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Enhanced extracellular recombinant keratinase activity in Bacillus subtilis SCK6 through signal peptide optimization and site-directed mutagenesis.

ABSTRACT: Keratinase has a great commercial value owing to its applications in the enzymatic dehairing of goatskins. In this study, we adopted a combined strategy to enhance the extracellular recombinant keratinase activity in Bacillus subtilis SCK6. First, nine signal peptides were screened to enhance the expression of extracellular keratinase. The recombinant strain with SP_LipA exhibited the highest extracellular keratinase activity of 739.03 U per mL, which was two-fold higher activity of the wild type. Second, based on the multiple sequence alignment with the bacterial alkaline proteases, the mutant (M123L/V149I/A242N) was introduced into the keratinase. Comparing with the wild type of keratinase, the mutant M123L/V149I/A242N showed an increase in the extracellular keratinase activity, which was about 1.2-fold higher activity of the wild type. Finally, the keratinase expression vector with SP_LipA and mutant M123L/V149I/A242N was constructed, and the extracellular keratinase activity reported at 830.91 U per mL was a 2.2-fold activity of the wild type. Then, the mutant keratinase was purified and characterized. The mutant exhibited properties similar to those of the wild type at an optimal temperature of 60 °C and pH 10.0. Conclusively, the extracellular expression of keratinase was enhanced via a combined strategy, and the mutant keratinase demonstrated properties similar to that of the wild type of keratinase.

SUBMITTER: Tian J

PROVIDER: S-EPMC9073338 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMenaquinone (MK-7) is a highly valuable vitamin K2 produced by Bacillus subtilis. Common static metabolic engineering approaches for promoting the production of MK-7 have been studied previously. However, these approaches caused an accumulation of toxic substances and reduced product yield. Hence, dynamic regulation by the quorum sensing (QS) system is a promising method for achieving a balance between product synthesis and cell growth.ResultsIn this study, the QS transcriptional regulator SinR, which plays a significant role in biofilm formation and MK production simultaneously, was selected, and its site-directed mutants were constructed. Among these mutants, sinR knock out strain (KO-SinR) increased the biofilm biomass by 2.8-fold compared to the wild-type. SinRquad maximized the yield of MK-7 (102.56 ± 2.84 mg/L). To decipher the mechanism of how this mutant regulates MK-7 synthesis and to find additional potential regulators that enhance MK-7 synthesis, RNA-seq was used to analyze expression changes in the QS system, biofilm formation, and MK-7 synthesis pathway. The results showed that the expressions of tapA, tasA and epsE were up-regulated 9.79-, 0.95-, and 4.42-fold, respectively. Therefore, SinRquad formed more wrinkly and smoother biofilms than BS168. The upregulated expressions of glpF, glpk, and glpD in this biofilm morphology facilitated the flow of glycerol through the biofilm. In addition, NADH dehydrogenases especially sdhA, sdhB, sdhC and glpD, increased 1.01-, 3.93-, 1.87-, and 1.11-fold, respectively. The increased expression levels of NADH dehydrogenases indicated that more electrons were produced for the electron transport system. Electrical hyperpolarization stimulated the synthesis of the electron transport chain components, such as cytochrome c and MK, to ensure the efficiency of electron transfer. Wrinkly and smooth biofilms formed a network of interconnected channels with a low resistance to liquid flow, which was beneficial for the uptake of glycerol, and facilitated the metabolic flux of four modules of the MK-7 synthesis pathway.ConclusionsIn this study, we report for the first time that SinRquad has significant effects on MK-7 synthesis by forming wrinkly and smooth biofilms, upregulating the expression level of most NADH dehydrogenases, and providing higher membrane potential to stimulate the accumulation of the components in the electron transport system.

Project description:BackgroundOur laboratory has constructed a Bacillus stearothermophilus α-amylase (AmyS) derivative with excellent enzymatic properties. Bacillus subtilis is generally regarded as safe and has excellent protein secretory capability, but heterologous extracellular production level of B. stearothermophilus α-amylase in B. subtilis is very low.ResultsIn this study, the extracellular production level of B. stearothermophilus α-amylase in B. subtilis was enhanced by signal peptide optimization, chaperone overexpression and α-amylase mutant selection. The α-amylase optimal signal peptide (SPYojL) was obtained by screening 173 B. subtilis signal peptides. Although the extracellular α-amylase activity that was produced by the resulting recombinant strain was 3.5-fold greater than that of the control, significant quantities of inclusion bodies were detected. Overexpressing intracellular molecular chaperones significantly reduced inclusion body formation and further increased α-amylase activity. Error-prone PCR produced an amylase mutant K82E/S405R (AmySA) with enzymatic activity superior to that of AmyS. Expression of the amySA gene with the SPYojL while overexpressing molecular chaperones resulted in a 7.1-fold improvement in α-amylase activity. When the final expression strain (WHS11YSA) was cultivated in a 3-L fermenter for 92 h, the α-amylase activity of the culture supernatant was 9201.1 U mL-1, which is the highest level that has been reported to date.ConclusionsThis is the first report that describes an improvement of B. stearothermophilus α-amylase extracellular production levels in B. subtilis using these strategies, and this represents the highest extracellular production level ever reported for α-amylase from B. stearothermophilus in B. subtilis. This high-level production provides a basis for enhanced industrial production of α-amylase. These extracellular production level improvement approaches are also expected to be valuable in the expression of other enzymes in B. subtilis.

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Enhanced extracellular recombinant keratinase activity in Bacillus subtilis SCK6 through signal peptide optimization and site-directed mutagenesis.

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