Kawano Yusuke, Onishi Fumito, Shiroyama Maeka, Miura Masashi, Tanaka Naoyuki, Oshiro Satoshi, Nonaka Gen, Nakanishi Tsuyoshi, Ohtsu Iwao
Innovation Medical Research Institute, University of Tsukuba, Tsukuba, Japan.
Research Institute for Bioscience Products and Fine Chemicals, Ajinomoto Co., Kawasaki, Japan.
Appl Microbiol Biotechnol. 2017 Sep;101(18):6879-6889. doi: 10.1007/s00253-017-8420-4. Epub 2017 Jul 29.
Sulfate (SO) is an often-utilized and well-understood inorganic sulfur source in microorganism culture. Recently, another inorganic sulfur source, thiosulfate (SO), was proposed to be more advantageous in microbial growth and biotechnological applications. Although its assimilation pathway is known to depend on O-acetyl-L-serine sulfhydrylase B (CysM in Escherichia coli), its metabolism has not been extensively investigated. Therefore, we aimed to explore another yet-unidentified CysM-independent thiosulfate assimilation pathway in E. coli. ΔcysM cells could accumulate essential L-cysteine from thiosulfate as the sole sulfur source and could grow, albeit slowly, demonstrating that a CysM-independent thiosulfate assimilation pathway is present in E. coli. This pathway is expected to consist of the initial part of the thiosulfate to sulfite (SO) conversion, and the latter part might be shared with the final part of the known sulfate assimilation pathway [sulfite → sulfide (S) → L-cysteine]. This is because thiosulfate-grown ΔcysM cells could accumulate a level of sulfite and sulfide equivalent to that of wild-type cells. The catalysis of thiosulfate to sulfite is at least partly mediated by thiosulfate sulfurtransferase (GlpE), because its overexpression could enhance cellular thiosulfate sulfurtransferase activity in vitro and complement the slow-growth phenotype of thiosulfate-grown ΔcysM cells in vivo. GlpE is therefore concluded to function in the novel CysM-independent thiosulfate assimilation pathway by catalyzing thiosulfate to sulfite. We applied this insight to L-cysteine overproduction in E. coli and succeeded in enhancing it by GlpE overexpression in media containing glucose or glycerol as the main carbon source, by up to ~1.7-fold (1207 mg/l) or ~1.5-fold (1529 mg/l), respectively.
硫酸盐（SO）是微生物培养中常用且已被充分了解的无机硫源。最近，另一种无机硫源硫代硫酸盐（SO）被认为在微生物生长和生物技术应用中更具优势。尽管已知其同化途径依赖于O-乙酰-L-丝氨酸巯基酶B（大肠杆菌中的CysM），但其代谢尚未得到广泛研究。因此，我们旨在探索大肠杆菌中另一种尚未确定的不依赖CysM的硫代硫酸盐同化途径。ΔcysM细胞能够以硫代硫酸盐作为唯一硫源积累必需的L-半胱氨酸并生长，尽管生长缓慢，这表明大肠杆菌中存在不依赖CysM的硫代硫酸盐同化途径。该途径预计由硫代硫酸盐转化为亚硫酸盐（SO）的初始部分组成，而后半部分可能与已知硫酸盐同化途径的最后部分共享[亚硫酸盐→硫化物（S）→L-半胱氨酸]。这是因为以硫代硫酸盐生长的ΔcysM细胞能够积累与野生型细胞相当水平的亚硫酸盐和硫化物。硫代硫酸盐向亚硫酸盐的催化至少部分由硫代硫酸盐硫转移酶（GlpE）介导，因为其过表达能够在体外增强细胞硫代硫酸盐硫转移酶活性，并在体内补充以硫代硫酸盐生长的ΔcysM细胞的缓慢生长表型。因此得出结论，GlpE通过催化硫代硫酸盐转化为亚硫酸盐而在新的不依赖CysM的硫代硫酸盐同化途径中发挥作用。我们将这一见解应用于大肠杆菌中L-半胱氨酸的过量生产，并通过在以葡萄糖或甘油作为主要碳源的培养基中过表达GlpE成功地将其提高，分别提高了约1.7倍（1207 mg/l）或约1.5倍（1529 mg/l）。
