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Development of a counterselectable seamless mutagenesis system in lactic acid bacteria.

ABSTRACT: Lactic acid bacteria (LAB) are receiving more attention to act as cell factories for the production of high-value metabolites. However, the molecular tools for genetic modifying these strains are mainly vector-based double-crossover strategies, which are laborious and inefficient. To address this problem, several counterselectable markers have been developed, while few of them could be used in the wild-type host cells without pretreatment.The pheS gene encoding phenylalanyl-tRNA synthetase alpha subunit was identified in Lactococcus lactis NZ9000 genome. When mutant pheS gene (pheS*) under the control of the Lc. lactis NZ9000 L-lactate dehydrogenase promoter (Pldh) was expressed from a plasmid, the resulted PheS* with an A312G substitution rendered cells sensitive to the phenylalanine analog p-chloro-phenylalanine (p-Cl-Phe). This result suggested pheS* was suitable to be used as a counterselectable marker in Lc. lactis. However, the expression level of pheS* from a chromosomal copy was too low to confer p-Cl-Phe sensitivity. Therefore, a strategy of cascading promoters was attempted for strengthening the expression level of pheS*. Expectedly, a cassette 5Pldh-pheS* with five tandem repetitive promoters Pldh resulted in a sensitivity to 15 mM p-Cl-Phe. Subsequently, a counterselectable seamless mutagenesis system PheS*/pG+host9 based on a temperature-sensitive plasmid pG+host9 harboring a 5Pldh-pheS* cassette was developed in Lc. lactis. We also demonstrated the possibility of applying pheS* to be a counterselectable marker in Lactobacillus casei BL23.As reported in E. coli, pheS* as a counterselectable marker has been demonstrated to be functional in targeted gene(s) deletion in Lc. lactis as well as in L. casei. Moreover, the efficiency and timesaving counterselectable seamless mutagenesis system PheS*/pG+host9 could be used in the wild-type host cells without pretreatment.

SUBMITTER: Xin Y

PROVIDER: S-EPMC5499019 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Development of a counterselectable seamless mutagenesis system in lactic acid bacteria.

Xin Yongping Y Guo Tingting T Mu Yingli Y Kong Jian J

Microbial cell factories 20170705 1

<h4>Background</h4>Lactic acid bacteria (LAB) are receiving more attention to act as cell factories for the production of high-value metabolites. However, the molecular tools for genetic modifying these strains are mainly vector-based double-crossover strategies, which are laborious and inefficient. To address this problem, several counterselectable markers have been developed, while few of them could be used in the wild-type host cells without pretreatment.<h4>Results</h4>The pheS gene encoding ...[more]

PMID: 28679374

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Project description:BACKGROUND: Lactic acid bacteria (LAB) are a group of gram-positive, lactic acid producing Firmicutes. They have been extensively used in food fermentations, including the production of various dairy products. The proteolytic system of LAB converts proteins to peptides and then to amino acids, which is essential for bacterial growth and also contributes significantly to flavor compounds as end-products. Recent developments in high-throughput genome sequencing and comparative genomics hybridization arrays provide us with opportunities to explore the diversity of the proteolytic system in various LAB strains. RESULTS: We performed a genome-wide comparative genomics analysis of proteolytic system components, including cell-wall bound proteinase, peptide transporters and peptidases, in 22 sequenced LAB strains. The peptidase families PepP/PepQ/PepM, PepD and PepI/PepR/PepL are described as examples of our in silico approach to refine the distinction of subfamilies with different enzymatic activities. Comparison of protein 3D structures of proline peptidases PepI/PepR/PepL and esterase A allowed identification of a conserved core structure, which was then used to improve phylogenetic analysis and functional annotation within this protein superfamily.The diversity of proteolytic system components in 39 Lactococcus lactis strains was explored using pangenome comparative genome hybridization analysis. Variations were observed in the proteinase PrtP and its maturation protein PrtM, in one of the Opp transport systems and in several peptidases between strains from different Lactococcus subspecies or from different origin. CONCLUSIONS: The improved functional annotation of the proteolytic system components provides an excellent framework for future experimental validations of predicted enzymatic activities. The genome sequence data can be coupled to other "omics" data e.g. transcriptomics and metabolomics for prediction of proteolytic and flavor-forming potential of LAB strains. Such an integrated approach can be used to tune the strain selection process in food fermentations.

Dataset Information

Development of a counterselectable seamless mutagenesis system in lactic acid bacteria.

Publications

Development of a counterselectable seamless mutagenesis system in lactic acid bacteria.

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