Construction of a highly active secretory expression system via an engineered dual promoter and a highly efficient signal peptide in Bacillus subtilis

Guan, Chengran; Cui, Wenjing; Cheng, Jintao; Li, Rui; Liu, Zhongmei; Zhou, Li; Zhou, Zhemin

doi:10.1016/j.nbt.2016.01.005

Cited by 68 publications

(67 citation statements)

References 37 publications

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“…An array of NK‐producing Bacillus strains was originally screened for producing NK under naturally fermented conditions (Inatsu et al ., ; Wang et al ., ; Wei et al ., ; Kumar et al ., ). In recent years, along with the cloning of the aprN gene from Bacillus natto, NK has been overexpressed in a variety of hosts, including Bacillus subtilis , Escherichia coli and Lactococcus lactis (Liang et al ., ,b; Chen et al ., ; Degering et al ., ; Nguyen et al ., ; Guan et al ., ,b). Although NK from a Douchi‐isolating B. subtilis YF38 has been successfully overexpressed in E. coli and transported to the periplasmic space, the yield of total enzyme was only 49 mg per litre of culture.…”

Section: Introductionmentioning

confidence: 99%

“…B. subtilis can naturally secrete heterologous proteins into the extracellular compartment, and thus this host has been broadly explored for the overproduction of numerous industrial and pharmaceutical proteins (Liu et al ., ,b). Bacillus , as well as Lactococcus , has been explored to overproduce recombinant NK as a workhorse (Liang et al ., ,b; Wu et al ., ; Wei et al ., ; Guan et al ., ,b).…”

Section: Introductionmentioning

confidence: 99%

“…To enhance the secretory capability and final yield of recombinant NK in B. subtilis , several strategies employing multiple biological and process engineering methods have been developed, including promoter engineering, signal peptide screening and optimization of fermentation process (Chen et al ., ; Cho et al ., ; Degering et al ., ; Kwon et al ., ; Wu et al ., ; Guan et al ., ,b). Genetic engineering of the promoter has great potential for the augmentation of NK expression.…”

Section: Introductionmentioning

confidence: 99%

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

Cui

Suo

Cheng

et al. 2018

Microbial Biotechnology

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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.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Cui

Suo

Cheng

et al. 2018

Microbial Biotechnology

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“…To comprehensively compare the expression levels between commonly used strong constitutive promoters and P43′-riboE1, three wild-type promoters P43, P aprE and P srfA , which have been characterized in previous studies [19, 20], were employed to express the GFP reporter. BSG43, BSG04, and BSG03 carrying the three strong promoters respectively, were cultured in LB for 31 h. The BSG11 harbouring pBSG11 was treated with 8 mM theophylline for 24 h. Then, the final expression level was determined by SDS-PAGE analysis.…”

Section: Resultsmentioning

confidence: 99%

“…To sub-clone the riboE1-mediated expression element into the E. coli – B. subtilis shuttle vector, the P aprE’ -riboE1 fragment and 36-bp flanking sequences with homology to the upstream and downstream regions of srfA promoter (P srfA ) on plasmid pBSG03 were amplified using primers G61F and G61R. This fragment was used as a mega-primer for whole-plasmid inverse PCR to insert the template into plasmid pBSG03 in place of P srfA [19], yielding shuttle vector pBSG61. The expression level from the native aprE promoter in pBSG04 was compared to expression from P srfA and P43.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2016

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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.

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A brief overview on the application and sources of α‐amylase and expression hosts properties in order to production of recombinant α‐amylase

Movahedpour

Asadi

Khatami

et al. 2021

Biotech and App Biochem

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By reducing the activation energy, enzymes accelerate the chemical reaction; therefore, they are good alternative for industrial catalysts. Amylase is a suitable enzyme as a catalyst for the chemical decomposition of starch. This enzyme is of great importance, and its production is highly profitable. α-Amylase is among the most important amylases produced naturally by animals, plants, and microorganisms. Still, the α-amylases produced by bacteria have a special place in industry and commerce. Moreover, a large volume of this enzyme can be produced by selecting an appropriate and optimized host to clone and express the α-amylase gene. The present study briefly reviews the structure, application, sources, and hosts used to produce recombinant α-amylase.

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Construction of a highly active secretory expression system via an engineered dual promoter and a highly efficient signal peptide in Bacillus subtilis

Cited by 68 publications

References 37 publications

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

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

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

A brief overview on the application and sources of α‐amylase and expression hosts properties in order to production of recombinant α‐amylase

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