Improvement of production yield of l-cysteine through in vitro metabolic pathway with thermophilic enzymes

The demand for the amino acid l-cysteine is increasing in the food, cosmetic, and pharmaceutical industries. Conventionally, the commercial production of l-cysteine is achieved by its extraction from the acid hydrolysate of hair and feathers. However, this production method is associated with the release of environmentally hazardous wastewater. Additionally, l-cysteine produced from animal sources cannot be halal-certified, which limits the market size. Although recent studies have developed an alternative commercial l-cysteine production method based on microbial fermentation, the production yield was insufficient owing to the cytotoxicity of l-cysteine against the host cells. In a previous study, we had developed an in vitro l-cysteine production method with a combination of 11 thermophilic enzymes, which yielded 10.5 mM l-cysteine from 20 mM glucose. In this study, we performed re-screening for enzymes catalyzing the rate-limiting steps of the in vitro pathway. Subsequently, the genes encoding enzymes necessary for the in vitro synthesis of l-cysteine were assembled in an expression vector and co-expressed in a single strain. To prevent the synthesis of hydrogen peroxide (H2O2), which is a byproduct and inhibits the enzyme activity, the redox balance in this biosynthetic pathway was maintained by replacing the H2O2-forming NADH oxidase with another enzymatic reaction in which pyruvate was used as a sacrificial substrate. The re-designed in vitro synthetic pathway resulted in the production of 28.2 mM l-cysteine from 20 mM glucose with a molar yield of 70.5%.