Efficient Extracellular Production of Recombinant Proteins in E. Coli Via Enhancing Expression of DacA on the Genome

Overview

Journal J Ind Microbiol Biotechnol

Publisher Oxford University Press

Specialty Biotechnology

Date 2022 Jun 1

PMID 35648451

Authors

Haiquan Yang

Haokun Wang

Fuxiang Wang

Kunjie Zhang

Jinfeng Qu

Jianmin Guan

Wei Shen

Yu Cao

Yuanyuan Xia

Xianzhong Chen

Affiliations

Soon will be listed here.

Abstract

D, D-carboxypeptidase DacA plays an important role in the synthesis and stabilization of Escherichia coli cell wall peptidoglycan. The production level of extracellular recombinant proteins in E. coli can be enhanced by high D, D-carboxypeptidase activity. Construction of expression systems under optimal promoters is one of the main strategies to realize high protein production in E. coli. In this study, the promoter PdacA-3 from DacA on the genome of E. coli BL21 (DE3) was verified to be efficient for recombinant green fluorescent protein using the plasmid mutant pET28a-PdacA with PdacA-3. Meanwhile, the promoter PdacA-3 was engineered to increase the production level of proteins via inserting one or two Shine-Dalgarno (SD) sequences between the promoter PdacA-3 and the target genes. The expression level of dacA on the genome was increased by the improved transcription of the engineered promoters (especially after inserting one additional SD sequence). The engineered promoters increased cell membrane permeabilities to significantly enhance the secretion production of extracellular recombinant proteins in E. coli. Among them, the extracellular recombinant amylase activities in E. coli BL21::1SD-pET28a-amyK and E. coli BL21::2SD-pET28a-amyK were increased by 2.0- and 1.6-fold that of the control (E. coli BL21-pET28a-amyK), respectively. Promoter engineering also affected the morphology and growth of the E. coli mutants. It was indicated that the engineered promoters enhanced the expression of dacA on the genome to disturb the synthesis and structural stability of cell wall peptidoglycans.

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References

Pang C, Liu S, Zhang G, Zhou J, Du G, Li J . Enhancing extracellular production of lipoxygenase in Escherichia coli by signal peptides and autolysis system. Microb Cell Fact. 2022; 21(1):42. PMC: 8933919. DOI: 10.1186/s12934-022-01772-x. View

Popham D, Young K . Role of penicillin-binding proteins in bacterial cell morphogenesis. Curr Opin Microbiol. 2003; 6(6):594-9. DOI: 10.1016/j.mib.2003.10.002. View

Blazeck J, Alper H . Promoter engineering: recent advances in controlling transcription at the most fundamental level. Biotechnol J. 2012; 8(1):46-58. DOI: 10.1002/biot.201200120. View

Meberg B, Paulson A, Priyadarshini R, Young K . Endopeptidase penicillin-binding proteins 4 and 7 play auxiliary roles in determining uniform morphology of Escherichia coli. J Bacteriol. 2004; 186(24):8326-36. PMC: 532442. DOI: 10.1128/JB.186.24.8326-8336.2004. View

Koster M, Bitter W, Tommassen J . Protein secretion mechanisms in Gram-negative bacteria. Int J Med Microbiol. 2000; 290(4-5):325-31. DOI: 10.1016/S1438-4221(00)80033-8. View

Selleck W, Tan S . Recombinant protein complex expression in E. coli. Curr Protoc Protein Sci. 2008; Chapter 5:Unit 5.21. PMC: 2582387. DOI: 10.1002/0471140864.ps0521s52. View

Baquero M, Bouzon M, Quintela J, Ayala J, Moreno F . dacD, an Escherichia coli gene encoding a novel penicillin-binding protein (PBP6b) with DD-carboxypeptidase activity. J Bacteriol. 1996; 178(24):7106-11. PMC: 178621. DOI: 10.1128/jb.178.24.7106-7111.1996. View

Wong W, Ali A, Ma M . Cloning, expression, and characterization of diuretic hormone Manduca diuresin from Manduca sexta in Escherichia coli. Protein Expr Purif. 2003; 29(1):51-7. DOI: 10.1016/s1046-5928(03)00007-x. View

Young K . Approaching the physiological functions of penicillin-binding proteins in Escherichia coli. Biochimie. 2001; 83(1):99-102. DOI: 10.1016/s0300-9084(00)01205-0. View

10.

Ding N, Yuan Z, Zhang X, Chen J, Zhou S, Deng Y . Programmable cross-ribosome-binding sites to fine-tune the dynamic range of transcription factor-based biosensor. Nucleic Acids Res. 2020; 48(18):10602-10613. PMC: 7544201. DOI: 10.1093/nar/gkaa786. View

11.

Loh B, Grant C, Hancock R . Use of the fluorescent probe 1-N-phenylnaphthylamine to study the interactions of aminoglycoside antibiotics with the outer membrane of Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1984; 26(4):546-51. PMC: 179961. DOI: 10.1128/AAC.26.4.546. View

12.

Terpe K . Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol. 2006; 72(2):211-22. DOI: 10.1007/s00253-006-0465-8. View

13.

Saier Jr M . Protein secretion and membrane insertion systems in gram-negative bacteria. J Membr Biol. 2007; 214(2):75-90. DOI: 10.1007/s00232-006-0049-7. View

14.

Tang H, Wu Y, Deng J, Chen N, Zheng Z, Wei Y . Promoter Architecture and Promoter Engineering in . Metabolites. 2020; 10(8). PMC: 7466126. DOI: 10.3390/metabo10080320. View

15.

Yang H, Hu J, Lu X, Wang F, Shen W, Hu W . Improving extracellular protein production in Escherichia coli by overexpressing D,D-carboxypeptidase to perturb peptidoglycan network synthesis and structure. Appl Microbiol Biotechnol. 2018; 103(2):793-806. DOI: 10.1007/s00253-018-9510-7. View

16.

Morra R, Del Carratore F, Muhamadali H, Horga L, Halliwell S, Goodacre R . Translation Stress Positively Regulates MscL-Dependent Excretion of Cytoplasmic Proteins. mBio. 2018; 9(1). PMC: 5790912. DOI: 10.1128/mBio.02118-17. View

17.

Barreteau H, Kovac A, Boniface A, Sova M, Gobec S, Blanot D . Cytoplasmic steps of peptidoglycan biosynthesis. FEMS Microbiol Rev. 2008; 32(2):168-207. DOI: 10.1111/j.1574-6976.2008.00104.x. View

18.

Liu L, Yang H, Shin H, Chen R, Li J, Du G . How to achieve high-level expression of microbial enzymes: strategies and perspectives. Bioengineered. 2013; 4(4):212-23. PMC: 3728192. DOI: 10.4161/bioe.24761. View

19.

Pan X, Sun C, Tang M, Liu C, Zhang J, You J . Loss of Serine-Type D-Ala-D-Ala Carboxypeptidase DacA Enhances Prodigiosin Production in . Front Bioeng Biotechnol. 2019; 7:367. PMC: 6901396. DOI: 10.3389/fbioe.2019.00367. View

20.

Voss M, Schroder B, Fluhrer R . Mechanism, specificity, and physiology of signal peptide peptidase (SPP) and SPP-like proteases. Biochim Biophys Acta. 2013; 1828(12):2828-39. DOI: 10.1016/j.bbamem.2013.03.033. View