Overexpression of a Thermostable α-Amylase through Genome Integration in Bacillus subtilis

A carbohydrate binding module 68 (CBM68) of pullulanase from Anoxybacillus sp. LM18-11 was used to enhance the secretory expression of a thermostable α-amylase (BLA702) in Bacillus subtilis, through an atypical secretion pathway. The extracellular activity of BLA702 guided by CBM68 was 1248 U/mL, which was 12.6 and 7.2 times higher than that of BLA702 guided by its original signal peptide and the endogenous signal peptide LipA, respectively. A single gene knockout strain library containing 51 genes encoding macromolecular transporters was constructed to detect the effect of each transporter on the secretory expression of CBM68-BLA702. The gene knockout strain 0127 increased the extracellular amylase activity by 2.5 times. On this basis, an engineered strain B. subtilis 0127 (AmyE::BLA702-NprB::CBM68-BLA702-PrsA) was constructed by integrating BLA702 and CBM68-BLA702 at the AmyE and NprB sites in the genome of B. subtilis 0127, respectively. The molecular chaperone PrsA was overexpressed, to reduce the inclusion body formation of the recombinant enzymes. The highest extracellular amylase activity produced by B. subtilis 0127 (AmyE::BLA702-NprB::CBM68-BLA702-PrsA) was 3745.7 U/mL, which was a little lower than that (3825.4 U/mL) of B. subtilis 0127 (pMAC68-BLA702), but showing a better stability of passage. This newly constructed strain has potential for the industrial production of BLA702.

[1]  G. Qin,et al.  Enhanced extracellular β‐mannanase production by overexpressing PrsA lipoprotein in Bacillus subtilis and optimizing culture conditions , 2022, Journal of basic microbiology.

[2]  Nadezhda T. Doncheva,et al.  The impact of PrsA over-expression on the Bacillus subtilis transcriptome during fed-batch fermentation of alpha-amylase production , 2022, bioRxiv.

[3]  U. Hellmich,et al.  Backbone NMR assignment of the nucleotide binding domain of the Bacillus subtilis ABC multidrug transporter BmrA in the post-hydrolysis state , 2022, Biomolecular NMR assignments.

[4]  Yanhe Ma,et al.  Regulate the hydrophobic motif to enhance the non-classical secretory expression of Pullulanase PulA in Bacillus subtilis. , 2021, International journal of biological macromolecules.

[5]  Jinfang Zhang,et al.  Reducing the cell lysis to enhance yield of acid-stable alpha amylase by deletion of multiple peptidoglycan hydrolase-related genes in Bacillus amyloliquefaciens. , 2020, International journal of biological macromolecules.

[6]  Xueli Zhang,et al.  New base editors change C to A in bacteria and C to G in mammalian cells. , 2020, Nature biotechnology.

[7]  S. Rosser,et al.  CRISPR-dCas9 mediated cytosine deaminase base editing in Bacillus subtilis. , 2020, ACS synthetic biology.

[8]  Xue Cai,et al.  Construction of a highly active secretory expression system in Bacillus subtilis of a recombinant amidase by promoter and signal peptide engineering. , 2019, International journal of biological macromolecules.

[9]  Peter F. Hallin,et al.  Identification and optimization of PrsA in Bacillus subtilis for improved yield of amylase , 2019, Microbial cell factories.

[10]  Bin Ye,et al.  Construction of second generation protease‐deficient hosts of Bacillus subtilis for secretion of foreign proteins , 2019, Biotechnology and bioengineering.

[11]  Huitu Zhang,et al.  Optimization of alkaline protease production by rational deletion of sporulation related genes in Bacillus licheniformis , 2019, Microbial cell factories.

[12]  Shihui Yang,et al.  CRISPR-assisted multi-dimensional regulation for fine-tuning gene expression in Bacillus subtilis , 2019, Nucleic acids research.

[13]  G. Shi,et al.  Development of an Inducible Secretory Expression System in Bacillus licheniformis Based on an Engineered Xylose Operon. , 2018, Journal of agricultural and food chemistry.

[14]  Dawei Zhang,et al.  Multimer recognition and secretion by the non-classical secretion pathway in Bacillus subtilis , 2017, Scientific Reports.

[15]  Guocheng Du,et al.  Metabolic engineering of Bacillus subtilis fueled by systems biology: Recent advances and future directions. , 2017, Biotechnology advances.

[16]  C. Harwood,et al.  Effect of Genome Position on Heterologous Gene Expression in Bacillus subtilis: An Unbiased Analysis. , 2016, ACS synthetic biology.

[17]  Zhengxiang Wang,et al.  A highly active alpha amylase from Bacillus licheniformis: directed evolution, enzyme characterization and structural analysis. , 2014, Journal of Microbiology and Biotechnology.

[18]  C. You,et al.  Transformation of Bacillus subtilis. , 2014, Methods in molecular biology.

[19]  B. Schwikowski,et al.  Condition-Dependent Transcriptome Reveals High-Level Regulatory Architecture in Bacillus subtilis , 2012, Science.

[20]  Yi Wang,et al.  Characterisation of mutagenised acid-resistant alpha-amylase expressed in Bacillus subtilis WB600 , 2008, Applied Microbiology and Biotechnology.

[21]  K. Maurer,et al.  Targeted deletion of genes encoding extracellular enzymes in Bacillus licheniformis and the impact on the secretion capability. , 2007, Journal of biotechnology.

[22]  P. Lu,et al.  Protein secretion pathways in Bacillus subtilis: implication for optimization of heterologous protein secretion. , 2007 .

[23]  Eduardo P C Rocha,et al.  Replication‐associated gene dosage effects shape the genomes of fast‐growing bacteria but only for transcription and translation genes , 2006, Molecular microbiology.

[24]  G. Fichant,et al.  Inventory, assembly and analysis of Bacillus subtilis ABC transport systems. , 1999, Journal of molecular biology.

[25]  S. Ehrlich,et al.  Integration of linear, heterologous DNA molecules into the Bacillus subtilis chromosome: mechanism and use in induction of predictable rearrangements , 1985, Journal of bacteriology.