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A facile and robust T7-promoter-based high-expression of heterologous proteins in Bacillus subtilis

A facile and robust T7-promoter-based high-expression of heterologous proteins in Bacillus subtilis Introduction 1989), Ralstonia eutropha (Barnard et  al. 2004), Rhodo- The bacteriophage T7-protomer protein expression sys - bacter capsulatus (Drepper et al. 2005), Streptomyces liv- tem is the most widely used technique of the production idans (Lussier et al. 2010), Shewanella oneidensis (Yi and of recombinant proteins because of its simple genetic Ng 2021), and so on. operation, high expression levels, and tightly regulated The Gram-positive bacterium B. subtilis is Generally expression of targeted genes (Terpe 2006; Ting et  al. Recognized As Safe (GRAS) microorganism due to its 2020). It was first developed in the Gram-negative bacte - lack of pathogenicity and absence of endotoxins as well rium Escherichia coli (Rosenberg et al. 1987). The T7–E. as its safe use as food and feed probiotics. It is one of coli expression system consists of two important parts: the most important industrial hosts for the production (1) an expression plasmid containing a gene of inter- of numerous proteins, especially homologous enzymes, est under the control of the T7 promoter and (2) a T7 such as α-amylase (Chen et  al. 2015), protease (Dong expression host, such as E. coli DE3, which has a chro- et  al. 2017; Wenzel et  al. 2011), xylanase (Helianti et  al. mosomal copy of the T7 RNA polymerase gene with 2016; Rashid and Sohail 2021), lipase (Lu et  al. 2010; the control of a lacUV5 promoter. The isopropyl-β-D- Wu et al. 2020), β-glucanase (Niu et al. 2018), and so on thiogalactopyranoside (IPTG)-induced T7 expression (Schallmey et  al. 2004; Terpe 2006). Also, a few hetero- can be regulated by co-expressing the lac repressor from geneous enzymes have been over-expressed by B. subtilis the plasmid and by co-expressing the T7 lysozyme, a nat- by using different promoters (Harwood et  al. 2002). For ural inhibitor of T7 RNA polymerase (Moffatt and Stud - example, the natural strictly regulated xylose-inducible ier 1987). Later, this system has been adapted to other promoter P in B. subtilis has been demonstrated to xylA organisms, such as Bacillus subtilis (Conrad et al. 1996), achieve modest expression levels (Bhavsar et  al. 2001; Bacillus megaterium (Gamer et  al. 2009), Lactococcus Kim et  al. 1996). Similarly, several natural inducible lactis (Wells et  al. 1993), Pseudomonas (Davison et  al. promoters of B. subtilis have been investigated, such as Table 1 The comparison of the protein expression systems of B. subtilis with different promoters Promoter Inducer Characteristics References P Xylose Strictly controlled by XylR repressor, and the protein expression was inhibited by (Bhavsar et al. 2001; Kim et al. 1996) xylA glucose P Maltose Positively regulated by the transcriptional regulator MalR, and was repressed by glucose (Yang et al. 2006; Yue et al. 2017) malA via CcpA and catabolite responsive elements P Subtilin The level of expression depended directly on the amount of inducer (subtilin) used, not (Bongers et al. 2005) spaS subject to catabolite control P Glycine The glycine tandem riboswitch was used to obtain regulatable expression of recombi‑ (Phan and Schumann 2007) gcv nant proteins IPTG Fused the 5ʹ‑sequence of a promoter from the B. subtilis phage SPO1 and the (Yansura and Henner 1984) spac 3ʹ‑sequences of the E. coli lac promoter including its operator region P IPTG Fused groES promoter and lacO operon and optimization of nucleotides at the con‑ (Phan et al. 2012, 2006; Tran et al. 2020) grac served regions of the groESL promoter Ye  et al. Bioresources and Bioprocessing (2022) 9:56 Page 3 of 12 P (Yang et  al. 2006; Yue et  al. 2017), P (Bongers as both the inducer and carbon source. However, this sys- glv spaS et  al. 2005), P (Phan and Schumann 2007), and so tem had two weaknesses, such as the genetic instability gcv on (Table  1). Furthermore, the heterologous P pro- because the comK gene was ON in the presence of xylose spac moter has been developed by fusing the 5ʹ-sequence because promoter P controlled its expression, and the xylA of a promoter from the B. subtilis phage SPO1 and the inducer was consumed continuously by the host. Another 3ʹ-sequences of the E. coli lac promoter including its strategy to address genetic instability was the insertion of operator region and this promoter was inducible by both the T7 RNA polymerase gene and the gene of inter- a factor of 50 in terms of 1–10  mM IPTG (Yansura est into the chromosome (Castillo-Hair et al. 2019; Chen and Henner 1984). The P promoter and its derived et  al. 2010). IPTG-inducible promoters (e.g., P or grac spac mutants based on the strong promoter of the groESL P ) were used to regulate the expression of T7 RNA hy-spank operon harboring the lac operator enabled the overex- polymerase and integrated the genes of interest into the pression of beta-galactosidase to achieve up to 53% of adjacent or distant site in the chromosome (Castillo-Hair the total cellular protein (Phan et  al. 2012, 2006; Tran et al. 2019; Chen et al. 2010). However, genetic modifica - et al. 2020). However, most of them did not have as high tion of the chromosome was time-consuming and suf- expression efficiencies as those of the T7–E. coli system fered from low biotransformation efficiency. (i.e., ~ 15–50%) and/or suffered from low transformation To develop a better T7-promoter expression system efficiency or time-consuming genetic operations. There - for B. subtilis, we need address several issues, such as fore, it is needed to develop a facile heterologous protein easy biotransformation of the host, facile preparation expression system in B. subtilis. of the expression plasmid, and a good expression host Several efforts have been conducted to adapt the whose major proteases were knocked out. In this study, T7-promoter expression system into B. subtilis. The ear - we developed a new B. subtilis host that featured (1) liest attempt was conducted by Conrad et  al. (Conrad an inducible comK gene (Zhang and Zhang 2011); (2) et  al. 1996). They designed an expression system com - the knock-out of some inherent protease genes, such posed of the T7 RNA polymerase under the control of as aprE and nprE (Kawamura and Doi 1984); (3) the xylose-inducible promoter P and the gene of inter- insertion of the constitutive expression of the T7 RNA xylA est under the control of the T7 promoter. In it, the T7 polymerase in the chromosome, and (4) the knock-out polymerase gene was inserted in the amyE site of the of the sporulation gene spoIIAC (Higgins and Dworkin chromosome, and the genes of interest (i.e., α-amylase, 2012) and surfactin synthase gene srfAC (Peypoux et al. β-1,4-glucosidase, and β-galactosidase) were inserted 1999) related to fermentation foam generation. The tar - into the respective plasmids. However, the heterolo- geted gene was placed in an episomal plasmid pHT01 gous enzymes were expressed only when an antibiotic under the control of a hybrid promoter T7-lac and its rifampicin was added to inhibit the host’s inherent RNA ribosome binding site sequence was derived from B. sub- polymerase. Recently, Sun and his coworkers further tilis. For any new targeted protein, the users can easily improved the T7 promoter system in an undomesticated prepare the plasmid by one-step genetic operation (i.e., B. subtilis strain ATCC6051a (Ji et al. 2021). The T7 RNA restriction enzyme-free and sequence-independent), polymerase gene under the control of the P promoter prolonged overlap extension polymerase chain reac- xylA was inserted in the aprE site chromosome for two pur- tion (POE-PCR) (You et  al. 2012) and easily transform poses: to introduce the T7 RNA polymerase expression the host with high transformation efficiencies. We cassette and to break the native protease gene of the host. tested heterologous expression of green fluorescent The expression frame of the target gene, which contains protein (GFP), α-glucan phosphorylase (αGP), inositol all the expression elements (i.e., T7 promoter, ribosome monophosphatase (IMP), phosphoglucomutase (PGM), binding site sequence (RBS), the gene of interest, termina- and 4-α-glucanotransferase (4GT) proteins in B. subti- tor) is highly similar to the pET21a vector, except that E. lis, and demonstrated its applicability in high-density coli RBS (i.e., AAGGA) was replaced with the B. subtilis fermentation. RBS sequence (i.e., AAG GAG G), and the whole expres- sion frame was located in E. coli–B. subtilis shuttle vector Materials and methods pMK4 (Ji et  al. 2021). To address the low transforma- Materials tion efficiency of B. subtilis, they inserted the comK gene All chemicals used were of analytical grade or higher responsible for the competence master regulator under quality and purchased from Sinopharm (Beijing, China), the control of the xylose promoter in the nprE site of the Aladdin (Shanghai, China), and Sigma-Aldrich (St. Louis, chromosome (Zhang and Zhang 2011). They expressed MO) unless specified. Taq DNA Polymerase was pur - approximately 1.0  g/L α-L-arabinofuranosidase in the LB chased from BioMed (Beijing, China), PrimeSTAR MAX media supplemented with 10  g/L xylose, which worked DNA Polymerase and Premixed Protein Marker (Low) Ye et al. Bioresources and Bioprocessing (2022) 9:56 Page 4 of 12 Table 2 Bacterial strains and plasmids Strain or plasmid Characteristics Source Bacillus subtilis SCK6 Erm , his nprR2 nprE18 △aprA3 △eglS102 △bglT bglSRV, lacA::P -comK Lab stock xylA SCK8 Erm , SCK6 derivate, △upp This work SCK9 Erm , SCK8 derivate, △upp△spoIIAC This work SCK10 Erm , SCK9 derivate, △upp△spoIIAC△srfAC This work SCK22 Erm , SCK10 derivate, △upp△spoIIAC△srfAC, amyE::P ‑ T7RNAP This work Plasmids R R pSS Amp, Cm , modular vector carrying upp‑ cassette Lab stock R R pHT01 Amp, Cm , E. coli-B. subtilis shuttle vector Lab stock pDG1730 Spc , integration vector contains a spectinomycin resistance gene sandwiched between amyE‑front and amyE‑back Lab stock pDG1730‑ T7RNAP Spc , pDG1730 derivate, used to integrate genes on the genome, with T7RNAP expression cassette (spc‑upp‑P ‑ This work T7RNAP) and amyE gene upstream and downstream homology arms R R pHT7 Amp, Cm , pHT01 derivate, with T7‑lac promoter This work R R pHT7‑ GFP Amp, Cm , pHT7 derivate, with GFP cloned This work R R pHT7‑αGP Amp, Cm , pHT7 derivate, with αGP cloned This work R R pHT7‑IMP Amp, Cm , pHT7 derivate, with IMP cloned This work R R pHT7‑PGM Amp, Cm , pHT7 derivate, with PGM cloned This work R R pHT7‑4GT Amp, Cm , pHT7 derivate, with 4GT cloned This work Table 3 Primers used in this work Primers Oligo sequences (5ʹ → 3ʹ) Primers used in construction of pHT7‑ GFP P1 gtttaactttaagaaaggaggatataccatgagtaaaggagaagaacttttc P2 gctttgttagcagccggatctcattatttgtatagttcatccatgccatg P3 catggcatggatgaactatacaaataatgagatccggctgctaacaaagc P4 gaaaagttcttctcctttactcaggtatatcctcctttcttaaagttaaac Primers used in construction of pHT7‑αGP P5 ctttaagaaaggaggatataccatggtgaacgtttccaatgccgttgaggatg P6 cgggctttgttagcagccggatcttagtcaagtcccttccacttgaccagac P7 catcctcaacggcattggaaacgttcaccatggtatatcctcctttcttaaag P8 gtctggtcaagtggaagggacttgactaagatccggctgctaacaaagcccg Primers used in construction of pHT7‑IMP P9 taactttaagaaaggaggatataccatgctggatcgcctggatttctctattaaactgctgcg P10 cgggctttgttagcagccggatctcatttaccgccgatttcttcaacaac P11 taactttaagaaaggaggatataccatgctggatcgcctggatttctctattaaactgctgcg P12 gttgttgaagaaatcggcggtaaatgagatccggctgctaacaaagcccg Primers used in construction of pHT7‑PGM P13 ctttaagaaaggaggatataccatggggaagctgtttggaacatttggag P14 cgggctttgttagcagccggatcttatgaaagcgctttctcaagtagctc P15 gagctacttgagaaagcgctttcataagatccggctgctaacaaagcccg P16 ctccaaatgttccaaacagcttccccatggtatatcctcctttcttaaag Primers used in construction of pHT7‑4GT P17 ctttaagaaaggaggatataccatggaacgtatcaacttcatcttcggtatcc P18 ggctttgttagcagccggatctcacagttcgcgaaaacgaacggtgaattcc P19 ggaattcaccgttcgttttcgcgaactgtgagatccggctgctaacaaagcc P20 ggataccgaagatgaagttgatacgttccatggtatatcctcctttcttaaag Ye  et al. Bioresources and Bioprocessing (2022) 9:56 Page 5 of 12 were purchased from Takara (Dalian, China). Plasmid cell death. Deletion of upp endows the mutant strain with extraction and PCR purification kit were purchased from resistance to 5-FU (Dong and Zhang 2014). Tiangen Biotech (Beijing, China). The knock-out of a gene in the chromosome was con - ducted by double-crossover homologous recombination Strains, plasmids, and cultivation conditions (Shi et  al. 2013). In brief, the upstream homology arm Strains and plasmids used are listed in Table  2. Plasmids of the target gene including the direct repeat DR region, were constructed using the Simple Cloning technol- the upp gene, the antibiotic gene, and the downstream ogy (You et  al. 2012). The primers (Table  3 and Addi- homology arm of the target gene including the DR region tional file  1: Table S1) used for PCR were synthesized by were sequentially connected to form large-size DNA GENEWIZ (Beijing, China). B. subtilis SCK6 which was multimers by prolonged overlap extension PCR and then derived from B. subtilis 1A751 contains a genetic cas- were transferred into B. subtilis. Primer sequences used sette expressing the comK gene in its genome (Zhang and for PCR are in the supplementary materials (Additional Zhang 2011). An E. coli–B. subtilis shuttle vector pHT01 file  1: Table S1).The first-round double- crossover homol - (Nguyen et  al. 2007) was used to clone and express the ogous recombination occurred with the upp gene and desired recombinant protein. The strains were cultured antibiotic gene integrated into the chromosome through in Luria–Bertani (LB) medium containing 0.5% yeast resistance plate screening. The correct transformants extract, 1% tryptone, and 1% NaCl at 37 °C. When neces- verified by PCR were cultured in the resistance-free sary, the medium was supplemented with 5  mg/L chlo- medium, and the second-round homologous recombina- ramphenicol or 0.3 mg/L erythromycin. tion occurred between the two DR regions. u Th s, the tar - get transformants, where the upp gene, resistance gene Construction of the B. subtilis SCK22 strain and the target gene were all deleted, could be obtained by Figure  1 shows the design of the T7 expression system using 5-FU plate screening. in B. subtilis. To construct a seamless knock-out system, For strains SCK8, SCK9 and SCK10, the upp, spoIIAC the upp gene was knocked out as a negative selection and srfAC genes were knocked out by using this seam- marker gene. The upp gene encodes uracil phosphoribo- less knock-out system, respectively. The pSS plasmid syltransferase (UPRTase), which can catalyze pyrimidine backbone, upstream homology arm of the target gene, analog 5-fluorouracil (5-FU) to 5-fluoro-dUMP, which is upp gene fragment, chloramphenicol resistance gene a strong inhibitor of thymidylate synthetase and leads to fragment, and downstream homology arm gene of the Fig. 1 Schematic representation of the industrial host SCK22 harboring plasmid pHT7. After xylose induction, under the control of xylose‑inducible promoter, comK gene was expressed and then super receptive cells were formed. Endogenous protein genes and fermentation‑related genes were knocked out on the genome to make it suitable for high‑ density fermentation. The P promoter enabled the constitutive expression of T7 RNA polymerase, and the expression plasmid encoded the target gene, where the T7 promoter was under the control of the lac operon Ye et al. Bioresources and Bioprocessing (2022) 9:56 Page 6 of 12 target gene were sequentially connected to obtain an sequence was subcloned into plasmid pHT01, yielding integration plasmid by prolonged overlap extension PCR plasmid pHT7. The gfp gene derived from Aequorea (Morimoto et al. 2008; Shi et al. 2013). Subsequently, the victoria was selected as the gene of interest and plasmid was transferred into B. subtilis and the target inserted after the RBS sequence of the pHT7 plasmid transformants were obtained by two rounds of homolo- (Fig. 2A and B). The insertion DNA fragment encoding gous recombination (Shi et al. 2013). green fluorescent protein (GFP) was amplified by PCR SCK22 was constructed based on the mutant strain with a pair of primers P1 and P2 (Table  3). The linear SCK10. The integration vector pDG1730 was used as it vector backbone was amplified from plasmid pHT7 contained a spectinomycin resistance gene sandwiched with a pair of primers P3 and P4. The two PCR prod - between amyE-front and amyE-back (Guerout-Fleury ucts were assembled by POE‐PCR. The POE‐ PCR prod- et  al. 1996). The pDG1730 plasmid backbone, upp gene uct was directly transformed into B. subtilis SCK22, fragment, the upstream homology arm DR region and yielding plasmid pHT7-GFP. The plasmid pHT7-GFP a DNA cassette encoding the T7 RNA polymerase with was used as the protein expression vector to verify the P promoter were sequentially connected to obtain and optimize the newly constructed T7 expression a new plasmid pDG1730-T7RNAP by using Simple Clon- system. The other four enzyme expression plasmids ing (You et  al. 2012). Then, the constructed integrated (i.e., α-glucan phosphorylase (αGP) from the thermo- plasmid pDG1730-T7RNAP was transformed into B. philic bacterium Thermococcus kodakarensis, inositol subtilis SCK10 to obtain SCK22. monophosphatase (IMP) from Thermotoga maritima, phosphoglucomutase (PGM) from T. kodakarensis, and Construction of the pHT7‑based expression vectors 4-α-glucanotransferase (4GT) from Thermococcus lito - The DNA fragment containing a T7-lac promoter, T7 ralis) were constructed in the same way (Fig.  2C). The terminator, and the B. subtilis RBS sequence (i.e., AAG gene sequences of these four enzymes were described GAG G) (Fig.  2A) was chemically synthesized. This elsewhere (You et al. 2017; Zhou et al. 2016). Fig. 2 Key DNA sequence before and after the gene of interest (A), plasmid map of pHT7‑ GFP (B) and scheme of plasmid multimerization (C). Plasmid pHT7‑ GFP contains the T7 promoter‑terminator cassette including a lacO lactose operator and a SD sequence, the replication origin ori in E. coli, the replication origin repA in B. subtilis, the lac repressor gene, chloramphenicol‑resistance gene and ampicillin‑resistance gene Ye  et al. Bioresources and Bioprocessing (2022) 9:56 Page 7 of 12 Transformation of the B. subtilis SCK22 strains according to the measured fluorescence intensity and The transformation of B. subtilis SCK22 was performed fluorescence curve (Additional file  1: Figure S1). Cell as described elsewhere (Zhang and Zhang 2011) with growth was monitored by measuring its absorbance at minor modifications. The B. subtilis SCKC22 strain was 600 nm. spread on solid LB medium containing 0.3  mg/L eryth- romycin and then cultured overnight in 37  °C. Single Fed‑batch fermentation colonies were picked from the plate and then inoculated The fermentation was carried out in a 5-L fermenter in 3  mL of the LB liquid medium containing 0.3  mg/L (T&J Bio-engineering Co., Shanghai, China). The fermen - erythromycin at 37  °C. The cell cultures were incubated tation medium consisted of the following components in a 250 rpm shaker for 4 h. The cultures were then trans - (per liter): 10 g of yeast extract, 0.2 g histidine, 20 g glyc- ferred to 50 mL of the LB medium and grew at 37 °C until erol, 5.12 g N a HPO ·12H O, 3.0 g K H PO , 0.5 g NaCl, 2 4 2 2 4 the absorbency at 600  nm was approximately 0.6–0.8. 0.5 g MgSO ·7H O, 0.011  g C aCl , 1.0  g N H Cl, 0.2  mL 4 2 2 4 Xylose (final concentration of 10 g/L) was added and cul - of 1% (w/v) vitamin B1, and 0.1 mL of the trace elements tured for 2  h. The resulting cell cultures were ready to solution. The stock solution of trace elements contained be transformed as super-competent cells or divided into the following (per liter) in 3  M HCl: 80  g FeSO ·7H O, 4 2 aliquots and stocked at −  80  °C with 10% (v/v) glycerol 10 g AlCl ·6H O, 2.0  g Z nSO ·7HO, 1.0  g C uCl ·2H O, 3 2 4 2 2 for future use (thawed for direct transformation). Then 2.0 g NaMoO ·2H O, 10 g MnS O ·H O, 4.0 g CoC l , and 4 2 4 2 2 1  μL of POE-PCR product was mixed with 100  μL of 0.5 g H BO . Appropriate antibiotics and defoamer were 3 4 the super-competent cells, and then was incubated in a added if necessary. rotary shaking incubator at 200  rpm for 1.5  h at 37  °C. The cryopreserved strains were inoculated into 50  mL Spread the transformed competent cells on solid LB plate of the LB medium containing 1% glucose and then cul- with the appropriate antibiotic and incubate the plate at tured at 37  °C for 12  h with vigorous shaking. Then the 37 °C for 8–12 h to select transformants. entire cell cultures were transferred into the fermenter. Dissolved oxygen (DO) was monitored using a DO sen- Heterologous protein expression sor and was maintained above 20% saturation by control- The SCK22 strains containing plasmids pHT7-GFP, ling both the aeration rate (2–18 L/min) and the agitation pHT7-αGP, pHT7-IMP, pHT7-PGM or pHT7-4GT were rate (200–1000  rpm). Foaming was controlled by the cultured in small culture tubes overnight and then inocu- addition of the Sigma anti-foaming agent. After about lated into 1-L shake flasks containing 200  mL of the LB 8 h cultivation, the DO shown a suddenly increased, indi- liquid medium at 37 °C. The inoculum size was adjusted cating the complete consumption of carbon source. The to allow the cell culture to have an absorbency of about feeding solution (i.e., 50% (g/g) glycerol, 5% (g/g) yeast 0.05 at 600  nm. When A was reached 0.8–1.0, IPTG extract, and 0.5% (g/g) histidine) was added slowly. The was added, followed by 4  h of cell cultivation. After fer- addition rate of the feeding solution was adjusted to be mentation, the broth was centrifuged. The cell pellets approximately 6–10  g/L/h according the growth rates were washed with the saline water once. The cell pellets of bacteria. The fermentation was performed at pH 6.8 were re-suspended in 50 mM HEPES buffer (pH 7.0) con - and 37  °C, whereas the pH was adjusted with 25% (v/v) taining 100 mM NaCl. After ultra-sonication and centrif- ammonia. The samples were collected at the indicated ugation, the supernatants containing all soluble proteins time intervals. including the target protein were analyzed by SDS-PAGE according to the standard procedure. The gels were Protein analysis by SDS‑PAGE stained by Coomassie brilliant blue R250 staining. Cell culture samples were harvested and centrifuged at 12,000×g for 5  min. The pellets were re-suspended in Fluorescence measurement and quantification of GFP 50 mM HEPES buffer (pH 7.0) containing 100 mM NaCl. Cell cultures of SCK22/pHT7-GFP were centrifuged at After ultra-sonication in an ice bath, cell debris were 12,000×g for 5  min to obtain bacterial cells and super- removed by centrifugation at 12,000×g for 5  min. After natants. After the bacterial cells were washed with adding the SDS-PAGE loading buffer, the cell lysates the saline water once, they were re-suspended in the and the supernatants of the cell lysates were incubated 50 mM HEPES buffer (pH 7.0) containing 100 mM NaCl in a boiling water bath for 10 min and equal amounts of prior to disruption by ultra-sonication on an ice bath. proteins were loaded into 12% SDS-PAGE gels. The Pre - The fluorescence intensities of the supernatants of cell mixed Protein Marker (Low covering the 14.3 to 97.2 kDa lysates and fermentation broth represented the intracel- range) (Takara Bio Inc., China) was used as a molecu- lular and extracellular GFP concentrations, respectively. lar mass marker. Following electrophoresis, proteins The expression level of GFP protein was calculated were visualized by Coomassie Brilliant Blue R250. The Ye et al. Bioresources and Bioprocessing (2022) 9:56 Page 8 of 12 SDS-PAGE results were imaged and analyzed by Bio-Rad Gel Doc XR + Imaging System. Other assays The concentrations of the proteins were determined by the Bradford with bovine serum albumin as the reference. All data were averaged from three independent samples. Results Construction of the T7 expression system in B. subtilis Figure  1 shows the design of the Bacillus T7 expression system. Similar to the E. coli T7 system, it had two parts: a plasmid encoding the gene of interest which was under control of the T7 promoter, and a Bacillus host whose chromosome had a T7 RNA polymerase gene under the control of a constitutive P promoter. First, because B. subtilis has much lower transformation efficiency than E. coli and it was time-consuming to prepare compe- tent cells, the DNA cassette containing the comK gene Fig. 3 Profile of fermentation of B. subtilis SCK22/pHT7‑ GFP (A) and under the control of the inducible P promoter was xylA SDS‑PAGE analysis of GFP over time (B) inserted into its chromosome, wherein the ComK of B. subtilis was the master regulator for competence devel- opment (Mironczuk et al. 2008; Zhang and Zhang 2011). The induced super-competent cells of B. subtilis exhib - ited transformation efficiencies of ~ 10 transformants per μg of multimeric plasmid DNA (Zhang and Zhang 2011). Second, similar to the ompT- and ion-deficient E. coli BL21, two Bacillus protease genes (i.e., aprE and nprE) were knocked out from the chromosome so that the host was suitable for the expression of recombinant protein. Third, to avoid sporulation during its fermenta - tion, the spoIIAC gene (RNA polymerase sigma-F factor) was knocked out (Zhang et al. 2016). Fourth, the surfac- tin synthase gene srfAC (surfactin synthase subunit 3) was also knocked out because its expression could form broth foam, impairing high-density fermentation (Zhang et  al. 2016). Last, the T7 RNA polymerase gene with its P promoter was inserted into the amyE gene of the Fig. 4 Eec ff ts of the inducer IPTG concentration on GFP expression chromosome, yielding the T7 expression host B. subtilis level and total GFP concentration SCK22. Expression plasmid pHT7 (Fig.  2) was constructed to contain a T7-lac hybrid promoter, B. subtilis RBS sequence (i.e., AAG GAG G), the gene of interest (e.g., operon followed by the synthesis of a large amount of the green fluorescent protein, gfp ) and the T7 terminator, targeted protein. based on pHT01 plasmid. The partial DNA sequence of plasmid pHT7 before and after the gene of interest is pre- sented in Fig.  2A. In the absence of the inducer IPTG, a Flask fermentation and optimization repressor protein (LacI) that repressed T7-lac promoter The strain SCK22 harboring plasmid pHT7-GFP was transcription prevented the target gene from being syn- cultured in 1-L a fl sks, wherein the amount of green fluo - thesized. When IPTG was added, it would bind to LacI rescent proteins could be quantitated by measuring the and release the tetrameric repressor from the lac opera- fluorescence intensity of GFP. The profile of the fermen - tor, thereby allowing the transcription of the T7-lac tation of strain SCK22/pHT7-GFP is shown in Fig.  3A. Ye  et al. Bioresources and Bioprocessing (2022) 9:56 Page 9 of 12 Fig. 5 SDS‑PAGE analysis of targeted protein expression in B. subtilis SCK22. Lanes: M the standard protein markers, T the total cell lysate, S the supernatant of the total cell lysate, αGP α‑ glucan phosphorylase, IMP inositol monophosphatase, PGM phosphoglucomutase, 4GT 4‑α‑ glucanotransferase We also tested the optimal IPTG concentration from 0 to 2.0  mM (Fig.  4). The maximum intracellular GFP concentration (0.146  g/L) was obtained when IPTG was 1.0 mM. The GFP content was 22.4% relative to the total cellular protein. Synthesis of four other heterologous proteins We tested the applicability of this expression to four other proteins. They were αGP from T. kodakarensis , IMP from T. maritima, PGM from T. kodakarensis, and 4GT from T. litoralis. These thermophilic enzymes were used to synthesize inositol from starch in  vitro (You et  al. 2017; Fig. 6 Profile of high‑ cell‑ density fermentation of B. subtilis SCK22/ Zhou et al. 2016). Their expression was initiated by add - pHT7‑IMP in a 5‑L fermenter ing 1.0 mM IPTG when A reached approximately 0.8– 1.0. SDS-PAGE (Fig.  5) shows that the expression levels of αGP, IMP, PGM, and 4GT were 31.7%, 26.3%, 24.3%, The absorbency of cells at A rose to 4.1 at the 7th hour and 40.3%, respectively. There were no inclusion bodies and then declined slowly. When the A was about 1.0, found for all cases (Fig.  5), which was confirmed by the 0.5  mM IPTG was added. GFP was synthesized after measuring the difference of protein concentrations in the IPTG addition and its concentration kept increasing to cell lysate and its supernatants. These results suggested 0.16  g/L. After 4-h induction, nearly all GFP was intra- that this Bacillus T7 expression system can be used to cellular and the GFP content relative to its total cellular express numerous heterologous proteins efficiently. protein was approximately 21%. Because some fraction of cells began to lyse after reaching the highest cell density, Fed‑batch high‑cell‑density fermentation approximately a third GFP was released to the broth at the To investigate whether the newly constructed T7 expres- end of fermentation. SDS-PAGE analysis also shows the sion system is suitable for high-density fermentation, B. increased GFP expression over time (Fig.  3B). The intra - subtilis SCK22/pHT7-IMP was tested in fed-batch fer- cellular GFP content gradually increased to 19.6% after mentation. As shown in Fig.  6, after 8  h fermentation, IPTG addition until it reached the maximum after 4 h. the feeding solution was added. With the feed addition, Ye et al. Bioresources and Bioprocessing (2022) 9:56 Page 10 of 12 the cell concentration continued to increase (A up to fermentation was constructed by double-crossover 129.6). When 1.0 mM IPTG was added at 10 h, the IMP homologous recombination, and the plasmids were was synthesized. After 30 h fermentation, the intracellu- constructed easily by simple cloning. The intracellu - lar IMP expression level reached a peak, accounting for lar expression level of heterologous proteins reached 27.2% of the total intracellular protein, and the IMP titer the highest level of 25% ~ 40% at 4  h after 1.0  mM IPTG was 4.78  g/L. Afterwards, the intracellular expression induction. The yield of IMP reached 4.78 g/L in high-den - level of IMP declined due to cell lysis. SDS-PAGE analysis sity fermentation. In summary, the Bacillus T7 expression was also conducted to obtain of the relative percentage of system has the advantages of simple genetic operation, intracellular IMP to the total cellular protein (Additional stable expression of heterologous proteins, wide applica- file  1: Figure S2). These results showed that this Bacillus bility, and suitable for high-density fermentation. T7 expression system was also suitable for high-density fermentation. Abbreviations RBS: Ribosome binding site; SDS: Sodium dodecyl sulfate; PAGE: Poly‑ acrylamide gel electrophoresis; IPTG: Isopropyl‑β‑D ‑thiogalactopyranoside; GRAS: Generally recognized as safe; POE‑PCR: Prolonged overlap extension Discussion polymerase chain reaction; GFP: Green fluorescent protein; αGP: α‑ Glucan In this study, we developed a simple Bacillus T7 protein phosphorylase; IMP: Inositol monophosphatase; PGM: Phosphoglucomutase; 4GT: 4‑α‑ Glucanotransferase; LB: Luria–Bertani; UPRTase: Uracil phosphoribo‑ expression system. This system contained a B. subtilis syltransferase; 5‑FU: 5‑Fluorouracil; DO: Dissolved oxygen. SCK22 host recombinant strain and a plasmid pHT7. The host was deficient in two major protease genes (i.e., Supplementary Information aprE and nprE), a sporulation gene and (spoIIAC), and a The online version contains supplementary material available at https:// doi. surfactin synthase genes srfAC. Two genes were inserted org/ 10. 1186/ s40643‑ 022‑ 00540‑4. its chromosome: the xylose-inducible comK gene for high transformation and the constitutive T7 RNA polymerase Additional file1: Figure S1. The standard curve of GFP protein concen‑ tration and its fluorescence intensity. Figure S2. SDS‑PAGE analysis of IMP gene. With an available B. subtilis SCK22, it was easy and of B. subtilis SCK22/pHT7‑IMP in a 5‑L fermenter over time. Lanes: M, pro ‑ fast to construct the expression plasmid by using POE- tein markers; T, the cell lysate; S, the supernatant of the cell lysate. Figure PCR and transform into the host with high transforma - S3. SDS‑PAGE analysis of expression of 4‑α‑ glucanotransferase (4GT ) from Thermococcus litoralis in E.coli BL21(DE3). Lanes: M, protein markers; T, the tion efficiency. Five heterologous proteins were expressed cell lysate; S, the supernatant of the cell lysate. Table S1. Primers for gene efficiently in this system. As compared to other Bacillus knockout. T7-derived expression systems (Castillo-Hair et al. 2019; Chen et  al. 2010; Conrad et  al. 1996; Ji et  al. 2021), this Acknowledgements system featured its wide applicability, easy genetic opera- Not applicable. tion, high expression capacity in both flask and fed-batch Author contributions fermentation, and tightly controlled synthesis of the tar- JY, YJL, YQB, TZ and WJ designed experiments, performed experiments, and get protein. analyzed data; YHZ, TS and ZJW conceived the idea and supervised the research. JY, YJL and YHZ wrote and revised the manuscript. All authors read It was notable that this Bacillus T7 expression synthesis and approved the final manuscript. could be superior to the E. coli counterpart for some pro- teins. It found out that at least a half of recombinant 4GT Funding This work was supported by Tianjin Synthetic Biotechnology Innovation synthesized was inclusion bodies when it was expressed Capacity Improvement Project ( TSBICIP‑KJGG‑003). in E. coli (Additional file  1: Figure S3) although its expres- sion conditions were intensively optimized, for example, Availability of data and materials The datasets supporting this article are included in the manuscript. decreased protein synthesis temperature, various IPTG concentration, codon optimization, co-expression of Declarations multiple chaperones (Duan et al. 2019). In contrast, there was not a significant amount of inclusion body observed Ethics approval and consent to participate when it was expressed in Bacillus. The reasons behind Not applicable. the better protein synthesis and folding in the Bacillus T7 Consent for publication expression could be under further investigation. All of the authors have read and approved to submit it to Bioresources and Bioprocessing. Competing interests Conclusion The authors declare that they have no competing interests. In conclusion, to develop a better T7-promoter expres- Received: 20 February 2022 Accepted: 27 April 2022 sion system for B. subtilis, the strain SCK22 with high transformation efficiency and suitable for high-density Ye  et al. 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A facile and robust T7-promoter-based high-expression of heterologous proteins in Bacillus subtilis

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Abstract

Introduction 1989), Ralstonia eutropha (Barnard et  al. 2004), Rhodo- The bacteriophage T7-protomer protein expression sys - bacter capsulatus (Drepper et al. 2005), Streptomyces liv- tem is the most widely used technique of the production idans (Lussier et al. 2010), Shewanella oneidensis (Yi and of recombinant proteins because of its simple genetic Ng 2021), and so on. operation, high expression levels, and tightly regulated The Gram-positive bacterium B. subtilis is Generally expression of targeted genes (Terpe 2006; Ting et  al. Recognized As Safe (GRAS) microorganism due to its 2020). It was first developed in the Gram-negative bacte - lack of pathogenicity and absence of endotoxins as well rium Escherichia coli (Rosenberg et al. 1987). The T7–E. as its safe use as food and feed probiotics. It is one of coli expression system consists of two important parts: the most important industrial hosts for the production (1) an expression plasmid containing a gene of inter- of numerous proteins, especially homologous enzymes, est under the control of the T7 promoter and (2) a T7 such as α-amylase (Chen et  al. 2015), protease (Dong expression host, such as E. coli DE3, which has a chro- et  al. 2017; Wenzel et  al. 2011), xylanase (Helianti et  al. mosomal copy of the T7 RNA polymerase gene with 2016; Rashid and Sohail 2021), lipase (Lu et  al. 2010; the control of a lacUV5 promoter. The isopropyl-β-D- Wu et al. 2020), β-glucanase (Niu et al. 2018), and so on thiogalactopyranoside (IPTG)-induced T7 expression (Schallmey et  al. 2004; Terpe 2006). Also, a few hetero- can be regulated by co-expressing the lac repressor from geneous enzymes have been over-expressed by B. subtilis the plasmid and by co-expressing the T7 lysozyme, a nat- by using different promoters (Harwood et  al. 2002). For ural inhibitor of T7 RNA polymerase (Moffatt and Stud - example, the natural strictly regulated xylose-inducible ier 1987). Later, this system has been adapted to other promoter P in B. subtilis has been demonstrated to xylA organisms, such as Bacillus subtilis (Conrad et al. 1996), achieve modest expression levels (Bhavsar et  al. 2001; Bacillus megaterium (Gamer et  al. 2009), Lactococcus Kim et  al. 1996). Similarly, several natural inducible lactis (Wells et  al. 1993), Pseudomonas (Davison et  al. promoters of B. subtilis have been investigated, such as Table 1 The comparison of the protein expression systems of B. subtilis with different promoters Promoter Inducer Characteristics References P Xylose Strictly controlled by XylR repressor, and the protein expression was inhibited by (Bhavsar et al. 2001; Kim et al. 1996) xylA glucose P Maltose Positively regulated by the transcriptional regulator MalR, and was repressed by glucose (Yang et al. 2006; Yue et al. 2017) malA via CcpA and catabolite responsive elements P Subtilin The level of expression depended directly on the amount of inducer (subtilin) used, not (Bongers et al. 2005) spaS subject to catabolite control P Glycine The glycine tandem riboswitch was used to obtain regulatable expression of recombi‑ (Phan and Schumann 2007) gcv nant proteins IPTG Fused the 5ʹ‑sequence of a promoter from the B. subtilis phage SPO1 and the (Yansura and Henner 1984) spac 3ʹ‑sequences of the E. coli lac promoter including its operator region P IPTG Fused groES promoter and lacO operon and optimization of nucleotides at the con‑ (Phan et al. 2012, 2006; Tran et al. 2020) grac served regions of the groESL promoter Ye  et al. Bioresources and Bioprocessing (2022) 9:56 Page 3 of 12 P (Yang et  al. 2006; Yue et  al. 2017), P (Bongers as both the inducer and carbon source. However, this sys- glv spaS et  al. 2005), P (Phan and Schumann 2007), and so tem had two weaknesses, such as the genetic instability gcv on (Table  1). Furthermore, the heterologous P pro- because the comK gene was ON in the presence of xylose spac moter has been developed by fusing the 5ʹ-sequence because promoter P controlled its expression, and the xylA of a promoter from the B. subtilis phage SPO1 and the inducer was consumed continuously by the host. Another 3ʹ-sequences of the E. coli lac promoter including its strategy to address genetic instability was the insertion of operator region and this promoter was inducible by both the T7 RNA polymerase gene and the gene of inter- a factor of 50 in terms of 1–10  mM IPTG (Yansura est into the chromosome (Castillo-Hair et al. 2019; Chen and Henner 1984). The P promoter and its derived et  al. 2010). IPTG-inducible promoters (e.g., P or grac spac mutants based on the strong promoter of the groESL P ) were used to regulate the expression of T7 RNA hy-spank operon harboring the lac operator enabled the overex- polymerase and integrated the genes of interest into the pression of beta-galactosidase to achieve up to 53% of adjacent or distant site in the chromosome (Castillo-Hair the total cellular protein (Phan et  al. 2012, 2006; Tran et al. 2019; Chen et al. 2010). However, genetic modifica - et al. 2020). However, most of them did not have as high tion of the chromosome was time-consuming and suf- expression efficiencies as those of the T7–E. coli system fered from low biotransformation efficiency. (i.e., ~ 15–50%) and/or suffered from low transformation To develop a better T7-promoter expression system efficiency or time-consuming genetic operations. There - for B. subtilis, we need address several issues, such as fore, it is needed to develop a facile heterologous protein easy biotransformation of the host, facile preparation expression system in B. subtilis. of the expression plasmid, and a good expression host Several efforts have been conducted to adapt the whose major proteases were knocked out. In this study, T7-promoter expression system into B. subtilis. The ear - we developed a new B. subtilis host that featured (1) liest attempt was conducted by Conrad et  al. (Conrad an inducible comK gene (Zhang and Zhang 2011); (2) et  al. 1996). They designed an expression system com - the knock-out of some inherent protease genes, such posed of the T7 RNA polymerase under the control of as aprE and nprE (Kawamura and Doi 1984); (3) the xylose-inducible promoter P and the gene of inter- insertion of the constitutive expression of the T7 RNA xylA est under the control of the T7 promoter. In it, the T7 polymerase in the chromosome, and (4) the knock-out polymerase gene was inserted in the amyE site of the of the sporulation gene spoIIAC (Higgins and Dworkin chromosome, and the genes of interest (i.e., α-amylase, 2012) and surfactin synthase gene srfAC (Peypoux et al. β-1,4-glucosidase, and β-galactosidase) were inserted 1999) related to fermentation foam generation. The tar - into the respective plasmids. However, the heterolo- geted gene was placed in an episomal plasmid pHT01 gous enzymes were expressed only when an antibiotic under the control of a hybrid promoter T7-lac and its rifampicin was added to inhibit the host’s inherent RNA ribosome binding site sequence was derived from B. sub- polymerase. Recently, Sun and his coworkers further tilis. For any new targeted protein, the users can easily improved the T7 promoter system in an undomesticated prepare the plasmid by one-step genetic operation (i.e., B. subtilis strain ATCC6051a (Ji et al. 2021). The T7 RNA restriction enzyme-free and sequence-independent), polymerase gene under the control of the P promoter prolonged overlap extension polymerase chain reac- xylA was inserted in the aprE site chromosome for two pur- tion (POE-PCR) (You et  al. 2012) and easily transform poses: to introduce the T7 RNA polymerase expression the host with high transformation efficiencies. We cassette and to break the native protease gene of the host. tested heterologous expression of green fluorescent The expression frame of the target gene, which contains protein (GFP), α-glucan phosphorylase (αGP), inositol all the expression elements (i.e., T7 promoter, ribosome monophosphatase (IMP), phosphoglucomutase (PGM), binding site sequence (RBS), the gene of interest, termina- and 4-α-glucanotransferase (4GT) proteins in B. subti- tor) is highly similar to the pET21a vector, except that E. lis, and demonstrated its applicability in high-density coli RBS (i.e., AAGGA) was replaced with the B. subtilis fermentation. RBS sequence (i.e., AAG GAG G), and the whole expres- sion frame was located in E. coli–B. subtilis shuttle vector Materials and methods pMK4 (Ji et  al. 2021). To address the low transforma- Materials tion efficiency of B. subtilis, they inserted the comK gene All chemicals used were of analytical grade or higher responsible for the competence master regulator under quality and purchased from Sinopharm (Beijing, China), the control of the xylose promoter in the nprE site of the Aladdin (Shanghai, China), and Sigma-Aldrich (St. Louis, chromosome (Zhang and Zhang 2011). They expressed MO) unless specified. Taq DNA Polymerase was pur - approximately 1.0  g/L α-L-arabinofuranosidase in the LB chased from BioMed (Beijing, China), PrimeSTAR MAX media supplemented with 10  g/L xylose, which worked DNA Polymerase and Premixed Protein Marker (Low) Ye et al. Bioresources and Bioprocessing (2022) 9:56 Page 4 of 12 Table 2 Bacterial strains and plasmids Strain or plasmid Characteristics Source Bacillus subtilis SCK6 Erm , his nprR2 nprE18 △aprA3 △eglS102 △bglT bglSRV, lacA::P -comK Lab stock xylA SCK8 Erm , SCK6 derivate, △upp This work SCK9 Erm , SCK8 derivate, △upp△spoIIAC This work SCK10 Erm , SCK9 derivate, △upp△spoIIAC△srfAC This work SCK22 Erm , SCK10 derivate, △upp△spoIIAC△srfAC, amyE::P ‑ T7RNAP This work Plasmids R R pSS Amp, Cm , modular vector carrying upp‑ cassette Lab stock R R pHT01 Amp, Cm , E. coli-B. subtilis shuttle vector Lab stock pDG1730 Spc , integration vector contains a spectinomycin resistance gene sandwiched between amyE‑front and amyE‑back Lab stock pDG1730‑ T7RNAP Spc , pDG1730 derivate, used to integrate genes on the genome, with T7RNAP expression cassette (spc‑upp‑P ‑ This work T7RNAP) and amyE gene upstream and downstream homology arms R R pHT7 Amp, Cm , pHT01 derivate, with T7‑lac promoter This work R R pHT7‑ GFP Amp, Cm , pHT7 derivate, with GFP cloned This work R R pHT7‑αGP Amp, Cm , pHT7 derivate, with αGP cloned This work R R pHT7‑IMP Amp, Cm , pHT7 derivate, with IMP cloned This work R R pHT7‑PGM Amp, Cm , pHT7 derivate, with PGM cloned This work R R pHT7‑4GT Amp, Cm , pHT7 derivate, with 4GT cloned This work Table 3 Primers used in this work Primers Oligo sequences (5ʹ → 3ʹ) Primers used in construction of pHT7‑ GFP P1 gtttaactttaagaaaggaggatataccatgagtaaaggagaagaacttttc P2 gctttgttagcagccggatctcattatttgtatagttcatccatgccatg P3 catggcatggatgaactatacaaataatgagatccggctgctaacaaagc P4 gaaaagttcttctcctttactcaggtatatcctcctttcttaaagttaaac Primers used in construction of pHT7‑αGP P5 ctttaagaaaggaggatataccatggtgaacgtttccaatgccgttgaggatg P6 cgggctttgttagcagccggatcttagtcaagtcccttccacttgaccagac P7 catcctcaacggcattggaaacgttcaccatggtatatcctcctttcttaaag P8 gtctggtcaagtggaagggacttgactaagatccggctgctaacaaagcccg Primers used in construction of pHT7‑IMP P9 taactttaagaaaggaggatataccatgctggatcgcctggatttctctattaaactgctgcg P10 cgggctttgttagcagccggatctcatttaccgccgatttcttcaacaac P11 taactttaagaaaggaggatataccatgctggatcgcctggatttctctattaaactgctgcg P12 gttgttgaagaaatcggcggtaaatgagatccggctgctaacaaagcccg Primers used in construction of pHT7‑PGM P13 ctttaagaaaggaggatataccatggggaagctgtttggaacatttggag P14 cgggctttgttagcagccggatcttatgaaagcgctttctcaagtagctc P15 gagctacttgagaaagcgctttcataagatccggctgctaacaaagcccg P16 ctccaaatgttccaaacagcttccccatggtatatcctcctttcttaaag Primers used in construction of pHT7‑4GT P17 ctttaagaaaggaggatataccatggaacgtatcaacttcatcttcggtatcc P18 ggctttgttagcagccggatctcacagttcgcgaaaacgaacggtgaattcc P19 ggaattcaccgttcgttttcgcgaactgtgagatccggctgctaacaaagcc P20 ggataccgaagatgaagttgatacgttccatggtatatcctcctttcttaaag Ye  et al. Bioresources and Bioprocessing (2022) 9:56 Page 5 of 12 were purchased from Takara (Dalian, China). Plasmid cell death. Deletion of upp endows the mutant strain with extraction and PCR purification kit were purchased from resistance to 5-FU (Dong and Zhang 2014). Tiangen Biotech (Beijing, China). The knock-out of a gene in the chromosome was con - ducted by double-crossover homologous recombination Strains, plasmids, and cultivation conditions (Shi et  al. 2013). In brief, the upstream homology arm Strains and plasmids used are listed in Table  2. Plasmids of the target gene including the direct repeat DR region, were constructed using the Simple Cloning technol- the upp gene, the antibiotic gene, and the downstream ogy (You et  al. 2012). The primers (Table  3 and Addi- homology arm of the target gene including the DR region tional file  1: Table S1) used for PCR were synthesized by were sequentially connected to form large-size DNA GENEWIZ (Beijing, China). B. subtilis SCK6 which was multimers by prolonged overlap extension PCR and then derived from B. subtilis 1A751 contains a genetic cas- were transferred into B. subtilis. Primer sequences used sette expressing the comK gene in its genome (Zhang and for PCR are in the supplementary materials (Additional Zhang 2011). An E. coli–B. subtilis shuttle vector pHT01 file  1: Table S1).The first-round double- crossover homol - (Nguyen et  al. 2007) was used to clone and express the ogous recombination occurred with the upp gene and desired recombinant protein. The strains were cultured antibiotic gene integrated into the chromosome through in Luria–Bertani (LB) medium containing 0.5% yeast resistance plate screening. The correct transformants extract, 1% tryptone, and 1% NaCl at 37 °C. When neces- verified by PCR were cultured in the resistance-free sary, the medium was supplemented with 5  mg/L chlo- medium, and the second-round homologous recombina- ramphenicol or 0.3 mg/L erythromycin. tion occurred between the two DR regions. u Th s, the tar - get transformants, where the upp gene, resistance gene Construction of the B. subtilis SCK22 strain and the target gene were all deleted, could be obtained by Figure  1 shows the design of the T7 expression system using 5-FU plate screening. in B. subtilis. To construct a seamless knock-out system, For strains SCK8, SCK9 and SCK10, the upp, spoIIAC the upp gene was knocked out as a negative selection and srfAC genes were knocked out by using this seam- marker gene. The upp gene encodes uracil phosphoribo- less knock-out system, respectively. The pSS plasmid syltransferase (UPRTase), which can catalyze pyrimidine backbone, upstream homology arm of the target gene, analog 5-fluorouracil (5-FU) to 5-fluoro-dUMP, which is upp gene fragment, chloramphenicol resistance gene a strong inhibitor of thymidylate synthetase and leads to fragment, and downstream homology arm gene of the Fig. 1 Schematic representation of the industrial host SCK22 harboring plasmid pHT7. After xylose induction, under the control of xylose‑inducible promoter, comK gene was expressed and then super receptive cells were formed. Endogenous protein genes and fermentation‑related genes were knocked out on the genome to make it suitable for high‑ density fermentation. The P promoter enabled the constitutive expression of T7 RNA polymerase, and the expression plasmid encoded the target gene, where the T7 promoter was under the control of the lac operon Ye et al. Bioresources and Bioprocessing (2022) 9:56 Page 6 of 12 target gene were sequentially connected to obtain an sequence was subcloned into plasmid pHT01, yielding integration plasmid by prolonged overlap extension PCR plasmid pHT7. The gfp gene derived from Aequorea (Morimoto et al. 2008; Shi et al. 2013). Subsequently, the victoria was selected as the gene of interest and plasmid was transferred into B. subtilis and the target inserted after the RBS sequence of the pHT7 plasmid transformants were obtained by two rounds of homolo- (Fig. 2A and B). The insertion DNA fragment encoding gous recombination (Shi et al. 2013). green fluorescent protein (GFP) was amplified by PCR SCK22 was constructed based on the mutant strain with a pair of primers P1 and P2 (Table  3). The linear SCK10. The integration vector pDG1730 was used as it vector backbone was amplified from plasmid pHT7 contained a spectinomycin resistance gene sandwiched with a pair of primers P3 and P4. The two PCR prod - between amyE-front and amyE-back (Guerout-Fleury ucts were assembled by POE‐PCR. The POE‐ PCR prod- et  al. 1996). The pDG1730 plasmid backbone, upp gene uct was directly transformed into B. subtilis SCK22, fragment, the upstream homology arm DR region and yielding plasmid pHT7-GFP. The plasmid pHT7-GFP a DNA cassette encoding the T7 RNA polymerase with was used as the protein expression vector to verify the P promoter were sequentially connected to obtain and optimize the newly constructed T7 expression a new plasmid pDG1730-T7RNAP by using Simple Clon- system. The other four enzyme expression plasmids ing (You et  al. 2012). Then, the constructed integrated (i.e., α-glucan phosphorylase (αGP) from the thermo- plasmid pDG1730-T7RNAP was transformed into B. philic bacterium Thermococcus kodakarensis, inositol subtilis SCK10 to obtain SCK22. monophosphatase (IMP) from Thermotoga maritima, phosphoglucomutase (PGM) from T. kodakarensis, and Construction of the pHT7‑based expression vectors 4-α-glucanotransferase (4GT) from Thermococcus lito - The DNA fragment containing a T7-lac promoter, T7 ralis) were constructed in the same way (Fig.  2C). The terminator, and the B. subtilis RBS sequence (i.e., AAG gene sequences of these four enzymes were described GAG G) (Fig.  2A) was chemically synthesized. This elsewhere (You et al. 2017; Zhou et al. 2016). Fig. 2 Key DNA sequence before and after the gene of interest (A), plasmid map of pHT7‑ GFP (B) and scheme of plasmid multimerization (C). Plasmid pHT7‑ GFP contains the T7 promoter‑terminator cassette including a lacO lactose operator and a SD sequence, the replication origin ori in E. coli, the replication origin repA in B. subtilis, the lac repressor gene, chloramphenicol‑resistance gene and ampicillin‑resistance gene Ye  et al. Bioresources and Bioprocessing (2022) 9:56 Page 7 of 12 Transformation of the B. subtilis SCK22 strains according to the measured fluorescence intensity and The transformation of B. subtilis SCK22 was performed fluorescence curve (Additional file  1: Figure S1). Cell as described elsewhere (Zhang and Zhang 2011) with growth was monitored by measuring its absorbance at minor modifications. The B. subtilis SCKC22 strain was 600 nm. spread on solid LB medium containing 0.3  mg/L eryth- romycin and then cultured overnight in 37  °C. Single Fed‑batch fermentation colonies were picked from the plate and then inoculated The fermentation was carried out in a 5-L fermenter in 3  mL of the LB liquid medium containing 0.3  mg/L (T&J Bio-engineering Co., Shanghai, China). The fermen - erythromycin at 37  °C. The cell cultures were incubated tation medium consisted of the following components in a 250 rpm shaker for 4 h. The cultures were then trans - (per liter): 10 g of yeast extract, 0.2 g histidine, 20 g glyc- ferred to 50 mL of the LB medium and grew at 37 °C until erol, 5.12 g N a HPO ·12H O, 3.0 g K H PO , 0.5 g NaCl, 2 4 2 2 4 the absorbency at 600  nm was approximately 0.6–0.8. 0.5 g MgSO ·7H O, 0.011  g C aCl , 1.0  g N H Cl, 0.2  mL 4 2 2 4 Xylose (final concentration of 10 g/L) was added and cul - of 1% (w/v) vitamin B1, and 0.1 mL of the trace elements tured for 2  h. The resulting cell cultures were ready to solution. The stock solution of trace elements contained be transformed as super-competent cells or divided into the following (per liter) in 3  M HCl: 80  g FeSO ·7H O, 4 2 aliquots and stocked at −  80  °C with 10% (v/v) glycerol 10 g AlCl ·6H O, 2.0  g Z nSO ·7HO, 1.0  g C uCl ·2H O, 3 2 4 2 2 for future use (thawed for direct transformation). Then 2.0 g NaMoO ·2H O, 10 g MnS O ·H O, 4.0 g CoC l , and 4 2 4 2 2 1  μL of POE-PCR product was mixed with 100  μL of 0.5 g H BO . Appropriate antibiotics and defoamer were 3 4 the super-competent cells, and then was incubated in a added if necessary. rotary shaking incubator at 200  rpm for 1.5  h at 37  °C. The cryopreserved strains were inoculated into 50  mL Spread the transformed competent cells on solid LB plate of the LB medium containing 1% glucose and then cul- with the appropriate antibiotic and incubate the plate at tured at 37  °C for 12  h with vigorous shaking. Then the 37 °C for 8–12 h to select transformants. entire cell cultures were transferred into the fermenter. Dissolved oxygen (DO) was monitored using a DO sen- Heterologous protein expression sor and was maintained above 20% saturation by control- The SCK22 strains containing plasmids pHT7-GFP, ling both the aeration rate (2–18 L/min) and the agitation pHT7-αGP, pHT7-IMP, pHT7-PGM or pHT7-4GT were rate (200–1000  rpm). Foaming was controlled by the cultured in small culture tubes overnight and then inocu- addition of the Sigma anti-foaming agent. After about lated into 1-L shake flasks containing 200  mL of the LB 8 h cultivation, the DO shown a suddenly increased, indi- liquid medium at 37 °C. The inoculum size was adjusted cating the complete consumption of carbon source. The to allow the cell culture to have an absorbency of about feeding solution (i.e., 50% (g/g) glycerol, 5% (g/g) yeast 0.05 at 600  nm. When A was reached 0.8–1.0, IPTG extract, and 0.5% (g/g) histidine) was added slowly. The was added, followed by 4  h of cell cultivation. After fer- addition rate of the feeding solution was adjusted to be mentation, the broth was centrifuged. The cell pellets approximately 6–10  g/L/h according the growth rates were washed with the saline water once. The cell pellets of bacteria. The fermentation was performed at pH 6.8 were re-suspended in 50 mM HEPES buffer (pH 7.0) con - and 37  °C, whereas the pH was adjusted with 25% (v/v) taining 100 mM NaCl. After ultra-sonication and centrif- ammonia. The samples were collected at the indicated ugation, the supernatants containing all soluble proteins time intervals. including the target protein were analyzed by SDS-PAGE according to the standard procedure. The gels were Protein analysis by SDS‑PAGE stained by Coomassie brilliant blue R250 staining. Cell culture samples were harvested and centrifuged at 12,000×g for 5  min. The pellets were re-suspended in Fluorescence measurement and quantification of GFP 50 mM HEPES buffer (pH 7.0) containing 100 mM NaCl. Cell cultures of SCK22/pHT7-GFP were centrifuged at After ultra-sonication in an ice bath, cell debris were 12,000×g for 5  min to obtain bacterial cells and super- removed by centrifugation at 12,000×g for 5  min. After natants. After the bacterial cells were washed with adding the SDS-PAGE loading buffer, the cell lysates the saline water once, they were re-suspended in the and the supernatants of the cell lysates were incubated 50 mM HEPES buffer (pH 7.0) containing 100 mM NaCl in a boiling water bath for 10 min and equal amounts of prior to disruption by ultra-sonication on an ice bath. proteins were loaded into 12% SDS-PAGE gels. The Pre - The fluorescence intensities of the supernatants of cell mixed Protein Marker (Low covering the 14.3 to 97.2 kDa lysates and fermentation broth represented the intracel- range) (Takara Bio Inc., China) was used as a molecu- lular and extracellular GFP concentrations, respectively. lar mass marker. Following electrophoresis, proteins The expression level of GFP protein was calculated were visualized by Coomassie Brilliant Blue R250. The Ye et al. Bioresources and Bioprocessing (2022) 9:56 Page 8 of 12 SDS-PAGE results were imaged and analyzed by Bio-Rad Gel Doc XR + Imaging System. Other assays The concentrations of the proteins were determined by the Bradford with bovine serum albumin as the reference. All data were averaged from three independent samples. Results Construction of the T7 expression system in B. subtilis Figure  1 shows the design of the Bacillus T7 expression system. Similar to the E. coli T7 system, it had two parts: a plasmid encoding the gene of interest which was under control of the T7 promoter, and a Bacillus host whose chromosome had a T7 RNA polymerase gene under the control of a constitutive P promoter. First, because B. subtilis has much lower transformation efficiency than E. coli and it was time-consuming to prepare compe- tent cells, the DNA cassette containing the comK gene Fig. 3 Profile of fermentation of B. subtilis SCK22/pHT7‑ GFP (A) and under the control of the inducible P promoter was xylA SDS‑PAGE analysis of GFP over time (B) inserted into its chromosome, wherein the ComK of B. subtilis was the master regulator for competence devel- opment (Mironczuk et al. 2008; Zhang and Zhang 2011). The induced super-competent cells of B. subtilis exhib - ited transformation efficiencies of ~ 10 transformants per μg of multimeric plasmid DNA (Zhang and Zhang 2011). Second, similar to the ompT- and ion-deficient E. coli BL21, two Bacillus protease genes (i.e., aprE and nprE) were knocked out from the chromosome so that the host was suitable for the expression of recombinant protein. Third, to avoid sporulation during its fermenta - tion, the spoIIAC gene (RNA polymerase sigma-F factor) was knocked out (Zhang et al. 2016). Fourth, the surfac- tin synthase gene srfAC (surfactin synthase subunit 3) was also knocked out because its expression could form broth foam, impairing high-density fermentation (Zhang et  al. 2016). Last, the T7 RNA polymerase gene with its P promoter was inserted into the amyE gene of the Fig. 4 Eec ff ts of the inducer IPTG concentration on GFP expression chromosome, yielding the T7 expression host B. subtilis level and total GFP concentration SCK22. Expression plasmid pHT7 (Fig.  2) was constructed to contain a T7-lac hybrid promoter, B. subtilis RBS sequence (i.e., AAG GAG G), the gene of interest (e.g., operon followed by the synthesis of a large amount of the green fluorescent protein, gfp ) and the T7 terminator, targeted protein. based on pHT01 plasmid. The partial DNA sequence of plasmid pHT7 before and after the gene of interest is pre- sented in Fig.  2A. In the absence of the inducer IPTG, a Flask fermentation and optimization repressor protein (LacI) that repressed T7-lac promoter The strain SCK22 harboring plasmid pHT7-GFP was transcription prevented the target gene from being syn- cultured in 1-L a fl sks, wherein the amount of green fluo - thesized. When IPTG was added, it would bind to LacI rescent proteins could be quantitated by measuring the and release the tetrameric repressor from the lac opera- fluorescence intensity of GFP. The profile of the fermen - tor, thereby allowing the transcription of the T7-lac tation of strain SCK22/pHT7-GFP is shown in Fig.  3A. Ye  et al. Bioresources and Bioprocessing (2022) 9:56 Page 9 of 12 Fig. 5 SDS‑PAGE analysis of targeted protein expression in B. subtilis SCK22. Lanes: M the standard protein markers, T the total cell lysate, S the supernatant of the total cell lysate, αGP α‑ glucan phosphorylase, IMP inositol monophosphatase, PGM phosphoglucomutase, 4GT 4‑α‑ glucanotransferase We also tested the optimal IPTG concentration from 0 to 2.0  mM (Fig.  4). The maximum intracellular GFP concentration (0.146  g/L) was obtained when IPTG was 1.0 mM. The GFP content was 22.4% relative to the total cellular protein. Synthesis of four other heterologous proteins We tested the applicability of this expression to four other proteins. They were αGP from T. kodakarensis , IMP from T. maritima, PGM from T. kodakarensis, and 4GT from T. litoralis. These thermophilic enzymes were used to synthesize inositol from starch in  vitro (You et  al. 2017; Fig. 6 Profile of high‑ cell‑ density fermentation of B. subtilis SCK22/ Zhou et al. 2016). Their expression was initiated by add - pHT7‑IMP in a 5‑L fermenter ing 1.0 mM IPTG when A reached approximately 0.8– 1.0. SDS-PAGE (Fig.  5) shows that the expression levels of αGP, IMP, PGM, and 4GT were 31.7%, 26.3%, 24.3%, The absorbency of cells at A rose to 4.1 at the 7th hour and 40.3%, respectively. There were no inclusion bodies and then declined slowly. When the A was about 1.0, found for all cases (Fig.  5), which was confirmed by the 0.5  mM IPTG was added. GFP was synthesized after measuring the difference of protein concentrations in the IPTG addition and its concentration kept increasing to cell lysate and its supernatants. These results suggested 0.16  g/L. After 4-h induction, nearly all GFP was intra- that this Bacillus T7 expression system can be used to cellular and the GFP content relative to its total cellular express numerous heterologous proteins efficiently. protein was approximately 21%. Because some fraction of cells began to lyse after reaching the highest cell density, Fed‑batch high‑cell‑density fermentation approximately a third GFP was released to the broth at the To investigate whether the newly constructed T7 expres- end of fermentation. SDS-PAGE analysis also shows the sion system is suitable for high-density fermentation, B. increased GFP expression over time (Fig.  3B). The intra - subtilis SCK22/pHT7-IMP was tested in fed-batch fer- cellular GFP content gradually increased to 19.6% after mentation. As shown in Fig.  6, after 8  h fermentation, IPTG addition until it reached the maximum after 4 h. the feeding solution was added. With the feed addition, Ye et al. Bioresources and Bioprocessing (2022) 9:56 Page 10 of 12 the cell concentration continued to increase (A up to fermentation was constructed by double-crossover 129.6). When 1.0 mM IPTG was added at 10 h, the IMP homologous recombination, and the plasmids were was synthesized. After 30 h fermentation, the intracellu- constructed easily by simple cloning. The intracellu - lar IMP expression level reached a peak, accounting for lar expression level of heterologous proteins reached 27.2% of the total intracellular protein, and the IMP titer the highest level of 25% ~ 40% at 4  h after 1.0  mM IPTG was 4.78  g/L. Afterwards, the intracellular expression induction. The yield of IMP reached 4.78 g/L in high-den - level of IMP declined due to cell lysis. SDS-PAGE analysis sity fermentation. In summary, the Bacillus T7 expression was also conducted to obtain of the relative percentage of system has the advantages of simple genetic operation, intracellular IMP to the total cellular protein (Additional stable expression of heterologous proteins, wide applica- file  1: Figure S2). These results showed that this Bacillus bility, and suitable for high-density fermentation. T7 expression system was also suitable for high-density fermentation. Abbreviations RBS: Ribosome binding site; SDS: Sodium dodecyl sulfate; PAGE: Poly‑ acrylamide gel electrophoresis; IPTG: Isopropyl‑β‑D ‑thiogalactopyranoside; GRAS: Generally recognized as safe; POE‑PCR: Prolonged overlap extension Discussion polymerase chain reaction; GFP: Green fluorescent protein; αGP: α‑ Glucan In this study, we developed a simple Bacillus T7 protein phosphorylase; IMP: Inositol monophosphatase; PGM: Phosphoglucomutase; 4GT: 4‑α‑ Glucanotransferase; LB: Luria–Bertani; UPRTase: Uracil phosphoribo‑ expression system. This system contained a B. subtilis syltransferase; 5‑FU: 5‑Fluorouracil; DO: Dissolved oxygen. SCK22 host recombinant strain and a plasmid pHT7. The host was deficient in two major protease genes (i.e., Supplementary Information aprE and nprE), a sporulation gene and (spoIIAC), and a The online version contains supplementary material available at https:// doi. surfactin synthase genes srfAC. Two genes were inserted org/ 10. 1186/ s40643‑ 022‑ 00540‑4. its chromosome: the xylose-inducible comK gene for high transformation and the constitutive T7 RNA polymerase Additional file1: Figure S1. The standard curve of GFP protein concen‑ tration and its fluorescence intensity. Figure S2. SDS‑PAGE analysis of IMP gene. With an available B. subtilis SCK22, it was easy and of B. subtilis SCK22/pHT7‑IMP in a 5‑L fermenter over time. Lanes: M, pro ‑ fast to construct the expression plasmid by using POE- tein markers; T, the cell lysate; S, the supernatant of the cell lysate. Figure PCR and transform into the host with high transforma - S3. SDS‑PAGE analysis of expression of 4‑α‑ glucanotransferase (4GT ) from Thermococcus litoralis in E.coli BL21(DE3). Lanes: M, protein markers; T, the tion efficiency. Five heterologous proteins were expressed cell lysate; S, the supernatant of the cell lysate. Table S1. Primers for gene efficiently in this system. As compared to other Bacillus knockout. T7-derived expression systems (Castillo-Hair et al. 2019; Chen et  al. 2010; Conrad et  al. 1996; Ji et  al. 2021), this Acknowledgements system featured its wide applicability, easy genetic opera- Not applicable. tion, high expression capacity in both flask and fed-batch Author contributions fermentation, and tightly controlled synthesis of the tar- JY, YJL, YQB, TZ and WJ designed experiments, performed experiments, and get protein. analyzed data; YHZ, TS and ZJW conceived the idea and supervised the research. JY, YJL and YHZ wrote and revised the manuscript. All authors read It was notable that this Bacillus T7 expression synthesis and approved the final manuscript. could be superior to the E. coli counterpart for some pro- teins. It found out that at least a half of recombinant 4GT Funding This work was supported by Tianjin Synthetic Biotechnology Innovation synthesized was inclusion bodies when it was expressed Capacity Improvement Project ( TSBICIP‑KJGG‑003). in E. coli (Additional file  1: Figure S3) although its expres- sion conditions were intensively optimized, for example, Availability of data and materials The datasets supporting this article are included in the manuscript. decreased protein synthesis temperature, various IPTG concentration, codon optimization, co-expression of Declarations multiple chaperones (Duan et al. 2019). In contrast, there was not a significant amount of inclusion body observed Ethics approval and consent to participate when it was expressed in Bacillus. The reasons behind Not applicable. the better protein synthesis and folding in the Bacillus T7 Consent for publication expression could be under further investigation. All of the authors have read and approved to submit it to Bioresources and Bioprocessing. Competing interests Conclusion The authors declare that they have no competing interests. In conclusion, to develop a better T7-promoter expres- Received: 20 February 2022 Accepted: 27 April 2022 sion system for B. subtilis, the strain SCK22 with high transformation efficiency and suitable for high-density Ye  et al. 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Bioresources and BioprocessingSpringer Journals

Published: May 18, 2022

Keywords: Bacillus subtilis; T7 expression system; Recombinant protein expression; High cell-density fermentation

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