No abstract

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.

l -Cysteine (Cys) is metabolically fundamental sulfur compound and important components in various cellular factors. Interestingly, free-form Cys itself as a simple monomeric amino acid was recently shown to function in a novel antioxidative system ( cysteine / cystine shuttle system ) in Escherichia coli . However, as for Cys-containing dipeptides, the biological functions, effects, and even contents have still remained largely elusive. The potential functions should be a part of cellular redox system and important in basic and applied biology. For its progress, establishment of reliable quantitation method is the first. However, such accurate analysis is unexpectedly difficult even in Cys, because thiol compounds convert through disulfide-exchange and air oxidation during sample preparation. Addressing this problem, in this study, thiol molecules like Cys-containing dipeptides were derivatized by using monobromobimane (thiol-specific alkylating reagent) and detected as S -bimanyl derivatives by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). Sample separation was processed with a C18 column (2.1 mm × 150 mm, 1.7 μm) and with water-acetonitrile gradient mobile phase containing 0.1% (v/v) formic acid at flow rate of 0.25 ml/min. The mass spectrometer was operated in the multiple reaction monitoring in positive/negative mode with electrospray ionization. The derivatization could indeed avoid the unfavorable reactions, namely, developed the method reflecting their correct contents on sampling. Furthermore, the method was successfully applied to monitoring Cys-containing dipeptides in E. coli Cys producer overexpressing bacD gene. This is the first report of the quantitative analysis of Cys-containing dipeptides, which should be useful for further study of fermentative production of Cys-containing dipeptides.

Euglena gracilis is a microalga, which has been used as a model organism for decades. Recent technological advances have enabled mass cultivation of this species for industrial applications such as feedstock in nutritional foods and cosmetics. E. gracilis degrades its storage polysaccharide (paramylon) under hypoxic conditions for energy acquisition by an oxygen-independent process and accumulates high amount of wax-ester as a by-product. Using this sequence of reactions referred to as wax-ester fermentation, E. gracilis is studied for its application in biofuel production. Although the wax-ester production pathway is well characterized, little is known regarding the biochemical reactions underlying the main metabolic route, especially, the existence of an unknown sulfur-compound metabolism implied by the nasty odor generation accompanying the wax-ester fermentation. In this study, we show sulfur-metabolomics of E. gracilis in aerobic and hypoxic conditions, to reveal the biochemical reactions that occur during wax-ester synthesis. Our results helped us in identifying hydrogen sulfide (H2S) as the nasty odor-producing component in wax-ester fermentation. In addition, the results indicate that glutathione and protein degrades during hypoxia, whereas cysteine, methionine, and their metabolites increase in the cells. This indicates that this shift of abundance in sulfur compounds is the cause of H2S synthesis.