Construction of Primary Chassis Cells with Efficient Protein Expression in Thermus thermophilus

Construction of Primary Chassis Cells with Efficient Protein Expression in Thermus thermophilus | 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 Construction of Primary Chassis Cells with Efficient Protein Expression in Thermus thermophilus Yuqian Liang¹, Mohamed Motawaa¹, Xuying Bu¹, Junwei Wei¹, Yuan Shao¹, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6092828/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Jul, 2025 Read the published version in Microbial Cell Factories → Version 1 posted 8 You are reading this latest preprint version Abstract Background: Thermus thermophilus HB27 is a promising thermophilic chassis for recombinant thermostable protein production, owing to its high optimal growth temperature, which can simplify downstream processing and reduce contamination risks. However, maximizing its potential requires optimized genetic tools and host strains. Key limitations include a shortage of well-characterized strong constitutive promoters and potential degradation of recombinant proteins by proteases. To address these, we established a β-galactosidase reporter system (endogenous TTP0042) to screen for strong constitutive promoters and investigated the impact of deleting specific protease genes on protein expression. Results: Screening of 13 endogenous promoter regions identified P0984 as exhibiting significantly 13-fold higher activity than the control promoter driving the reporter gene. Constructing a plasmid-free strain (HB27ΔpTT27) successfully minimized 270 kb of the genome; it exhibited auxotrophy for cobalamin (requiring 0.1 μg/ml AdoCbl for growth) and a slightly reduced growth rate compared to the wild-type, while its transformation efficiency remained comparable. Notably, a CRISPR-deficient precursor strain (HB27ΔIII-ABΔI-CΔ CRF3 ) showed a significant (~100-fold) increase in transformation efficiency compared to the wild-type, facilitating subsequent genetic manipulations. Systematic knockout of 16 predicted non-essential protease loci was performed. Characterization revealed that deletion of TTC0264 (putative ClpY/HslU) and TTC1905 (putative HhoB) significantly reduced extracellular proteolytic activity. Iterative deletion based on phenotypic analysis led to strain DSP9 (10 protease loci deletions), which maintained robust growth and exhibited enhanced accumulation of the β-galactosidase reporter protein compared to the parental strains. Conclusions: This study provides foundational advancements for T. thermophilus HB27 chassis development, and genetic tools represent valuable resources for optimizing T. thermophilus as a platform for heterologous thermostable protein production and ideas for antibiotic-free systems. Thermus thermophilus HB27 Chassis Cell Promoter Library β-galactosidase Monitor System pTT27-Plasmid Protease Activity Figures Figure 1 Figure 2 Figure 3 Figure 4 1 Background The burgeoning emphasis on renewable energy sources and sustainable industrial practices significantly drives the escalating global demand for thermostable proteins. Thermostable enzymes (e.g., cellulases, xylanases, α-amylases, laccases) are crucial for applications requiring elevated temperatures, such as biofuel production, bioleaching, wastewater treatment, and agricultural biotechnology [ 1 – 7 ]. Utilizing thermostable proteins offers advantages like reduced cooling costs, lower contamination risks, and increased reaction rates [ 1 – 7 ]. However, producing thermostable proteins efficiently often faces challenges in conventional mesophilic hosts like E. coli . Issues include protein misfolding, aggregation, degradation, and lack of necessary post-translational modifications due to differences in cellular machinery and environment [ 8 , 9 ]. The unique structural features conferring thermostability (e.g., specific hydrophobic interactions, salt bridges) may not be correctly established in mesophilic cytoplasm, potentially requiring specialized chaperone systems or protein engineering approaches [ 10 – 14 ]. Thermus thermophilus , particularly strain HB27, presents a compelling alternative host for producing thermostable proteins [ 9 , 15 ]. Its optimal growth at high temperatures (65–75°C) inherently favors the stability and correct folding of many thermostable target proteins. Furthermore, high-temperature cultivation simplifies downstream purification, as many endogenous mesophilic contaminants are eliminated [ 10 , 16 , 17 ]. T. thermophilus HB27 offers additional advantages like rapid growth, high cell density potential, natural competence for transformation, and available genetic tools, including CRISPR-Cas systems for genome editing [ 4 , 5 , 18 – 23 ]. Consequently, HB27 is an attractive chassis for producing various thermostable enzymes and proteins [ 4 , 24 – 26 ]. Developing T. thermophilus as a robust chassis aligns sustainable bioproduction goals by potentially reducing energy inputs for cooling and simplifying processing [ 27 – 29 ]. However, it is important to note that this platform is primarily suited for thermostable proteins; expressing inherently thermolabile proteins (e.g., many mammalian biopharmaceuticals) remains a significant challenge due to the high cultivation temperatures [ 4 , 30 ]. Despite its potential, optimizing T. thermophilus for industrial-scale production requires addressing key limitations. One is the limited availability of strong, well-characterized constitutive promoters for driving high-level, stable gene expression. While some inducible promoters exist (e.g., P arg , P dnaK , P nar , P pilA4 ), they often suffer from drawbacks like complex induction strategies, growth inhibition, or unintended regulation [ 31 – 34 ]. Plasmid-based expression systems can also face instability and metabolic burden [ 35 , 36 ]. Another significant challenge is the potential degradation of recombinant proteins by endogenous host proteases, which can severely reduce yields [ 37 – 39 ]. While protease activity is essential for cellular health, specific proteases can target heterologous proteins. Previous work in T. thermophilus indicated that deleting certain proteases (e.g., Lon-type) could improve yields, but the roles of many other proteases remain uncharacterized [ 39 ]. Therefore, this study aimed to enhance T. thermophilus HB27 as a microbial chassis through a multi-pronged strategy: (1) Screening of 13 endogenous genomic regions to identify strong constitutive promoters. (2) Constructing and characterizing a plasmid-free strain (HB27ΔpTT27) to reduce genome size and explore its potential as an auxotrophy-based selection. (3) Systematically deleted 16 predicted non-essential protease genes to assess their impact on growth, extracellular proteolytic activity, and reporter protein accumulation. We aim to generate characterized strains and genetic tools to facilitate the development of T. thermophilus HB27 for improved recombinant protein production, particularly focusing on thermostable targets. 2 Materials and Methods 2.1 Bacterial strain and cultivation E. coli DH5α was used as a host for plasmid engineering and multiplication, cultivated in LB medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl) at 37°C with 200 rpm in a shaker incubator (BLabotery, ZQPL-200). T. thermophilus HB27 (Wildtype), HB27∆TTP0042 ( T. thermophilus HB27 with β-galactosidase gene knockout), HB27∆III-AB∆I-C∆CRE3 ( T. thermophilus HB27 with I-C, III-A & B CRISPR-Cas system and CRF3 (CRISPR-associated Rossmann Fold) genes knockouts), and T. thermophilus stains were cultivated at 65°C with 200 rpm shaking in Ttᶧ medium (8 g/L tryptone, 4 g/L yeast extract, 3 g/L NaCl, 4.36 mM NaHCO₃,0.26 mM MgSO₄, 0.739 mM MgCl₂.6H₂O, 0.012 mM KCl, and 0.249 mM CaCl₂.2H₂O) [ 23 , 34 ]. Petri dishes of T. thermophilus were wrapped in sterilized bags to maintain plate humidity in the static incubator (BLabotery, SPL-250). Strains were listed in the supplementary data Table S1 . 2.2 Plasmid construction To construct plasmids for endogenous promoter screening , promoter regions were isolated from the HB27 genome utilizing PCR and primers with about 20 nt at the 5'-end complementary to the plasmid ligation side. Then, the TTP0042 codon (β-glucosidase reporter gene) amplified from the pTT27 plasmid was cloned into pRKP31, which was pre-digested with SalI and NheI. Then, the P31 promoter was replaced with the next screened promoters into the pRKP31-TTP0042 plasmid utilizing 2X CE Mix V3 (Vazyme, C117-01-AA). To construct the knockout plasmids for replicon repT and the 16 protease loci based on the endogenous CRISPR system type I-B , a spacer that matched the target loci was cloned within two BbsI restriction sites on the pRKP31-AC3 shuttle vector by T4 polynucleotide (BioLabs, M0201S). A spacer fragment was generated by annealing two complementary single-stranded oligo DNA 42bp in length with 4nt at the 5`-end complementary to the BbsI sticky ends, at 95°C for 5 min, and slowly cooled down at room temperature to form the double-stranded inserts. The two arms of the homology direct repair (HDR) template were separately amplified using the HB27 genome as template and connected using splicing by 20 bp overhang extension PCR (SOE PCR). Plasmids were constructed in two steps. First, spacers that matched the double-strand break (DSB) site were cloned. Then, the plasmid was linearized by PCR, followed by DpnI (Thermo Scientific, FD1704) enzyme digestion, and the HDR was cloned by 20 bp overhang homology utilizing ABclonal MultiF Seamless Assembly Mix (ABclonal, RK21020) [ 23 ]. Fig. S1 illustrates the schematic procedure of endogenous CRISPR-Cas-based gene editing technology. Plasmids were validated by sequencing (Sangon, Shanghai, China) before being transformed into the HB27 cells. Plasmids used or constructed are listed in Table S3. Primers used for PCR amplification and validation are listed in Table S2. Primers and single-stranded oligo DNA were synthesized by Sangon Co., Shanghai, China. PCR was performed using the Phanta Max Super‐Fidelity DNA polymerase (Vazyme, P505). The amplification was performed at 95°C for 5 min, 95°C for 15 sec, Tm-5°C for 15 sec, 72°C for 1 min/1 kb, and 72°C for 5 min, for 30 cycles. The amplicons and digested fragments were purified with the E.Z.N.A. Cycle-Pure kit (Omega BIO-TEK, D6492- 02). Plasmid extracted utilizing E.Z.N.A. Plasmid Mini Kit I (Omega BIO-TEK, D6943- 02). 2.3 Transformation, cell morphology, and growth rate estimation The heat shock transformation method was used for DH5α competent cells. 50 µL DH5α was thawed on ice for 5 min, 10 µL recombinant plasmid was gently mixed, and placed on ice for 25 min. Heat shock at 42℃ for 45 sec, followed by ice incubation for 2 min, then 900 µL LB medium was pipetted and incubated at 37°C, 200 rpm for 1 h, and finally spread on LB selection medium [ 23 ]. T. thermophilus utilizes a natural competence system to take up exogenous DNA during the mid-log phase. 1 µg plasmid was mixed with 500 µL mid-log phase cells, incubated for 2.5 h at 65℃, 180 rpm, and then spread on a Ttᶧ selection medium [ 23 ]. The pRKP31 and pRKP31-AC3 Shuttle plasmids were used as backbones for construction with a kanamycin-selectable marker, under selection pressure of 30 µg/mL and 20 µg/mL for E. coli and T. thermophilus , respectively. For microscopic cell morphology of T. thermophilus , a fuchsin-dyed smear was prepared and observed using the immersion oil 100X objective lens [ 40 ]. For the growth curve , bacteria were acquired by measuring the optical turbidity at an absorbance of 600 nm. Since the optical density of the culture is proportional to the cell density, measuring the turbidity of the culture can be used to estimate the number of bacterial cells. Overnight cultures were diluted 1:100 v/v into 50 mL Ttᶧ medium in a shaker incubator at 65℃, 180 r/min. Samples were taken every 2 to 4 h intervals for 24 to 48 h. The strains were cultured in three biological repeats [ 41 ]. 2.4 Quantitative and qualitative β-galactosidase assay Quantitative assessment of β-galactosidase activity was performed based on the established O-Nitrophenyl-β-D-galactopyranoside (ONPG) assay as described [ 42 , 43 ]. Reporter plasmids were introduced into the T. thermophilus HB27 ΔTTP0042. Three distinct single colonies harboring the respective reporter plasmid were randomly selected for each independent experimental replicate. These transformed strains were cultivated in Ttᶧ medium under the optimum conditions. Upon reaching the log growth phase, as determined by an optical density at (OD 600 ~ 1.0), bacterial cells were harvested via centrifugation. The resulting cellular pellets were resuspended in 10 mM Tris-HCl buffer (pH 8.0). To obtain crude cellular lysates, the resuspended cells underwent controlled sonication. Following lysis, cellular debris was eliminated through centrifugation at 13,000 × g for 30 min at 20°C to obtain clarified supernatants. The protein concentration within these clarified cellular extracts was precisely quantified using the Bradford reagent (Tiangen, PA102). A 50 µL aliquot of the clarified supernatant was combined with 450 µL of reaction buffer (2.8 mM ONPG in 50 mM sodium phosphate, pH 6.5). Samples were incubated at 65°C for a precisely controlled duration of 20 minutes, after which the enzymatic reaction was terminated by adding an equal volume of 1 M Na 2 CO 3 . The absorbance of the o-nitrophenol was spectroscopically estimated at 420 nm using a microplate reader (Victor Nivo 3S; PerkinElmer). One unit of specific β-galactosidase activity is operationally defined as 1 nmol ρ-nitrophenol produced per min per mg total protein. In the qualitative plate assays , 5 µL of mid-log phase (OD 600 ~ 1.0) screened strains were spotted on Ttᶧ plates supplemented with 100 mg/L X-gal. Plates were incubated at 65℃ for 2 days to observe the color change [ 44 ]. 2.5 Extracellular protease assay The protease mutations were screened for extracellular protease activity by skim milk plate assay [ 45 ]. 2.5 µL of mid-log phase (OD 600 ~ 1.0) mutants were spotted on Ttᶧ plates supplemented with 10% (v/v) skimmed milk. The clear zone diameter was measured 3 days after incubation at 65°C by flooding the plates with 10% trichloroacetic acid (TCA). The relative enzyme activity was calculated using the following formula: $$\:REA=\frac{CZD}{CD}$$ Where: REA is relative enzyme activity, CZD is clear zone diameter, and CD is colony diameter. 2.6 Statistical analysis All histogram drawings and statistical analysis were completed using the GraphPad Prism 8 software (GraphPad Software Inc.). Data are expressed as the mean ± standard deviation (SD). The significance of differences between groups' mean values was analyzed using Duncan’s Multiple Range Test (DMRT) at a P < 0.05 significance level. 3 Results 3.1 Construction of promoter screening reporter vector T. thermophilus HB27 β-galactosidase is involved in lactose metabolism and acts as a prevalent reporter enzyme used in studying the activities of promoters [32, 34]. T. thermophilus HB27 β-galactosidase transcript by three loci (TTP0042, TTP0220, TTP0222) located in the pTT27 plasmid [19]. The TTP0042 locus was detected as the main source of β-galactosidase activity in T. thermophilus HB27. Thus, the β-galactosidase-deletion strain (HB27ΔTTP0042) was used as a competent cell for promoter screening. Thirteen promoter regions were inserted into pRKP-TTP0042 by homology ligation. Promoters named as follows: Pslp (206 bp), Pago (300 bp), Pnqo (414 bp), P31 (216 bp), P031 (214 bp), P43 (195 bp), P214 (160 bp), P215 (184 bp), P0416 (853 bp), P0984 (858 bp), P1578 (842 bp), P1706 (849 bp), P1798 (861 bp). The pRKP-TTP0042 screening vector contains the promoter-less endogenous TTP0042 β-galactosidase, heat–stable Kanamycin resistance selectable marker derived with Pslp promoter, E. coli replicon puc-ori, and T. thermophilus replicon repA ori (Fig. 1A). Transformants grown up on Ttᶧ selection plates were picked up for verification using specific primers (Fig. S2 and Table S2). 3.2 Effect of different promoters on β-galactosidase expression To analyze the potential transcription level of the 13 promoters. The HB27ΔTTP0042 transformants were examined qualitatively and quantitatively by X-Gal plates and ONPG assay (Fig. 1B, C). Transformants, Control (HB27ΔTTP0042), and the HB27 (wild-type) were grown in Ttᶧ medium under optimal growth conditions to mid-log phase, and then the β-glucosidase activities were examined. The Pago, P031, P43, P0416, and P1578 promoters have a significantly lower activity as appears in yellow colonies on the X-Gal plates (Fig. 1B). Interestingly, the quality and quantity assay show that Pslp, Pnqo, P31, P214, P215, P0984, P1706, and P1798 promoters were significantly higher than the wild-type and control as shows in Fig. 1B, C. Among the 13 promoters, the P0984 promoter shows significantly 13-fold higher activity, which recommends the utilization of the P0984 promoter for the subsequent thermostable protein production. 3.3 Construction of HB27 plasmid-free strain To step forward chassis cell construction and minimize the T. thermophilus HB27 genome. We deleted the pTT27 plasmid (~270 kb) [19]. We utilized the CRISPR-deficient strain HB27ΔIII-ABΔI-CΔ CRF3 , previously constructed by our lab as a competent cell due to its enhanced transformability, lack CRISPR type III subtype A&B, type I subtype C, and CRISPR-associated Rossmannfold (64 kbp deletion) as mapped in Fig. 2A. The pTT27 plasmid replication origin (repT) was identified, 42nts were selected after PAM sequence (5′-TTC-3′) in the middle of the repT locus as a specific region for DSB, and an approximately 500 bp region flanking repT locus were cloned in the pRKP31-AC3 vector for HDR (Fig S1). Colonies grown up in the Ttᶧ selection medium were screened by colony PCR with three pairs of primers flanking the target deletion region (Table S2, Fig. S3A). PCR results confirmed the DSB sandwiched by the HDR; control strain was 3319 bp, 2214 bp, and 3322 bp, while those of the plasmid-free strain showed no bands, suggesting a successful deletion was established (Fig. S3B). As pTT27 encodes key carotenoid biosynthesis genes ( crtI, crtB ) [19], the resulting HB27ΔpTT27 colonies were white, in contrast to the wild-type strain (Fig. S3C). 3.4 Characterization of HB27 plasmid-free strain To evaluate the plasmid-free strain (HB27ΔpTT27) as a potential chassis, its phenotypic properties (growth rate, morphology, transformation efficiency) were compared with the control (HB27ΔIII-ABΔI-CΔ CRF3 ) and the wild-type HB27. Growth rate: As shown in Fig. 2B, the growth of HB27ΔIII-ABΔI-CΔ CRF3 was comparable to wild-type HB27, reaching stationary phase (OD 600 ~5) around 30 h. However, HB27ΔpTT27 failed to grow significantly in the standard Ttᶧ medium. Since pTT27 harbors the cobalamin (Vitamin B12) biosynthesis gene cluster (TTP0001-TTP0023) [19, 46]. We hypothesized that this deletion caused auxotrophy. Supplementation with 0.1 μg/mL AdoCbl rescued growth, although the growth rate remained slightly slower, and the final OD was lower than wild-type and competent strains. This confirms that pTT27 encodes essential genes for cobalamin synthesis and potentially other factors affecting optimal growth under these conditions. This auxotrophy could potentially be applied to antibiotic-free selection systems in future applications [47]. Transformation efficiency: We assessed natural transformation efficiency using the pRKP0984-TTP0042 plasmid. Wild-type HB27 exhibited a baseline efficiency of approximately 2.02×10⁴ CFU/μg. The plasmid-free HB27ΔpTT27 strain showed a similar efficiency (2.13×10⁴ CFU/μg), indicating that removal of the pTT27 plasmid did not significantly impair DNA uptake under these conditions. In contrast, the competent CRISPR-deficient strain HB27ΔIII-ABΔI-CΔ CRF3 displayed significantly enhanced efficiency (1.78×10⁶ CFU/μg), nearly a 100-fold increase over the wild-type (Fig. 2C). This pronounced increase is likely due to the removal of CRISPR-Cas systems that target foreign DNA. Such enhancement is consistent with observations in other bacteria, where inactivation of defense systems like restriction-modification (R-M), StySA, or BREX systems improves transformation efficiencies [48-52]. This high transformability proved advantageous for the subsequent protease gene deletions. Cell morphology: Microscopic examination revealed that HB27ΔIII-ABΔI-CΔ CRF3 morphology was indistinguishable from wild-type bacilli. However, the plasmid-free HB27ΔpTT27 strain consistently displayed a slightly elongated cell shape compared to the wild-type (Fig. 2D). This subtle morphological change might be linked to the altered growth kinetics or potential cell envelope modifications resulting from the large deletion [53], warranting further investigation. 3.5 Construction of protease-encoding loci knockout strains Bioinformatic analysis identified 16 predicted nonessential protease-encoding genes within the T. thermophilus HB27 genome distributed across the chromosomal loci as listed in Table 1. We knocked out these 16 loci to optimize the highly transformable HB27ΔIII-ABΔI-CΔ CRF3 strain. To ensure precise locus deletion without cluster gene interruption, we targeted the protein-coding sequences while preserving promoter elements and regulatory regions (Fig. 3A). Utilizing the pRKP31-AC3 plasmid-mediated HDR recombination system based on the endogenous CRISPR-Cas9 Type I-B machinery. We successfully constructed 16 individual protease knockout strains. In vivo transcript of the designed crRNA generates ribonucleoprotein (RNP) complexes. These RNPs effectively cleaved the protease locus, with precise repair mediated by homologous recombination utilizing an HDR template, thus introducing the designed null mutations. As illustrated in Fig. 3A, B, Table S2, the PCR-based validation strategy confirmed the homogeneity of the mutant population. Validated strains were then subjected to iterative rounds of subculturing in an antibiotic-free Ttᶧ medium to facilitate plasmid loss, yielding marker-free protease deletion strains for future editing and ensuring a complete gene editing mutant. As illustrated in Fig 3A 'exam-genome-F/R' primer pair confirmed the target deletion by amplifying across the modified region, yielding amplicons of reduced size depending on the target locus compared to wild-type allele (Fig. 3B), 'exam-sp-F/exam-genome-R' primer combination detect the presence of any residual wild-type alleles, 'exam-plasmid-F/R' primer confirm the subsequent loss of the editing plasmid. This multi-faceted screening approach generates 16 individual protease deletion loci, designated DP1 through DP16, each carrying a precise deletion in the intended protease-encoding locus. 3.6 C haracterization of protease-encoding loci knockout strains Growth rate: The 16 individual protease deletion strains (DP1-DP16) were monitored in a liquid Ttᶧ medium, revealing distinct growth phenotypes among strains. DP6-DP11, exhibited growth profiles indistinguishable from the wild-type strain, reaching stationary phase by 30 h of incubation with a comparable OD 600 ~5 (Fig. 3D). DP1-DP4, DP12-DP16 displayed subtle variations in growth rates relative to the wild-type, while reached the stationary phase by 30 h, with slightly lower OD than the wild-type (Fig. 3C, E). DP5 (TTC0265 deletion, putative FtsH) manifested a significantly attenuated growth rate with extended incubation period of 40 h to reach the stationary phase (Fig. 3C). Notably, DP1 (TTC0035 deletion), DP5 (TTC0265 deletion), and DP16 (TTC1905 deletion) consistently demonstrated a reduction in maximal biomass accumulation. Extracellular proteolytic activity was assessed using Ttᶧ skim milk plates incubated for 72 h at 65°C (Fig. 3F) to compare the resulting hydrolysis zones of the strains (Table 2). Notably, DP2, DP3, DP9, DP11-DP15, exhibited hydrolytic zones with no statistically significant difference compared to HB27 and HB27ΔIII-ABΔI-CΔ CRF3 , indicating unimpaired extracellular protease function. In contrast, DP1, DP5-DP8, and DP10 generated significantly smaller zones of hydrolysis, suggesting a reduction in secreted protease activity. Remarkably, DP4 (TTC0264 deletion, putative ClpY/HslU) and DP16 (TTC1905 deletion, putative HhoB) displayed severely attenuated hydrolytic activity (1.61 ± 0.58 mm in diameter). These observations strongly implicate the proteases encoded by TTC0264 and TTC1905 as key contributors to overall secreted proteolytic activity under these conditions. Reporter protein accumulation assesses the impact of protease deletions on heterologous protein expression capabilities, using the pRKP0984-TTP0042 reporter plasmid. DP1, DP2, DP3, DP5, and DP8 exhibited levels statistically indistinguishable from the competent strain HB27ΔIII-ABΔI-CΔ CRF3 (Fig. 3G). Conversely, the remaining protease deletion strains demonstrated a statistically significant reduction in β-galactosidase activity. Based on integrated analysis (growth, protease activity, reporter expression), a subset of 10 loci (TTC0174, TTC0251, TTC0264, TTC0417, TTC0481, TTC0492, TTC0663, TTC0687, TTC0950, TTC0974) were selected for multiple knockout construction, starting from DP6 (ΔTTC0417) which showed wild-type-like growth and slightly reduced reporter expression. 3.7 Construction of multiple protease-encoding loci knockout strains To develop T. thermophilus HB27 chassis cells with enhanced heterologous protein expression capabilities, we generated a series of the above-mentioned knockout loci using the DP6 strain (HB27ΔIII-ABΔI-CΔ CRF3 ΔTTC0417) as a competent cell with subsequent rounds of gene editing. We constructed nine multiple protease loci knockout strains, named DSP1-DSP9 (Fig. S4), and confirmed colony PCR (Fig. S5). The successful construction of these iterative knockout strains (DSP1-DSP9) represents a step toward optimizing T. thermophilus HB27. However, knockout efficiency progressively decreased with each successive deletion, ultimately limiting further gene editing using the I-B system, and underscores the need for alternative strategies to further refine this chassis. 3.8 Characterization of multiple protease-encoding loci knockout strains Growth rate: All multiple-deletion strains (DSP1-DSP9) exhibited growth profiles generally comparable to the wild-type strain, reaching log phase around 24 h, though final biomass was slightly reduced (OD 600 3.57-3.88 vs ~4.2) (Fig. 4A). DSP9 showed the least growth impairment among the multiple-deletion strains. Extracellular protease activity: DSP1-DSP3 showed markedly smaller hydrolysis zones compared to the parental strains, consistent with the accumulation of deletions including TTC0264 (putative ClpY/HslU). Other strains (DSP4-DSP6, DSP8-DSP9) showed activity closer to the parental strain, suggesting compensatory effects or that the deleted proteases in these combinations contribute less to casein hydrolysis under these conditions (Table 3). Reporter gene expression: β-galactosidase activity varied among the multiple deletion strains (Fig. 4B). DSP1-DSP2 and DSP4-DSP5 showed activity similar to the parental strains. DSP3 showed reduced activity. Significantly, strains DSP6 through DSP9 exhibited enhanced β-galactosidase activity compared to the starting strain, with DSP6 showing the highest (11,480 U/mg) and DSP9 also showing strong activity (10,844 U/mg). This suggests that the combined deletion of these specific proteases reduces degradation or turnover of the intracellular β-galactosidase reporter. Based on this characterization, DSP9 emerged as a promising first-generation chassis candidate. Its genome size is reduced compared to wild-type (~3.5% deletion from chromosome relative to WT), it maintains robust growth kinetics, and shows enhanced reporter protein accumulation (approximately twofold higher activity than the wild-type strain under these conditions). 4 Discussion This study aimed to develop improved chassis strain and genetic tools for T. thermophilus HB27, focusing on enhancing its utility for heterologous protein production, particularly thermostable proteins. We successfully identified a strong native constitutive promoter (P0984), generated and characterized a plasmid-free strain (HB27ΔpTT27), demonstrated significantly improved transformation efficiency by deleting specific CRISPR-Cas loci, and systematically evaluated the impact of single and multiple protease loci deletions. The identification of strong constitutive promoters as Pslp, Pnqo, P31, P214, P215, P0984, P1706, and P1798 expands the limited promoter toolbox for T. thermophilus , providing valuable elements for achieving high-level gene expression without complex induction strategies, contrasting with previously characterized inducible or regulated promoters [ 31 – 34 ]. The construction of the plasmid-free HB27ΔpTT27 strain represents a significant genome reduction (~ 11% of total genome size). While this strain exhibited cobalamin auxotrophy and slightly impaired growth, necessitating supplementation, it offers potential advantages. These include potentially increased genetic stability in further recombinant thermostable protein production. Its comparable transformation efficiency suggests basic competence machinery is unaffected. The auxotrophy presents an opportunity for developing antibiotic-free selection systems, a desirable trait for industrial applications [ 47 ], which is currently under investigation. However, the limitations (auxotrophy, slower growth) must be considered, as adding a specific supplement quantity is incomparable to a naturally occurring genetic system. The dramatic (~ 100-fold) increase in transformation efficiency observed in the CRISPR-deficient strain (HB27ΔIII-ABΔI-CΔ CRF3 ) is a key finding. This highlights the significant barrier that native CRISPR-Cas systems pose to the introduction of foreign DNA in this strain. This result aligns with findings in other bacteria where disabling defense systems (CRISPR-Cas, R-M, BREX) enhances genetic accessibility [ 48 – 52 ]. This improved transformability was instrumental in enabling the subsequent efficient construction of multiple protease knockout strains. Our systematic protease knockout strategy provided insights into their roles in HB27. Deletion of TTC0264 (putative ClpY/HslU) and TTC1905 (putative HhoB) markedly reduced extracellular caseinolytic activity, identifying them as major secreted proteases under these conditions. The varied effects of single deletions on growth (e.g., significant impairment in ΔTTC0265/DP5) and reporter expression highlight the complex roles and potential redundancy of proteases in cellular physiology [ 59 ]. A notable observation was the enhanced accumulation of the intracellular β-galactosidase reporter in strains DSP6-DSP9, which harbor multiple protease deletions, including TTC0264 (putative ClpY/HslU). This occurred despite TTC0264 being implicated primarily in extracellular activity based on the skim milk plate assay. TTC0264 and TTC1905 are predicted extracellular proteases; their knockout indirectly stabilizes intracellular proteins by reducing stress-induced misfolding, which involves significant metabolic reorganization [ 60 – 62 ]. Thus, we emphasize that extracellular protease knockouts can indirectly stabilize intracellular proteins by reducing stress responses that lead to misfolding. As well as the deletion of major proteases might trigger complex regulatory responses (e.g., stress responses, altered expression of other intracellular proteases) that indirectly affect β-galactosidase stability or synthesis [ 63 , 64 ]. While the exact mechanism requires further study, the empirical result of increased reporter accumulation in these multiple knockout strains (DSP6-DSP9) supports their potential benefit for improving yields of certain intracellular proteins. The deletions performed target homologs of key protease systems known to be involved in protein quality control (e.g., Clp system components like ClpY/HslU, FtsH-like proteases, HhoB serine protease). In other organisms, these systems degrade misfolded or damaged proteins, regulate protein turnover, and are crucial for stress responses [ 60 , 61 , 65 – 71 ]. Our results, such as the growth defect in DP5 (ΔTTC0265/FtsH-like) and the enhanced reporter stability in DSP6-DSP9 (lacking TTC0264/ClpY/HslU among others), are broadly consistent with these general roles. However, this study focused on the phenotypic outcomes (growth, extracellular activity, reporter expression) rather than a detailed mechanistic analysis of protein quality control pathways. The observed effects likely stem from altering the balance of protein synthesis, folding, and degradation, but the specific substrates and regulatory consequences within T. thermophilus require further investigation. Limitations and Future Directions: This study provides foundational tools and strains. A key limitation, as highlighted by the reviewers, is the reliance on β-galactosidase as the primary reporter. While useful for comparative purposes within this study (e.g., promoter strength, effect of protease knockouts), demonstrating the practical utility of these strains requires testing the expression of industrially relevant target proteins, particularly thermostable enzymes [ 5 ]. Direct quantitative comparisons of protein yields between these optimized T. thermophilus strains and established mesophilic hosts ( E. coli, Pichia sp ) were beyond the scope of this initial work but represent an important future direction, especially for targets where Thermus offers intrinsic advantages. Furthermore, the observed drawbacks of the plasmid-free strain (growth defect, auxotrophy) need to be addressed or accounted for in specific applications. Evaluating the performance of the engineered strains (especially DSP9) under realistic fermentation conditions is crucial. Future work could also involve proteomic analysis to confirm protease depletion and investigate global cellular responses, as well as exploring combinatorial effects of the developed tools (e.g., using promoter P0984 in strain DSP9). 5 Conclusions This study successfully generated and characterized valuable resources for engineering T. thermophilus HB27 as a host for heterologous protein production. Key contributions include the identification of the strong constitutive promoter P0984, the construction and characterization of a plasmid-free strain (HB27ΔpTT27) revealing its auxotrophic nature and potential for genome streamlining, the demonstration that deleting specific CRISPR-Cas loci dramatically enhances transformation efficiency, and the development of protease deletion strains (DP series and multi-deletion DSP series). Strain DSP9, harboring 10 targeted protease gene deletions built upon the CRISPR-deficient background, emerged as a promising first-generation chassis, exhibiting robust growth and enhanced intracellular accumulation of reporter protein. While this work establishes a strong foundation, further validation using industrially relevant thermostable proteins and optimization under bioprocess conditions are necessary next steps. Declarations Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This study was supported by the National Key Research and Development Program of China (2022YFA0912200), National Natural Science Foundation of China (32170096), and Key Research and Development Project of Hubei Province (2023BBB025). Acknowledgements The authors would like to thank lab members for their continued support. Contributions Y.Q.L. & M.M.: Conceptualization, Conducted experiment, Data curation, Formal analysis, Visualization, Investigation, Writing–original draft, review & editing; J.W.W. & X.Y.B. & Y.S.: Resources, Methodology; Y.J.L.: Conceptualization, Supervision, Proofreading, Funding, Project administration, Resources. References Ajeje SB, Hu Y, Song G, Peter SB, Afful RG, Sun F, Asadollahi MA, Amiri H, Abdulkhani A, Sun H. Thermostable Cellulases / Xylanases From Thermophilic and Hyperthermophilic Microorganisms: Current Perspective. Front Bioeng Biotechnol. 2021;9:794304. Jaiswal N, Jaiswal P. Thermostable α-Amylases and Laccases: Paving the Way for Sustainable Industrial Applications. Processes 2024, 12. Gomes E, de Souza AR, Orjuela GL, Da Silva R, de Oliveira TB, Rodrigues A. Applications and Benefits of Thermophilic Microorganisms and Their Enzymes for Industrial Biotechnology. In Gene Expression Syst Fungi: Advancements Appl 2016: 459–92.[ Fungal Biology ].. 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S: Proteomic profiling of Deinococcus radiodurans with response to thioredoxin reductase inhibitor and ionizing radiation treatment. J Proteom 2022, 267. Wickner S, Maurizi MR, Gottesman S. Posttranslational Quality Control: Folding, Refolding, and Degrading Proteins. Science. 1999;286:1888–93. Clausen T, Southan C, Ehrmann M. The HtrA Family of Proteases. Mol Cell. 2002;10:443–55. Kim D-Y, Kim K-K. Structure and Function of HtrA Family Proteins, the Key Players in Protein Quality Control. BMB Rep. 2005;38:266–74. Imlay JA. The molecular mechanisms and physiological consequences of oxidative stress: lessons from a model bacterium. Nat Rev Microbiol. 2013;11:443–54. Alba BM, Gross CA. Regulation of the Escherichia coli σE-dependent envelope stress response. Mol Microbiol. 2004;52:613–9. Tables Table 1. Selected protases identified in the genome sequence of T. thermophilus HB27. Locus Annotation General Function TTC0035 ATP-dependent zinc metalloprotease FtsH, EC:3.4.24.- (DP1) Acts as a processive, ATP-dependent zinc metallopeptidase for both cytoplasmic and membrane proteins. Plays a role in the quality control of integral membrane proteins. TTC0174 ATP-dependent clp protease ATP-binding subunit clpA / Negative regulator of genetic competence Clpc/MecB. (DP2) chaperone and an integral component of the ATP-dependent ClpAP protease, participates in regulatory protein degradation and the dissolution and degradation of protein aggregate. TTC0251 ATP-dependent Clp protease ATP-binding subunit ClpX. (DP3) ATP-dependent specificity component of the Clp protease. It directs the protease to specific substrates. Can perform chaperone functions in the absence of ClpP. TTC0264 ATP-dependent hsl protease ATP-binding subunit hslU. (DP4) The HslU subunit of the HslU-HslV complex functions as an ATP dependent 'unfoldase'. The binding of ATP and its subsequent hydrolysis by HslU are essential for unfolding of protein substrates subsequently hydrolysed by HslV [48]. HslU recognizes the N-terminal part of its protein substrates and unfolds these before they are guided to HslV for hydrolysis in peptidase family T1 [49]. TTC0265 ATP-dependent protease hslV, EC:3.4.25.- (DP5) TTC0417 S1C family serine proteases. (DP6) Catalysis of the hydrolysis of internal, alpha-peptide bonds in a polypeptide chain by a catalytic mechanism that involves a catalytic triad consisting of a serine nucleophile that is activated by a proton relay involving an acidic residue (e.g. aspartate or glutamate) and a basic residue (usually histidine). TTC0481 Membrane metalloprotease. (DP7) Catalysis of the hydrolysis of peptide bonds by a mechanism in which water acts as a nucleophile, one or two metal ions hold the water molecule in place, and charged amino acid side chains are ligands for the metal ions. TTC0492 CPBP family of intramembrane metalloproteinases, Abortive infection protein. (DP8) Catalysis of the hydrolysis of internal, alpha-peptide bonds in a polypeptide chain by a mechanism in which water acts as a nucleophile, one or two metal ions hold the water molecule in place, and charged amino acid side chains are ligands for the metal ions. TTC0663 M66 family metalloproteinases. (DP9) This family of metallopeptidases contains StcE, a virulence factor found in Shiga toxigenic Escherichia coli organisms. StcE peptidase cleaves C1 esterase inhibitor [50]. TTC0687 ClpP/crotonase-like domain superfamily. (DP10) ClpP is an ATP-dependent protease that cleaves a number of proteins, such as casein and albumin. It exists as a heterodimer of ATP-binding regulatory A and catalytic P subunits, both of which are required for effective levels of protease activity in the presence of ATP [51]. TTC0950 M50B family Zinc metalloprotease. (DP11) Catalysis of the hydrolysis of peptide bonds by a mechanism in which water acts as a nucleophile, one or two metal ions hold the water molecule in place, and charged amino acid side chains are ligands for the metal ions. TTC0974 PrcB C-terminal domain-containing protein. (DP12) This domain is found at the C terminus of Treponema denticola PrcB. PrcB interacts with the PrtP protease (dentilisin) and is required for the stability of the protease complex [52]. TTC1110 Rhomboid family intramembrane serine protease. (DP13) Catalysis of the hydrolysis of internal, alpha-peptide bonds in a polypeptide chain by a catalytic mechanism that involves a catalytic triad consisting of a serine nucleophile that is activated by a proton relay involving an acidic residue (e.g. aspartate or glutamate) and a basic residue (usually histidine). TTC1111 Periplasmic serine protease. (DP14) Catalysis of the hydrolysis of a peptide bond. A peptide bond is a covalent bond formed when the carbon atom from the carboxyl group of one amino acid shares electrons with the nitrogen atom from the amino group of a second amino acid. TTC1128 ATP-dependent zinc metalloprotease FtsH, EC:3.4.24.- (DP15) Homologous to TTC0035. TTC1905 S1C family serine protease. (DP16) Homologous to TTC0417. Table 2. Relative extracellular protease activity for T. thermophilus HB27 and deletion strains. Strain Relative Enzyme Activity Strain Relative Enzyme Activity HB27 2.57±0.50 a HB27ΔIII-ABΔI-CΔ CRF3 2.5±0.87 ab DP1 2.19±0.29 de DP2 2.5±0.50 ab DP3 2.52±0.29 a DP4 1.64±0.50 f DP5 1.95±1.26 e DP6 2.26±1.15 cd DP7 2.23±0.76 cd DP8 2.23±0.29 cd DP9 2.47±0.76 ab DP10 2.14±0.50 de DP11 2.40±0.58 abc DP12 2.43±1.32 abc DP13 2.31±0.58 bcd DP14 2.57±0.50 a DP15 2.52±0.29 a DP16 1.61±0.58 f Bars represent ±SD of three replicates. Different letters indicate significant differences according to Duncan’s multiple range tests at P < 0.05 . Table 3. Relative extracellular protease activity for T. thermophilus HB27 and multiple deletion strains. Strain Relative Enzyme Activity Strain Relative Enzyme Activity HB27 3.83±0.76 bcd HB27ΔIII-ABΔI-CΔ CRF3 4.83±1.04 b DSP1 1.00±0.50 e DSP2 1.50±0.50 e DSP3 1.17±0.76 e DSP4 4.33±0.76 bc DSP5 3.17±0.76 cd DSP6 4.33±0.29 bc DSP7 6.83±0.29 a DSP8 3.00±0.50 d DSP9 4.83±0.58 b Bars represent ±SD of three replicates. Different letters indicate significant differences according to Duncan’s multiple range tests at P < 0.05 . Additional Declarations No competing interests reported. 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03:08:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6092828/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6092828/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12934-025-02785-y","type":"published","date":"2025-07-10T15:57:25+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82083680,"identity":"ba5c6130-cd2f-4533-9d2a-6a288ebdb3cd","added_by":"auto","created_at":"2025-05-06 14:51:45","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":861617,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figures1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6092828/v1/8e07f9fc76e15e4178d27d57.jpg"},{"id":82084828,"identity":"2ede0e8d-e56e-4b64-9129-fa69178404cb","added_by":"auto","created_at":"2025-05-06 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legend.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figures3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6092828/v1/7bd2be524beeb3738c0e0cc9.jpg"},{"id":82083681,"identity":"4e8f9f39-b43e-4e26-8c1c-4c4c00721d35","added_by":"auto","created_at":"2025-05-06 14:51:45","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":550023,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figures4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6092828/v1/dc3eaf392668ebd7e54e8139.jpg"},{"id":86700196,"identity":"15a805ec-5239-449b-887e-577eb613d913","added_by":"auto","created_at":"2025-07-14 16:11:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4253037,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6092828/v1/62bca459-bac9-43a6-92ab-55eaf8fc113d.pdf"},{"id":82083187,"identity":"39b45073-3407-482b-af12-626437485b3a","added_by":"auto","created_at":"2025-05-06 14:43:45","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4140665,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryData.docx","url":"https://assets-eu.researchsquare.com/files/rs-6092828/v1/7e6858ada3855ce3b52c2bfd.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Construction of Primary Chassis Cells with Efficient Protein Expression in Thermus thermophilus","fulltext":[{"header":"1 Background","content":"\u003cp\u003eThe burgeoning emphasis on renewable energy sources and sustainable industrial practices significantly drives the escalating global demand for thermostable proteins. Thermostable enzymes (e.g., cellulases, xylanases, α-amylases, laccases) are crucial for applications requiring elevated temperatures, such as biofuel production, bioleaching, wastewater treatment, and agricultural biotechnology [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Utilizing thermostable proteins offers advantages like reduced cooling costs, lower contamination risks, and increased reaction rates [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, producing thermostable proteins efficiently often faces challenges in conventional mesophilic hosts like \u003cem\u003eE. coli\u003c/em\u003e. Issues include protein misfolding, aggregation, degradation, and lack of necessary post-translational modifications due to differences in cellular machinery and environment [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The unique structural features conferring thermostability (e.g., specific hydrophobic interactions, salt bridges) may not be correctly established in mesophilic cytoplasm, potentially requiring specialized chaperone systems or protein engineering approaches [\u003cspan additionalcitationids=\"CR11 CR12 CR13\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eThermus thermophilus\u003c/em\u003e, particularly strain HB27, presents a compelling alternative host for producing thermostable proteins [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Its optimal growth at high temperatures (65\u0026ndash;75\u0026deg;C) inherently favors the stability and correct folding of many thermostable target proteins. Furthermore, high-temperature cultivation simplifies downstream purification, as many endogenous mesophilic contaminants are eliminated [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. \u003cem\u003eT. thermophilus\u003c/em\u003e HB27 offers additional advantages like rapid growth, high cell density potential, natural competence for transformation, and available genetic tools, including CRISPR-Cas systems for genome editing [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Consequently, HB27 is an attractive chassis for producing various thermostable enzymes and proteins [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Developing \u003cem\u003eT. thermophilus\u003c/em\u003e as a robust chassis aligns sustainable bioproduction goals by potentially reducing energy inputs for cooling and simplifying processing [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, it is important to note that this platform is primarily suited for thermostable proteins; expressing inherently thermolabile proteins (e.g., many mammalian biopharmaceuticals) remains a significant challenge due to the high cultivation temperatures [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite its potential, optimizing \u003cem\u003eT. thermophilus\u003c/em\u003e for industrial-scale production requires addressing key limitations. One is the limited availability of strong, well-characterized constitutive promoters for driving high-level, stable gene expression. While some inducible promoters exist (e.g., P\u003csub\u003earg\u003c/sub\u003e, P\u003csub\u003ednaK\u003c/sub\u003e, P\u003csub\u003enar\u003c/sub\u003e, P\u003csub\u003epilA4\u003c/sub\u003e), they often suffer from drawbacks like complex induction strategies, growth inhibition, or unintended regulation [\u003cspan additionalcitationids=\"CR32 CR33\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Plasmid-based expression systems can also face instability and metabolic burden [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Another significant challenge is the potential degradation of recombinant proteins by endogenous host proteases, which can severely reduce yields [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. While protease activity is essential for cellular health, specific proteases can target heterologous proteins. Previous work in \u003cem\u003eT. thermophilus\u003c/em\u003e indicated that deleting certain proteases (e.g., Lon-type) could improve yields, but the roles of many other proteases remain uncharacterized [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTherefore, this study aimed to enhance \u003cem\u003eT. thermophilus\u003c/em\u003e HB27 as a microbial chassis through a multi-pronged strategy: (1) Screening of 13 endogenous genomic regions to identify strong constitutive promoters. (2) Constructing and characterizing a plasmid-free strain (HB27ΔpTT27) to reduce genome size and explore its potential as an auxotrophy-based selection. (3) Systematically deleted 16 predicted non-essential protease genes to assess their impact on growth, extracellular proteolytic activity, and reporter protein accumulation. We aim to generate characterized strains and genetic tools to facilitate the development of \u003cem\u003eT. thermophilus\u003c/em\u003e HB27 for improved recombinant protein production, particularly focusing on thermostable targets.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Bacterial strain and cultivation\u003c/h2\u003e \u003cp\u003e \u003cem\u003eE. coli\u003c/em\u003e DH5α was used as a host for plasmid engineering and multiplication, cultivated in LB medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl) at 37\u0026deg;C with 200 rpm in a shaker incubator (BLabotery, ZQPL-200). \u003cem\u003eT. thermophilus\u003c/em\u003e HB27 (Wildtype), HB27∆TTP0042 (\u003cem\u003eT. thermophilus\u003c/em\u003e HB27 with β-galactosidase gene knockout), HB27∆III-AB∆I-C∆CRE3 (\u003cem\u003eT. thermophilus\u003c/em\u003e HB27 with I-C, III-A \u0026amp; B CRISPR-Cas system and \u003cem\u003eCRF3\u003c/em\u003e (CRISPR-associated Rossmann Fold) genes knockouts), and \u003cem\u003eT. thermophilus stains\u003c/em\u003e were cultivated at 65\u0026deg;C with 200 rpm shaking in Ttᶧ medium (8 g/L tryptone, 4 g/L yeast extract, 3 g/L NaCl, 4.36 mM NaHCO₃,0.26 mM MgSO₄, 0.739 mM MgCl₂.6H₂O, 0.012 mM KCl, and 0.249 mM CaCl₂.2H₂O) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Petri dishes of \u003cem\u003eT. thermophilus\u003c/em\u003e were wrapped in sterilized bags to maintain plate humidity in the static incubator (BLabotery, SPL-250). Strains were listed in the supplementary data Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Plasmid construction\u003c/h2\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eTo construct plasmids for endogenous promoter screening\u003c/span\u003e, promoter regions were isolated from the HB27 genome utilizing PCR and primers with about 20 nt at the 5'-end complementary to the plasmid ligation side. Then, the TTP0042 codon (β-glucosidase reporter gene) amplified from the pTT27 plasmid was cloned into pRKP31, which was pre-digested with SalI and NheI. Then, the P31 promoter was replaced with the next screened promoters into the pRKP31-TTP0042 plasmid utilizing 2X CE Mix V3 (Vazyme, C117-01-AA).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eTo construct the knockout plasmids for replicon repT and the 16 protease loci based on the endogenous CRISPR system type I-B\u003c/span\u003e, a spacer that matched the target loci was cloned within two BbsI restriction sites on the pRKP31-AC3 shuttle vector by T4 polynucleotide (BioLabs, M0201S). A spacer fragment was generated by annealing two complementary single-stranded oligo DNA 42bp in length with 4nt at the 5`-end complementary to the BbsI sticky ends, at 95\u0026deg;C for 5 min, and slowly cooled down at room temperature to form the double-stranded inserts. The two arms of the homology direct repair (HDR) template were separately amplified using the HB27 genome as template and connected using splicing by 20 bp overhang extension PCR (SOE PCR). Plasmids were constructed in two steps. First, spacers that matched the double-strand break (DSB) site were cloned. Then, the plasmid was linearized by PCR, followed by DpnI (Thermo Scientific, FD1704) enzyme digestion, and the HDR was cloned by 20 bp overhang homology utilizing ABclonal MultiF Seamless Assembly Mix (ABclonal, RK21020) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e illustrates the schematic procedure of endogenous CRISPR-Cas-based gene editing technology.\u003c/p\u003e \u003cp\u003ePlasmids were validated by sequencing (Sangon, Shanghai, China) before being transformed into the HB27 cells. Plasmids used or constructed are listed in Table S3. Primers used for PCR amplification and validation are listed in Table S2. Primers and single-stranded oligo DNA were synthesized by Sangon Co., Shanghai, China. PCR was performed using the Phanta Max Super‐Fidelity DNA polymerase (Vazyme, P505). The amplification was performed at 95\u0026deg;C for 5 min, 95\u0026deg;C for 15 sec, Tm-5\u0026deg;C for 15 sec, 72\u0026deg;C for 1 min/1 kb, and 72\u0026deg;C for 5 min, for 30 cycles. The amplicons and digested fragments were purified with the E.Z.N.A. Cycle-Pure kit (Omega BIO-TEK, D6492- 02). Plasmid extracted utilizing E.Z.N.A. Plasmid Mini Kit I (Omega BIO-TEK, D6943- 02).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Transformation, cell morphology, and growth rate estimation\u003c/h2\u003e \u003cp\u003eThe heat shock transformation method was used for DH5α competent cells. 50 \u0026micro;L DH5α was thawed on ice for 5 min, 10 \u0026micro;L recombinant plasmid was gently mixed, and placed on ice for 25 min. Heat shock at 42℃ for 45 sec, followed by ice incubation for 2 min, then 900 \u0026micro;L LB medium was pipetted and incubated at 37\u0026deg;C, 200 rpm for 1 h, and finally spread on LB selection medium [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eT. thermophilus\u003c/em\u003e utilizes a natural competence system to take up exogenous DNA during the mid-log phase. 1 \u0026micro;g plasmid was mixed with 500 \u0026micro;L mid-log phase cells, incubated for 2.5 h at 65℃, 180 rpm, and then spread on a Ttᶧ selection medium [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe pRKP31 and pRKP31-AC3 Shuttle plasmids were used as backbones for construction with a kanamycin-selectable marker, under selection pressure of 30 \u0026micro;g/mL and 20 \u0026micro;g/mL for \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eT. thermophilus\u003c/em\u003e, respectively.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eFor microscopic cell morphology of\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eT. thermophilus\u003c/span\u003e, a fuchsin-dyed smear was prepared and observed using the immersion oil 100X objective lens [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eFor the growth curve\u003c/span\u003e, bacteria were acquired by measuring the optical turbidity at an absorbance of 600 nm. Since the optical density of the culture is proportional to the cell density, measuring the turbidity of the culture can be used to estimate the number of bacterial cells. Overnight cultures were diluted 1:100 v/v into 50 mL Ttᶧ medium in a shaker incubator at 65℃, 180 r/min. Samples were taken every 2 to 4 h intervals for 24 to 48 h. The strains were cultured in three biological repeats [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Quantitative and qualitative β-galactosidase assay\u003c/h2\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eQuantitative assessment of β-galactosidase activity\u003c/span\u003e was performed based on the established O-Nitrophenyl-β-D-galactopyranoside (ONPG) assay as described [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Reporter plasmids were introduced into the \u003cem\u003eT. thermophilus\u003c/em\u003e HB27 ΔTTP0042. Three distinct single colonies harboring the respective reporter plasmid were randomly selected for each independent experimental replicate. These transformed strains were cultivated in Ttᶧ medium under the optimum conditions. Upon reaching the log growth phase, as determined by an optical density at (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;~\u0026thinsp;1.0), bacterial cells were harvested via centrifugation. The resulting cellular pellets were resuspended in 10 mM Tris-HCl buffer (pH 8.0). To obtain crude cellular lysates, the resuspended cells underwent controlled sonication. Following lysis, cellular debris was eliminated through centrifugation at 13,000 \u0026times; g for 30 min at 20\u0026deg;C to obtain clarified supernatants. The protein concentration within these clarified cellular extracts was precisely quantified using the Bradford reagent (Tiangen, PA102). A 50 \u0026micro;L aliquot of the clarified supernatant was combined with 450 \u0026micro;L of reaction buffer (2.8 mM ONPG in 50 mM sodium phosphate, pH 6.5). Samples were incubated at 65\u0026deg;C for a precisely controlled duration of 20 minutes, after which the enzymatic reaction was terminated by adding an equal volume of 1 M Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e. The absorbance of the o-nitrophenol was spectroscopically estimated at 420 nm using a microplate reader (Victor Nivo 3S; PerkinElmer). One unit of specific β-galactosidase activity is operationally defined as 1 nmol ρ-nitrophenol produced per min per mg total protein.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eIn the qualitative plate assays\u003c/span\u003e, 5 \u0026micro;L of mid-log phase (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;~\u0026thinsp;1.0) screened strains were spotted on Ttᶧ plates supplemented with 100 mg/L X-gal. Plates were incubated at 65℃ for 2 days to observe the color change [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Extracellular protease assay\u003c/h2\u003e \u003cp\u003eThe protease mutations were screened for extracellular protease activity by skim milk plate assay [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. 2.5 \u0026micro;L of mid-log phase (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;~\u0026thinsp;1.0) mutants were spotted on Ttᶧ plates supplemented with 10% (v/v) skimmed milk. The clear zone diameter was measured 3 days after incubation at 65\u0026deg;C by flooding the plates with 10% trichloroacetic acid (TCA). The relative enzyme activity was calculated using the following formula:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:REA=\\frac{CZD}{CD}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere: REA is relative enzyme activity, CZD is clear zone diameter, and CD is colony diameter.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll histogram drawings and statistical analysis were completed using the GraphPad Prism 8 software (GraphPad Software Inc.). Data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). The significance of differences between groups' mean values was analyzed using Duncan\u0026rsquo;s Multiple Range Test (DMRT) at a \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e significance level.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Construction of promoter screening reporter vector\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eT. thermophilus\u003c/em\u003e HB27 β-galactosidase is involved in lactose metabolism and acts as a prevalent reporter enzyme used in studying the activities of promoters [32, 34]. \u003cem\u003eT. thermophilus\u003c/em\u003e HB27 β-galactosidase transcript by three loci (TTP0042, TTP0220, TTP0222) located in the pTT27 plasmid [19]. The TTP0042 locus was detected as the main source of β-galactosidase activity in \u003cem\u003eT. thermophilus\u003c/em\u003e HB27. Thus, the β-galactosidase-deletion strain (HB27ΔTTP0042) was used as a competent cell for promoter screening. Thirteen promoter regions were inserted into pRKP-TTP0042 by homology ligation. Promoters named as follows: Pslp (206 bp), Pago (300 bp), Pnqo (414 bp), P31 (216 bp), P031 (214 bp), P43 (195 bp), P214 (160 bp), P215 (184 bp), P0416 (853 bp), P0984 (858 bp), P1578 (842 bp), P1706 (849 bp), P1798 (861 bp). The pRKP-TTP0042 screening vector contains the promoter-less endogenous TTP0042 β-galactosidase, heat–stable Kanamycin resistance selectable marker derived with Pslp promoter, \u003cem\u003eE. coli\u003c/em\u003e replicon puc-ori, and \u003cem\u003eT. thermophilus\u003c/em\u003e replicon repA ori (Fig. 1A). Transformants grown up on Ttᶧ selection plates were picked up for verification using specific primers (Fig. S2 and Table S2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Effect of different promoters on β-galactosidase expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo analyze the potential transcription level of the 13 promoters. The HB27ΔTTP0042 transformants were examined qualitatively and quantitatively by X-Gal plates and ONPG assay (Fig. 1B, C). Transformants, Control (HB27ΔTTP0042), and the HB27 (wild-type) were grown in Ttᶧ medium under optimal growth conditions to mid-log phase, and then the β-glucosidase activities were examined. The Pago, P031, P43, P0416, and P1578 promoters have a significantly lower activity as appears in yellow colonies on the X-Gal plates (Fig. 1B). Interestingly, the quality and quantity assay show that Pslp, Pnqo, P31, P214, P215, P0984, P1706, and P1798 promoters were significantly higher than the wild-type and control as shows in Fig. 1B, C. Among the 13 promoters, the P0984 promoter shows significantly 13-fold higher activity, which recommends the utilization of the P0984 promoter for the subsequent thermostable protein production.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Construction of HB27 plasmid-free strain\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo step forward chassis cell construction and minimize the \u003cem\u003eT. thermophilus\u003c/em\u003e HB27 genome. We deleted the pTT27 plasmid (~270 kb) [19]. We utilized the CRISPR-deficient strain HB27ΔIII-ABΔI-CΔ\u003cem\u003eCRF3\u003c/em\u003e, previously constructed by our lab as a competent cell due to its enhanced transformability, lack CRISPR type III subtype A\u0026amp;B, type I subtype C, and CRISPR-associated Rossmannfold (64 kbp deletion) as mapped in Fig. 2A. The pTT27 plasmid replication origin (repT) was identified, 42nts were selected after PAM sequence (5′-TTC-3′) in the middle of the repT locus as a specific region for DSB, and an approximately 500 bp region flanking repT locus were cloned in the pRKP31-AC3 vector for HDR (Fig S1). Colonies grown up in the Ttᶧ selection medium were screened by colony PCR with three pairs of primers flanking the target deletion region (Table S2, Fig. S3A). PCR results confirmed the DSB sandwiched by the HDR; control strain was 3319 bp, 2214 bp, and 3322 bp, while those of the plasmid-free strain showed no bands, suggesting a successful deletion was established (Fig. S3B). As pTT27 encodes key carotenoid biosynthesis genes (\u003cem\u003ecrtI, crtB\u003c/em\u003e) [19], the resulting HB27ΔpTT27 colonies were white, in contrast to the wild-type strain (Fig. S3C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Characterization of HB27\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;plasmid-free strain\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the plasmid-free strain (HB27ΔpTT27) as a potential chassis, its phenotypic properties (growth rate, morphology, transformation efficiency) were compared with the control (HB27ΔIII-ABΔI-CΔ\u003cem\u003eCRF3\u003c/em\u003e) and the wild-type HB27.\u003c/p\u003e\n\u003cp\u003eGrowth rate: As shown in Fig. 2B, the growth of HB27ΔIII-ABΔI-CΔ\u003cem\u003eCRF3\u003c/em\u003e was comparable to wild-type HB27, reaching stationary phase (OD\u003csub\u003e600\u003c/sub\u003e~5) around 30 h. However, HB27ΔpTT27 failed to grow significantly in the standard Ttᶧ medium. Since pTT27 harbors the cobalamin (Vitamin B12) biosynthesis gene cluster (TTP0001-TTP0023) [19, 46]. We hypothesized that this deletion caused auxotrophy. Supplementation with 0.1 μg/mL AdoCbl rescued growth, although the growth rate remained slightly slower, and the final OD was lower than wild-type and competent strains. This confirms that pTT27 encodes essential genes for cobalamin synthesis and potentially other factors affecting optimal growth under these conditions. This auxotrophy could potentially be applied to antibiotic-free selection systems in future applications [47].\u003c/p\u003e\n\u003cp\u003eTransformation efficiency: We assessed natural transformation efficiency using the pRKP0984-TTP0042 plasmid. Wild-type HB27 exhibited a baseline efficiency of approximately 2.02×10⁴ CFU/μg. The plasmid-free HB27ΔpTT27 strain showed a similar efficiency (2.13×10⁴ CFU/μg), indicating that removal of the pTT27 plasmid did not significantly impair DNA uptake under these conditions. In contrast, the competent CRISPR-deficient strain HB27ΔIII-ABΔI-CΔ\u003cem\u003eCRF3\u003c/em\u003e displayed significantly enhanced efficiency (1.78×10⁶ CFU/μg), nearly a 100-fold increase over the wild-type (Fig. 2C). This pronounced increase is likely due to the removal of CRISPR-Cas systems that target foreign DNA. Such enhancement is consistent with observations in other bacteria, where inactivation of defense systems like restriction-modification (R-M), StySA, or BREX systems improves transformation efficiencies [48-52]. This high transformability proved advantageous for the subsequent protease gene deletions.\u003c/p\u003e\n\u003cp\u003eCell morphology: Microscopic examination revealed that HB27ΔIII-ABΔI-CΔ\u003cem\u003eCRF3\u003c/em\u003e morphology was indistinguishable from wild-type bacilli. However, the plasmid-free HB27ΔpTT27 strain consistently displayed a slightly elongated cell shape compared to the wild-type (Fig. 2D). This subtle morphological change might be linked to the altered growth kinetics or potential cell envelope modifications resulting from the large deletion [53], warranting further investigation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Construction of protease-encoding loci knockout strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBioinformatic analysis identified 16 predicted nonessential protease-encoding genes within the \u003cem\u003eT. thermophilus\u0026nbsp;\u003c/em\u003eHB27 genome distributed across the chromosomal loci as listed in Table 1. We knocked out these 16 loci to optimize the highly transformable HB27ΔIII-ABΔI-CΔ\u003cem\u003eCRF3\u003c/em\u003e strain. To ensure precise locus deletion without cluster gene interruption, we targeted the protein-coding sequences while preserving promoter elements and regulatory regions (Fig. 3A). Utilizing the pRKP31-AC3 plasmid-mediated HDR recombination system based on the endogenous CRISPR-Cas9 Type I-B machinery. We successfully constructed 16 individual protease knockout strains. \u003cem\u003eIn vivo\u003c/em\u003e transcript of the designed crRNA generates ribonucleoprotein (RNP) complexes. These RNPs effectively cleaved the protease locus, with precise repair mediated by homologous recombination utilizing an HDR template, thus introducing the designed null mutations. As illustrated in Fig. 3A, B, Table S2, the PCR-based validation strategy confirmed the homogeneity of the mutant population. Validated strains were then subjected to iterative rounds of subculturing in an antibiotic-free Ttᶧ medium to facilitate plasmid loss, yielding marker-free protease deletion strains for future editing and ensuring a complete gene editing mutant. As illustrated in Fig 3A 'exam-genome-F/R' primer pair confirmed the target deletion by amplifying across the modified region, yielding amplicons of reduced size depending on the target locus compared to wild-type allele (Fig. 3B), 'exam-sp-F/exam-genome-R' primer combination detect the presence of any residual wild-type alleles, 'exam-plasmid-F/R' primer confirm the subsequent loss of the editing plasmid. This multi-faceted screening approach generates 16 individual protease deletion loci, designated DP1 through DP16, each carrying a precise deletion in the intended protease-encoding locus.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 C\u003c/strong\u003e\u003cstrong\u003eharacterization of protease-encoding loci knockout strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGrowth rate: The 16 individual protease deletion strains (DP1-DP16) were monitored in a liquid Ttᶧ medium, revealing distinct growth phenotypes among strains. DP6-DP11, exhibited growth profiles indistinguishable from the wild-type strain, reaching stationary phase by 30 h of incubation with a comparable OD\u003csub\u003e600\u003c/sub\u003e~5 (Fig. 3D). DP1-DP4, DP12-DP16 displayed subtle variations in growth rates relative to the wild-type, while reached the stationary phase by 30 h, with slightly lower OD than the wild-type (Fig. 3C, E). DP5 (TTC0265 deletion, putative FtsH) manifested a significantly attenuated growth rate with extended incubation period of 40 h to reach the stationary phase (Fig. 3C). Notably, DP1 (TTC0035 deletion), DP5 (TTC0265 deletion), and DP16 (TTC1905 deletion) consistently demonstrated a reduction in maximal biomass accumulation.\u003c/p\u003e\n\u003cp\u003eExtracellular proteolytic activity was assessed using Ttᶧ skim milk plates incubated for 72 h at 65°C (Fig. 3F) to compare the resulting hydrolysis zones of the strains (Table 2). Notably, DP2, DP3, DP9, DP11-DP15, exhibited hydrolytic zones with no statistically significant difference compared to HB27 and HB27ΔIII-ABΔI-CΔ\u003cem\u003eCRF3\u003c/em\u003e, indicating unimpaired extracellular protease function. In contrast, DP1, DP5-DP8, and DP10 generated significantly smaller zones of hydrolysis, suggesting a reduction in secreted protease activity. Remarkably, DP4 (TTC0264 deletion, putative ClpY/HslU) and DP16 (TTC1905 deletion, putative HhoB) displayed severely attenuated hydrolytic activity (1.61 ± 0.58 mm in diameter). These observations strongly implicate the proteases encoded by TTC0264 and TTC1905 as key contributors to overall secreted proteolytic activity under these conditions.\u003c/p\u003e\n\u003cp\u003eReporter protein accumulation assesses the impact of protease deletions on heterologous protein expression capabilities, using the pRKP0984-TTP0042 reporter plasmid. DP1, DP2, DP3, DP5, and DP8 exhibited levels statistically indistinguishable from the competent strain HB27ΔIII-ABΔI-CΔ\u003cem\u003eCRF3\u003c/em\u003e (Fig. 3G). Conversely, the remaining protease deletion strains demonstrated a statistically significant reduction in β-galactosidase activity.\u003c/p\u003e\n\u003cp\u003eBased on integrated analysis (growth, protease activity, reporter expression), a subset of 10 loci (TTC0174, TTC0251, TTC0264, TTC0417, TTC0481, TTC0492, TTC0663, TTC0687, TTC0950, TTC0974) were selected for multiple knockout construction, starting from DP6 (ΔTTC0417) which showed wild-type-like growth and slightly reduced reporter expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7 Construction of multiple protease-encoding loci knockout strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo develop \u003cem\u003eT. thermophilus\u003c/em\u003e HB27 chassis cells with enhanced heterologous protein expression capabilities, we generated a series of the above-mentioned knockout loci using the DP6 strain (HB27ΔIII-ABΔI-CΔ\u003cem\u003eCRF3\u003c/em\u003eΔTTC0417) as a competent cell with subsequent rounds of gene editing. We constructed nine multiple protease loci knockout strains, named DSP1-DSP9 (Fig. S4), and confirmed colony PCR (Fig. S5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe successful construction of these iterative knockout strains (DSP1-DSP9) represents a step toward optimizing \u003cem\u003eT. thermophilus\u003c/em\u003e HB27. However, knockout efficiency progressively decreased with each successive deletion, ultimately limiting further gene editing using the I-B system, and underscores the need for alternative strategies to further refine this chassis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.8 Characterization of multiple protease-encoding loci knockout strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGrowth rate: All multiple-deletion strains (DSP1-DSP9) exhibited growth profiles generally comparable to the wild-type strain, reaching log phase around 24 h, though final biomass was slightly reduced (OD\u003csub\u003e600\u003c/sub\u003e 3.57-3.88 vs ~4.2) (Fig. 4A). DSP9 showed the least growth impairment among the multiple-deletion strains.\u003c/p\u003e\n\u003cp\u003eExtracellular protease activity: DSP1-DSP3 showed markedly smaller hydrolysis zones compared to the parental strains, consistent with the accumulation of deletions including TTC0264 (putative ClpY/HslU). Other strains (DSP4-DSP6, DSP8-DSP9) showed activity closer to the parental strain, suggesting compensatory effects or that the deleted proteases in these combinations contribute less to casein hydrolysis under these conditions (Table 3).\u003c/p\u003e\n\u003cp\u003eReporter gene expression: β-galactosidase activity varied among the multiple deletion strains (Fig. 4B). DSP1-DSP2 and DSP4-DSP5 showed activity similar to the parental strains. DSP3 showed reduced activity. Significantly, strains DSP6 through DSP9 exhibited enhanced β-galactosidase activity compared to the starting strain, with DSP6 showing the highest (11,480 U/mg) and DSP9 also showing strong activity (10,844 U/mg). This suggests that the combined deletion of these specific proteases reduces degradation or turnover of the intracellular β-galactosidase reporter.\u003c/p\u003e\n\u003cp\u003eBased on this characterization, DSP9 emerged as a promising first-generation chassis candidate. Its genome size is reduced compared to wild-type (~3.5% deletion from chromosome relative to WT), it maintains robust growth kinetics, and shows enhanced reporter protein accumulation (approximately twofold higher activity than the wild-type strain under these conditions).\u003c/p\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eThis study aimed to develop improved chassis strain and genetic tools for \u003cem\u003eT. thermophilus\u003c/em\u003e HB27, focusing on enhancing its utility for heterologous protein production, particularly thermostable proteins. We successfully identified a strong native constitutive promoter (P0984), generated and characterized a plasmid-free strain (HB27ΔpTT27), demonstrated significantly improved transformation efficiency by deleting specific CRISPR-Cas loci, and systematically evaluated the impact of single and multiple protease loci deletions.\u003c/p\u003e \u003cp\u003eThe identification of strong constitutive promoters as Pslp, Pnqo, P31, P214, P215, P0984, P1706, and P1798 expands the limited promoter toolbox for \u003cem\u003eT. thermophilus\u003c/em\u003e, providing valuable elements for achieving high-level gene expression without complex induction strategies, contrasting with previously characterized inducible or regulated promoters [\u003cspan additionalcitationids=\"CR32 CR33\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe construction of the plasmid-free HB27ΔpTT27 strain represents a significant genome reduction (~\u0026thinsp;11% of total genome size). While this strain exhibited cobalamin auxotrophy and slightly impaired growth, necessitating supplementation, it offers potential advantages. These include potentially increased genetic stability in further recombinant thermostable protein production. Its comparable transformation efficiency suggests basic competence machinery is unaffected. The auxotrophy presents an opportunity for developing antibiotic-free selection systems, a desirable trait for industrial applications [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], which is currently under investigation. However, the limitations (auxotrophy, slower growth) must be considered, as adding a specific supplement quantity is incomparable to a naturally occurring genetic system.\u003c/p\u003e \u003cp\u003eThe dramatic (~\u0026thinsp;100-fold) increase in transformation efficiency observed in the CRISPR-deficient strain (HB27ΔIII-ABΔI-CΔ\u003cem\u003eCRF3\u003c/em\u003e) is a key finding. This highlights the significant barrier that native CRISPR-Cas systems pose to the introduction of foreign DNA in this strain. This result aligns with findings in other bacteria where disabling defense systems (CRISPR-Cas, R-M, BREX) enhances genetic accessibility [\u003cspan additionalcitationids=\"CR49 CR50 CR51\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. This improved transformability was instrumental in enabling the subsequent efficient construction of multiple protease knockout strains.\u003c/p\u003e \u003cp\u003eOur systematic protease knockout strategy provided insights into their roles in HB27. Deletion of TTC0264 (putative ClpY/HslU) and TTC1905 (putative HhoB) markedly reduced extracellular caseinolytic activity, identifying them as major secreted proteases under these conditions. The varied effects of single deletions on growth (e.g., significant impairment in ΔTTC0265/DP5) and reporter expression highlight the complex roles and potential redundancy of proteases in cellular physiology [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA notable observation was the enhanced accumulation of the intracellular β-galactosidase reporter in strains DSP6-DSP9, which harbor multiple protease deletions, including TTC0264 (putative ClpY/HslU). This occurred despite TTC0264 being implicated primarily in extracellular activity based on the skim milk plate assay. TTC0264 and TTC1905 are predicted extracellular proteases; their knockout indirectly stabilizes intracellular proteins by reducing stress-induced misfolding, which involves significant metabolic reorganization [\u003cspan additionalcitationids=\"CR61\" citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Thus, we emphasize that extracellular protease knockouts can indirectly stabilize intracellular proteins by reducing stress responses that lead to misfolding. As well as the deletion of major proteases might trigger complex regulatory responses (e.g., stress responses, altered expression of other intracellular proteases) that indirectly affect β-galactosidase stability or synthesis [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. While the exact mechanism requires further study, the empirical result of increased reporter accumulation in these multiple knockout strains (DSP6-DSP9) supports their potential benefit for improving yields of certain intracellular proteins.\u003c/p\u003e \u003cp\u003eThe deletions performed target homologs of key protease systems known to be involved in protein quality control (e.g., Clp system components like ClpY/HslU, FtsH-like proteases, HhoB serine protease). In other organisms, these systems degrade misfolded or damaged proteins, regulate protein turnover, and are crucial for stress responses [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan additionalcitationids=\"CR66 CR67 CR68 CR69 CR70\" citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Our results, such as the growth defect in DP5 (ΔTTC0265/FtsH-like) and the enhanced reporter stability in DSP6-DSP9 (lacking TTC0264/ClpY/HslU among others), are broadly consistent with these general roles. However, this study focused on the phenotypic outcomes (growth, extracellular activity, reporter expression) rather than a detailed mechanistic analysis of protein quality control pathways. The observed effects likely stem from altering the balance of protein synthesis, folding, and degradation, but the specific substrates and regulatory consequences within \u003cem\u003eT. thermophilus\u003c/em\u003e require further investigation.\u003c/p\u003e \u003cp\u003eLimitations and Future Directions: This study provides foundational tools and strains. A key limitation, as highlighted by the reviewers, is the reliance on β-galactosidase as the primary reporter. While useful for comparative purposes within this study (e.g., promoter strength, effect of protease knockouts), demonstrating the practical utility of these strains requires testing the expression of industrially relevant target proteins, particularly thermostable enzymes [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Direct quantitative comparisons of protein yields between these optimized \u003cem\u003eT. thermophilus\u003c/em\u003e strains and established mesophilic hosts (\u003cem\u003eE. coli, Pichia sp\u003c/em\u003e) were beyond the scope of this initial work but represent an important future direction, especially for targets where Thermus offers intrinsic advantages. Furthermore, the observed drawbacks of the plasmid-free strain (growth defect, auxotrophy) need to be addressed or accounted for in specific applications. Evaluating the performance of the engineered strains (especially DSP9) under realistic fermentation conditions is crucial. Future work could also involve proteomic analysis to confirm protease depletion and investigate global cellular responses, as well as exploring combinatorial effects of the developed tools (e.g., using promoter P0984 in strain DSP9).\u003c/p\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003eThis study successfully generated and characterized valuable resources for engineering \u003cem\u003eT. thermophilus\u003c/em\u003e HB27 as a host for heterologous protein production. Key contributions include the identification of the strong constitutive promoter P0984, the construction and characterization of a plasmid-free strain (HB27ΔpTT27) revealing its auxotrophic nature and potential for genome streamlining, the demonstration that deleting specific CRISPR-Cas loci dramatically enhances transformation efficiency, and the development of protease deletion strains (DP series and multi-deletion DSP series). Strain DSP9, harboring 10 targeted protease gene deletions built upon the CRISPR-deficient background, emerged as a promising first-generation chassis, exhibiting robust growth and enhanced intracellular accumulation of reporter protein. While this work establishes a strong foundation, further validation using industrially relevant thermostable proteins and optimization under bioprocess conditions are necessary next steps.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Key Research and Development Program of China (2022YFA0912200), National Natural Science Foundation of China (32170096), and Key Research and Development Project of Hubei Province (2023BBB025).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank lab members for their continued support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.Q.L. \u0026amp; M.M.: Conceptualization, Conducted experiment, Data curation, Formal analysis, Visualization, Investigation, Writing–original draft, review \u0026amp; editing; J.W.W. \u0026amp; X.Y.B. \u0026amp; Y.S.: Resources, Methodology; Y.J.L.: Conceptualization, Supervision, Proofreading, Funding, Project administration, Resources.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAjeje SB, Hu Y, Song G, Peter SB, Afful RG, Sun F, Asadollahi MA, Amiri H, Abdulkhani A, Sun H. 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Mol Microbiol. 2004;52:613\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Selected protases identified in the genome sequence of \u003cem\u003eT. thermophilus\u003c/em\u003e HB27.\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"712\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLocus\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 209px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAnnotation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 428px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGeneral Function\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eTTC0035\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 209px;\"\u003e\n \u003cp\u003eATP-dependent zinc metalloprotease FtsH, EC:3.4.24.- (DP1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 428px;\"\u003e\n \u003cp\u003eActs as a processive, ATP-dependent zinc metallopeptidase for both cytoplasmic and membrane proteins. Plays a role in the quality control of integral membrane proteins.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eTTC0174\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 209px;\"\u003e\n \u003cp\u003eATP-dependent clp protease ATP-binding subunit clpA / Negative regulator of genetic competence Clpc/MecB. (DP2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 428px;\"\u003e\n \u003cp\u003echaperone and an integral component of the ATP-dependent ClpAP protease, participates in regulatory protein degradation and the dissolution and degradation of protein aggregate.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eTTC0251\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 209px;\"\u003e\n \u003cp\u003eATP-dependent Clp protease ATP-binding subunit ClpX. (DP3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 428px;\"\u003e\n \u003cp\u003eATP-dependent specificity component of the Clp protease. It directs the protease to specific substrates. Can perform chaperone functions in the absence of ClpP.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eTTC0264\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 209px;\"\u003e\n \u003cp\u003eATP-dependent hsl protease ATP-binding subunit hslU. (DP4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 428px;\"\u003e\n \u003cp\u003eThe HslU subunit of the HslU-HslV complex functions as an ATP dependent \u0026apos;unfoldase\u0026apos;. The binding of ATP and its subsequent hydrolysis by HslU are essential for unfolding of protein substrates subsequently hydrolysed by HslV [48]. HslU recognizes the N-terminal part of its protein substrates and unfolds these before they are guided to HslV for hydrolysis in peptidase family T1 [49].\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eTTC0265\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 209px;\"\u003e\n \u003cp\u003eATP-dependent protease hslV, EC:3.4.25.- (DP5)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eTTC0417\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 209px;\"\u003e\n \u003cp\u003eS1C family serine proteases. (DP6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 428px;\"\u003e\n \u003cp\u003eCatalysis of the hydrolysis of internal, alpha-peptide bonds in a polypeptide chain by a catalytic mechanism that involves a catalytic triad consisting of a serine nucleophile that is activated by a proton relay involving an acidic residue (e.g. aspartate or glutamate) and a basic residue (usually histidine).\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eTTC0481\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 209px;\"\u003e\n \u003cp\u003eMembrane metalloprotease. (DP7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 428px;\"\u003e\n \u003cp\u003eCatalysis of the hydrolysis of peptide bonds by a mechanism in which water acts as a nucleophile, one or two metal ions hold the water molecule in place, and charged amino acid side chains are ligands for the metal ions.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eTTC0492\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 209px;\"\u003e\n \u003cp\u003eCPBP family of intramembrane metalloproteinases, Abortive infection protein. (DP8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 428px;\"\u003e\n \u003cp\u003eCatalysis of the hydrolysis of internal, alpha-peptide bonds in a polypeptide chain by a mechanism in which water acts as a nucleophile, one or two metal ions hold the water molecule in place, and charged amino acid side chains are ligands for the metal ions.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eTTC0663\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 209px;\"\u003e\n \u003cp\u003eM66 family metalloproteinases. (DP9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 428px;\"\u003e\n \u003cp\u003eThis family of metallopeptidases contains StcE, a virulence factor found in Shiga toxigenic Escherichia coli organisms. StcE peptidase cleaves C1 esterase inhibitor [50].\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eTTC0687\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 209px;\"\u003e\n \u003cp\u003eClpP/crotonase-like domain superfamily. (DP10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 428px;\"\u003e\n \u003cp\u003eClpP is an ATP-dependent protease that cleaves a number of proteins, such as casein and albumin. It exists as a heterodimer of ATP-binding regulatory A and catalytic P subunits, both of which are required for effective levels of protease activity in the presence of ATP [51].\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eTTC0950\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 209px;\"\u003e\n \u003cp\u003eM50B family Zinc metalloprotease. (DP11)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 428px;\"\u003e\n \u003cp\u003eCatalysis of the hydrolysis of peptide bonds by a mechanism in which water acts as a nucleophile, one or two metal ions hold the water molecule in place, and charged amino acid side chains are ligands for the metal ions.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eTTC0974\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 209px;\"\u003e\n \u003cp\u003ePrcB C-terminal domain-containing protein. (DP12)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 428px;\"\u003e\n \u003cp\u003eThis domain is found at the C terminus of \u003cem\u003eTreponema denticola\u003c/em\u003e PrcB. PrcB interacts with the PrtP protease (dentilisin) and is required for the stability of the protease complex [52].\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eTTC1110\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 209px;\"\u003e\n \u003cp\u003eRhomboid family intramembrane serine protease. (DP13)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 428px;\"\u003e\n \u003cp\u003eCatalysis of the hydrolysis of internal, alpha-peptide bonds in a polypeptide chain by a catalytic mechanism that involves a catalytic triad consisting of a serine nucleophile that is activated by a proton relay involving an acidic residue (e.g. aspartate or glutamate) and a basic residue (usually histidine).\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eTTC1111\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 209px;\"\u003e\n \u003cp\u003ePeriplasmic serine protease. (DP14)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 428px;\"\u003e\n \u003cp\u003eCatalysis of the hydrolysis of a peptide bond. A peptide bond is a covalent bond formed when the carbon atom from the carboxyl group of one amino acid shares electrons with the nitrogen atom from the amino group of a second amino acid.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eTTC1128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 209px;\"\u003e\n \u003cp\u003eATP-dependent zinc metalloprotease FtsH, EC:3.4.24.- (DP15)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 428px;\"\u003e\n \u003cp\u003eHomologous to TTC0035.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eTTC1905\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 209px;\"\u003e\n \u003cp\u003eS1C family serine protease. (DP16)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 428px;\"\u003e\n \u003cp\u003eHomologous to TTC0417.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003eRelative extracellular protease activity for \u003cem\u003eT. thermophilus\u003c/em\u003e HB27 and deletion strains.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrain\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 194px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRelative Enzyme Activity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 209px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrain\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 192px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRelative Enzyme Activity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003eHB27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 194px;\"\u003e\n \u003cp\u003e2.57\u0026plusmn;0.50\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 209px;\"\u003e\n \u003cp\u003eHB27\u0026Delta;III-AB\u0026Delta;I-C\u0026Delta;\u003cem\u003eCRF3\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 192px;\"\u003e\n \u003cp\u003e2.5\u0026plusmn;0.87\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003eDP1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 194px;\"\u003e\n \u003cp\u003e2.19\u0026plusmn;0.29\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 209px;\"\u003e\n \u003cp\u003eDP2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 192px;\"\u003e\n \u003cp\u003e2.5\u0026plusmn;0.50\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003eDP3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 194px;\"\u003e\n \u003cp\u003e2.52\u0026plusmn;0.29\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 209px;\"\u003e\n \u003cp\u003eDP4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 192px;\"\u003e\n \u003cp\u003e1.64\u0026plusmn;0.50\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003eDP5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 194px;\"\u003e\n \u003cp\u003e1.95\u0026plusmn;1.26\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 209px;\"\u003e\n \u003cp\u003eDP6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 192px;\"\u003e\n \u003cp\u003e2.26\u0026plusmn;1.15\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003eDP7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 194px;\"\u003e\n \u003cp\u003e2.23\u0026plusmn;0.76\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 209px;\"\u003e\n \u003cp\u003eDP8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 192px;\"\u003e\n \u003cp\u003e2.23\u0026plusmn;0.29\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003eDP9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 194px;\"\u003e\n \u003cp\u003e2.47\u0026plusmn;0.76\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 209px;\"\u003e\n \u003cp\u003eDP10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 192px;\"\u003e\n \u003cp\u003e2.14\u0026plusmn;0.50\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003eDP11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 194px;\"\u003e\n \u003cp\u003e2.40\u0026plusmn;0.58\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 209px;\"\u003e\n \u003cp\u003eDP12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 192px;\"\u003e\n \u003cp\u003e2.43\u0026plusmn;1.32\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003eDP13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 194px;\"\u003e\n \u003cp\u003e2.31\u0026plusmn;0.58\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 209px;\"\u003e\n \u003cp\u003eDP14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 192px;\"\u003e\n \u003cp\u003e2.57\u0026plusmn;0.50\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003eDP15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 194px;\"\u003e\n \u003cp\u003e2.52\u0026plusmn;0.29\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 209px;\"\u003e\n \u003cp\u003eDP16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 192px;\"\u003e\n \u003cp\u003e1.61\u0026plusmn;0.58\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eBars represent \u0026plusmn;SD of three replicates. Different letters indicate significant differences according to Duncan\u0026rsquo;s multiple range tests at \u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u0026nbsp;\u003c/strong\u003eRelative extracellular protease activity for \u003cem\u003eT. thermophilus\u003c/em\u003e HB27 and multiple deletion strains.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrain\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRelative Enzyme\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eActivity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrain\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 187px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRelative Enzyme\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eActivity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003eHB27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e3.83\u0026plusmn;0.76\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003eHB27\u0026Delta;III-AB\u0026Delta;I-C\u0026Delta;\u003cem\u003eCRF3\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 187px;\"\u003e\n \u003cp\u003e4.83\u0026plusmn;1.04\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003eDSP1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e1.00\u0026plusmn;0.50\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003eDSP2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 187px;\"\u003e\n \u003cp\u003e1.50\u0026plusmn;0.50\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003eDSP3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e1.17\u0026plusmn;0.76\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003eDSP4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 187px;\"\u003e\n \u003cp\u003e4.33\u0026plusmn;0.76\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003eDSP5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e3.17\u0026plusmn;0.76\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003eDSP6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 187px;\"\u003e\n \u003cp\u003e4.33\u0026plusmn;0.29\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003eDSP7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e6.83\u0026plusmn;0.29\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003eDSP8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 187px;\"\u003e\n \u003cp\u003e3.00\u0026plusmn;0.50\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003eDSP9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e4.83\u0026plusmn;0.58\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 187px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eBars represent \u0026plusmn;SD of three replicates. Different letters indicate significant differences according to Duncan\u0026rsquo;s multiple range tests at \u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Thermus thermophilus HB27, Chassis Cell, Promoter Library, β-galactosidase Monitor System, pTT27-Plasmid, Protease Activity","lastPublishedDoi":"10.21203/rs.3.rs-6092828/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6092828/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003e\u003cem\u003eThermus thermophilus\u003c/em\u003e HB27 is a promising thermophilic chassis for recombinant thermostable protein production, owing to its high optimal growth temperature, which can simplify downstream processing and reduce contamination risks. However, maximizing its potential requires optimized genetic tools and host strains. Key limitations include a shortage of well-characterized strong constitutive promoters and potential degradation of recombinant proteins by proteases. To address these, we established a β-galactosidase reporter system (endogenous TTP0042) to screen for strong constitutive promoters and investigated the impact of deleting specific protease genes on protein expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eScreening of 13 endogenous promoter regions identified P0984 as exhibiting significantly 13-fold higher activity than the control promoter driving the reporter gene. Constructing a plasmid-free strain (HB27ΔpTT27) successfully minimized 270 kb of the genome; it exhibited auxotrophy for cobalamin (requiring 0.1 μg/ml AdoCbl for growth) and a slightly reduced growth rate compared to the wild-type, while its transformation efficiency remained comparable. Notably, a CRISPR-deficient precursor strain (HB27ΔIII-ABΔI-CΔ\u003cem\u003eCRF3\u003c/em\u003e) showed a significant (~100-fold) increase in transformation efficiency compared to the wild-type, facilitating subsequent genetic manipulations. Systematic knockout of 16 predicted non-essential protease loci was performed. Characterization revealed that deletion of TTC0264 (putative ClpY/HslU) and TTC1905 (putative HhoB) significantly reduced extracellular proteolytic activity. Iterative deletion based on phenotypic analysis led to strain DSP9 (10 protease loci deletions), which maintained robust growth and exhibited enhanced accumulation of the β-galactosidase reporter protein compared to the parental strains.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e This study provides foundational advancements for \u003cem\u003eT. thermophilus\u003c/em\u003eHB27 chassis development, and genetic tools represent valuable resources for optimizing \u003cem\u003eT. thermophilus\u003c/em\u003e as a platform for heterologous thermostable protein production and ideas for antibiotic-free systems.\u003c/p\u003e","manuscriptTitle":"Construction of Primary Chassis Cells with Efficient Protein Expression in Thermus thermophilus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-06 14:43:40","doi":"10.21203/rs.3.rs-6092828/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accepted","date":"2025-06-30T12:49:21+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"147937266841891872065344986136370903859","date":"2025-05-31T12:31:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"178670957358461459399826660747857736703","date":"2025-05-27T00:15:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-10T04:37:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"67331356900724613858982018792031414428","date":"2025-05-06T09:27:30+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-05T11:32:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-03T15:02:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microbial Cell Factories","date":"2025-04-29T09:01:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"07f44259-da2e-42fb-b93d-7549b0d37e59","owner":[],"postedDate":"May 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-14T16:10:31+00:00","versionOfRecord":{"articleIdentity":"rs-6092828","link":"https://doi.org/10.1186/s12934-025-02785-y","journal":{"identity":"microbial-cell-factories","isVorOnly":false,"title":"Microbial Cell Factories"},"publishedOn":"2025-07-10 15:57:25","publishedOnDateReadable":"July 10th, 2025"},"versionCreatedAt":"2025-05-06 14:43:40","video":"","vorDoi":"10.1186/s12934-025-02785-y","vorDoiUrl":"https://doi.org/10.1186/s12934-025-02785-y","workflowStages":[]},"version":"v1","identity":"rs-6092828","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6092828","identity":"rs-6092828","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}