Nattokinase (NK) is a serine protease of the subtilisin family; as a potent fibrinolytic enzyme, it is potentially useful for thrombosis therapy. For NK to be applied as an oral medicine for the treatment of cardiovascular diseases, it must overcome the extremely acidic environments of the gastrointestinal tract despite its limited acidic stability. In this study, three strategies were adopted to improve the acid resistance of NK: (a) Surface charge engineering, (b) sequence alignment, and (c) mutation based on the literature. Eleven variants were constructed and four single-point mutations were screened out for their distinctive catalytic properties: Q59E increased the specific activity; S78T improved the acid stability; Y217K enhanced the acid and thermal stabilities; and N218D improved the thermostability. Based on these observations, multipoint variants were constructed and characterized, and one variant with better acid stability, catalytic efficiency, and thermostability was obtained. Molecular dynamics simulation was carried out to clarify the molecular mechanism of the increased stability of S78T and Y217K mutants under acidic conditions. This study explored effective strategies to engineer acid resistance of NK; moreover, the NK variants with better catalytic properties found in this study have potential applications for the medical industry.

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...

As a natural sesquiterpene compound with numerous biological activities, patchoulol has extensive applications in the cosmetic industry and potential usage in pharmaceuticals. Although several patchoulol-producing microbial strains have been constructed, the low productivity still hampers large-scale fermentation. Escherichia coli possesses the ease of genetic manipulation and simple nutritional requirements and does not comprise competing pathways for the farnesyl diphosphate (FPP) precursor, showing its potential for patchoulol biosynthesis. Here, combinatorial strategies were applied to produce patchoulol in E. coli. The initial strain was constructed, and it produced 14 mg/L patchoulol after fermentation optimization. Patchoulol synthase (PTS) was engineered by semirational design, resulting in improved substrate binding affinity and a patchoulol titer of 40.3 mg/L; the patchoulol titer reached 66.2 mg/L after fusing of PTS with FPP synthase. To further improve the patchoulol production, the genome of an efficient chassis strain was engineered by deleting the competitive routes for acetate, lactate, ethanol, and succinate synthesis and cumulatively enhancing the expression of efflux transporters, which improved patchoulol production to 338.6 mg/L. When tested in a bioreactor, the patchoulol titer and productivity were further improved to 970.1 mg/L and 199 mg/L/d, respectively, and were among the highest levels reported using mineral salt medium.

Programmable dual-base editor: a propellent for evolving bacterial chassis.

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.

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Fructosyltransferase is a key enzyme in fructo-oligosaccharide production, while the highly demanding conditions of industrial processes may reduce its stability and activity. This study employs sequence alignment and structural analysis to target three potential residues (Gln38, Ile39, and Cys43) around the active center of FruSG from Aspergillus niger, and mutants with greatly improved activity and stability were obtained through site-directed mutagenesis. The K m values of C43N and Q38Y were, respectively, reduced to 60.8 and 93.1% compared to those of WT. Meanwhile, the k cat of C43N was increased by 21.2-fold compared to that of WT. These imply that both the affinity and catalytic efficiency of C43N were significantly enhanced compared to WT. The Glide docking score of sucrose inside C43N was calculated to be −5.980, which was lower than that of WT (−4.887). What is more, the proposed general acid/base catalyst Glu273 with a lower pK a value of C43N calculated by PROPKA might contribute to an easier catalytic reaction compared to that of WT. The thermal stability and pH stability of the mutant C43N were significantly enhanced compared to those of WT, and more hydrogen bonds formed during molecular dynamics simulations might contribute to the improved stability of C43N.

L-2-Aminobutyric acid (L-ABA) is a chiral precursor of several important drugs. L-Glutamate is an ideal material for the production of L-ABA through γ-decarboxylation; however, enzyme-mediated γ-decarboxylation of L-glutamate has not yet been reported. Here, this goal was achieved through isomerization of L-glutamate to (2S,3S)-3-methylaspartate by glutamate mutase (GM) and β-decarboxylation of 3-methylaspartate by L-aspartate-β-decarboxylase (Asd). Although the activity of wild-type Asd toward 3-methylaspartate could not be detected, a mutant Asd exhibiting high activity was isolated on the basis of enzyme engineering. The conversion of L-glutamate to L-ABA achieved 98.9%, which provided a more cost-effective process for L-ABA industrial production.