Kazuhiro Takumi

Enhancement of L-cysteine production by disruption of yciW in Escherichia coli.

Using in silico analysis, the yciW gene of Escherichia coli was identified as a novel L-cysteine regulon that may be regulated by the transcriptional activator CysB for sulfur metabolic genes. We found that overexpression of yciW conferred tolerance to L-cysteine, but disruption of yciW increased L-cysteine production in E. coli.

Involvement of the yciW gene in l-cysteine and l-methionine metabolism in Escherichia coli

We here analyzed a sulfur index of Escherichia coli using LC-MS/MS combined with thiol-specific derivatization by monobromobimane. The obtained sulfur index was then applied to evaluate the L-cysteine producer. E. coli cells overexpressing the yciW gene, a novel Cys regulon, accumulated l-homocysteine, suggesting that YciW is involved in L-methionine biosynthesis.

Bacterial Cysteine-Inducible Cysteine Resistance Systems.

UNLABELLED 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 byccdR(formerly PAJ_0332 orybaO), 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 IMPORTANCE Because 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.

Induction of the Escherichia coli yijE gene expression by cystine

Cystine is formed from two molecules of the cysteine under oxidized conditions, but is reversibly converted to cysteine by reduction. Growth of Escherichia coli is retarded in the presence of excess cystine. Transcriptome analysis showed 11 up-regulated and 26 down-regulated genes upon exposure to excess cystine. The reporter assay confirmed regulation by cystine of the expression of one up-regulated membrane gene, yijE, and two down-regulated membrane genes, yhdT and yihN. In order to identify the as yet unidentified gene encoding cystine efflux transporter, the putative cystine efflux candidate, yijE gene, was over-expressed. Expression of the yijE gene suppressed the slow growth of E. coli in the presence of high concentration of extracellular cystine. In good agreement, the knock-out of yijE gene increased the sensibility to cystine. These observations altogether imply that the yijE gene is involved in response to cystine in E. coli. Graphical Abstract The Escherichia coli yijE gene, induced by cystine, is involved in response to excess cystine.

Fermentative production of cysteine by Pantoea ananatis

ABSTRACT 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.

Cysteine degradation gene yhaM, encoding cysteine desulfidase, serves as a genetic engineering target to improve cysteine production in Escherichia coli

Cysteine is an important amino acid for various industries; however, there is no efficient microbial fermentation-based production method available. Owing to its cytotoxicity, bacterial intracellular levels of cysteine are stringently controlled via several modes of regulation, including cysteine degradation by cysteine desulfhydrases and cysteine desulfidases. In Escherichia coli, several metabolic enzymes are known to exhibit cysteine degradative activities, however, their specificity and physiological significance for cysteine detoxification via degradation are unclear. Relaxing the strict regulation of cysteine is crucial for its overproduction; therefore, identifying and modulating the major degradative activity could facilitate the genetic engineering of a cysteine-producing strain. In the present study, we used genetic screening to identify genes that confer cysteine resistance in E. coli and we identified yhaM, which encodes cysteine desulfidase and decomposes cysteine into hydrogen sulfide, pyruvate, and ammonium. Phenotypic characterization of a yhaM mutant via growth under toxic concentrations of cysteine followed by transcriptional analysis of its response to cysteine showed that yhaM is cysteine-inducible, and its physiological role is associated with resisting the deleterious effects of cysteine in E. coli. In addition, we confirmed the effects of this gene on the fermentative production of cysteine using E. coli-based cysteine-producing strains. We propose that yhaM encodes the major cysteine-degrading enzyme and it has the most significant role in cysteine detoxification among the numerous enzymes reported in E. coli, thereby providing a core target for genetic engineering to improve cysteine production in this bacterium.