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Pantoea ananatis carotenoid production confers toxoflavin tolerance and is regulated by Hfq-controlled quorum sensing.

ABSTRACT: Carotenoids are widely used in functional foods, cosmetics, and health supplements, and their importance and scope of use are continuously expanding. Here, we characterized carotenoid biosynthetic genes of the plant-pathogenic bacterium Pantoea ananatis, which carries a carotenoid biosynthetic gene cluster (including crtE, X, Y, I, B, and Z) on a plasmid. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that the crtEXYIB gene cluster is transcribed as a single transcript and crtZ is independently transcribed in the opposite direction. Using splicing by overlap extension with polymerase chain reaction (SOE by PCR) based on asymmetric amplification, we reassembled crtE-B, crtE-B-I, and crtE-B-I-Y. High-performance liquid chromatography confirmed that Escherichia coli expressing the reassembled crtE-B, crtE-B-I, and crtE-B-I-Y operons produced phytoene, lycopene, and ?-carotene, respectively. We found that the carotenoids conferred tolerance to UV radiation and toxoflavin. Pantoea ananatis shares rice environments with the toxoflavin producer Burkholderia glumae and is considered to be the first reported example of producing and using carotenoids to withstand toxoflavin. We confirmed that carotenoid production by P. ananatis depends on RpoS, which is positively regulated by Hfq/ArcZ and negatively regulated by ClpP, similar to an important regulatory network of E. coli (Hfq^ArcZ ?RpoS ? ClpXP). We also demonstrated that Hfq-controlled quorum signaling de-represses EanR to activate RpoS, thereby initiating carotenoid production. Survival genes such as those responsible for the production of carotenoids of the plant-pathogenic P. ananatis must be expressed promptly to overcome stressful environments and compete with other microorganisms. This mechanism is likely maintained by a brake with excellent performance, such as EanR.

SUBMITTER: Choi O

PROVIDER: S-EPMC7883899 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Pantoea ananatis carotenoid production confers toxoflavin tolerance and is regulated by Hfq-controlled quorum sensing.

Choi Okhee O Kang Byeongsam B Lee Yongsang Y Lee Yeyeong Y Kim Jinwoo J

MicrobiologyOpen 20201202 1

Carotenoids are widely used in functional foods, cosmetics, and health supplements, and their importance and scope of use are continuously expanding. Here, we characterized carotenoid biosynthetic genes of the plant-pathogenic bacterium Pantoea ananatis, which carries a carotenoid biosynthetic gene cluster (including crtE, X, Y, I, B, and Z) on a plasmid. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that the crtEXYIB gene cluster is transcribed as a single transcrip ...[more]

PMID: 33269542

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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

Pantoea ananatis carotenoid production confers toxoflavin tolerance and is regulated by Hfq-controlled quorum sensing.

Publications

Pantoea ananatis carotenoid production confers toxoflavin tolerance and is regulated by Hfq-controlled quorum sensing.

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