An operator-based expression toolkit for Bacillus subtilis enables fine-tuning of gene expression and biosynthetic pathway regulation

Abstract

Significance A gene regulatory system is an important tool for the engineering of biosynthetic pathways of organisms. Here, we report the development of an inducible-ON/OFF regulatory system using a malO operator as a key element. We identified and modulated sequence, position, numbers, and spacing distance of malO operators, generating a series of activating or repressive promoters with tunable strength. The stringency and robustness are both guaranteed in this system, a maximal induction factor of 790-fold was achieved, and nine proteins from different organisms were expressed with high yields. This system can be utilized as a gene switch, promoter enhancer, or metabolic valve in synthetic biology applications. This operator-based engineering strategy can be employed for developing similar regulatory systems in different microorganisms.

Boosting the Heterologous Expression of d-Allulose 3-Epimerase in Bacillus subtilis through Protein Engineering and Catabolite-Responsive Element Box Engineering

“…In contrast, it is more efficient to alleviate the CCR effect by genetic engineering. The common methods are knockout of the genes related to CcpA in the host and modification of the CRE box to block the regulating function of CcpA. , Mutation on the CRE box that could destroy its palindromic sequence would have a concise and effective impact on the prevention of CcpA binding to suppress the CCR effect. In 2015, Bruder et al found that the mutations of nucleotides in the conserved regions performed well in weakening the CCR effect, indicating that the highly conserved nucleotides were more suitable for mutagenesis to reduce the CCR effect.…”

“…With seryl-phosphorylation Hpr (HPr-Ser-P) acting as its cofactor, CcpA can bind with the catabolite-responsive element (CRE) box to regulate numerous catabolic genes . Alleviating the CCR effect can enhance the utilization of nondominant carbon sources. − …”

Improving prodigiosin production by transcription factor engineering and promoter engineering in Serratia marcescens

“…Promoter engineering was widely used to enhance gene expression at the transcription level and achieved high yields production of target metabolites in different microorganisms ( Tang et al, 2020 ; Fu et al, 2022 ; Liu et al, 2022 ). To improve the production of prodigiosin in S. marcescens , a panel of strong promoters must be available for fine-tune gene expression in S. marcescens .…”

“…Promoter engineering is a method widely used to enhance gene expression at the transcription level. And to improve the yield of target products, numbers of homologous or heterologous promoters have been developed for constitutive or inducible gene expression in model strain such as Escherichia coli ( Song et al, 2015 ; Tang et al, 2020 ), Bacillus subtilis ( Cui et al, 2019 ; Fu et al, 2022 ), and Corynebacterium glutamicum ( Huang et al, 2021 ; Liu et al, 2022 ) and non-model strain such as Streptococcus thermophilus ( Kong et al, 2019 ), Corynebacterium ammoniagenes ( Hou et al, 2019 ), and Schlegelella brevitalea ( Ouyang et al, 2020 ). However, the choice of strong constitutive natural promoters in prodigiosin-producing strain S. marcescens is still limited.…”

Magnetothermal Control of Temperature-Sensitive Repressors in Superparamagnetic Iron Nanoparticle-Coated Bacillus subtilis

“…Bacillus subtilis is a model Gram-positive organism widely used in synthetic biology with numerous strategies available for manipulating gene expression, , global metabolic networks, and its genome − making it well-suited for engineering systems for spatial and temporal regulation , High heat resistance and well characterized protein production pathways make B. subtilis an ideal chassis organism for investigating thermal energy-controlled protein production as therapeutics. − B. subtilis also has multiple well-characterized inducible genetic systems including several sugar-regulated elements . For these reasons, B. subtilis has been used in a variety of in vitro industry applications in pharmaceutical/nutraceutical production, recombinant protein production, and production of functional peptides and oligopeptides. − However, the available inducible systems have limited control for both in vitro and in vivo applications due to potential host toxicity, cost, and carbon-source dependence …”