Characterization of vector elements and soluble expression of PCV3  cap protein in Kluyveromyces marxianus

Characterization of vector elements and soluble expression of PCV3 cap protein in Kluyveromyces marxianus | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL Yeast This is a preprint and has not been peer reviewed. Data may be preliminary. 5 April 2025 V1 Latest version Share on Characterization of vector elements and soluble expression of PCV3 cap protein in Kluyveromyces marxianus Authors : Jiamei Li , Xuechen Yang , Mingyue Xu , Wenjing Yin , Guoyu Yang , Yueting Zheng , Wei Yang , and Zhang Wei [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174381314.49571039/v1 Published Yeast Version of record Peer review timeline 245 views 190 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Due to thermotolerance and rapid growth yeast Kluyveromyces marxianus ( K.marxianus ) is emerging as a new platform organism for production of recombinant proteins. We constructed an expression vector to express exogenous proteins. Firstly, qPCR was used to validate the expression efficiency of endogenous promoters. Selecting PDC1 as the promoter to initiate the expression of EGFP for validation, the vector could successfully express foreign proteins. With Hygromycin in the medium the vector can accumulate in the cell. Further construction the cap of porcine circovirus type 3 ( PCV3 ) was carried out on the vector, and soluble PCV3 cap protein was obtained. Under electron microscopy, PCV3 cap formed viral like particles(VLP). This study successfully constructed the expression vector and elements of K.marxianus , which can further promote the research on the expression of exogenous proteins in K.marxianus . The soluble expression of PCV3 cap and formed VLP structure laid the foundation for further developing PCV3 vaccines. Introduction Yeast has been greatly used in food, enzymes, biofuels, flavors and pharmaceutials. Some of the commonly employed yeasts for research as well as industrial processes are Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Kluyveromyces lactis and Yarrowia lipolytica. Recently another yeast K. marxianus , a sister species of K. lactis , has also emerged as a valuable strain for biotechnological applications (Karim, Gerliani and Aider 2020). K.marxianus possesses various desirable properties which is absent in many other commonly used yeast, and thus the strain is a potential substitute for biotechnological needs. It’s one of the fastest growing organism as compared to any of the other known eukaryotes(Groeneveld, Stouthamer and Westerhoff 2009). It’s a thermotolerant yeast that could grow up to 52 ℃(Banat, Nigam and Marchant 1992) . K. marxianus can assimilate various sugars such as glucose, lactose, mannose, galactose and xylose(Fonseca, de Carvalho and Gombert 2013, Rodrussamee et al. 2011). K. marxianus have been broadly used in biotechnoloy including native enzymes production. K. marxianus possess natural ability to secrete enzymes such as inulinase to hydrolyze sucrose and inulin(Rouwenhorst et al. 1988). Inulinase comprises more than 60% of total excreted proteins in the culture fluid for certain K. marxianus strains, indicating that the inulinase is expressed from a strong promoter and its secretion is instigated by an effffective signal sequence(Zhou et al. 2018). PCV3 belongs to the Circovirus genus of the Circoviridae family(Ouyang et al. 2019, Liu et al. 2020).The virus was first identified in American domestic pigs with porcine dermatitis and nephropathy syndrome (PDNS)-like clinical signs, cardiac and multisystemic inflammation in 2016(Phan et al. 2016). Recent studies on epidemiology and pathology of PCV3 have further demonstrated that PCV3 infection is related to the respiratory, digestive and/or reproductive disorders, and multisystemic inflammation(Opriessnig et al. 2020). There are two major open reading frames (ORFs) in PCV3 complete genome. ORF1 encodes a viral replication-associated protein (Rep). ORF2, which orients in the direction opposite to ORF1, encodes cap protein(Palinski et al. 2017). Cap protein is the only structural protein of PCVs . PCV3 cap protein has been considered as the main diagnostic target and vaccine candidate(Deng et al. 2018, Li et al. 2018, Zhang et al. 2019) In this study, we carried out analysis of available transcriptomic profile of K.marxianus , and identified novel promoters for high expression of proteins. we tested the promoter and vector by expression green Green fluorescent protein. We futher used the promoter cassette to engineer K.marxianus with PCV3 cap production. We obtained soluble PCV3 cap with VLP structure that further suited for vaccines development. Also the vector with the promoter can be used to express other heterologous proteins and thus cantributes to novel genetic tools for biotechnological applications of K.marxianus . Materials and methods Microorganisms and plasmids The host strain K. marxianus was from China General Microbiological Culture Collection Center, (CGMCC No.2.3940), broad-host-range Cas9/gRNA co-expression backbone plasmid pUDP002 was from addgene (Plasmid #103872)(Juergens et al. 2018). pEGFP-N1 vector was derived from laboratory preservation. pUDP-pdc1,pUDP-pdc1-EGFP, pUDP-pgk1-EGFP, pUDP-gdh1-EGFP and pUDP-pdc1-cap were constructed in this experiment. YPD media is composed of 1% yeast extract, 2% peptone and 2% dextrose (w/v). 200μg/mL hygromycin(A600230, sangon) was added into the medium for culturing K. marxianus with plasmid. Promoter analysis We first analyzed the promoter strength from available RNA-seq data(Lertwattanasakul et al. 2015), high transcription genes were futher verified by quantitative-PCR. Yeast, grown in YPD media at 30℃ and 200 rpm until mid-log phase, were harvested and total RNA was isolated using Yeast RNA Purifification Kit (IB03KA7017,sangon). The 1μg of isolated RNA was used to prepare cDNA using a cDNA synthesis kit (RR037A,Takara). For quantitative RealTime PCR (qRT-PCR),1μl cDNA was amplifified by using gene specifific primer (Table S1) and following manufacturer’s protocol. The reaction was subjected to an initial denaturation at 95℃ for 30s and 40 repetitive cycles of 10s at 95℃, 30s at 60℃ in CFX96 Touch Real-Time PCR System (BIO-RED). Vector design and construction The pUDP002 vector was used as a backbone for construction of recombinant yeast expression vector. pUDP002 was cut by enzyme AflII and BclI(R0520V,R3160V,NEB) and about 5700bp fragment was recovered by DNA Gel recovery kit(B518131,Sangon). PDC1 promoter and terminator with overlap sequences were amplified respectively from genome of K.marxianus , then were joined together by infusion amplification. The PDC1 with recombination sequence was inserted into pUDP002 backbone fragment by Seamless Cloning Kit(C11301,Vazyme). The new constructed vector with PDC1 promoter and terminator was named pUDP-pdc1. EGFP gene fused 6 His tag at C terminal with recombinant sequence was amplified from pEGFP-N1 vector and then inserted into pUDP-pdc1 vector with EcoRI and XbaI digested fragment, and the new vector was named pUDP-pdc1-EGFP. The promoter PGK1 and GDH1 were cloned from genome of K.marxianus, And followed inserted into pUDP-pdc1-EGFP( Afe Ⅰ and Eco R Ⅰ digested) replaced PDC1. The new constructed vector named pUDP-pgk1-EGFP and pUDP-gdh1-EGFP respectly. The cap gene of PCV3 (BJ-YH2016) fused 6 His tag at C terminal was optimized on the basis of codon usage for K.marxianus and Synthesized by general biol Corporation(china). By the same manner, PCV3 cap gene was amplified and inserted into EcoRI and XbaI digested fragment of pUDP-pdc1, and the new vector was named pUDP-pdc1-cap. Determination of plasmid copy number pUDP-pdc1-EGFP, pUDP-pdc1-cap were electrically transformed into K.marxianus . Three different colonies from each transformation were grown in the YPD medium with 200μg/ml hygromycin overnight and then grown in the YPD liquid medium without hygromycin for different time. Genomic DNA was extracted from the culture by Yeast Genomic DNA Purification Kit ( B518227, Sangon) and then subjected to the quantitative PCR by using SYBR qPCR Master Mix（Q711-02,Vazyme). The copy number of plasmid was determined by comparing the level of PUC ORI in the plasmid with that of endogenous LEU2 promoter region in the chromosome. Vector with PDC1 promoter driving target gene transcription analysis K.marxianus with pUDP-pdc1-EGFP or pUDP-pdc1-cap were cultured with liquid YPD with 200μg/mL hygromycin at 30°C for 24h, 48h and 72h. the total RNA isolation, cDNA synthesis and qRT-PCR methods were same as above mentioned. For detection EGFP and PCV3 cap the different primers (Table S1) were used. And to discriminate inner vector DNA contamination the primers in vector ORI region were designed and amplified the same samples. Fluorometric analysis the expression of EGFP K.marxianus with pUDP-pdc1-EGFP, pUDP-pgk1-EGFP or pUDP-gdh1-EGFP were cultured with liquid YPD with 200μg/mL hygromycin at 30°C for 24h, 48h and 72h. EGFP fluorescence was measured on VArioskan Flash(Thermo Scientific) with excitation and emission set at 485 and 525nm respectively.The ratio of fluorescence intensity to OD600 determined the expression level of GFP. EGFP and PCV3 cap expression and Immunoblot analysis K.marxianus with pUDP-pdc1-EGFP, pUDP-pgk1-EGFP, pUDP-gdh1-EGFP or pUDP-pdc1-cap was inoculated into 100 ml of liquid YPD medium with 200μg/mL hygromycin and cultured at 30 °C for 24h, 48h and 64h. The cells were harvested by centrifugation and washed with PBS buffer. The cells were lysed, and cell-debris was removed by centrifugation at 3000g for 2 min. The lysate supernatant was used for immunoblotting. About 10–15μg of total protein from cell lysate was subjected to electrophoresis onto a 12% SDS–polyacrylamide gel. The proteins were then transferred onto PVDF membrane. A 1:5000 dilution of an anti-GFP (Mouse anti GFP-Tag mAb,AE012,ABclonal) or anti-His6 (Mouse anti His-Tag mAb,AE003,ABclonal) antibody was used for detection. Transmission electronic microscopy The formation of PCV2 VLPs was examined by transmission electron microscopy. The recombinant protein was eluted by Q-column with salt ion concentration gradient. Then protein was dialyzed against 50 mM of Tris with different concentrations of NaCl (100 mM, 150 mM, 300 mM) and pH7.5. then samples were spotted onto carbon-coated copper grids. After adsorption for 5 min at room temperature, the copper grids were dried with filter papers followed by staining with 3% of phosphotungstic acid (PTA) for 5 min, and then imaged at an accelerating voltage of 120 kV. Result Promoter analysis and choice The promoter is the dominant factor that regulates gene expression in vectors. Based on a previous RNA-seq of the K. marxianus (Lertwattanasakul et al. 2015),high expression genes were retested by Q-PCR,the PDC1 gene shown the high expression level in different culture time, and the promoter and terminator of PDC1 was chosen to continue construct expression vector. Upstream 0 to 2000bp of the start codon ATG is defined as the promoter region and amplified from the genome. Insert Fig.1 Vector construction and test Both PDC1 promoter and terminator was amplified from the genome of K .marxianus , and then linked into one fragment. The pUDP002 vector was digested by both AflII and BclI. Fragment with Hygromycin resistance gene, yeast replication origin, E.coli replication origin and ampicillin resistance gene were recovered as backbone. By seamless cloning the PDC1 promoter and terminator was inserted into the backbone to form a new expression vector named pUDP-pdc1. Then EGFP gene and PCV3 cap gene was inserted into the vector pUDP-pdc1 respectively for subsequent protein expression. Insert Fig.2 Copy number and stability of plasmid pUDP-pdc1-EGFP were electrically transformed into K.marxianus . K.marxianus was cultured without hygromycin conditions for different time. Q-PCR primers specific for INU promoter and plasmid ORI region were designed both for genome and plasmid copy number detection. For 24h without hygromycin the plasmid about 27.5 fold of genome and 72h without hygromycin the plasmid still stable copied about 5.2 fold of K.marxianus genome. Insert Fig.3 Vector with pDC1 promoter driving EGFP and PCV3 cap transcription pUDP-pdc1-EGFP and pUDP-pdc1-cap vectors were electrically transferred to the K.marxianus respectively according the previous method. The RNA transcription level were tested by Q-PCR, to wipe off the plasmid DNA contaminate, primers for plasmid ORI region also amplified as control. The reslut shown PDC1 could drive exogenous gene transcription properly. For PDC1 also transcribes endogenous PDC1 gene. So we compared the endogenous and exogenous gene transcription level both driven by PDC1 promoter. The result shown with the pUDP-pdc1-EGFP plasmid, at 24h, 48h and 72h EGFP transcription level compared with endogenous gene PDC1 about 0.6 fold,1.3 fold and 3.0 fold respectively. The result showed that both endogenous PDC1 gene and vector pUDP-pdc1-EGFP competed for PDC1-associated transcription factors. And the transcription level of EGFP driven by PDC1 promoter in plasmid was 1.1 fold of transcription of LAC4 gene at 72h. And compared with LAC4 gene driven by LAC4 promoter, K.marxianus with pUDP-pdc1-EGFP endogenous pDC1 gene transcription dicreased 2.4 fold, 3.7 fold and 6.7 fold at 24h, 48h and 72h respectively. Insert Fig.4 EGFP and PCV3 cap expression K.marxianus with pUDP-pdc1-EGFP was cultured in YPD medium with hygromycin for different time. Then the cells were harvested and EGFP fluorescence was measured with excitation and emission set at 485 and 525nm. The fluorescent signal of K.marxianus with pUDP-pdc1-EGFP was about 12.8fold, 14.5fold and 15.6fold of wild type at 24h, 48h and 72h. The results showed that the pdc1 promoter could drive the soluble expression of EGFP in K.marxianus . K.marxianus with pudp-pdc1-EGFP or pudp-pdc1-cap vectors were cultured in YPD medium with hygromycin for different time then the cell were harvested and disrupted. The supernatant were subjected to SDS-PAGE, and analysed by western blotting with antibody for His tag. About 25kd band showed EGFP expression both by SDS-PAGE and western blotting. About 35kd band revealed PCV3 cap expression in supernatant of K.marxianus . The results showed that PCV3 cap can express in soluble manner in K.marxianus . Insert Fig.5 To futher verify if the soluble cap can assemble to virus like particle (VLP), the whole supernatant of K.marxianus with PCV3 cap expression were precipitated by salt and resolved in PBS, then was eluted by Q-column with salt ion concentration gradient to futher purify protein. The PCV3 cap solution were subjected to transmission electronic microscopy. The result shown that the cap protein can assembled to VLP by itself. Compare different promoter expression EGFP protein level K.marxianus with pUDP-pdc1-EGFP, pUDP-pgk1-EGFP or pUDP-gdh1-EGFP was cultured in YPD medium with hygromycin for different time. Then the cells were harvested and EGFP fluorescence was measured with excitation and emission set at 485 and 525nm,The fluorescent signal of cell with pUDP-pdc1-EGFP was about 1.9 fold of cell pUDP-pgk1-EGFP and 2.6 fold of cell pUDP-gdh1-EGFP. Aforementioned cell were harvested and disrupted. The supernatant were subjected to SDS-PAGE, and analysed by western blotting with antibody for His tag andβ-actin.The results of protein expression showed that promoter strength was consistent with that of q-PCR. Insert Fig.6 Discussion K.marxianus have property of thermotolerant and assimilate various sugars as carbon source over common used yeast system. To explore this unconventional yeast for metabolic engineering and recombinant protein expression systems, We established a vector system suited this yeast. This vector has a hygromycin screening marker that makes it easy to screen by adding hygromycin to the culture medium. While other vector always use auxotroph restoring gene such as URA3 and HIS3 gene as screening marker that make the screening troublesome for need medium with clear chemical composition(Gombert et al. 2016). And if the vector is not integrated to the genome the large scale application is limited by the restricted medium. Another advantage of hygromycin screening marker over auxotroph restoring gene marker is it can apply to different species of nature K.marxianus that don’t need genetically disrupt of metabolic gene of the host. As an episomal vector it can self replicate in the host cell with the yeast self replication element, and can integrated into the host genome by non-homologous end joining (NHEJ) in the yeast K.marxianus (Suzuki et al. 2015). By adding recombinant sequeces It can site-specifically integrated to the host genome that can make target gene expression more stable at high level. For the target gene expression the promoter and terminator is the important element. Previous research always use the exogenous promoter,such as plasmid expression of Cas9 or deactivated Cas9 (dCas9) was accomplished using the heterologous S. cerevisiae TEF1 promoter(Lobs, Schwartz and Wheeldon 2017). Here we want to find the endogenous promoter with high expression strength. Because the strength of promoter determine the transcription level of target gene. we analyzed the transcriptome of K.marxianus from the available RNA sequence data. And the target gene RNA retested by the qPCR to confirm the transcription level. As the result we chose the PDC1 promoter and terminator to construct the expression vector. With the expression vector pSUP-pdc1,We further tested the vector by insert EGFP gene to downstream of PDC1 promoter. After the vector was electrotransformed into the host, We first analyzed the stable of the vector in the host by qPCR. And the plasmid can stable replication in the host without hygromycin in the medium for 72 hours culture. Then qPCR of EGFP mRNA shown that the EGFP gene could transcript with normal level by the PDC1 promoter, and western blot of EGFP protein further verified the vector can expression target gene in the k.marxianus. PCV3 is prevalent in swine industry recently. Studies on epidemiology and pathology of PCV3 have further demonstrated that PCV3 infection is related to the respiratory, digestive and/or reproductive disorders, and multisystemic inflammation[12].The cap protein is the major immunogen of PCV3 and is considered as the vaccine candidate.The E.coli is a cheap and easy host for exogenous proteins expression, but eukaryocyte source gene always expressed in insoluble form of inclusion body in E.coli. Here we inserted the PCV3 cap gene into the pUDP-pdc1 vector and electrotransformed into the host. qPCR result shown PCV3 cap could transcript at normal level in the host, and western blot also shown the cap protein could express in soluble form. The soluble cap of PCV3 can self assemble to VLP structure. VLP structure can enhance immune reaction of the host better than subunit vaccine and can be further applied in PCV3 vaccine development. We tested broad range of K.marxianus expression gene and chosen PDC1 promoter to constructed a transient expression vector. This vector can replicate itself in host stably and can driven proteins expression in the high level by the strong promoter PDC1.We successfully expressed PCV3 cap protein by this vector in soluble manner and the PCV3 cap protein can assemble to VLP structure autonomously.This work help advance K.marxianus as a thermotolerant and broad medium source host for exogenous protein expression. Acknowledgements This work was supported by National Natural Science Foundation of China (32302838), the Science and Technology Innovation Fund project of Henan Agricultural University (no. 30500577). Funding This work was supported by National Natural Science Foundation of China (32302838), the Science and Technology Innovation Fund project of Henan Agricultural University (no. 30500577). Declarations Confict of interest The authors declare no competing fnancial interest. References Banat, I. M., P. Nigam & R. Marchant (1992) Isolation of thermotolerant, fermentative yeasts growing at 52 degrees C and producing ethanol at 45 degrees C and 50 degrees C. World J Microbiol Biotechnol, 8 , 259-63.Deng, J., X. Li, D. Zheng, Y. Wang, L. Chen, H. Song, T. Wang, Y. Huang, W. Pang & K. Tian (2018) Establishment and application of an indirect ELISA for porcine circovirus 3. Arch Virol, 163 , 479-482.Fonseca, G. G., N. M. de Carvalho & A. K. Gombert (2013) Growth of the yeast Kluyveromyces marxianus CBS 6556 on different sugar combinations as sole carbon and energy source. Appl Microbiol Biotechnol, 97 , 5055-67.Gombert, A. K., J. V. Madeira, Jr., M. E. Cerdan & M. I. Gonzalez-Siso (2016) Kluyveromyces marxianus as a host for heterologous protein synthesis. Appl Microbiol Biotechnol, 100 , 6193-6208.Groeneveld, P., A. H. Stouthamer & H. V. Westerhoff (2009) Super life–how and why ’cell selection’ leads to the fastest-growing eukaryote. FEBS J, 276 , 254-70.Juergens, H., J. A. Varela, A. R. Gorter de Vries, T. Perli, V. J. M. Gast, N. Y. Gyurchev, A. S. Rajkumar, R. Mans, J. T. Pronk, J. P. Morrissey & J. G. Daran (2018) Genome editing in Kluyveromyces and Ogataea yeasts using a broad-host-range Cas9/gRNA co-expression plasmid. FEMS Yeast Res, 18.Karim, A., N. Gerliani & M. Aider (2020) Kluyveromyces marxianus: An emerging yeast cell factory for applications in food and biotechnology. Int J Food Microbiol, 333 , 108818.Lertwattanasakul, N., T. Kosaka, A. Hosoyama, Y. Suzuki, N. Rodrussamee, M. Matsutani, M. Murata, N. Fujimoto, Suprayogi, K. Tsuchikane, S. Limtong, N. Fujita & M. Yamada (2015) Genetic basis of the highly efficient yeast Kluyveromyces marxianus: complete genome sequence and transcriptome analyses. Biotechnol Biofuels, 8 , 47.Li, X., Y. Bai, H. Zhang, D. Zheng, T. Wang, Y. Wang, J. Deng, Z. Sun & K. Tian (2018) Production of a monoclonal antibody against Porcine circovirus type 3 cap protein. J Virol Methods, 261 , 10-13.Liu, X., X. Zhang, X. Li, T. Ouyang, G. Niu, H. Ouyang & L. Ren (2020) Genotyping based on complete coding sequences of porcine circovirus type 3 is stable and reliable. Infect Genet Evol, 78 , 104116.Lobs, A. K., C. Schwartz & I. Wheeldon (2017) Genome and metabolic engineering in non-conventional yeasts: Current advances and applications. Synth Syst Biotechnol, 2 , 198-207.Opriessnig, T., A. K. Karuppannan, A. Castro & C. T. Xiao (2020) Porcine circoviruses: current status, knowledge gaps and challenges. Virus Res, 286 , 198044.Ouyang, T., G. Niu, X. Liu, X. Zhang, Y. Zhang & L. Ren (2019) Recent progress on porcine circovirus type 3. Infect Genet Evol, 73 , 227-233.Palinski, R., P. Pineyro, P. Shang, F. Yuan, R. Guo, Y. Fang, E. Byers & B. M. Hause (2017) A Novel Porcine Circovirus Distantly Related to Known Circoviruses Is Associated with Porcine Dermatitis and Nephropathy Syndrome and Reproductive Failure. J Virol, 91.Phan, T. G., F. Giannitti, S. Rossow, D. Marthaler, T. P. Knutson, L. Li, X. Deng, T. Resende, F. Vannucci & E. Delwart (2016) Detection of a novel circovirus PCV3 in pigs with cardiac and multi-systemic inflammation. Virol J, 13 , 184.Rodrussamee, N., N. Lertwattanasakul, K. Hirata, Suprayogi, S. Limtong, T. Kosaka & M. Yamada (2011) Growth and ethanol fermentation ability on hexose and pentose sugars and glucose effect under various conditions in thermotolerant yeast Kluyveromyces marxianus. Appl Microbiol Biotechnol, 90 , 1573-86.Rouwenhorst, R. J., L. E. Visser, A. A. Van Der Baan, W. A. Scheffers & J. P. Van Dijken (1988) Production, Distribution, and Kinetic Properties of Inulinase in Continuous Cultures of Kluyveromyces marxianus CBS 6556. Appl Environ Microbiol, 54 , 1131-7.Suzuki, A., H. Fujii, H. Hoshida & R. Akada (2015) Gene expression analysis using strains constructed by NHEJ-mediated one-step promoter cloning in the yeast Kluyveromyces marxianus. FEMS Yeast Res, 15.Zhang, S., D. Wang, Y. Jiang, Z. Li, Y. Zou, M. Li, H. Yu, K. Huang, Y. Yang & N. Wang (2019) Development and application of a baculovirus-expressed capsid protein-based indirect ELISA for detection of porcine circovirus 3 IgG antibodies. BMC Vet Res, 15 , 79.Zhou, J., P. Zhu, X. Hu, H. Lu & Y. Yu (2018) Improved secretory expression of lignocellulolytic enzymes in Kluyveromyces marxianus by promoter and signal sequence engineering. Biotechnol Biofuels, 11 , 235. Fig.1 Evaluation of promoters for RNA transcription. Specific mRNA was amplified by Q-PCR to test promoter transcription strength. Primers used is shown in S1. Values were the means±standard error of mean (SEM) from biological triplicates. Fig.2 Flow chart of expression vector construction. pUDP002 vector was digested by AflII and BclI, PDC1 promoter amplification from K.marxianus genome was insert into the vector by infusion manner, then the target gene PCV3 cap and EGFP were inserted into the downstream of PDC1 promoter respectively. The final vectors named pUDP-pdc1-cap and pUDP-pdc1-EGFP respectively. Fig.3 Anaylsis of pUDP-pdc1-EGFP copy number and stable in K.marxianus . K.marxianus with pudp-pdc1-EGFP plasmid was cultured different time without hygromycin and Q-PCR of plasmid ORI region and genome INU1 promoter region was done, the result was ORI divided by genome number. Fig.4 Vector with PDC1 promoter driven exogenous mRNA transcription. A and B. K.marxianus with pUDP-pdc1-EGFP or pUDP-pdc1-cap was cultured for different time and Q-PCR was done with EGFP specific primers, plasmid ori region as control to distinguish DNA contamination. C. K.marxianus with pUDP-pdc1-EGFP was cultured for different time and Q-PCR was done with EGFP, LAC4 gene, PDC1 gene specific primers. Fig.5 assays for the protein expression in K. marxianus . A and B. EGFP and PCV3 cap expression in K.marxianus . Lane M: PageRuler Prestained Protein Ladder; lane 1:the supernatant of cell lysate from K. marxianus with vector culturing for 24 hours; lane 2: the supernatant of cell lysate from K. marxianus with vector culturing for 48 hours; lane3: the supernatant of cell lysate from K. marxianus with vector culturing for 72 hours; lane 4: the total of cell lysate from K.marxianus without vector. Anti -His tag antibody was used for Western blotting. C.Measure the fluorescence of the pudp-pdc1-EGFP K.marxianus and wild type.the ratio of fluorescence to OD600nm determined the expression level of EGFP. D.TEM image of the PCV3 cap VLPs assembled intracellularly in the K.marxianus with pUDP-pdc1-cap vector Scale bar, 200 nm. Fig.6 Compare different promoter expression of EGFP. A. Different promoter expression of EGFP in K.marxianus . Lane M: PageRuler Prestained Protein Ladder; lane 1-4:the supernatant of cell lysate from K. marxianus with vector of PGK1, PDC1, GDH1 promoter repectively and without vector culturing for 24 hours; Anti-His tag antibody and anti-β-actin antibody was used for Western blotting. B. Measure the fluorescence of the K.marxianus with vector of different promoter and wild type. the ratio of fluorescence to OD600nm determined the expression level of EGFP. Information & Authors Information Version history V1 Version 1 05 April 2025 Peer review timeline Published Yeast Version of Record 22 Oct 2025 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Collection Yeast Keyword saccharomyces Authors Affiliations Jiamei Li Henan Agricultural University View all articles by this author Xuechen Yang Henan Agricultural University View all articles by this author Mingyue Xu Henan Agricultural University View all articles by this author Wenjing Yin Henan Agricultural University View all articles by this author Guoyu Yang Henan Agricultural University View all articles by this author Yueting Zheng Henan Agricultural University View all articles by this author Wei Yang ChongQing Academy of Animal Sciences View all articles by this author Zhang Wei [email protected] Henan Agricultural University View all articles by this author Metrics & Citations Metrics Article Usage 245 views 190 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Jiamei Li, Xuechen Yang, Mingyue Xu, et al. Characterization of vector elements and soluble expression of PCV3 cap protein in Kluyveromyces marxianus. Authorea . 05 April 2025. DOI: https://doi.org/10.22541/au.174381314.49571039/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . Format Please select one from the list RIS (ProCite, Reference Manager) EndNote BibTex Medlars RefWorks Direct import Tips for downloading citations document.getElementById('citMgrHelpLink').addEventListener('click', function() { popupHelp(this.href); return false; }); $(".js__slcInclude").on("change", function(e){ if ($(this).val() == 'refworks') $('#direct').prop("checked", false); $('#direct').prop("disabled", ($(this).val() == 'refworks')); }); View Options View options PDF View PDF Figures Tables Media Share Share Share article link Copy Link Copied! Copying failed. Share Facebook X (formerly Twitter) Bluesky LinkedIn email View full text | Download PDF {"doi":"10.22541/au.174381314.49571039/v1","type":"Article"} Now Reading: Share Figures Tables Close figure viewer Back to article Figure title goes here Change zoom level Go to figure location within the article Download figure Toggle share panel Toggle share panel Share Toggle information panel Toggle information panel Go to previous graphic Go to next graphic Go to previous table Go to next table All figures All tables View all material View all material xrefBack.goTo xrefBack.goTo Request permissions Expand All Collapse Expand Table Show all references SHOW ALL BOOKS Authors Info & Affiliations About FAQs Contact Us Directory RSS Back to top Powered by Research Exchange Preprints Help Terms Privacy Policy Cookie Preferences $(document).ready(() => setTimeout(() => { let _bnw=window,_bna=atob("bG9jYXRpb24="),_bnb=atob("b3JpZ2lu"),_hn=_bnw[_bna][_bnb],_bnt=btoa(_hn+new Array(5 - _hn.length % 4).join(" ")); $.get("/resource/lodash?t="+_bnt); },4000)); (function(){function c(){var b=a.contentDocument||a.contentWindow.document;if(b){var d=b.createElement('script');d.innerHTML="window.__CF$cv$params={r:'9fea185dcfa44807',t:'MTc3OTI2NzA0MA=='};var a=document.createElement('script');a.src='/cdn-cgi/challenge-platform/scripts/jsd/main.js';document.getElementsByTagName('head')[0].appendChild(a);";b.getElementsByTagName('head')[0].appendChild(d)}}if(document.body){var a=document.createElement('iframe');a.height=1;a.width=1;a.style.position='absolute';a.style.top=0;a.style.left=0;a.style.border='none';a.style.visibility='hidden';document.body.appendChild(a);if('loading'!==document.readyState)c();else if(window.addEventListener)document.addEventListener('DOMContentLoaded',c);else{var e=document.onreadystatechange||function(){};document.onreadystatechange=function(b){e(b);'loading'!==document.readyState&&(document.onreadystatechange=e,c())}}}})();