Engineering of Escherichia colil-serine O-acetyltransferase on the basis of crystal structure: desensitization to feedback inhibition by l-cysteine

Yan, Yaw Kai; Kashiwagi, T.; Ishikawa, K.; Ziyatdinov, Mikhail Kharisovich; Redkina, E.I.; Kiriukhin, Michael Y.; Gusyatiner, M. M.; Kobayashi, Shinichiro; Takagi, Hiroshi; Suzuki, E

doi:10.1093/protein/gzj015

Cited by 25 publications

(21 citation statements)

References 11 publications

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“…To investigate the importance of the major CD in P. ananatis for cysteine production and to explore its possible applications in fermentative cysteine production, we overexpressed and deleted ccdA in a model P. ananatis cysteineproducing strain (CYS2-1 and CYS3-1 for overexpression and deletion, respectively) carrying the feedback-insensitive mutant cysE gene, i.e., cysE5 encoding a mutant SAT (containing amino acid substitutions V95R and D96P) from E. coli (19). We observed strong negative effects on the production of cysteine when ccdA was overexpressed by use of pACYC-ccdA (strain CYS2-2), which is consistent with its functional characteristic as a dominant CD with a major role in cysteine degradation (Table 1).…”

Section: Resultsmentioning

confidence: 99%

“…We constructed a P. ananatis-based producer strain, AG4854 (engineered from SC17), for further analysis. This strain AG4854 was deregulated with respect to two important key enzymes, SAT and 3-PGDH, by introducing genes encoding the feedback resistant mutants cysE5 (19) from E. coli harboring amino acid substitutions V95R and D96P, and serA348 (18) from P. ananatis harboring an amino acid substitution, N348A. We employed the mutant allele cysE5 instead of cysEX because it had advantages in terms of its enzymatic parameters in vitro as well as its growth phenotypes when introduced into the producer strains (see the Discussion for further details).…”

Section: Resultsmentioning

confidence: 99%

“…Enhancing cysteine efflux pumps is also crucial for downregulating the intracellular level of cysteine to avoid any toxic effects and for secreting cysteine into the fermentation medium to facilitate further isolation processes (14)(15)(16)(17). E. coli has been extensively studied as a host strain for the fermentative production of cysteine, where previous studies have identified core factors related to cysteine production, i.e., the deregulation of feedback loops by introducing a feedback inhibition-insensitive mutant for 3-PGDH (18) and SAT (19,20), overexpression of cysteine efflux pumps (14)(15)(16)(17)21), and disruption of CDs (12,13).…”

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confidence: 99%

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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%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Fermentative Production of Cysteine by Pantoea ananatis

Takumi

Ziyatdinov

Samsonov

et al. 2017

Appl Environ Microbiol

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“…To determine whether cefA gene product-mediated efflux of cysteine affected the enhancement of cefA expression on the overproduction of cysteine, we employed cysteine-producing strains. 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).…”

Section: Resultsmentioning

confidence: 99%

“…Several modes of regulation at different levels have been identified, reflecting its significance as a thiol-containing molecule for cellular functions, as well as its deleterious effects, requiring careful handling. Escherichia coli regulates cysteine levels through multiple mechanisms that primarily involve biosynthesis through feedback inhibition of serine acetyltransferase (SAT) and 3-phosphoglycerate dehydrogenase (3-PGDH) by cysteine and serine, respectively (5,6). The master regulator CysB organizes the CysB regulon by controlling the transcription of most of the genes that encode proteins involved in sulfur assimilation, including the components of sulfur transport systems and the cysteine biosynthetic pathway (7)(8)(9).…”

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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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Selective Drug Targets in Parasites

Kohler

Marhöfer²

2009

Antiparasitic and Antibacterial Drug Discovery

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Engineering of Escherichia colil-serine O-acetyltransferase on the basis of crystal structure: desensitization to feedback inhibition by l-cysteine

Cited by 25 publications

References 11 publications

Fermentative Production of Cysteine by Pantoea ananatis

Fermentative Production of Cysteine by Pantoea ananatis

Bacterial Cysteine-Inducible Cysteine Resistance Systems

Selective Drug Targets in Parasites

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