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Use of the lambda Red-recombineering method for genetic engineering of Pantoea ananatis.

ABSTRACT: BACKGROUND: Pantoea ananatis, a member of the Enterobacteriacea family, is a new and promising subject for biotechnological research. Over recent years, impressive progress in its application to L-glutamate production has been achieved. Nevertheless, genetic and biotechnological studies of Pantoea ananatis have been impeded because of the absence of genetic tools for rapid construction of direct mutations in this bacterium. The lambda Red-recombineering technique previously developed in E. coli and used for gene inactivation in several other bacteria is a high-performance tool for rapid construction of precise genome modifications. RESULTS: In this study, the expression of lambda Red genes in P. ananatis was found to be highly toxic. A screening was performed to select mutants of P. ananatis that were resistant to the toxic affects of lambda Red. A mutant strain, SC17(0) was identified that grew well under conditions of simultaneous expression of lambda gam, bet, and exo genes. Using this strain, procedures for fast introduction of multiple rearrangements to the Pantoea ananatis genome based on the lambda Red-dependent integration of the PCR-generated DNA fragments with as short as 40 bp flanking homologies have been demonstrated. CONCLUSION: The lambda Red-recombineering technology was successfully used for rapid generation of chromosomal modifications in the specially selected P. ananatis recipient strain. The procedure of electro-transformation with chromosomal DNA has been developed for transfer of the marked mutation between different P. ananatis strains. Combination of these techniques with lambda Int/Xis-dependent excision of selective markers significantly accelerates basic research and construction of producing strains.

SUBMITTER: Katashkina JI

PROVIDER: S-EPMC2682490 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Use of the lambda Red-recombineering method for genetic engineering of Pantoea ananatis.

Katashkina Joanna I JI Hara Yoshihiko Y Golubeva Lyubov I LI Andreeva Irina G IG Kuvaeva Tatiana M TM Mashko Sergey V SV

BMC molecular biology 20090423

<h4>Background</h4>Pantoea ananatis, a member of the Enterobacteriacea family, is a new and promising subject for biotechnological research. Over recent years, impressive progress in its application to L-glutamate production has been achieved. Nevertheless, genetic and biotechnological studies of Pantoea ananatis have been impeded because of the absence of genetic tools for rapid construction of direct mutations in this bacterium. The lambda Red-recombineering technique previously developed in E ...[more]

PMID: 19389224

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Project description:Cysteine is a commercially important amino acid; however, it lacks an efficient fermentative production method. Due to its cytotoxicity, intracellular cysteine levels are stringently controlled via several regulatory modes. Managing its toxic effects as well as understanding and deregulating the complexities of regulation are crucial for establishing the fermentative production of cysteine. The regulatory modes include feedback inhibition of key metabolic enzymes, degradation, efflux pumps, and the transcriptional regulation of biosynthetic genes by a master cysteine regulator, CysB. These processes have been extensively studied using Escherichia coli for overproducing cysteine by fermentation. In this study, we genetically engineered Pantoea ananatis, an emerging host for the fermentative production of bio-based materials, to identify key factors required for cysteine production. According to this and our previous studies, we identified a major cysteine desulfhydrase gene, ccdA (formerly PAJ_0331), involved in cysteine degradation, and the cysteine efflux pump genes cefA and cefB (formerly PAJ_3026 and PAJ_p0018, respectively), which may be responsible for downregulating the intracellular cysteine level. Our findings revealed that ccdA deletion and cefA and cefB overexpression are crucial factors for establishing fermentative cysteine production in P. ananatis and for obtaining a higher cysteine yield when combined with genes in the cysteine biosynthetic pathway. To our knowledge, this is the first demonstration of cysteine production in P. ananatis, which has fundamental implications for establishing overproduction in this microbe.IMPORTANCE The efficient production of cysteine is a major challenge in the amino acid fermentation industry. In this study, we identified cysteine efflux pumps and degradation pathways as essential elements and genetically engineered Pantoea ananatis, an emerging host for the fermentative production of bio-based materials, to establish the fermentative production of cysteine. This study provides crucial insights into the design and construction of cysteine-producing strains, which may play central roles in realizing commercial basis production.

Dataset Information

Use of the lambda Red-recombineering method for genetic engineering of Pantoea ananatis.

Publications

Use of the lambda Red-recombineering method for genetic engineering of Pantoea ananatis.

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