Enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from S-sulfocysteine increases L-cysteine production in Escherichia coli

Abstract

BackgroundEscherichia coli has two L-cysteine biosynthetic pathways; one is synthesized from O-acetyl L-serine (OAS) and sulfate by L-cysteine synthase (CysK), and another is produced via S-sulfocysteine (SSC) from OAS and thiosulfate by SSC synthase (CysM). SSC is converted into L-cysteine and sulfite by an uncharacterized reaction. As thioredoxins (Trx1 and Trx2) and glutaredoxins (Grx1, Grx2, Grx3, Grx4, and NrdH) are known as reductases of peptidyl disulfides, overexpression of such reductases might be a good way for improving L-cysteine production to accelerate the reduction of SSC in E. coli.ResultsBecause the redox enzymes can reduce the disulfide that forms on proteins, we first tested whether these enzymes catalyze the reduction of SSC to L-cysteine. All His-tagged recombinant enzymes, except for Grx4, efficiently convert SSC into L-cysteine in vitro. Overexpression of Grx1 and NrdH enhanced a 15-40% increase in the E. coliL-cysteine production. On the other hand, disruption of the cysM gene cancelled the effect caused by the overexpression of Grx1 and NrdH, suggesting that its improvement was due to the efficient reduction of SSC under the fermentative conditions. Moreover, L-cysteine production in knockout mutants of the sulfite reductase genes (ΔcysI and ΔcysJ) and the L-cysteine synthase gene (ΔcysK) each decreased to about 50% of that in the wild-type strain. Interestingly, there was no significant difference in L-cysteine production between wild-type strain and gene deletion mutant of the upstream pathway of sulfite (ΔcysC or ΔcysH). These results indicate that sulfite generated from the SSC reduction is available as the sulfur source to produce additional L-cysteine molecule. It was finally found that in the E. coliL-cysteine producer that co-overexpress glutaredoxin (NrdH), sulfite reductase (CysI), and L-cysteine synthase (CysK), there was the highest amount of L-cysteine produced per cell.ConclusionsIn this work, we showed that Grx1 and NrdH reduce SSC to L-cysteine, and the generated sulfite is then utilized as the sulfur source to produce additional L-cysteine molecule through the sulfate pathway in E. coli. We also found that co-overexpression of NrdH, CysI, and CysK increases L-cysteine production. Our results propose that the enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from SSC is a novel method for improvement of L-cysteine production.

Isozyme-Specific Ligands for O-acetylserine sulfhydrylase, a Novel Antibiotic Target

“…The knowledge of the relative abundance and regulation by effectors of the two isozymes during infection is a relevant missing information that can contribute to the pharmacological exploitation of these targets. For example, it has been recently shown that the activity of OASS-B on thiosulfate could represent an energy saving path to cysteine biosynthesis and could be preferred in metabolic conditions where the conservation of ATP and NADPH is important [117]. In addition, very recently, works by Hayes and coworkers [118], [119], identified OASS as the activating factor for a toxin that controls contact-dependent growth inhibition in E.coli .…”

CysK2 from Mycobacterium tuberculosis Is an O -Phospho- l -Serine-Dependent S -Sulfocysteine Synthase

“…Formation of S-sulfocysteine from thiosulfate would thus provide a salvage pathway whereby some of the sulfur could be rescued by incorporation into L-cysteine. S-sulfocysteine, generated from thiosulfate by S-sulfocysteine synthases, has been postulated as an intermediate of an alternative route to L-cysteine biosynthesis in Rhodospirillum tenue (31), Salmonella enterica serovar Typhimurium (32), and E. coli (33). In the last case, it was shown that thioredoxin and/or NrdH is involved in the reduction of S-sulfocysteine to cysteine and sulfite.…”

Cysteine synthase A overexpression in Corynebacterium glutamicum enhances l‐isoleucine production

“…CysK catalyzes the final reaction of l ‐cysteine synthesis , and its overexpression could enhance the productivity of l ‐cysteine and serine‐rich proteins . On the other hand, CysK is also related to multiple physiological processes, including contact‐dependent growth inhibition , quorum sensing , and stress response .…”

“…In E. coli and Salmonella typhimurium , CysK is composed of two identical 34 kDa subunits ; cysK and other genes relevant to the l ‐cysteine biosynthesis are scattered on the chromosome . Overexpression of CysK could enhance productivity of l ‐cysteine and serine‐rich proteins in E. coli . CysK also plays an essential role in bacterial contact‐dependent growth inhibition .…”