Stepwise modifications of genetic parts reinforce the secretory production of nattokinase in <i>Bacillus subtilis</i>

Cui, Wenjing; Suo, Feiya; Cheng, Jintao; Hao, Wenliang; Guo, Junling; Zhou, Zhemin

doi:10.1111/1751-7915.13298

Cited by 18 publications

(12 citation statements)

References 47 publications

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“…4d). The P BH4 synthetic promoter obtained in this study by SETarSCoP derived from the P srfA is stronger than the previously reported mutants derived from the same parental template by only using a semi-rational design strategy targeting the conserved and non-conservative sequences, upon which the highest yielding level of NK controlled by that evolved promoter was 292 FU/mL at the similar culture condition [65]. Therefore, SETarSCoP is superior to several previous strategies for promoter evolution on different aspects.…”

Section: Resultsmentioning

confidence: 94%

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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

et al. 2019

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Background Promoter 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. Results The native promoter, P srfA , 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 P srfA . 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 P srfA . 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 P BH4 was identified and verified to be a highly evolved synthetic promoter. The strength of P BH4 was higher than P srfA by approximately 3 times. Moreover, P BH4 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. Conclusion Comparing 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. Electronic supplementary material The online version of this article (10.1186/s12934-019-1148-3) contains suppleme...

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Section: Resultsmentioning

confidence: 94%

“…The activity of GusA was measured with 4-nitrophenyl β- d -glucuronide (PNPG) as described previously [7]. Strains BSNK and BSBHNK were used to verify the expression level of NK, driven by P srfA and P BH4 and NK activity was determined by a modified fibrin degradation assay [65].…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2019

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“…We chose to express the Douchi fibrinolytic enzyme. The enzyme exhibits high thrombolytic activity and it has great application prospects in the prevention and treatment of cardiovascular and cerebrovascular diseases [ 25 ].…”

Section: Resultsmentioning

confidence: 99%

Development and application of a rapid all-in-one plasmid CRISPR-Cas9 system for iterative genome editing in Bacillus subtilis

et al. 2022

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Background Bacillus subtilis, an important industrial microorganism, is commonly used in the production of industrial enzymes. Genome modification is often necessary to improve the production performance of cell. The dual-plasmid CRISPR-Cas9 system suitable for iterative genome editing has been applied in Bacillus subtilis. However, it is limited by the selection of knockout genes, long editing cycle and instability. Results To address these problems, we constructed an all-in-one plasmid CRISPR-Cas9 system, which was suitable for iterative genome editing of B. subtilis. The PEG4000-assisted monomer plasmid ligation (PAMPL) method greatly improved the transformation efficiency of B. subtilis SCK6. Self-targeting sgRNArep transcription was tightly controlled by rigorous promoter PacoR, which could induce the elimination of plasmids after genome editing and prepare for next round of genome editing. Our system achieved 100% efficiency for single gene deletions and point mutations, 96% efficiency for gene insertions, and at least 90% efficiency for plasmid curing. As a proof of concept, two extracellular protease genes epr and bpr were continuously knocked out using this system, and it only took 2.5 days to complete one round of genome editing. The engineering strain was used to express Douchi fibrinolytic enzyme DFE27, and its extracellular enzyme activity reached 159.5 FU/mL. Conclusions We developed and applied a rapid all-in-one plasmid CRISPR-Cas9 system for iterative genome editing in B. subtilis, which required only one plasmid transformation and curing, and accelerated the cycle of genome editing. To the best of our knowledge, this is the rapidest iterative genome editing system for B. subtilis. We hope that the system can be used to reconstruct the B. subtilis cell factory for the production of various biological molecules.

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“…It has been demonstrated that a few posttranslationally dehydrated peptides and a thioetherbridged azurin peptide fragment could be transported via the Sec pathway in L. lactis (30,31). In our initial attempt, five widely used B. subtilis signal peptides, including three Sec signal peptides, SP AmyE (32), SP AprE (33), and SP Epr (34), and two Tat signal peptides, SP PhoD (35) and SP YwbN (36), were each fused to the N terminus of NisA. The fusion peptides were coexpressed with NisB and NisC in B. subtilis WB800.…”

Section: Resultsmentioning

confidence: 99%

Analysis of Cross-Functionality within LanBTC Synthetase Complexes from Different Bacterial Sources with Respect to Production of Fully Modified Lanthipeptides

Chen

Kuipers

2022

Appl Environ Microbiol

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Lanthipeptides belong to a family of ribosomally synthesized and posttranslationally modified peptides (RiPPs) containing (methyl)lanthionine residues. Commonly, class I lanthipeptides are synthesized by a gene cluster encoding a precursor peptide (LanA), a biosynthetic machinery (LanBTC), a protease (LanP), a two-component regulatory system (LanRK), and an immunity system (LanI and LanFEG). Although nisin and subtilin are highly similar class I lanthipeptides, the cross-regulation by LanRK and the cross-immunity by LanI and LanFEG between the nisin and subtilin systems have been proven very low. Here, the possibility of the cross-functionality by LanBTC to modify and transport nisin precursor (NisA) and subtilin precursor (SpaS) was evaluated in Bacillus subtilis and Lactococcus lactis . Interestingly, we found that a promiscuous NisBC-SpaT complex is able to synthesize and export nisin precursor, as efficiently as the native nisin biosynthetic machinery NisBTC, in L. lactis , but not in B. subtilis . The assembly of the NisBC-SpaT complex at a single microdomain, close to the old cell pole, was observed by fluorescence microscopy in L. lactis . In contrast, such a complex was not formed in B. subtilis . Furthermore, the isolation of the NisBC-SpaT complex and its subcomplexes from the cytoplasmic membrane of L. lactis by pull-down assays was successfully conducted. Our work demonstrates that the association of LanBC with LanT is critical for the efficient biosynthesis and secretion of the lanthipeptide precursor with complete modifications, and suggests a cooperative mechanism between LanBC and LanT in the modification and transport processes. IMPORTANCE A multimeric synthetase LanBTC complex has been proposed for the in vivo production of class I lanthipeptides. However, it has been demonstrated that LanB, LanC, and LanT can perform their functionality in vivo and in vitro , independently of other Lan proteins. The role of protein-protein interactions, especially between the modification complex LanBC and the transport system LanT, in the biosynthesis process of lanthipeptides is still unclear. In this study, the importance of the presence of a well-installed LanBTC complex in the cell membrane for lanthipeptide biosynthesis and transport was reinforced. In L. lactis , the recruitment of SpaT from the peripheral cell membrane to the cell poles by the NisBC complex was observed, which may explain the mechanism by which secretion of premature peptide is prevented.

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

Cited by 18 publications

References 47 publications

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

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

Development and application of a rapid all-in-one plasmid CRISPR-Cas9 system for iterative genome editing in Bacillus subtilis

Analysis of Cross-Functionality within LanBTC Synthetase Complexes from Different Bacterial Sources with Respect to Production of Fully Modified Lanthipeptides

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