Enhancement of a high efficient autoinducible expression system in Bacillus subtilis by promoter engineering

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

doi:10.1016/j.pep.2016.07.008

Cited by 35 publications

(23 citation statements)

References 34 publications

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“…D). Notably, the mutant promoter mediating the highest level of overproduction of NK differs from that mediating the production of GFP compared to our previous reports (Cheng et al ., ; Guan et al ., ,b). These data demonstrate that it is impractical to fabricate a universal promoter mediating equivalent expression levels for diverse target proteins since the expression of heterologous protein is determined by two genetic steps: transcription and translation.…”

Section: Resultsmentioning

confidence: 99%

“…In our recent reports, a series of mutant promoters based on P srfA was fabricated by semi-rational design via combinatorial modification of the À10 box and the À35 box. The expression levels driven by these novel synthetic promoters were characterized by the reporter gene, and the compatibility to the target proteins was validated by testing the expression of amino peptidase (Cheng et al, 2016). However, it was still unclear whether higher expression levels of NK could be achieved using these promoter variants.…”

Section: Selection Of a Suitable Signal Peptide For The Secretion Of mentioning

confidence: 99%

“…Two areas, the À10 box and À35 box, located at the bacterial promoter, are critical to the level of gene transcription (Lee et al, 2012;Blazeck and Alper, 2013). According to some reports, modification of these two elements may be a feasible strategy to augment the transcription levels of downstream heterologous genes (Phan et al, 2012;Cheng et al, 2016). However, it is difficult to equip a universal signal peptide with a specific promoter, by which the recombinant system harbouring the two combined genetic elements could express and secrete the heterologous element at the desired level.…”

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: Resultsmentioning

confidence: 99%

Section: Selection Of a Suitable Signal Peptide For The Secretion Of mentioning

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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“…The inducible promoter P xyl is driven by xylose, P spac is induced by IPTG. Auto-inducible promoters (e.g., P pst and P cry3Aa ) can be used to express the target gene from the late log phase to the stationary phase [50]. P srf is an autoinducible promoter which is triggered by signal molecules acting in a quorum sensing pathway.…”

Section: Strategies For Enhancing Surfactin Productionmentioning

confidence: 99%

Rational strain improvement for surfactin production: enhancing the yield and generating novel structures

Liu

2019

Microb Cell Fact

110

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Surfactin, one of the most powerful microbial surfactants, is a lipopeptide-type biosurfactant which combines interesting physicochemical properties and biological activities. However, the high cost caused by its low productivity largely limits the commercial application of surfactin. Hence, many engineered bacterium have also been used to enhance surfactin biosynthesis. This review briefly summarizes the mechanism of surfactin biosynthesis, highlighting the synthesis pathway of N-terminally attached fatty acids, and outlines the main genetic engineering strategies for improving the yield and generating novel structures of surfactin, including promoter engineering, enhancing efflux systems, modifying the transcriptional regulatory genes of surfactin synthase (srfA), genomics and transcriptomics analysis, non ribosomal peptide synthetase (NRPS) domain and combinatorial biosynthesis. Finally, we discuss the future prospects of the research on surfactin.

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“…The inducible strong promoter is an important factor to achieve high level expression of target gene in B. subtilis . The inducible systems take the advantage that gene expression can be switched on at the proper time during fermentation [ 58 ], which is sometimes essential, for example, when the product is toxic to the host cell. Therefore, this work is also devoted to construction of inducible expression system based on our strong promoter.…”

Section: Discussionmentioning

confidence: 99%

Construction of a High-Expression System in Bacillus through Transcriptomic Profiling and Promoter Engineering

Miao

Liang

et al. 2020

Microorganisms

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Bacillus subtilis is an ideal host for secretion and expression of foreign proteins. The promoter is one of the most important elements to facilitate the high-level production of recombinant protein. To expand the repertoire of strong promoters for biotechnological applications in Bacillus species, 14 highly transcribed genes based on transcriptome profiling of B. pumilus BA06 were selected and evaluated for their promoter strength in B. subtilis. Consequently, a strong promoter P2069 was obtained, which could drive the genes encoding alkaline protease (aprE) and green fluorescent protein (GFP) to express more efficiency by an increase of 3.65-fold and 18.40-fold in comparison with the control promoter (PaprE), respectively. Further, promoter engineering was applied to P2069, leading to a mutation promoter (P2069M) that could increase GFP expression by 3.67-fold over the wild-type promoter (P2069). Moreover, the IPTG-inducible expression systems were constructed using the lac operon based on the strong promoters of P2069 and P2069M, which could work well both in B. subtilis and B. pumilus. In this study, highly efficient expression system for Bacillus was constructed based on transcriptome data and promoter engineering, which provide not only a new option for recombinant expression in B. subtilis, but also novel genetic tool for B. pumilus.

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Enhancement of a high efficient autoinducible expression system in Bacillus subtilis by promoter engineering

Cited by 35 publications

References 34 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

Rational strain improvement for surfactin production: enhancing the yield and generating novel structures

Construction of a High-Expression System in Bacillus through Transcriptomic Profiling and Promoter Engineering

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