Enhanced extracellular production of raw starch-degrading α-amylase in Bacillus subtilis through expression regulatory element modification and fermentation optimization

Microbial Cell Factories

Research

Dongbang Yao¹  Huanhuan Gao¹  Bin Wang¹  Xudong Han¹  Zemin Fang¹  Yazhong Xiao¹  Wei Fang¹  He Li¹ 

[1] School of Life Sciences, Anhui University, 230601, Hefei, China;Anhui Key Laboratory of Modern Biomanufacturing, 230601, Hefei, China;Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis, 230601, Hefei, China;AHU Green Industry Innovation Research Institute, 230088, Hefei, China;

关键词: Raw starch-degrading α-amylase;    Bacillus subtilis;    Promoter;    Signal peptide;    RBS sequence;    Fermentation optimization;   

DOI  :  10.1186/s12934-023-02116-z

 received in 2023-02-07, accepted in 2023-05-17,  发布年份 2023

来源: Springer

【 摘 要 】

BackgroundRaw starch-degrading α-amylase (RSDA) can hydrolyze raw starch at moderate temperatures, thus contributing to savings in starch processing costs. However, the low production level of RSDA limits its industrial application. Therefore, improving the extracellular expression of RSDA in Bacillus subtilis, a commonly used industrial expression host, has great value.ResultsIn this study, the extracellular production level of Pontibacillus sp. ZY raw starch-degrading α-amylase (AmyZ1) in B. subtilis was enhanced by expression regulatory element modification and fermentation optimization. As an important regulatory element of gene expression, the promoter, signal peptide, and ribosome binding site (RBS) sequences upstream of the amyZ1 gene were sequentially optimized. Initially, based on five single promoters, the dual-promoter Pveg-PylB was constructed by tandem promoter engineering. Afterward, the optimal signal peptide SPNucB was obtained by screening 173 B. subtilis signal peptides. Then, the RBS sequence was optimized using the RBS Calculator to obtain the optimal RBS1. The resulting recombinant strain WBZ-VY-B-R1 showed an extracellular AmyZ1 activity of 4824.2 and 41251.3 U/mL during shake-flask cultivation and 3-L fermenter fermentation, which were 2.6- and 2.5-fold greater than those of the original strain WBZ-Y, respectively. Finally, the extracellular AmyZ1 activity of WBZ-VY-B-R1 was increased to 5733.5 U/mL in shake flask by optimizing the type and concentration of carbon source, nitrogen source, and metal ions in the fermentation medium. On this basis, its extracellular AmyZ1 activity was increased to 49082.1 U/mL in 3-L fermenter by optimizing the basic medium components as well as the ratio of carbon and nitrogen sources in the feed solution. This is the highest production level reported to date for recombinant RSDA production.ConclusionsThis study represents a report on the extracellular production of AmyZ1 using B. subtilis as a host strain, and achieved the current highest expression level. The results of this study will lay a foundation for the industrial application of RSDA. In addition, the strategies employed here also provide a promising way for improving other protein production in B. subtilis.

【 授权许可】

CC BY   
© The Author(s) 2023

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