High-efficient Extracellular Expression Of Bacillus Stearothermophilus ?-amylase In Genus Of Bacillus

?-Amylase(EC 3.2.1.1)is a kind of hydrolase that can hydrolyze the internal?-1,4-glycosidic bonds of starch to produce low molecular weight dextrin,malt oligosaccharide,maltose,glucose and other products.It is widely used in food,textile,paper,detergent and medical industries,with a large market demand.Bacillus licheniformis?-amylase is widely used in starch liquefaction process due to its excellent thermal stability.However,it is mainly produced and sold by foreign companies,domestic enzyme companies are difficult to compete with it,resulting in relatively high price.A Bacillus stearothermophilus?-amylase,which has the advantages of low Ca²⁺dependence and high conversion rate(approximately 8-fold greater than that of B.licheniformis?-amylase),was obtained in our previous study.In addition,a mutant(Amy S)with similar thermal stability to B.licheniformis?-amylase was obtained through molecular modification,which has high industrial application value.However,when the Amy S was expressed in Escherichia coli,there were some problems such as poor extracellular secretion and prone to produce inclusion bodies,which limited its application in industry.To solve these problems,the Amy S was expressed extracellular in genus of Bacillus with high protein secretion ability.First,the Amy S was expressed in Brevibacillus choshinensis with high-efficient protein synthesis ability,and its extracellular expression level was enhanced by knocking out extracellular protease,coexpressing extracellular chaperone and selecting endogenous strong promoters.Then,the Amy S was expressed in Bacillus subtilis with high density fermentation.Based on the previous research results of this study,we first investigated the influence of the promoter on the extracellular recombinant expression of?-amylase in B.subtilis,and then optimized the signal peptide,?-amylase amino acid sequence,host extracellular protease,fermentation process and the Sec secretion pathway.Finally,the high-efficient extracellular expression of B.stearothermophilus?-amylase in B.subtilis was achieved.This research accelerates the research process of our country's industrialized low-cost production of high-temperature?-amylase,which meets the requirements of practical applications and has independent property rights.What's more,this study provides many important strategies for promoting the efficient extracellular expression of other target proteins in genus of Bacillus.The main research results are listed as follows:(1)The Amy S extracellular expression in B.choshinensis was investigated,and the expression level was improved by knocking out extracellular protease and coexpressing extracellular chaperone.Using B.choshinensis HPD31-SP3 as host,a recombinant strain BCWPS containing amy S gene was constructed,and the extracellular protease gene bcp in its genome was excavated.A CRISPR/Cas9n gene editing system for B.choshinensis was established.Finally,on the basis of knocking out the extracellular protease gene bcp,the recombinant strain BCPPSQ obtained by coexpressing extracellular chaperone protein gene prs Q.After fermentation in shake flask and 3-L fermenter,the extracellular Amy S activities of BCPPSQ were 6940.9 and 17925.6 U·mL^-1,respectively,which were 2.1-and 7.6-fold higher than those of BCWPS.In addition,the 3-L fermenter fermentation enzyme activity of the BCCPSQ was the highest expression level of B.stearothermophilus?-amylase in B.choshinensis reported in the current literature.(2)The new endogenous strong promoters were identified and used to enhance the Amy S extracellular expression in B.choshinensis.Using RNA-seq technology,the B.choshinensis transcriptome data was obtained.Based on gene transcription level and gene function,the high expression target genes were screened.Through prediction and screening of their promoters,two new endogenous strong promoters PE and PF were obtained.In shaker flask fermentation,promoters PE and PF mediated Amy S activities were 2.3-and 1.3-fold higher than that of control promoter P2,respectively.In addition,the effect and versatility of PE promoter were verified by 3-L fermenter fermentation and new reporter proteins,respectively.At the 3-L fermenter fermentation level,the extracellular Amy S activity mediated by promoter PE was2.4-fold greater than that of promoter P2.When sucrose isomerase and cutinase were used as new reporter proteins,these reporter proteins activities mediated by promoter PE were 2.3-and 1.3-fold higher than those of promoter P2,respectively.(3)The Amy S extracellular expression in B.subtilis was investigated,and the expression level was improved by optimizing the expression elements.By optimizing the promoter,the extracellular Amy S activity was increased to 210.4 U·mL^-1.By optimizing the signal peptide,the extracellular Amy S activity was increased to 732.0 U·mL^-1,while there are obvious inclusion bodies.It was found that the optimized signal peptide reduced the intracellular Amy S precursor transmembrane transport efficiency,which was the reason for recombinant strain to produce inclusion bodies.Overexpression of intracellular chaperones reduced the inclusion bodies production and increased the extracellular Amy S activity to 1039.4 U·mL^-1.By optimizing the Amy S amino acid sequence(Amy SA),the extracellular?-amylase activity was increased to 1496.8 U·mL^-1,which was 11.2-fold greater than the original enzyme activity.(4)The effect of the host strain extracellular protease on Amy SA extracellular expression was investigated,and the expression level in B.subtilis was improved through fermentation optimization.The Amy SA was recombinant expressed in host strain with different extracellular protease deletion types,and it was found that the host strain B.subtilis WS9,which retains the extracellular protease genes wpr A and vpr,was most suitable for Amy SA extracellular expression.The fermentation medium composition and fermentation conditions were optimized in shake flask,and the recombinant strain extracellular Amy SA activity was increased by 40%.The fermentation conditions,the feed medium carbon sources types,the concentration ratio as well as total concentration of carbon source and nitrogen source in the feed medium were optimized in 3-L fermenter.As result,the recombinant strain extracellular Amy SA activity was increased to 23801.3 U·mL^-1,which was 2.9-fold greater than the original enzyme activity.(5)The high-efficient extracellular expression of Amy SA in B.subtilis was achieved by optimizing the Sec secretion process.The effects of each process in the Sec pathway on the Amy SA extracellular expression were investigated separately.The results showed that maintaining intracellular Amy SA precursor in a unfolded transportable state and its transmembrane transport efficiency were the limiting factors affecting the Amy SA extracellular expression.On the basis of overexpression of intracellular chaperone proteins and coexpression of membrane transport channel components Sec YEG,the entire Sec secretion process of Amy SA was optimized by screening signal peptides.The obtained signal peptide SP_{rpm G}increased the recombinant strain extracellular Amy SA activity by 93%.After 3-L fermenter fermentation,the resulting recombinant strain WHS9GSAB extracellular Amy SA activity and production efficiency were 35779.5 U·mL^-1 and 384.7 U·mL^-1·h^-1,respectively,which were 7.0-and 5.0-fold greater than those of the highest recombinant expression level of B.stearothermophilus?-amylase in B.subtilis reported in the current literature.