilization on porous magnetic nanoparticles.\u003c/strong\u003e \u003cem\u003eFrontiers in Microbiology \u003c/em\u003e2019, \u003cstrong\u003e10:\u003c/strong\u003e1675.\u003c/li\u003e\n\u003cli\u003eBinbin C: \u003cstrong\u003eCloning, expression, characteristics andapplication of bifunctional glutathione synthetase.\u003c/strong\u003e Zhejiang University, College Of Chemical\u0026amp;Biological Engineering; 2015.\u003c/li\u003e\n\u003cli\u003eThom\u0026eacute; R, Gust A, Toci R, Mendel R, Bittner F, Magalon A, Walburger A: \u003cstrong\u003eA sulfurtransferase is essential for activity of formate dehydrogenases in Escherichia coli.\u003c/strong\u003e \u003cem\u003eJournal of Biological Chemistry \u003c/em\u003e2012, \u003cstrong\u003e287:\u003c/strong\u003e4671-4678.\u003c/li\u003e\n\u003cli\u003eAxley M, Grahame DA, Stadtman TC: \u003cstrong\u003eEscherichia coli formate-hydrogen lyase. Purification and properties of the selenium-dependent formate dehydrogenase component.\u003c/strong\u003e \u003cem\u003eJournal of Biological Chemistry \u003c/em\u003e1990, \u003cstrong\u003e265:\u003c/strong\u003e18213-18218.\u003c/li\u003e\n\u003cli\u003eAshrafi H, Sadeghi AA, Chamani M: \u003cstrong\u003eEffect of Organic Selenium Supplementation on the Antioxidant Status, Immune Response, and the Relative Expression of IL-2 and IFN-\u0026gamma; Genes in Ewes During the Hot Season.\u003c/strong\u003e \u003cem\u003eBiological Trace Element Research \u003c/em\u003e2024, \u003cstrong\u003e202:\u003c/strong\u003e2052-2061.\u003c/li\u003e\n\u003cli\u003eCaiyi S, Yuting L, Xiaomin L, Yihua W, Guangzhi F, Yu S: \u003cstrong\u003eEffect of Selenium on Extracellular Enzyme Activities of Pleurotus ostreatus.\u003c/strong\u003e \u003cem\u003eJournal of Anhui Agricultural Sciences \u003c/em\u003e2023, \u003cstrong\u003e51:\u003c/strong\u003e42-44.\u003c/li\u003e\n\u003cli\u003eAraiso Y, Palioura S, Ishitani R, Sherrer RL, O\u0026rsquo;Donoghue P, Yuan J, Oshikane H, Domae N, DeFranco J, S\u0026ouml;ll D: \u003cstrong\u003eStructural insights into RNA-dependent eukaryal and archaeal selenocysteine formation.\u003c/strong\u003e \u003cem\u003eNucleic Acids Research \u003c/em\u003e2008, \u003cstrong\u003e36:\u003c/strong\u003e1187-1199.\u003c/li\u003e\n\u003cli\u003eGanichkin OM, Xu XM, Carlson BA, Mix H, Hatfield DL, Gladyshev VN, Wahl MC: \u003cstrong\u003eStructure and catalytic mechanism of eukaryotic selenocysteine synthase.\u003c/strong\u003e \u003cem\u003eJournal of Biological Chemistry \u003c/em\u003e2008, \u003cstrong\u003e283:\u003c/strong\u003e5849-5865.\u003c/li\u003e\n\u003cli\u003eYan Z, Romero H, Salinas G, Gladyshev VN: \u003cstrong\u003eDynamic evolution of selenocysteine utilization in bacteria: a balance between selenoprotein loss and evolution of selenocysteine from redox active cysteine residues.\u003c/strong\u003e \u003cem\u003eGenome Biology \u003c/em\u003e2006, \u003cstrong\u003e7:\u003c/strong\u003eR94.\u003c/li\u003e\n\u003cli\u003eMorales AE, Gonz\u0026aacute;lez L, Contreras EL, Serrano GR: \u003cstrong\u003eSelA and SelD genes involved in selenium absorption metabolism in lactic acid bacteria isolated from Mexican cheeses.\u003c/strong\u003e \u003cem\u003eInternational Dairy Journal \u003c/em\u003e2019, \u003cstrong\u003e103:\u003c/strong\u003e104629.\u003c/li\u003e\n\u003cli\u003eLinares DM, Kok J, Poolman B: \u003cstrong\u003eGenome Sequences of Lactococcus lactis MG1363 (Revised) and NZ9000 and Comparative Physiological Studies.\u003c/strong\u003e \u003cem\u003eJournal of Bacteriology \u003c/em\u003e2010, \u003cstrong\u003e192:\u003c/strong\u003e5806-5812.\u003c/li\u003e\n\u003cli\u003eHuang C, Cheng Y, Lixin MA, Yan H: \u003cstrong\u003eThe expression of methyl parathion hydrolase fusion with gfp in E. coli.\u003c/strong\u003e \u003cem\u003eJournal of Hubei University(Natural Science) \u003c/em\u003e2019.\u003c/li\u003e\n\u003cli\u003eZimmer M: \u003cstrong\u003eGreen fluorescent protein (GFP): applications, structure, and related photophysical behavior.\u003c/strong\u003e \u003cem\u003eCheminform \u003c/em\u003e2002, \u003cstrong\u003e102:\u003c/strong\u003e759-781.\u003c/li\u003e\n\u003cli\u003eSchaefer SK, Heidemann J, Puchert A, Koelbel K: \u003cstrong\u003eCrystal structure of a domain-swapped photoactivatable sfGFP variant provides evidence for GFP folding pathway.\u003c/strong\u003e \u003cem\u003eFEBS Journal \u003c/em\u003e2019, \u003cstrong\u003e286:\u003c/strong\u003e2329-2340.\u003c/li\u003e\n\u003cli\u003eSolovyov KV, Kern AM, Grudinina NA, Aleynikova TD: \u003cstrong\u003eGenetic Structures and Conditions of their Expression, which Allow Receiving Native Recombinant Proteins with High Output.\u003c/strong\u003e \u003cem\u003eInternational Journal of Biomedicine \u003c/em\u003e2012, \u003cstrong\u003e2:\u003c/strong\u003e78-81.\u003c/li\u003e\n\u003cli\u003eHannig G, Makrides SC: \u003cstrong\u003eStrategies for optimizing heterologous protein expression in Escherichia coli.\u003c/strong\u003e \u003cem\u003eTrends in Biotechnology \u003c/em\u003e1998, \u003cstrong\u003e16:\u003c/strong\u003e54-60.\u003c/li\u003e\n\u003cli\u003eMutalik VK, Guimaraes JC, Cambray G, Mai QA, Christoffersen MJ, Martin L, Yu A, Lam C, Rodriguez C, Bennett G: \u003cstrong\u003eQuantitative estimation of activity and quality for collections of functional genetic elements.\u003c/strong\u003e \u003cem\u003eNature Methods \u003c/em\u003e2013, \u003cstrong\u003e10:\u003c/strong\u003e347.\u003c/li\u003e\n\u003cli\u003eLiang X, Sun Z, Zhong J, Zhang Q, Huan L: \u003cstrong\u003eAdverse effect of nisin resistance protein on nisin-induced expression system in Lactococcus lactis.\u003c/strong\u003e \u003cem\u003eMicrobiological Research \u003c/em\u003e2010, \u003cstrong\u003e165:\u003c/strong\u003e458-465.\u003c/li\u003e\n\u003cli\u003eJiaHui L: \u003cstrong\u003eExpression and Optimization of the Novel Bifunctional.Glutathione Synthetase in Pichia pastoris and Escherichia coli.\u003c/strong\u003e Zhejiang University2019.\u003c/li\u003e\n\u003cli\u003eFang Y, Zhi H, Zhenjiang G, Wenjie Z: \u003cstrong\u003eOxidation of phycocyanin molecules by Se(IV) and formation of nano-Se(0).\u003c/strong\u003e \u003cem\u003eMarine Sciences \u003c/em\u003e2010, \u003cstrong\u003e34:\u003c/strong\u003e55-65.\u003c/li\u003e\n\u003cli\u003eSun Y, Wang H, Zhou L, Chang M, Yue T, Yuan Y, Shi Y: \u003cstrong\u003eDistribution characteristics of organic selenium in Se-enriched Lactobacillus (Lactobacillus paracasei).\u003c/strong\u003e \u003cem\u003eLWT Food Science and Technology \u003c/em\u003e2022, \u003cstrong\u003e165:\u003c/strong\u003e113699.\u003c/li\u003e\n\u003cli\u003eHolo H, Nes IF: \u003cstrong\u003eHigh-Frequency Transformation, by Electroporation, of Lactococcus lactis subsp. cremoris Grown with Glycine in Osmotically Stabilized Media.\u003c/strong\u003e \u003cem\u003eApplied and Environmental Microbiology \u003c/em\u003e1990, \u003cstrong\u003e55:\u003c/strong\u003e3119-3123.\u003c/li\u003e\n\u003cli\u003eSchaefer A, Tauch A, Jaeger W, Kalinowski J: \u003cstrong\u003eSmall mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum.\u003c/strong\u003e \u003cem\u003eGene \u003c/em\u003e1994, \u003cstrong\u003e145:\u003c/strong\u003e69-73.\u003c/li\u003e\n\u003cli\u003eHoffman KS, Chung CZ, Sll TMK-KJBOD: \u003cstrong\u003eRecoding UAG to selenocysteine in Saccharomyces cerevisiae.\u003c/strong\u003e \u003cem\u003eRNA \u003c/em\u003e2023, \u003cstrong\u003e29:\u003c/strong\u003e1400-1410.\u003c/li\u003e\n\u003cli\u003eKrahn N, Tharp JM, Crnković A, S\u0026ouml;ll D: \u003cstrong\u003eEngineering aminoacyl-tRNA synthetases for use in synthetic biology.\u003c/strong\u003e \u003cem\u003eThe Enzymes \u003c/em\u003e2020.\u003c/li\u003e\n\u003cli\u003eAldag C, Br\u0026ouml;cker MJ, Hohn MJ, Prat L, Hammond G, Plummer A, S\u0026ouml;ll D: \u003cstrong\u003eRewiring Translation for Elongation Factor Tu‐Dependent Selenocysteine Incorporation.\u003c/strong\u003e \u003cem\u003eAngewandte Chemie \u003c/em\u003e2013, \u003cstrong\u003e52:\u003c/strong\u003e1441-1445.\u003c/li\u003e\n\u003cli\u003eHankore ED, Zhang L, Chen Y, Liu K, Niu W, Guo J: \u003cstrong\u003eGenetic Incorporation of Noncanonical Amino Acids Using Two Mutually Orthogonal Quadruplet Codons.\u003c/strong\u003e \u003cem\u003eACS Synthetic Biology \u003c/em\u003e2019.\u003c/li\u003e\n\u003cli\u003eSuppmann S, Persson BC, Bck A: \u003cstrong\u003eDynamics and efficiency in vivo of UGA-directed selenocysteine insertion at the ribosome.\u003c/strong\u003e \u003cem\u003eThe EMBO Journal \u003c/em\u003e1999, \u003cstrong\u003e18:\u003c/strong\u003e2284-2293.\u003c/li\u003e\n\u003cli\u003eBr\u0026ouml;cker DMJ, Ho JML, Church PGM, S\u0026ouml;ll PD, O\u0026apos;Donoghue PP: \u003cstrong\u003eRecoding the Genetic Code with Selenocysteine.\u003c/strong\u003e \u003cem\u003eAngewandte Chemie International Edition \u003c/em\u003e2014.\u003c/li\u003e\n\u003cli\u003eCurran J, Yarus M: \u003cstrong\u003eReading frame selection and transfer RNA anticodon loop stacking.\u003c/strong\u003e \u003cem\u003eScience \u003c/em\u003e1987, \u003cstrong\u003e238:\u003c/strong\u003e1545-1550.\u003c/li\u003e\n\u003cli\u003eQi Y: \u003cstrong\u003eExpression and characterization of novel bifunctional antioxidant enzymes and exploration of a new encoding method for Sec in vitro.\u003c/strong\u003e Jilin University2024.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Selenocysteine, Lactococcus lactis, Formate dehydrogenase, Bifunctional glutathione synthetase, selenium enrichment","lastPublishedDoi":"10.21203/rs.3.rs-5428752/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5428752/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSe-enriched \u003cem\u003elactic acid bacteria\u003c/em\u003e (LAB) exist unclear metabolic flow, unstable composition of selenium spectrum and low selenoprotein content such prominent problems caused by complex metabolic pathway and non-specific incorporation of selenium currently. Accordingly, this study reports how to introduce the firstly proposed Selenocysteine Biosynthesis and Insertion Pathway (SBIP) into \u003cem\u003eLactococcus lactis\u003c/em\u003e (\u003cem\u003eL. lactis\u003c/em\u003e) and specifically guide selenium metabolic flow to direct synthesis of specific selenoprotein with employed multi-level metabolic engineering strategies. In result, the integration of these key factors turned out to facilitate the establishment of SBIP in NZ9000: SelA, SelB, SelC, SelD, GshF and FDH from NZ9000/SBIP up-regulated 8.01, 19.03, 925982.32, 34.51, 31879.16 and 28367.04 multiples compared with NZ9000/p-p; FI/OD\u003csub\u003e600\u003c/sub\u003e of NZ9000/SBIP-sfGFP was 362.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43; FDH enzyme activity of NZ9000/SBIP reached 28.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 mU/mg, and GshF 219.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79 mU/mg under the optimal expression. This first successful implementation of directed synthesis of selenoprotein FDH would indicate a whole new direction to supply Sec-contained proteins through biosynthesis in LAB factory.\u003c/p\u003e","manuscriptTitle":"Metabolic engineering of Selenocysteine Biosynthesis and Insertion Pathway in Lactococcus lactis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-13 10:38:41","doi":"10.21203/rs.3.rs-5428752/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"de7920f3-1092-4704-a7c8-bc34fedf1e6f","owner":[],"postedDate":"December 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-17T00:23:08+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-13 10:38:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5428752","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5428752","identity":"rs-5428752","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}