Effect of cysteine desulfhydrase gene disruption on l-cysteine overproduction in Escherichia coli

Awano, Naoki; Wada, Masaaki; Kohdoh, A.; Oikawa, Tadao; Takagi, Hiroshi; Nakamori, Shigeru

doi:10.1007/s00253-003-1262-2

Cited by 63 publications

(65 citation statements)

References 23 publications

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“…With the others, a correlation to L-serine is probably less apparent (see Discussion). Most interestingly, the cystathionine ␤-lyase mRNA level (metC) is increased 2.4-fold, and the respective enzyme of L-methionine synthesis (24), catalyzing a ␤-elimination reaction, has been reported to have a broad substrate specificity in C. glutamicum and in E. coli, also reacting with L-cysteine, which is structurally related to L-serine (1,45,59).…”

Section: Resultsmentioning

confidence: 99%

Cometabolism of a Nongrowth Substrate:l-Serine Utilization byCorynebacterium glutamicum

Netzer

Peters‐Wendisch

Eggeling

et al. 2004

Appl Environ Microbiol

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C-labeled L-serine and analysis of cellularly derived metabolites by nuclear magnetic resonance spectroscopy revealed that the carbon skeleton of L-serine is mainly converted to pyruvate-derived metabolites such as L-alanine. The sdaA gene was identified in the genome of C. glutamicum, and overexpression of sdaA resulted in (i) functional L-serine dehydratase (L-SerDH) activity, and therefore conversion of L-serine to pyruvate, and (ii) growth of the recombinant strain on L-serine as the single substrate. In contrast, deletion of sdaA decreased the L-serine cometabolism rate with glucose by 47% but still resulted in degradation of L-serine to pyruvate. Cystathionine ␤-lyase was additionally found to convert L-serine to pyruvate, and the respective metC gene was induced 2.4-fold under high internal L-serine concentrations. Upon sdaA overexpression, the growth rate on glucose is reduced 36% from that of the wild type, illustrating that even with glucose as a single substrate, intracellular L-serine conversion to pyruvate might occur, although probably the weak affinity of L-SerDH (apparent K m , 11 mM) prevents substantial L-serine degradation.Cometabolism is often observed for xenobiotic compounds which do not enable growth as a single carbon-and energy source (21). This is due, for example, to long degradation pathways and unnatural structures. On the other hand, microorganisms with a restricted metabolism, such as Lactococcus lactis, are dependent on cometabolism of essential natural compounds, e.g., amino acids (35). The amino acid L-serine is characterized by the fact that several organisms have the ability to introduce it into the central metabolism via pyruvate (35,2,16). Another distinguishing feature is its high cellular demand, exceeding the simple provision of L-serine for protein synthesis, since L-serine is additionally required for glycine, cysteine, tryptophan, and phospholipid synthesis as well as for 1-carbonunit generation (52). Glycine, in turn, is a precursor for purines and heme. In Corynebacterium glutamicum about 7.5% of the total carbon flux toward L-serine is utilized for these purposes (28). Prior estimates for Escherichia coli determined that as much as 15% of the carbon assimilated from glucose involves L-serine (42). Due to the high demand and its key position in the precursor supply, L-serine has to be regarded as an intermediate of the central metabolism (52).In spite of the presence of L-serine dehydratase (L-SerDH) (47) and the key position of L-serine, growth of E. coli on L-serine as a carbon source is very poor, allowing doubling times of about 60 h only (63). When the organism is additionally exposed to low concentrations of glycine, isoleucine, and threonine, growth is enhanced (36). Interestingly, during growth on tryptone broth, where a number of amino acids are present, L-serine is utilized immediately and earlier than any other amino acid (43). Taken together, L-serine is clearly a poor growth substrate for E. coli and is preferably cometabolized. Klebsiella aerogenes and ...

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Section: Resultsmentioning

confidence: 99%

Cometabolism of a Nongrowth Substrate:l-Serine Utilization byCorynebacterium glutamicum

Netzer

Peters‐Wendisch

Eggeling

et al. 2004

Appl Environ Microbiol

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“…The cultures were supplemented with 30 mM cysteine as required. Native PAGE and analysis of CD activity were performed as described previously (28). Total protein concentrations were analyzed using a standard Coomassie protein assay system; 2 g of the total protein was loaded in each lane.…”

Section: Methodsmentioning

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 assay was specific for cysteine, since methionine, glutathione, cystine, and dithiothreitol (DTT) did not generate sulfide. Only FeSO 4 , and not FeCl 3 or FeCl 2 , generated sulfide. Sulfide did not result from sulfate reduction, since mutants blocked in the reduction pathways (TO5 [⌬asrA], TO6 [⌬cysJ], and TO7 [⌬sbp]) still generated sulfide.…”

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confidence: 99%

“…Assay 1 measured methylene blue formation at 670 nm (58). Methylene blue forms when N=,N=-dimethyl-p-phenylenediamine reacts with sulfide in the presence of FeCl 3 . One unit is defined as the amount catalyzing the formation of 1 mol sulfide min Ϫ1 .…”

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confidence: 99%

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Cysteine Catabolism and Cysteine Desulfhydrase (CdsH/STM0458) in Salmonella enterica Serovar Typhimurium

2012

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Cysteine is potentially toxic and can affect diverse functions such as oxidative stress, antibiotic resistance, and swarming motility. The contribution of cysteine catabolism in modulating responses to cysteine has not been examined, in part because the genes have not been identified and mutants lacking these genes have not been isolated or characterized. We identified the gene for a previously described cysteine desulfhydrase, which we designated cdsH (formerly STM0458). We also identified a divergently transcribed gene that regulates cdsH expression, which we designated cutR (formerly ybaO, or STM0459). CdsH appears to be the major cysteine-degrading and sulfide-producing enzyme aerobically but not anaerobically. Mutants with deletions of cdsH and ybaO exhibited increased sensitivity to cysteine toxicity and altered swarming motility but unaltered cysteine-enhanced antibiotic resistance and survival in macrophages. Cysteine desulfhydrase (CDS) degrades cysteine to pyruvate, ammonia, and sulfide (19,26,43). The major CDS in Escherichia coli appears to be tryptophanase (TnaA), which has an apparent K m for cysteine of 11 mM (56). TnaA primarily catabolizes tryptophan, which appears to be abundant in the intestinal tract (29). In E. coli, TnaA can become 10% of soluble protein, which suggests the potential to also degrade cysteine in vivo, despite an unfavorable K m (56). Several factors regulate TnaA expression. TnaA is repressed by glucose, pyruvate, and acetate (6, 9, 10, 31). Expression requires cyclic AMP and is induced by tryptophan, cysteine, indole, growth in alkaline broth, depletion of heme and phosphatidyl glycerol, and anaerobic growth with an alternate electron acceptor (9, 51, 52). From these factors, it could be speculated that TnaA contributes to energy generation by degrading tryptophan and possibly cysteine to pyruvate.Salmonella enterica does not have TnaA, yet cysteine induces a powerful CDS (19,26,43). This CDS has been purified from S. enterica, and similarly to TnaA, it contains pyridoxal-5=-phosphate (42). The k cat /K m ratio for L-cysteine, a measure of catalytic efficiency, is 10,340 mM Ϫ1 s Ϫ1 for S. enterica CDS (calculated from values reported by Kredich et al. [42,43]) and 6.7 mM Ϫ1 s Ϫ1for E. coli TnaA (calculated from values reported by Snell [56]). In other words, the S. enterica CDS appears to be more than three orders of magnitude more efficient than TnaA. To explore the function of the S. enterica CDS and cysteine catabolism, we identified the gene for the major CDS and characterized its regulation and the phenotype of mutants lacking this enzyme. MATERIALS AND METHODSStrains and plasmids. Table 1 lists the strains and plasmids used in this study. All gene deletions and replacements were performed originally into the TT22971 background strain as described previously (21), followed by P22 transduction into the appropriate genetic background, and the products were ensured to be phage free by cross-streaking against P22-H5 on green plate agar (15). The following codons were delet...

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Effect of cysteine desulfhydrase gene disruption on l-cysteine overproduction in Escherichia coli

Cited by 63 publications

References 23 publications

Cometabolism of a Nongrowth Substrate:l-Serine Utilization byCorynebacterium glutamicum

Cometabolism of a Nongrowth Substrate:l-Serine Utilization byCorynebacterium glutamicum

Bacterial Cysteine-Inducible Cysteine Resistance Systems

Cysteine Catabolism and Cysteine Desulfhydrase (CdsH/STM0458) in Salmonella enterica Serovar Typhimurium

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