BackgroundBacillus subtilis is an all-important Gram-positive bacterium of valuable biotechnological utility that has been widely used to over-produce industrially and pharmaceutically relevant proteins. There are a variety of expression systems in terms of types of transcriptional patterns, among which the auto-inducible and growth-phase-dependent promoters are gaining increasing favor due to their inducer-independent feature, allowing for the potential to industrially scale-up. To expand the applicability of the auto-inducible expression system, a novel auto-regulatory expression system coupled with cell density was constructed and developed in B. subtilis using the quorum-sensing related promoter srfA (PsrfA).ResultsThe promoter of the srf operon was used to construct an expression plasmid with the green fluorescent protein (GFP) downstream of PsrfA. The expression displayed a cell-density-dependent pattern in that GFP had a fairly low expression level at the early exponential stage and was highly expressed at the late exponential as well as the stationary stages. Moreover, the recombinant system had a similar expression pattern in wild-type B. subtilis 168, WB600, and WB800, as well as in B. subtilis 168 derivative strain 1681, with the complete deletion of PsrfA, indicating the excellent compatibility of this system. Noticeably, the expression strength of PsrfA was enhanced by optimizing the −10 and −35 core sequence by substituting both sequences with consensus sequences. Importantly, the expression pattern was successfully developed in an auto-regulatory cell-density coupling system by the simple addition of glucose in which GFP could not be strongly expressed until glucose was depleted, resulting in a greater amount of the GFP product and increased cell density. The expression system was eventually tested by the successful over-production of aminopeptidase to a desired level.ConclusionThe auto-regulatory cell density coupling system that is mediated by PsrfA is a novel expression system that has an expression pattern that is split between cell-growth and over-expression, leading to an increase in cell density and elevating the overall expression levels of heterologously expressed proteins. The broad applicability of this system and inducer-free expression property in B. subtilis facilitate the industrial scale-up and medical applications for the over-production of a variety of desired proteins.

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.

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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Precise regulation of gene expression is fundamental for tailor-made gene circuit design in synthetic biology. Current strategies for this type of development are mainly based on directed evolution beginning with a native promoter template. The performances of engineered promoters are usually limited by the growth phase because only one promoter is recognized by one type of sigma factor (σ). Here, we constructed multiple-σ recognizable artificial hybrid promoters (AHPs) composed of tandems of dual and triple natural minimal promoters (NMPs). These NMPs, which use σ A , σ H and σ W , had stable functions in different growth phases. The functions of these NMPs resulted from an effect called transcription compensation, in which AHPs sequentially use one type of σ in the corresponding growth phase. The strength of the AHPs was influenced by the combinatorial order of each NMP and the length of the spacers between the NMPs. More importantly, the output of the precise regulation was achieved by equipping AHPs with synthetic ribosome binding sites and by redesigning them for induced systems. This strategy might offer promising applications to rationally design robust synthetic promoters in diverse chassis to spur the construction of more complex gene circuits, which will further the development of synthetic biology.