Fabrication and characterization of a robust and strong bacterial promoter from a semi-rationally engineered promoter library in Bacillus subtilis

Han, Laichuang; Suo, Feiya; Cui, Jiang; Gu, Jiao; Li, Ningna; Zhang, Naixin; Cui, Wenjing; Zhou, Zhemin

doi:10.1016/j.procbio.2017.06.024

“…There are two kinds of promoters: the constitutive promoter that is active in all circumstances and the regulated promoter that become active only in response to specific stimulation in the cell. Because the promoter is a crucial aspect of the expression system, many strong promoters have been screened and characterized in B. subtilis [23–26]. Recent studies have increasingly focused on the strategy to improve the expression level of recombinant proteins or peptides by the construction of tandem promoters and promoter engineering.…”

Section: Introductionmentioning

confidence: 99%

High-level extracellular production of recombinant nattokinase in Bacillus subtilis WB800 by multiple tandem promoters

Liu

¹

,

Zheng

²

,

Ge

³

et al. 2019

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Background Nattokinase (NK), which is a member of the subtilisin family, is a potent fibrinolytic enzyme that might be useful for thrombosis therapy. Extensive work has been done to improve its production for the food industry. The aim of our study was to enhance NK production by tandem promoters in Bacillus subtilis WB800. Results Six recombinant strains harboring different plasmids with a single promoter (P P43 , P HpaII , P BcaprE , P gsiB , P yxiE or P luxS ) were constructed, and the analysis of the fibrinolytic activity showed that P P43 and P HpaII exhibited a higher expression activity than that of the others. The NK yield that was mediated by P P43 and P HpaII reached 140.5 ± 3.9 FU/ml and 110.8 ± 3.6 FU/ml, respectively. These promoters were arranged in tandem to enhance the expression level of NK, and our results indicated that the arrangement of promoters in tandem has intrinsic effects on the NK expression level. As the number of repetitive P P43 or P HpaII increased, the expression level of NK was enhanced up to the triple-promoter, but did not increase unconditionally. In addition, the repetitive core region of P P43 or P HpaII could effectively enhance NK production. Eight triple-promoters with P P43 and P HpaII in different orders were constructed, and the highest yield of NK finally reached 264.2 ± 7.0 FU/ml, which was mediated by the promoter P HpaII -P HpaII -P P43 . The scale-up production of NK that was promoted by P HpaII -P HpaII -P P43 was also carried out in a 5-L fermenter, and the NK activity reached 816.7 ± 30.0 FU/mL. Conclusions Our studies demonstrated that NK was efficiently overproduced by tandem promoters in Bacillus subtilis . The highest fibrinolytic activity was promoted by P HpaII -P HpaII -P P43 , which was much higher than that had been reported in previous studies. These multiple tandem promoters were used successfully to control NK expression and might be useful for improving the expression level of the other genes. Electronic supplementary material The online version of this article (10.1186/s12866-019-1461-3) contains supplementary material, which is av...

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“…Specifically, it requires the state-of-the-art equipment, fluorescent activated cell sorting (FACS). In the process of the development of robust and synthetic promoters in B. subtilis in our previous studies [6, 55–57], a critical issue concerning the effective pipeline for the fabrication of highly efficient promoters spurred us to exploit distinct reliable pipelines that are more convenient to perform than the ordinary strategies. Importantly, previous studies indicate that the optimization of the − 16 region, which is located upstream of the − 10 box, substantially influences the transcription level of a bacterial promoter [58].…”

Section: Resultsmentioning

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

Han

¹

,

Suo

²

,

Miao

³

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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“…16) was derived from P SG35.1 where the -35 sequence (TACTAA) was replaced with the consensus sequence (TTGACA) [16]. P srfA, has the same -35 (GTGATA) and -10 sequences (TAAACT) of promoter of srfA [17]. These promoters were chosen because they do not require any specific inducer, which is expensive for large-scale cultivation and inconvenient, too.…”

Section: Construction Of Apre3-5 Genes With Its Promoter Replaced With Other Promotermentioning

confidence: 99%

Increase of a Fibrinolytic Enzyme Production through Promoter Replacement ofaprE3-5fromBacillus subtilisCH3-5

Zhuang

¹

,

Meng

²

,

Lê

³

et al. 2021

J. Microbiol. Biotechnol.

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Bio-Rad, USA). pHY3-5 (pHY300PLK containing aprE3-5) was used as the template DNA [13]. The reaction mixture (50 μl) consisted of 1 μl of template DNA, 1 μl of each primer (10 μM), 5 μl of dNTPs (0.25 mM), and 0.5 μl of ExTaq DNA polymerase (Takara, Japan). Amplification conditions were as follows: 94 o C for 5 min, 30 cycles of 94 o C for 30 s, 64 o C for 30 s, 72 o C for 40 s, and a final extension at 72 o C for 5 min.

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Fabrication and characterization of a robust and strong bacterial promoter from a semi-rationally engineered promoter library in Bacillus subtilis

Cited by 9 publications

References 41 publications

High-level extracellular production of recombinant nattokinase in Bacillus subtilis WB800 by multiple tandem promoters

High-level extracellular production of recombinant nattokinase in Bacillus subtilis WB800 by multiple tandem promoters

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

Increase of a Fibrinolytic Enzyme Production through Promoter Replacement ofaprE3-5fromBacillus subtilisCH3-5

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