Enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from S-sulfocysteine increases L-cysteine production in Escherichia coli.
Enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from S-sulfocysteine increases L-cysteine production in Escherichia coli.
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DOI：
10.1186/1475-2859-11-62
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发表时间：
2012-05-18
影响因子：
6.4
通讯作者：
Takagi H
中科院分区：
文献类型：
--
作者：
Nakatani T;Ohtsu I;Nonaka G;Wiriyathanawudhiwong N;Morigasaki S;Takagi H
大肠杆菌有两条l -半胱氨酸生物合成途径；一种是l -半胱氨酸合成酶（CysK）由o -乙酰基l -丝氨酸（OAS）和硫酸盐合成，另一种是SSC合成酶（CysM）由OAS和硫代硫酸盐合成s -硫代半胱氨酸（SSC）。SSC通过未表征的反应转化为l -半胱氨酸和亚硫酸盐。由于硫氧还毒素（Trx1和Trx2）和戊氧还毒素（Grx1、Grx2、Grx3、Grx4和NrdH）是肽基二硫化物的还原酶，因此过表达这些还原酶可能是提高l -半胱氨酸产量以加速大肠杆菌SSC还原的良好途径。由于氧化还原酶可以减少蛋白质上形成的二硫，我们首先测试了这些酶是否催化SSC还原为l -半胱氨酸。除Grx4外，所有his标记的重组酶都能在体外有效地将SSC转化为l -半胱氨酸。Grx1和NrdH的过表达使大肠杆菌的半胱氨酸产量增加了15-40%。另一方面，cysM基因的破坏抵消了Grx1和NrdH过表达造成的影响，表明其改善是由于发酵条件下SSC的有效还原。此外，亚硫酸盐还原酶基因（ΔcysI和ΔcysJ）和l -半胱氨酸合酶基因（ΔcysK）的敲除突变体的l -半胱氨酸产量均下降至野生型菌株的50%左右。有趣的是，野生型菌株与亚硫酸盐上游途径基因缺失突变体（ΔcysC或ΔcysH）之间的l -半胱氨酸产量没有显著差异。这些结果表明，SSC还原生成的亚硫酸盐可以作为硫源生成额外的l -半胱氨酸分子。最后发现，在共过表达glutaredoxin （NrdH）、亚硫酸盐还原酶（CysI）和l -半胱氨酸合成酶（CysK）的大肠杆菌中，每个细胞产生的l -半胱氨酸量最高。在这项工作中，我们发现Grx1和NrdH将SSC还原为l -半胱氨酸，然后产生的亚硫酸盐作为硫源，在大肠杆菌中通过硫酸盐途径产生额外的l -半胱氨酸分子。我们还发现NrdH、CysI和CysK的共同过表达增加了l -半胱氨酸的产生。我们的研究结果表明，增强硫氧还蛋白/谷胱甘肽介导的l -半胱氨酸合成是一种提高l -半胱氨酸产量的新方法。
Escherichia 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. Because 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. In 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.