Frontiers in Bioengineering and Biotechnology
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2019
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IF 5.122
Pan Xuewei,Sun Changhao,Tang Mi,Liu Chao,Zhang Jianing,You Jiajia,Osire Tolbert,Sun Yang,Zhao Youxi,Xu Meijuan,Yang Taowei,Rao Zhiming
Serratia marcescens, a gram-negative bacterium, found in a wide range of ecological niches can produce several high-value products, including prodigiosin, althiomycin, and serratamolide. Among them, prodigiosin has attracted attention due to its immunosuppressive, antimicrobial, and anticancer properties. However, the regulatory mechanisms behind prodigiosin synthesis in Serratia marcescens remains limited. Here, a transposon mutant library was constructed to identify the genes related to prodigiosin synthesis, and BVG90_02415 gene encoding a peptidoglycan synthesizing enzyme D-Ala-D-Ala carboxypeptidase DacA was found to negatively regulates prodigiosin synthesis. Quantitative measurements revealed that disruption of dacA increased prodigiosin production 1.46-fold that of the wild-type strain JNB5-1 in fermentation medium. By comparing differences in cell growth, pigA gene expression level, cell morphology, membrane permeability, and intracellular prodigiosin concentration between wild-type strain JNB5-1 and dacA mutant SK4-72, results revealed that the mechanism for hyper-producing of prodigiosin by the dacA mutant was probably that dacA disruption enhanced prodigiosin leakage, which in turn alleviated feedback inhibition of prodigiosin and increased expression of pig gene cluster. Collectively, this work provides a novel insight into regulatory mechanisms of prodigiosin synthesis and uncovers new roles of DacA protein in regulating cell growth, cell morphology, and membrane permeability in Serratia marcescens. Finally, this study offers a new strategy for improving production of high-value compounds in Serratia marcescens.
研究领域:
微生物学
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生物化学
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分子生物学
✓ 已验证
DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2018
·Vol. 84
·期 20
·
IF 3.633
Haiquan Yang,Xiao Lu,Jinyuan Hu,Yuan Chen,Wei Shen,Long Liu
Escherichia coli is one of the most widely used host microorganisms for recombinant protein expression and metabolic engineering, but it cannot efficiently secrete recombinant proteins to extracellular space. Here, extracellular protein secretion was enhanced in E. coli by deleting two d,d-carboxypeptidase genes (dacA and dacB, single and double deletions) to perturb the cell wall peptidoglycan network. Deletion of dacA and dacB enhanced the accumulation of intracellular soluble peptidoglycan in E. coli and affected cell morphology, resulting in a more irregular cell shape and the appearance of transparent bulges. Deletion of dacA and dacB appears to disrupt the normal rigid structure, presumably due to perturbation and destruction of the cell wall peptidoglycan network. The extracellular green fluorescent protein (GFP) fluorescence intensity of deletion mutants was increased by >2.0-fold compared with that of control cells, and that of the double deletion mutant was increased by 2.7-fold. Extracellular recombinant fibroblast growth factor receptor 2 (FGFR2) and collagen E4 secretion in deletion mutants was also enhanced compared with that in the control cells. Additionally, the extracellular recombinant amylase activity of single-deletion mutants BL21 ΔdacA pETDuet-amyk and BL21 ΔdacB pETDuet-amyk was increased 2.5- and 3.1-fold, respectively. The extracellular distribution of α-galactosidase by deletion mutants was also increased by >2.0-fold. Deletion of dacA and dacB increased outer membrane permeability, which could explain the enhanced extracellular protein secretion.IMPORTANCE Cell surface structure stabilization is important for extracellular secretion of proteins in Escherichia coli. As the main constituent of the cell wall, peptidoglycan contributes to cell structure robustness and stability. Here, we perturbed the peptidoglycan network by deleting dacA and dacB genes encoding d,d-carboxypeptidase enzymes to improve extracellular protein secretion. This new strategy could enhance the capacity of E. coli as a microbial cell factory for extracellular secretion of proteins and chemicals.
研究领域:
生物工程
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微生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
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2018
·Vol. 103
·期 2
·793-806
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IF 3.34
Yang Haiquan,Hu Jinyuan,Lu Xiao,Wang Fuxiang,Shen Wei,Hu Wei,Wang Lingling,Chen Xianzhong,Liu Long
Most recombinant proteins in Escherichia coli are not efficiently secreted to the extracellular space. Structural stabilisation of the cell wall is essential for extracellular protein production in E. coli , for which D,D-carboxypeptidases are essential. Herein, we perturbed the peptidoglycan structure of the E. coli cell wall by overexpressing D,D-carboxypeptidase genes dacA or dacB , and investigated the effect on extracellular protein production. Overexpression of dacA or dacB promoted the accumulation of intracellular soluble peptidoglycan, altered cell morphology (shape and size) and led to the formation of transparent globular structures in E. coli cells. Compared with controls (CK), extracellular production of recombinant green fluorescent protein (GFP) was increased by 1.7- and 2.3-fold upon overexpression of dacA and dacB , respectively. Similarly, extracellular production of recombinant amylase and α-galactosidase was increased by 4.5- and 2.8-fold, respectively, upon overexpression of dacA , and by 11.9- and 2.5-fold, respectively, upon overexpression of dacB . Overexpression of dacA or dacB enhanced both the outer and inner membrane permeability of E. coli . This cell wall engineering strategy opens up a new direction for enhancing extracellular protein and chemical production in E. coli .
研究领域:
微生物学
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生物技术
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分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
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