P43 Promoter Research Articles

Development of a novel strategy for robust synthetic bacterial promoters based on a stepwise evolution targeting the spacer region of the core promoter in Bacillus subtilis

BackgroundPromoter evolution by synthetic promoter library (SPL) is a powerful approach to development of functional synthetic promoters to synthetic biology. However, it requires much tedious and time-consuming screenings because of the plethora of different variants in SPL. Actually, a large proportion of mutants in the SPL are significantly lower in strength, which contributes only to fabrication of a promoter library with a continuum of strength. Thus, to effectively obtain the evolved synthetic promoter exhibiting higher strength, it is essential to develop novel strategies to construct mutant library targeting the pivotal region rather than the arbitrary region of the template promoter. In this study, a strategy termed stepwise evolution targeting the spacer of core promoter (SETarSCoP) was established in Bacillus subtilis to effectively evolve the strength of bacterial promoter.ResultsThe native promoter, PsrfA, from B. subtilis, which exhibits higher strength than the strong promoter P43, was set as the parental template. According to the comparison of conservation of the spacer sequences between − 35 box and − 10 box among a set of strong and weak native promoter, it revealed that 7-bp sequence immediately upstream of the − 10 box featured in the regulation of promoter strength. Based on the conservative feature, two rounds of consecutive evolution were performed targeting the hot region of PsrfA. In the first round, a primary promoter mutation library (pPML) was constructed by mutagenesis targeting the 3-bp sequence immediately upstream of the − 10 box of the PsrfA. Subsequently, four evolved mutants from pPML were selected to construction of four secondary promoter mutation libraries (sPMLs) based on mutagenesis of the 4-bp sequence upstream of the first-round target. After the consecutive two-step evolution, the mutant PBH4 was identified and verified to be a highly evolved synthetic promoter. The strength of PBH4 was higher than PsrfA by approximately 3 times. Moreover, PBH4 also exhibited broad suitability for different cargo proteins, such as β-glucuronidase and nattokinase. The proof-of-principle test showed that SETarSCoP successfully evolved both constitutive and inducible promoters.ConclusionComparing with the commonly used SPL strategy, SETarSCoP facilitates the evolution process to obtain strength-evolved synthetic bacterial promoter through fabrication and screening of small-scale mutation libraries. This strategy will be a promising method to evolve diverse bacterial promoters to expand the toolbox for synthetic biology.

Improvement of stress tolerance and riboflavin production of Bacillus subtilis by introduction of heat shock proteins from thermophilic bacillus strains.

In this study, stress tolerance devices consisting of heat shock protein (HSP) genes from thermophiles Geobacillus and Parageobacillus were introduced into riboflavin-producing strain Bacillus subtilis 446 to improve its stress tolerance and riboflavin production. The 12 HSP homologs were selected from 28 Geobacillus and Parageobacillus genomes according to their sequence clustering and phylogenetically analysis which represents the diversity of HSPs from thermophilic bacillus. The 12 HSP genes and 2 combinations of them (PtdnaK-PtdnaJ-PtgrpE and PtgroeL-PtgroeS) were heterologously expressed in B. subtilis 446 under the control of a strong constitutive promoter P43. Most of the 14 engineered strains showed increased cell density at 44 to 48°C and less cell death at 50°C compared with the control strains. Among them, strains B.s446-HSP20-3, B.s446-HSP20-2, and B.s446-PtDnaK-PtDnaJ-PtGrpE increased their cell densities over 25% at 44 to 48°C. They also showed 5-, 4-, and 4-fold improved cell survivals after the 10-h heat shock treatment at 50°C, respectively. These three strains also showed reduced cell death rates under osmotic stress of 10% NaCl, indicating that the introduction of HSPs improved not only the heat tolerance of B. subtilis 446 but also its osmotic tolerance. Fermentation of these three strains at higher temperatures of 39 and 43°C showed 23-66% improved riboflavin titers, as well as 24-h shortened fermentation period. These results indicated that implanting HSPs from thermophiles to B. subtilis 446 would be an efficient approach to improve its stress tolerance and riboflavin production.

Construction, Model-Based Analysis, and Characterization of a Promoter Library for Fine-Tuned Gene Expression in Bacillus subtilis.

Promoters are among the most-important and most-basic tools for the control of metabolic pathways. However, previous research mainly focused on the screening and characterization of some native promoters in Bacillus subtilis. To develop a broadly applicable promoter system for this important platform organism, we created a de novo synthetic promoter library (SPL) based on consensus sequences by analyzing the microarray transcriptome data of B. subtilis 168. A total of 214 potential promoters spanning a gradient of strengths was isolated and characterized by a green fluorescence assay. Among these, a detailed intensity analysis was conducted on nine promoters with different strengths by reverse-transcription polymerase chain reaction (RT-PCR)and sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). Furthermore, reconstructed promoters and promoter cassettes (tandem promoter cluster) were designed via statistical model-based analysis and tandem dual promoters, which showed strength that was increased 1.2- and 2.77-fold compared to that of promoter P43, respectively. Finally, the SPL was employed in the production of inosine and acetoin by repressing and over-expressing the relevant metabolic pathways, yielding a 700% and 44% increase relative to the respective control strains. This is the first report of a de novo synthetic promoter library for B. subtilis, which is independent of any native promoter. The strategy of improving and fine-tuning promoter strengths will contribute to future metabolic engineering and synthetic biology projects in B. subtilis.

One-Step Bioprocess of Inulin to Product Inulo-Oligosaccharides Using Bacillus subtilis Secreting an Extracellular Endo-Inulinase.

Inulo-oligosaccharides (IOSs), a novel food additive and health product, represent a promising alternative to antibiotics. As prebiotics, IOSs can be obtained from inulin by endo-inulinase-mediated hydrolysis. Nonetheless, enzymatic catalysis is not feasible industrially because of the required catalytic conditions and cost. In this study, a 2331-bp optimized gene inuQ (from Pseudomonas mucidolens) encoding endo-inulinase was cloned into shuttle vector PHY300PLK and transfected into Bacillus subtilis WB800-R, with the simultaneous deletion of gene sacC encoding levanase. The maximal IOS yield after hydrolysis of the crude extract of inulin was 67.84 ± 0.72g/L for a recombinant strain with the signal peptide nprB from alkaline protease and promoter P43. The conversion rate reached 75.38%. For the major IOSs, the degree of polymerization was between 3 and 5. This study offers a simple and efficient one-step bioprocess for IOS production from inulin through secretion of an extracellular heterologous endo-inulinase by B. subtilis.

High-level extracellular protein expression in Bacillus subtilis by optimizing strong promoters based on the transcriptome of Bacillus subtilis and Bacillus megaterium

Bacillus subtilis is widely used for the large-scale industrial production of proteins. In this study, the transcriptomes of B. subtilis 168 and B. megaterium DSM319 cells grown in stationary phase were analyzed to expand the repertoire of highly-active promoters for high-level protein expression based on the transcriptomes of these Bacillus strains. 24 genes with the highest expression levels among 2048 highly expressed gene families were chosen to examine promoter activity. The activities of four promoters with the beta-galactosidase (bgaB) gene as a reporter were stronger than those of the well-characterized strong promoter P43. The expression level of recombinant Pro-transglutaminase (pro-MTG) from Streptomyces mobaraensis achieved 87.6 U/mL and 70.7 U/mL under the control of two constitutive promoter PsodA and PydzA, respectively, compared to the promoter P43. Our study provides a basis for further studies on the Bacillus transcriptome by identifying strong promoters for industrial uses.

Development of an Efficient Genome Editing Tool in Bacillus licheniformis Using CRISPR-Cas9 Nickase.

Bacillus strains are important industrial bacteria that can produce various biochemical products. However, low transformation efficiencies and a lack of effective genome editing tools have hindered its widespread application. Recently, clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 techniques have been utilized in many organisms as genome editing tools because of their high efficiency and easy manipulation. In this study, an efficient genome editing method was developed for Bacillus licheniformis using a CRISPR-Cas9 nickase integrated into the genome of B. licheniformis DW2 with overexpression driven by the P43 promoter. The yvmC gene was deleted using the CRISPR-Cas9n technique with homology arms of 1.0 kb as a representative example, and an efficiency of 100% was achieved. In addition, two genes were simultaneously disrupted with an efficiency of 11.6%, and the large DNA fragment bacABC (42.7 kb) was deleted with an efficiency of 79.0%. Furthermore, the heterologous reporter gene aprN, which codes for nattokinase in Bacillus subtilis, was inserted into the chromosome of B. licheniformis with an efficiency of 76.5%. The activity of nattokinase in the DWc9nΔ7/pP43SNT-SsacC strain reached 59.7 fibrinolytic units (FU)/ml, which was 25.7% higher than that of DWc9n/pP43SNT-SsacC Finally, the engineered strain DWc9nΔ7 (Δepr ΔwprA Δmpr ΔaprE Δvpr ΔbprA ΔbacABC), with multiple disrupted genes, was constructed using the CRISPR-Cas9n technique. Taken together, we have developed an efficient genome editing tool based on CRISPR-Cas9n in B. licheniformis This tool could be applied to strain improvement for future research.IMPORTANCE As important industrial bacteria, Bacillus strains have attracted significant attention due to their production of biological products. However, genetic manipulation of these bacteria is difficult. The CRISPR-Cas9 system has been applied to genome editing in some bacteria, and CRISPR-Cas9n was proven to be an efficient and precise tool in previous reports. The significance of our research is the development of an efficient, more precise, and systematic genome editing method for single-gene deletion, multiple-gene disruption, large DNA fragment deletion, and single-gene integration in Bacillus licheniformis via Cas9 nickase. We also applied this method to the genetic engineering of the host strain for protein expression.

Combinatorial promoter engineering of glucokinase and phosphoglucoisomerase for improved N-acetylglucosamine production in Bacillus subtilis

In previous work, a recombinant Bacillus subtilis strain was successfully constructed for microbial production of N-acetylglucosamine (GlcNAc). In this study, GlcNAc titer was further improved by combinatorial promoter engineering of key genes glck encoding glucokinase and pgi encoding phosphoglucoisomerase. First, three promoters including constitutive promoter P43, xylose inducible promoter PxylA, and isopropyl-β-d-thiogalactoside inducible Pgrac were used to replace the native promoters of glcK and pgi, yielding 12 recombinant strains. It was found that when glcK and pgi were both under the control of promoter PxylA, the highest GlcNAc titer in 3-L fed-batch bioreactor reached 35.12g/L, which was 52.6% higher than that of the initial host. Next, the transcriptional levels of the related genes in glycolysis, GlcNAc synthesis pathway, peptidoglycan synthesis pathway, and pentose phosphate pathway were investigated by quantitative real-time PCR analysis. Fine-tuning upper GlcNAc synthesis pathway by combinatorial promoter substitution significantly enhanced GlcNAc production in engineered B. subtilis.

Overproduction of Rummeliibacillus pycnus arginase with multi-copy insertion of the arg R.pyc cassette into the Bacillus subtilis chromosome.

A plasmid-less and marker-less strain with multi-copy integration of the arginase gene from Rummeliibacillus pycnus was constructed using Bacillus subtilis 168 as a host. A total of nine copies of the arg R.pyc cassettes, in which the R. pycnus arginase gene was fused with the strong promoter P43, were inserted into the recipient chromosome. These multiple insertions were completed via step-by-step integrations into designed (2 copies) and random (9 copies) sites, respectively. A strategy for random site integration was developed based on the construction of the arg R.pyc cassette sandwiched between "front" and "back" homologous arms which were randomly restricted from chromosomal DNA. An antibiotic resistance marker was applied in transformant selection and was eliminated via the Cre/lox system. Performance showed that the highest enzyme activity (14.5U/mL) was obtained after culture in flasks, and this segregation stable strain could efficiently hydrolyze L-arginine with a 97.2% molar yield, showing potential application in the food industry.

Identification of strong promoters based on the transcriptome of Bacillus licheniformis.

To expand the repertoire of strong promoters for high level expression of proteins based on the transcriptome of Bacillus licheniformis. The transcriptome of B. licheniformis ATCC14580 grown to the early stationary phase was analyzed and the top 10 highly expressed genes/operons out of the 3959 genes and 1249 operons identified were chosen for study promoter activity. Using beta-galactosidase gene as a reporter, the candidate promoter pBL9 exhibited the strongest activity which was comparable to that of the widely used strong promoter p43. Furthermore, the pro-transglutaminase from Streptomyces mobaraensis (pro-MTG) was expressed under the control of promoter pBL9 and the activity of pro-MTG reached 82 U/ml after 36h, which is 23% higher than that of promoter p43 (66.8 U/ml). In our analyses of the transcriptome of B. licheniformis, we have identified a strong promoter pBL9, which could be adapted for high level expression of proteins in the host Bacillus subtilis.

Characterization and application of endogenous phase-dependent promoters in Bacillus subtilis

Bacillus subtilis as an important host has been widely used in synthetic biology, metabolic engineering, and production of industrial enzymes. To fully take advantage of this organism, 114 endogenous putative promoters were measured with a green fluorescent protein reporter and four classes of phase-dependent promoters (class I: exponential phase; class II: middle-log and early stationary phases; class III: lag-log and stationary phases; class IV: stationary phase) with different strengths were identified. The transcriptional strengths ranged from 0.03 to 2.03-fold of that of the commonly used strong promoter P43. On this basis, the temperature (for instance P bltD , P ydaD , and P gerBC ) and pH (such as P abrB , P ydjO , and P opuE ) inducible phase-dependent promoters were further identified and characterized. In comparison, P abrB (class I), P spoVG (class II), and P lytR (class III) achieved the highest expression levels of esterase, keratinase, and alkaline polygalacturonate lyase, respectively. The constructed phase-dependent promoter library should have great application potentials for enzyme production, metabolic engineering, and synthetic biology.

Engineering an inducible gene expression system for Bacillus subtilis from a strong constitutive promoter and a theophylline-activated synthetic riboswitch.

BackgroundSynthetic riboswitches have been increasingly used to control and tune gene expression in diverse organisms. Although a set of theophylline-responsive riboswitches have been developed for bacteria, fully functional expression elements mediated by synthetic riboswitches in Bacillus subtilis are rarely used because of the host-dependent compatibility between the promoters and riboswitches.ResultsA novel genetic element composed of the promoter P43 and a theophylline-riboswitch was developed and characterized in B. subtilis. When combined with a P43 promoter (P43′-riboE1), the theophylline-riboswitch successfully switched the constitutive expression pattern of P43 to an induced pattern. The expression mediated by the novel element could be activated at the translational level by theophylline with a relatively high induction ratio. The induction ratios for P43′-riboE1 by 4-mM theophylline were elevated during the induction period. The level of induced expression was dependent on the theophylline dose. Correspondingly, the induction ratios gradually increased in parallel with the elevated dose of theophylline. Importantly, the induced expression level was higher than three other strong constitutive promoters including PsrfA, PaprE, and the native P43. It was found that the distance between the SD sequence within the expression element and the start codon significantly influenced both the level of induced expression and the induction ratio. A 9-bp spacer was suitable for producing desirable expression level and induction ratio. Longer spacer reduced the activation efficiency. Importantly, the system successfully overexpressed β-glucuronidase at equal levels, and induction ratio was similar to that of GFP.ConclusionThe constructed theophylline-inducible gene expression system has broad compatibility and robustness, which has great potential in over-production of pharmaceutical and industrial proteins and utilization in building more complex gene circuits.

Enhanced extracellular production of L-asparaginase from Bacillus subtilis 168 by B. subtilis WB600 through a combined strategy.

L-asparaginase (EC 3.5.1.1, ASN) exhibits great commercial value due to its uses in the food and medicine industry. In this study, we reported the enhanced expression of type II ASN from Bacillus subtilis 168 in B. subtilis WB600 through a combined strategy. First, eight signal peptides (the signal peptide of the ASN, ywbN, yvgO, amyE, oppA, vpr, lipA, and wapA) were used for ASN secretion in B. subtilis by using Hpa II promoter, respectively. The signal peptide wapA achieved the highest extracellular ASN activity (28.91U/mL). Second, Hpa II promoter was replaced by a strong promoter, P43 promoter, resulting in 38.1% enhanced ASN activity. By two rounds of error-prone PCR mutation, the P43 promoter variants with remarkably enhanced strength (D7, E2, H6, B2, and F3) were identified. B2 (-28: A→G, -13: A→G) achieved ASN activity up to 51.13U/mL. Third, after deletion of the N-terminal 25-residues, ASN activity reached 102.41U/mL, which was 100% higher than that of the intact ASN. At last, the extracellular ASN of the B. subtilis arrived at 407.6U/mL (2.5g/L of ASN protein) in a 3-L bioreactor by using a fed-batch strategy. The purified ASN showed maximal activity at 65°C and its half-life at 65°C was 61min. The K m and k cat of the ASN were 5.29mM and 54.4s-1, respectively. To the best of our knowledge, we obtained the highest yield of ASN in a food-grade host ever reported, which may benefit the industrial production and application of ASN.

Secretory expression of a heterologous protein, Aiio-AIO6BS, in Bacillus subtilis via a non-classical secretion pathway

The quenching enzyme AIO6 (AiiO-AIO6) has been reported as a feed additive preparation for application in aquaculture and biological control of pathogenic Aeromonas hydrophila. We developed an economical strategy to express AIO6BS (AiiO-AIO6BS, codon optimized AIO6 in Bacillus subtilis) in Bacillus subtilis for facilitating its widespread application. Promoter p43 without the signal peptide was used for secretory expression of AIO6BS in B. subtilis. Western blotting analysis demonstrated that AIO6BS was successfully expressed and secreted into the cell culture. Expression analysis of AIO6BS in the single or double mutant of the lytC and lytD genes for cell autolysis in B. subtilis 1A751 and cell autolysis-resistant engineered strain LM2531 derived from the wild type 168 indicated that the release of the heterologous protein AIO6BS was not simply mediated by cell lysis. Expression level of AIO6BS did not change in the mutants of B. subtilis that harbored mutations in the secA, tatAC, or ecsA genes compared with that in the parent wild type strain. These results suggested the AIO6BS protein was likely secreted via a non-classical secretion pathway. The expression analysis of the various N- or C-terminal truncated gene products indicated that AIO6BS probably acts as an export signal to direct its self-secretion across the cell membrane.

High-level expression of nattokinase in Bacillus licheniformis by manipulating signal peptide and signal peptidase.

Nattokinase is an enzyme produced by Bacillus licheniformis and has potential to be used as a drug for treating cardiovascular disease due to its beneficial effects of preventing fibrin clots etc. However, the low activity and titre of this protein produced by B.licheniformis often hinders its application of commercial production. The aim of this work is to improve the nattokinase production by manipulating signal peptides and signal peptidases in B.licheniformis. The P43 promoter, amyL terminator and AprN target gene were used to form the nattokinase expression vector, pHY-SP-NK, which was transformed into B.licheniformis and nattokinase was expressed successfully. A library containing 81 predicted signal peptides was constructed for nattokinase expression in B.licheniformis, with the maximum activity being obtained under the signal peptide of AprE. Among four type I signal peptidases genes (sipS, sipT, sipV, sipW) in B.licheniformis, the deletion of sipV resulted in a highest decrease in nattokinase activity. Overexpression of sipV in B.licheniformis led to a nattokinase activity of 35·60FUml(-1) , a 4·68-fold improvement over activity produced by the initial strain. This work demonstrates the potential of B.licheniformis for industrial production of nattokinase through manipulation of signal peptides and signal peptidases expression. This study has screened the signal peptides of extracellular proteins of B.licheniformis for nattokinase production. Four kinds of Type I signal peptidases genes have been detected respectively in B.licheniformis to identify which one played the vital role for nattokinase production. This study provided a promising strain for industry production of nattokinase.

Food-Grade Expression of d-Psicose 3-Epimerase with Tandem Repeat Genes in Bacillus subtilis.

An integrative food-grade expression system with tandem repeat target genes was constructed for the expression of d-psicose 3-epimerase (DPEase; EC 5.1.3.30). The DPEase gene fused with the P43 promoter constituted an independent monomeric expression cassette. Multimers of the expression cassette were constructed in vitro using the isocaudamer strategy. The recombinant integration plasmids pDG-nDPE (n = 1, 2, 3), which contained one, two, or three consecutive P43-DPEase tandem repeats, were integrated into the genome of B. subtilis. Then, the antibiotic resistance gene was deleted by the Cre/lox system, and the food-grade recombinant B. subtilis 1A751-nDPE (n = 1, 2, 3) with integrated tandem repeats of the P43-DPEase expression cassette were generated. The specific activity of the B. subtilis 1A751-3DPE was the highest among the recombinant strains and was ∼2.2-fold that of the 1A751-1DPE strain. Under the optimal conditions, 8 g/L of freeze-dried enzyme powder could convert 20% d-fructose (300 g/L) into d-allulose after 1 h.

Promoter Screening from Bacillus subtilis in Various Conditions Hunting for Synthetic Biology and Industrial Applications.

The use of Bacillus subtilis in synthetic biology and metabolic engineering is highly desirable to take advantage of the unique metabolic pathways present in this organism. To do this, an evaluation of B. subtilis’ intrinsic biological parts is required to determine the best strategies to accurately regulate metabolic circuits and expression of target proteins. The strengths of promoter candidates were evaluated by measuring relative fluorescence units of a green fluorescent protein reporter, integrated into B. subtilis’ chromosome. A total of 84 predicted promoter sequences located upstream of different classes of proteins including heat shock proteins, cell-envelope proteins, and proteins resistant against toxic metals (based on similarity) and other kinds of genes were tested. The expression levels measured ranged from 0.0023 to 4.53-fold of the activity of the well-characterized strong promoter P43. No significant shifts were observed when strains, carrying different promoter candidates, were cultured at high temperature or in media with ethanol, but some strains showed increased activity when cultured under high osmotic pressure. Randomly selected promoter candidates were tested and found to activate transcription of thermostable β-galactosidase (bgaB) at a similar level, implying the ability of these sequences to function as promoter elements in multiple genetic contexts. In addition, selected promoters elevated the final production of both cytoplasmic bgaB and secreted protein α-amylase to about fourfold and twofold, respectively. The generated data allows a deeper understanding of B. subtilis’ metabolism and will facilitate future work to develop this organism for synthetic biology.

Construction of plasmid-free Escherichia coli for the production of arabitol-free xylitol from corncob hemicellulosic hydrolysate.

High costs and low production efficiency are a serious constraint to bio-based xylitol production. For industrial-scale production of xylitol, a plasmid-free Escherichia coli for arabitol-free xylitol production from corncob hemicellulosic hydrolysate has been constructed. Instead of being plasmid and inducer dependent, this strain relied on multiple-copy integration of xylose reductase (XR) genes into the chromosome, where their expression was controlled by the constitutive promoter P43. In addition, to minimize the flux from L-arabinose to arabitol, two strategies including low XR total activity and high selectivity of XR has been adopted. Arabitol was significantly decreased using plasmid-free strain which had lower XR total activity and an eight point-mutations of XR with a 27-fold lower enzyme activity toward L-arabinose was achieved. The plasmid-free strain in conjunction with this mutant XR can completely eliminate arabitol formation in xylitol production. In fed-batch fermentation, this plasmid-free strain produced 143.8 g L−1 xylitol at 1.84 g L−1 h−1 from corncob hemicellulosic hydrolysate. From these results, we conclude that this route by plasmid-free E. coli has potential to become a commercially viable process for xylitol production.

Enhancement of extracellular expression of Bacillus naganoensis pullulanase from recombinant Bacillus subtilis: Effects of promoter and host

Pullulanase plays an important role in industrial applications of starch processing. However, extracellular production of pullulanase from recombinant Bacillus subtilis is yet limited due to the issues on regulatory elements of B. subtilis expression system. In this study, the gene encoding B. naganoensis pullulanase (PUL) was expressed in B. subtilis WB800 under the promoter PHpaII in the shuttle vector pMA0911. The extracellular activity of expressed pullulanase was 3.9 U ml−1 from the recombinant B. subtilis WB800/pMA0911-PHpaII-pul. To further enhance the yield of PUL, the promoter PHpaII in pMA0911 was replaced by a stronger constitutive promoter P43. Then the activity was increased to 8.7 U ml−1 from the recombinant B. subtilis WB800/pMA0911-P43-pul. Effect of host on pullulanase expression was further investigated by comparison between B. subtilis WB600 and B. subtilis WB800. In addition to the available B. subtilis WB800 recombinants, the constructed plasmids pMA0911-PHpaII-pul and pMA0911-P43-pul were transformed into B. subtilis WB600, respectively. Consequently, the extracellular production of PUL was significantly enhanced by B. subtilis WB600/pMA0911-P43-pul, resulting in the extracellular pullulanase activity of 24.5 U ml−1. Therefore, promoter and host had an impact on pullulanase expression and their optimization would be useful to improve heterologous protein expression in B. subtilis.

Cloning and Expression of the γ-Polyglutamic Acid Synthetase Gene pgsBCA in Bacillus subtilis WB600.

To clone and express the γ-polyglutamic acid (γ-PGA) synthetase gene pgsBCA in Bacillus subtilis, a pWB980 plasmid was used to construct and transfect the recombinant expression vector pWB980-pgsBCA into Bacillus subtilis WB600. PgsBCA was expressed under the action of a P43 promoter in the pWB980 plasmid. Our results showed that the recombinant bacteria had the capacity to synthesize γ-PGA. The expression product was secreted extracellularly into the fermentation broth, with a product yield of 1.74 g/L or higher. γ-PGA samples from the fermentation broth were purified and characterized. Hydrolysates of γ-PGA presented in single form, constituting simple glutamic acid only, which matched the characteristics of the infrared spectra of the γ-PGA standard, and presented as multimolecular aggregates with a molecular weight within the range of 500–600 kDa. Expressing the γ-PGA synthetase gene pgsBCA in B. subtilis system has potential industrial applications.

Degradation of chlorothalonil through a hydrolytic dehalogenase secreted from Bacillus subtilis WB800

To achieve the secretory expression of chlorothalonil hydrolytic dehalogenase (Chd), the chd gene was cloned into the vector pP43NMK under the control of the P43 promoter and the NprB signal peptide-encoding sequence and extracellularly expressed in the protease-deficient strain Bacillus subtilis WB800. The optimization of Chd production in submerged culture through Plackett-Burman and Box-Behnken designs enhanced the activity of Chd from 6.80 to 14.50 U l−1 and the protein expression amount from 1.93 μg ml−1 to 5.65 μg ml−1. The obtained Chd catalyzed a hydroxyl substitution at the 4-chlorine atom of chlorothalonil to form 4-hydroxy-trichloroisophthalonitrile, which is more soluble in water and easier to remove from the surface of vegetables. Six types of vegetables classified by the edible organ were selected for the chlorothalonil enzymatic degradation assay. After enzymatic reaction for 120 min, almost all of the 25 mg kg−1 chlorothalonil on cherry tomatoes was removed, approximately 82% of the 45 mg kg−1 chlorothalonil on lactuca sativa was removed, and approximately 40%–67% of the chlorothalonil on all other vegetables was removed. Moreover, the chlorothalonil eluted from the vegetables could be degraded to <1 mg l−1 after 3 h. The results of the present study demonstrated the potential of Chd to clean up chlorothalonil residue on the surfaces of vegetables.