Enhancement of l-cysteine production by disruption of yciW in Escherichia coli

Kawano, Yusuke; Ohtsu, Iwao; Takumi, Kazuhiro; Tamakoshi, Ai; Nonaka, Gen; Funahashi, Eri; Ihara, Masaki; Takagi, Hiroshi

doi:10.1016/j.jbiosc.2014.07.006

Cited by 38 publications

(29 citation statements)

References 14 publications

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“…To characterize the activity of cefB as a cysteine efflux pump and to explore its possible application in fermentative cysteine production, we overexpressed cefB in a model P. ananatis cysteine-producing strain. We constructed a model strain that carried a feedback-insensitive mutant cysE gene, i.e., cysEX encoding a mutant SAT (harboring an amino acid substitution, T167A) from E. coli (20), which has been widely utilized for cysteine production (21,37,38). We employed cysEX among the many known mutant alleles of cysE to replicate the experimental conditions used in a previous study of other cysteine efflux pumps, EamA and EamB (15,39).…”

Section: Resultsmentioning

confidence: 99%

Fermentative Production of Cysteine by Pantoea ananatis

Takumi

Ziyatdinov

Samsonov

et al. 2017

Appl Environ Microbiol

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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.

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Section: Resultsmentioning

confidence: 99%

Fermentative Production of Cysteine by Pantoea ananatis

Takumi

Ziyatdinov

Samsonov

et al. 2017

Appl Environ Microbiol

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“…In E. coli, cysteine synthesis is regulated by SAT that is encoded by cysE and is subject to feedback inhibition by cysteine (5,6). Therefore, cells must express a feedback-insensitive SAT to overproduce cysteine (37)(38)(39). Moreover, enhancing the activity of efflux pumps exerts additional effects on the overproduction of cysteine (14,16,40).…”

Section: Resultsmentioning

confidence: 99%

Bacterial Cysteine-Inducible Cysteine Resistance Systems

Takumi

Nonaka

2016

J Bacteriol

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Cysteine donates sulfur to macromolecules and occurs naturally in many proteins. Because low concentrations of cysteine are cytotoxic, its intracellular concentration is stringently controlled. In bacteria, cysteine biosynthesis is regulated by feedback inhibition of the activities of serine acetyltransferase (SAT) and 3-phosphoglycerate dehydrogenase (3-PGDH) and is also regulated at the transcriptional level by inducing the cysteine regulon using the master regulator CysB. Here, we describe two novel cysteine-inducible systems that regulate the cysteine resistance of Pantoea ananatis, a member of the family Enterobacteriaceae that shows great potential for producing substances useful for biotechnological, medical, and industrial purposes. One locus, designated ccdA (formerly PAJ_0331), encodes a novel cysteine-inducible cysteine desulfhydrase (CD) that degrades cysteine, and its expression is controlled by the transcriptional regulator encoded by ccdR (formerly PAJ_0332 or ybaO), located just upstream of ccdA. The other locus, designated cefA (formerly PAJ_3026), encodes a novel cysteine-inducible cysteine efflux pump that is controlled by the transcriptional regulator cefR (formerly PAJ_3027), located just upstream of cefA. To our knowledge, this is the first example where the expression of CD and an efflux pump is regulated in response to cysteine and is directly involved in imparting resistance to excess levels of cysteine. We propose that ccdA and cefA function as safety valves that maintain homeostasis when the intra-or extracellular cysteine concentration fluctuates. Our findings contribute important insights into optimizing the production of cysteine and related biomaterials by P. ananatis. IMPORTANCEBecause of its toxicity, the bacterial intracellular cysteine level is stringently regulated at biosynthesis. This work describes the identification and characterization of two novel cysteine-inducible systems that regulate, through degradation and efflux, the cysteine resistance of Pantoea ananatis, a member of the family Enterobacteriaceae that shows great potential for producing substances useful for industrial purposes. We propose that this novel mechanism for sensing and regulating cysteine levels is a safety valve enabling adaptation to sudden changes in intra-or extracellular cysteine levels in bacteria. Our findings provide important insights into optimizing the production of cysteine and related biomaterials by P. ananatis and also a deep understanding of sulfur/cysteine metabolism and regulation in this plant pathogen and related bacteria.

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“…The yciW overexpressing cells showed tolerance in medium including excess Cys. Furthermore, the gene disruption improved the productivity of Cys in Cys-overproducing E. coli cells (8). These results suggest that the yciW product, YciW, converts Cys into Met or L-glutathione (GS).…”

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confidence: 93%

“…Also, most of the genes involved in the sulfur assimilation pathway are known to be coordinately regulated by a transcriptional factor CysB, which activates the regulons in response to cellular Cys shortage (7). Recently, we identified the yciW gene as a member of new CysB regulon gene by in silico analysis (8). To obtain the yciW gene (a DNA fragment of about 1140 bp), a polymerase chain reaction (PCR) was performed with a set of primers 5 0 -TCCGCTCGAGATGGAACAACGCCACATCAC-3 0 and 5 0 -GCTCTAGATTAATATGTCTCACCCAACGCG-3 0 (the underlining indicates the position of XhoI and XbaI, respectively) with the chromosomal DNA of the MG1655 strain (ATCC No.…”

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confidence: 99%