A novel strategy to improve protein secretion via overexpression of the SppA signal peptide peptidase in Bacillus licheniformis

Cai, Dongbo; Wang, Hao; He, Penghui; Zhu, Chengjun; Wang, Qin; Wei, Xuetuan; Nomura, Christopher T.; Chen, Shouwen

doi:10.1186/s12934-017-0688-7

Cited by 44 publications

(36 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…All samples were analyzed in triplicate, and the data were presented as the mean ± standard deviation for each sample point. All data were conducted to analyze the variance at p < 0.05 and p < 0.01, and a t test was applied to compare the mean values using the software package Statistica 6.0 (Invitrogen, California) (Cai, Wang, et al, ).…”

Section: Methodsmentioning

confidence: 99%

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

Cai

Chen

et al. 2018

Biotech & Bioengineering

Self Cite

View full text Add to dashboard Cite

Poly-γ-glutamic acid (γ-PGA) is an important multifunctional biopolymer with various applications, for which adenosine triphosphate (ATP) supply plays a vital role in biosynthesis. In this study, the enhancement of γ-PGA production was attempted through various approaches of improving ATP supply in the engineered strains of Bacillus licheniformis. The first approach is to engineer respiration chain branches of B. licheniformis, elimination of cytochrome bd oxidase branch reduced the maintenance coefficient, leading to a 19.27% increase of γ-PGA yield. The second approach is to introduce Vitreoscilla hemoglobin (VHB) into recombinant B. licheniformis, led to a 13.32% increase of γ-PGA yield. In the third approach, the genes purB and adK in ATP-biosynthetic pathway were respectively overexpressed, with the AdK overexpressed strain increased γ-PGA yield by 14.69%. Our study also confirmed that the respiratory nitrate reductase, NarGHIJ, is responsible for the conversion of nitrate to nitrite, and assimilatory nitrate reductase NasBC is for conversion of nitrite to ammonia. Both NarGHIJ and NasBC were positively regulated by the two-component system ResD-ResE, and overexpression of NarG, NasC, and ResD also improved the ATP supply and the consequent γ-PGA yield. Based on the above individual methods, a method of combining the deletion of cydBC gene and overexpression of genes vgB, adK, and resD were used to enhance ATP content of the cells to 3.53 μmol/g of DCW, the mutant WX-BCVAR with this enhancement produced 43.81 g/L of γ-PGA, a 38.64% improvement compared to wild-type strain WX-02. Collectively, our results demonstrate that improving ATP content in B. licheniformis is an efficient strategy to improve γ-PGA production.

show abstract

Section: Methodsmentioning

confidence: 99%

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

Cai

Chen

et al. 2018

Biotech & Bioengineering

Self Cite

View full text Add to dashboard Cite

show abstract

“…According to the Japan Natto Kinase Association (http://j-nattokinase.org/en/jnka_nattou_01.html), the activity of NK was defined as fibrinolytic units (FU) because this method has excellent quantitative performance using fibrin as the substrate. The activity of NK was measured and determined according to previously reported protocols (Guan et al ., ,b; Cai et al ., ,b). In brief, 0.7 ml of Tris–HCl (50 mM, pH 8.0) was first mixed with 0.2 ml of fibrinogen solution (0.72%, w/v).…”

Section: Methodsmentioning

confidence: 99%

“…These two parts are joined to the N ‐terminus of the mature polypeptide (Nakamura et al ., ). Compared to other fibrinolytic enzymes (urokinase, t‐PA and streprokinase), NK has the advantage of no side effects, low cost and long lifetime, has the potential to be used as a drug for treating cardiovascular disease and serves as a functional food additive (Cai et al ., ,b; Weng et al ., ).…”

Section: Introductionmentioning

confidence: 99%

Stepwise modifications of genetic parts reinforce the secretory production of nattokinase in Bacillus subtilis

Cui

Suo

Cheng

et al. 2018

Microbial Biotechnology

View full text Add to dashboard Cite

SummaryNattokinase (NK) is an important serine‐protease with direct fibrinolytic activity involving the prevention of cardiovascular disease as an antithrombotic agent. Dozens of studies have focused on the characterization of intrinsic novel promoters and signal peptides to the secretory production of recombinant proteins in Bacillus subtilis. However, intrinsic genetic elements have several drawbacks, which cannot mediate the production of NK to the desired level. In this study, the genetic elements, which were used to overproduce the recombinant secretory NK, were rationally modified in B. subtilis in a stepwise manner. The first step was to select a suitable signal peptide for the highly efficient secretion of NK. By comparison of the secretory levels mediated by two different signal peptides, which were encoded by the genes of a minor extracellular protease epr (SP epr) and cell‐wall associated protease wapA (SP wapA), respectively, SP wapA was verified as the superior secretory element. Second, P04, which was a synthetic promoter screened from an array of mutants based on the promoter cloned from the operon of a quorum‐sensing associated gene srfA (PsrfA), was paired to SP wapA. The secretory level of NK was obviously augmented by the combination of these two genetic elements. Third, the cis‐acting element CodY‐binding sequence positioned at the 5′UTR was deleted (yielding P08), and thus the secretory level was significantly elevated. The activity of NK, which was defined as fibrinolytic units (FU), reached to a level of 270 FU ml−1. Finally, the superior genetic element composed of P08 and SP wapA was utilized to overproduce NK in the host B. subtilis WB800, which was able to produce the secretory NK at 292 FU ml−1. The strategy established in this study can not only be used to overproduce NK in B. subtilis but also might be a promising pipeline to modify the genetic element for the synthetic secretory system.

show abstract

“…There are two signal peptide peptidases (SppA and TepA) in Bacillus , among which SppA was proven to mainly function as SPPs, and overexpression of SppA improved the yields of nattokinase and α‐amylase in B. licheniformis (Cai et al . ).…”

Section: Optimizing Secretion Pathwaymentioning

confidence: 97%

“…), signal peptide peptidases (Cai et al . ), chaperones (Chen et al . 2015d), proteases (Pummi et al .…”

Section: Introductionmentioning

confidence: 99%

EngineeringBacillusfor efficient production of heterologous protein: current progress, challenge and prospect

et al. 2019

View full text Add to dashboard Cite

Summary Bacillus species are widely recognized as good industrial platform strains, which can produce a variety of valuable products including enzymes and functional proteins. Bacillus expression systems gain various competitive edges over other expression systems, with respect to nontoxicity, convenience for gene modification and high yield of target proteins. Recently, a number of Bacillus expression systems have been developed, and various strategies were conducted to improve the yields of target proteins. In this review, we focused on the strategies of host strain optimization for heterologous protein production, including secretion pathway optimization, RNA and protein stability enhancement, cell growth facilitation, genome streamlining and transcriptional regulator engineering. In addition, Bacillus spore surface display and food‐grade expression systems were developed to expand the application of Bacillus expression system in recent years. Finally, the challenges and prospects of Bacillus expression system were discussed regarding the recent progresses, challenges and trends in this field.

show abstract

A novel strategy to improve protein secretion via overexpression of the SppA signal peptide peptidase in Bacillus licheniformis

Cited by 44 publications

References 39 publications

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

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

Stepwise modifications of genetic parts reinforce the secretory production of nattokinase in Bacillus subtilis

EngineeringBacillusfor efficient production of heterologous protein: current progress, challenge and prospect

Contact Info

Product

Resources

About