The secretion of biotechnologically or pharmaceutically relevant recombinant proteins into the culture supernatant of a bacterial expression host greatly facilitates their downstream processing and significantly reduces the production costs. The first step during the secretion of a desired target protein into the growth medium is its transport across the cytoplasmic membrane. In bacteria, two major export pathways, the general secretion or Sec pathway and the twin-arginine translocation or Tat pathway, exist for the transport of proteins across the plasma membrane. The routing into one of these alternative protein export systems requires the fusion of a Sec- or Tat-specific signal peptide to the amino-terminal end of the desired target protein. Since signal peptides, besides being required for the targeting to and membrane translocation by the respective protein translocases, also have additional influences on the biosynthesis, the folding kinetics, and the stability of the respective target proteins, it is not possible so far to predict in advance which signal peptide will perform best in the context of a given target protein and a given bacterial expression host. As outlined in this review, the most promising way to find the optimal signal peptide for a desired protein is to screen the largest possible diversity of signal peptides, either generated by signal peptide variation using large signal peptide libraries or, alternatively, by optimization of a given signal peptide using site-directed or random mutagenesis strategies.

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Signal peptides for recombinant protein secretion in bacterial expression systems

Bacillus subtilis, belonging to the type species of Bacillus, is a type of soil-derived, low %G+C, endospore-forming Gram-positive bacterium. After the discovery of B. subtilis 168 that displayed natural competence, this bacterium has been intensively considered to be an ideal model organism and a robust host to study several basic mechanisms, such as metabolism, gene regulation, bacterial differentiation, and application for industrial purposes, such as heterologous protein expression and the overproduction of an array of bioactive molecules. Since the first report of heterologous overproduction of recombinant proteins in this strain, the bulk production of a multitude of valuable enzymes, especially industrial enzymes, has been performed on a relatively large scale. Since B. subtilis can non-specifically secrete recombinant proteins using various signal peptides, it has tremendous advantages over Gram-negative bacterial hosts. Along with the report of the complete genome sequence of B. subtilis, a number of genetic tools, including diverse types of plasmids, bacterial promoters, regulatory elements, and signal peptides, have been developed and characterized. These novel genetic elements tremendously accelerated genetic engineering in B. subtilis recombinant systems. In addition, with the development of several complex gene expression systems, B. subtilis has performed a number of more complex functions. This ability enables it to be a substantial chassis in synthetic biology rather than just a workhorse for the overproduction of recombinant proteins. In this review, we review the progress in the development of B. subtilis as a universal platform to overproduce heterologous diverse high-value enzymes. This progress has occurred from the development of biological parts, including the characterization and utilization of native promoters, the fabrication of synthetic promoters and regulatory elements, and the assembly and optimization of genetic systems. Some important industrial enzymes that have been produced in large quantities in this host are also summarized in this review. Furthermore, the ability of B. subtilis to serve as a cellular tool was also briefly recapitulated and reviewed.

Exploitation of Bacillus subtilis as a robust workhorse for production of heterologous proteins and beyond.

Poly-γ-glutamic acid (γ-PGA) is an important biochemical product with a variety of applications. This work reports a novel approach to improve γ-PGA through over expression of key enzymes in cofactor NADPH generating process for NADPH pool. Six genes encoding the key enzymes in NADPH generation were over-expressed in the γ-PGA producing strain B. licheniformis WX-02. Among various recombinants, the strain over-expressing zwf gene (coding for glucose-6-phosphate dehydrogenase), WX-zwf, produced the highest γ-PGA concentration (9.13 g/L), 35% improvement compared to the control strain WX-pHY300. However, the growth rates and glucose uptake rates of the mutant WX-zwf were decreased. The transcriptional levels of the genes pgsB and pgsC responsible for γ-PGA biosynthesis were increased by 8.21- and 5.26-fold, respectively. The Zwf activity of the zwf over expression strain increased by 9.28-fold, which led to the improvement of the NADPH generation, and decrease of accumulation of by-products acetoin and 2,3-butanediol. Collectively, these results demonstrated that NADPH generation via over-expression of Zwf is as an effective strategy to improve the γ-PGA production in B. licheniformis.

/pdf/a-novel-approach-to-improve-poly-g-glutamic-acid-production-3h822xmfmf.pdf

A novel approach to improve poly-γ-glutamic acid production by NADPH Regeneration in Bacillus licheniformis WX-02.

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.

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

The respiratory chain is optimized to reduce metabolism coefficient, and the VHB and AdK are strengthened to improve ATP supply for enhanced γ‐PGA production. The roles of NarGHIJ, NasBC and ResD∼E are identified in the BFCNA medium during γ‐PGA production, and Strengthening of NarG, NasC and ResD improve the nitrate metabolism, ATP supply and γ‐PGA yield. The γ‐PGA yield produced by WX‐BCVAR is increased by 38.64% via improvement of ATP supply

Enhanced production of poly-γ-glutamic acid by improving ATP supply in metabolically engineered Bacillus licheniformis.

Nattokinase (NK) possesses the potential for prevention and treatment of thrombus-related diseases. In this study, high-level expression of nattokinase was achieved in Bacillus licheniformis WX-02 via host strain construction and signal peptides optimization. First, ten genes (mpr, vpr, aprX, epr, bpr, wprA, aprE, bprA, hag, amyl) encoding for eight extracellular proteases, a flagellin and an amylase were deleted to obtain B. licheniformis BL10, which showed no extracellular proteases activity in gelatin zymography. Second, the gene fragments of P43 promoter, Svpr, nattokinase and TamyL were combined into pHY300PLK to form the expression vector pP43SNT. In BL10 (pP43SNT), the fermentation activity and product activity per unit of biomass of nattokinase reached 14.33 FU/mL and 2,187.71 FU/g respectively, which increased by 39 and 156 % compared to WX-02 (pP43SNT). Last, Svpr was replaced with SsacC and SbprA, and the maximum fermentation activity (33.83 FU/mL) was achieved using SsacC, which was 229 % higher than that of WX-02 (pP43SNT). The maximum NK fermentation activity in this study reaches the commercial production level of solid state fermentation, and this study provides a promising engineered strain for industrial production of nattokinase, as well as a potential platform host for expression of other target proteins.

Efficient expression of nattokinase in Bacillus licheniformis: host strain construction and signal peptide optimization

Aims
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.

Methods and Results
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 35·60 FU ml−1, a 4·68-fold improvement over activity produced by the initial strain.

Conclusions
This work demonstrates the potential of B. licheniformis for industrial production of nattokinase through manipulation of signal peptides and signal peptidases expression.

Significance and Impact of the Study
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.

https://sfamjournals.onlinelibrary.wiley.com/doi/pdf/10.1111/jam.13175

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

Disclosed are bacillus licheniformis engineering bacteria for nattokinase production and a method for producing nattokinase by using the bacillus licheniformis engineering bacteria, which belong to the technical fields of microorganism genetic engineering and enzyme engineering. According to the invention, a nattokinase gene of bacillus subtilis MBS04-6 is obtained through PCR technology augmentation. Bacillus licheniformis BL10 is utilized as an expression host; promoter P43 of bacillus subtilis 168 is taken as a promoter; a signal peptide of extracellular serine protease Vpr of bacillus licheniformis WX-02 is taken a signal peptide; and a terminator sequence of alpha-amylase of bacillus licheniformis WX-02 is taken as a terminator. The expression elements are connected to shuttle vector pHY300PLK to form an expression vector, and the expression vector is transformed into the bacillus licheniformis BL10 to obtain the bacillus licheniformis engineering bacteria BL10 (pP43SNT) for nattokinase production. The bacterial strain has been deposited with the China Center for Type Culture Collection (CCTCC) at September 10th, 2013, and the accession number is CCTCC NO:M2013401. The invention also provides a method of the bacterial strain for producing nattokinase, and the maximum enzyme activity can reach 11.37 FU/mL in a liquid fermentation medium.

Bacillus licheniformis engineering bacteria for nattokinase production and method for producing nattokinase by using bacillus licheniformis engineering bacteria

The invention discloses a bacillus licheniformis host bacterium BL10 with multiple gene deletion, the accession number of which is CCTCCNO:M2013400. The host bacterium derives from a bacillus licheniformis WX-02 which partially or completely has deletion of ten genes. The ten genes comprises eight protease genes (mpr encoding a metalloprotease; vpr encoding a serine protease; aprX encoding an intracellular serine protease; epr encoding a minimal extracellular protease; bpr encoding a bacillus peptidase F; wprA encoding a protease combined with cell walls; aprE encoding an extracellular alkaline serine protease; and bprA encoding a bacillus peptidase F) and two extracellular secretory protein genes (hag encoding flagellin; and amyL encoding alpha-amylase). The BL10 has completely no extracellular protease activity and can reduce the degradation effect of a protease on a target protein. When the target protein is expressed by the expression host, the expression level is higher, and the host bacterium benefits protein expression enhancement.

Bacillus licheniformis expression host

The invention relates to a Bacillus licheniformis engineering bacterium capable of efficiently secreting nattokinase, and belongs to the technical fields of enzyme engineering and microbes. The method adopts a molecular biology technology to screen and replace signal peptides with important effects in an exogenous protein transhipment process, and the signal peptides are replaced by signal peptides from levanase SacC in Bacillus subtillis 168 from signal peptides from extracellular serine proteinase Vpr in Bacillus licheniformis WX-02 on the basis of nattokinase producing Bacillus licheniformis engineering bacterium BL10 (pP43SNT) preserved in a laboratory in advance in order to afresh construct the nattokinase producing Bacillus licheniformis engineering bacterium BL10 (pP43SacCNK). The bacterium disclosed in the invention can substantially improve the nattokinase secreting level under liquid fermentation conditions, and the maximum enzyme activity can reach 33.83 FU/mL. The bacterium is preserved in China Center for Type Culture Collection on June 16, 2014 with the preservation number of CCTCC NO: M2014253.

Chen Jingbang

Papers

Efficient expression of nattokinase in Bacillus licheniformis: host strain construction and signal peptide optimization

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

Bacillus licheniformis engineering bacteria for nattokinase production and method for producing nattokinase by using bacillus licheniformis engineering bacteria

Bacillus licheniformis expression host

Bacillus licheniformis engineering bacterium capable of efficiently secreting nattokinase