Enhanced Extracellular α-amylase Production in Brevibacillus Choshinensis by Optimizing Extracellular Degradation and Folding Environment

Overview

Journal J Ind Microbiol Biotechnol

Publisher Oxford University Press

Specialty Biotechnology

Date 2021 Oct 3

PMID 34601573

Citations 1

Authors

Dongbang Yao

Kang Zhang

Xuyang Zhu

Lingqia Su

Jing Wu

Affiliations

Soon will be listed here.

Abstract

A strategy for optimizing the extracellular degradation and folding environment of Brevibacillus choshinensis has been used to enhance the extracellular production of recombinant α-amylase. First, a gene (bcp) encoding an extracellular protease and another encoding an extracellular chaperone (prsC) were identified in the genome of B. choshinensis HPD31-SP3. Then, the effect of extracellular protein degradation on recombinant α-amylase production was investigated by establishing a CRISPR/Cas9n system to knock out bcp. The effect of extracellular folding capacity was investigated separately by coexpressing extracellular chaperones genes from different sources (prsA, prsC, prsL, prsQ) in B. choshinensis. The final recombinant strain (BCPPSQ), which coexpressed prsQ in a genetic background lacking bcp, produced an extracellular α-amylase activity of 6940.9 U/ml during shake-flask cultivation. This was 2.1-fold greater than that of the original strain BCWPS (3367.9 U/ml). Cultivation of BCPPSQ in a 3-l fermenter produced an extracellular α-amylase activity of 17925.6 U/ml at 72 h, which was 7.6-fold greater than that of BCWPS (2358.1 U/ml). This strategy demonstrates its great potential in enhancing extracellular α-amylase production in B. choshinensis. What's more, this study provides a strategic reference for improving the extracellular production of other recombinant proteins in B. choshinensis.

Citing Articles

Addition of arginine hydrochloride and proline to the culture medium enhances recombinant protein expression in Brevibacillus choshinensis: The case of RBD of SARS-CoV-2 spike protein and its antibody.

Matsunaga R, Tsumoto K Protein Expr Purif. 2022; 194:106075.

PMID: 35231586 PMC: 8881763. DOI: 10.1016/j.pep.2022.106075.

References

El-Sayed A, Abou-Dobara M, El-Fallal A, Omar N . Heterologous expression, purification, immobilization and characterization of recombinant α-amylase AmyLa from Laceyella sp. DS3. Int J Biol Macromol. 2019; 132:1274-1281. DOI: 10.1016/j.ijbiomac.2019.04.010. View

Jiang W, Bikard D, Cox D, Zhang F, Marraffini L . RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol. 2013; 31(3):233-9. PMC: 3748948. DOI: 10.1038/nbt.2508. View

Chen J, Fu G, Gai Y, Zheng P, Zhang D, Wen J . Combinatorial Sec pathway analysis for improved heterologous protein secretion in Bacillus subtilis: identification of bottlenecks by systematic gene overexpression. Microb Cell Fact. 2015; 14:92. PMC: 4482152. DOI: 10.1186/s12934-015-0282-9. View

Vitikainen M, Hyyrylainen H, Kivimaki A, Kontinen V, Sarvas M . Secretion of heterologous proteins in Bacillus subtilis can be improved by engineering cell components affecting post-translocational protein folding and degradation. J Appl Microbiol. 2005; 99(2):363-75. DOI: 10.1111/j.1365-2672.2005.02572.x. View

Ebisu S, Tsuboi A, Takagi H, Naruse Y, Yamagata H, Tsukagoshi N . Conserved structures of cell wall protein genes among protein-producing Bacillus brevis strains. J Bacteriol. 1990; 172(3):1312-20. PMC: 208600. DOI: 10.1128/jb.172.3.1312-1320.1990. View

Li K, Cai D, Wang Z, He Z, Chen S . Development of an Efficient Genome Editing Tool in Bacillus licheniformis Using CRISPR-Cas9 Nickase. Appl Environ Microbiol. 2018; 84(6). PMC: 5835740. DOI: 10.1128/AEM.02608-17. View

DUrzo N, Martinelli M, Nenci C, Brettoni C, Telford J, Maione D . High-level intracellular expression of heterologous proteins in Brevibacillus choshinensis SP3 under the control of a xylose inducible promoter. Microb Cell Fact. 2013; 12:12. PMC: 3582527. DOI: 10.1186/1475-2859-12-12. View

Hu W, Xiang J, Kong P, Liu L, Xie Q, Xiang H . Expression and Characterization of a Single-Chain Variable Fragment against Human LOX-1 in and . J Microbiol Biotechnol. 2017; 27(5):965-974. DOI: 10.4014/jmb.1702.02007. View

Li Z, Duan X, Wu J . Improving the thermostability and enhancing the Ca(2+) binding of the maltohexaose-forming α-amylase from Bacillus stearothermophilus. J Biotechnol. 2016; 222:65-72. DOI: 10.1016/j.jbiotec.2016.02.013. View

10.

Zou C, Duan X, Wu J . Efficient extracellular expression of Bacillus deramificans pullulanase in Brevibacillus choshinensis. J Ind Microbiol Biotechnol. 2015; 43(4):495-504. DOI: 10.1007/s10295-015-1719-1. View

11.

Millet J . Characterization of proteinases excreted by Bacillus subtilis Marburg strain during sporulation. J Appl Bacteriol. 1970; 33(1):207-19. DOI: 10.1111/j.1365-2672.1970.tb05245.x. View

12.

Quesada-Ganuza A, Antelo-Varela M, Mouritzen J, Bartel J, Becher D, Gjermansen M . Identification and optimization of PrsA in Bacillus subtilis for improved yield of amylase. Microb Cell Fact. 2019; 18(1):158. PMC: 6749698. DOI: 10.1186/s12934-019-1203-0. View

13.

Cong L, Ran F, Cox D, Lin S, Barretto R, Habib N . Multiplex genome engineering using CRISPR/Cas systems. Science. 2013; 339(6121):819-23. PMC: 3795411. DOI: 10.1126/science.1231143. View

14.

Li X, Wang Y, Park J, Gu L, Li D . An extremely thermostable maltogenic amylase from Staphylothermus marinus: Bacillus expression of the gene and its application in genistin glycosylation. Int J Biol Macromol. 2017; 107(Pt A):413-417. DOI: 10.1016/j.ijbiomac.2017.09.007. View

15.

Pohl S, Harwood C . Heterologous protein secretion by bacillus species from the cradle to the grave. Adv Appl Microbiol. 2010; 73:1-25. DOI: 10.1016/S0065-2164(10)73001-X. View

16.

Stahl M, Ferrari E . Replacement of the Bacillus subtilis subtilisin structural gene with an In vitro-derived deletion mutation. J Bacteriol. 1984; 158(2):411-8. PMC: 215443. DOI: 10.1128/jb.158.2.411-418.1984. View

17.

Yang T, Irene K, Liu H, Liu S, Zhang X, Xu M . Enhanced extracellular gamma glutamyl transpeptidase production by overexpressing of PrsA lipoproteins and improving its mRNA stability in Bacillus subtilis and application in biosynthesis of L-theanine. J Biotechnol. 2019; 302:85-91. DOI: 10.1016/j.jbiotec.2019.06.302. View

18.

Wang H, Zhang X, Qiu J, Wang K, Meng K, Luo H . Development of Bacillus amyloliquefaciens as a high-level recombinant protein expression system. J Ind Microbiol Biotechnol. 2018; 46(1):113-123. DOI: 10.1007/s10295-018-2089-2. View

19.

Tjalsma H, Antelmann H, Jongbloed J, Braun P, Darmon E, Dorenbos R . Proteomics of protein secretion by Bacillus subtilis: separating the "secrets" of the secretome. Microbiol Mol Biol Rev. 2004; 68(2):207-33. PMC: 419921. DOI: 10.1128/MMBR.68.2.207-233.2004. View

20.

Wei X, Zhou Y, Chen J, Cai D, Wang D, Qi G . Efficient expression of nattokinase in Bacillus licheniformis: host strain construction and signal peptide optimization. J Ind Microbiol Biotechnol. 2014; 42(2):287-95. DOI: 10.1007/s10295-014-1559-4. View