Ergothioneine (EGT), a histidine derivative with diverse physiological functions, has broad applications in food, cosmetics, and pharmaceuticals. Fermentative production of EGT using recombinant Saccharomyces cerevisiae is widely considered the most cost-effective and direct method. However, no previous studies have explored improving EGT production in S. cerevisiae through protein fusion technology. In this study, we present the first successful construction of a fungal EGT biosynthesis pathway in S. cerevisiae , coupled with enzyme fusion techniques and optimization of gene copy integration. The engineered strain, SZA011, achieved a record EGT production of 122.5 mg/L under optimized shake flask fermentation conditions. Moreover, in a 10 L fed-batch fermentation system, SZA011 produced 626.24 mg/L of EGT. This study introduces a novel strategy for enhancing microbial EGT generation through protein and genetic engineering, potentially enabling industrial applications.
To improve the fermentation production of transglutaminase (TGase) from Streptomyces mobaraensis for applications in the food industry, the atmospheric and room-temperature plasma (ARTP) mutagenesis was applied to breed S. mobaraensis mutants with increased TGase production.After eight rounds of iterative ARTP mutagenesis, four genetically stable mutants, Sm5-V1, Sm6-V13, Sm2-V10, and Sm7-V12, were identified, which showed increased TGase production by 27, 24, 24, and 19%, respectively. The best mutant Sm5-V1 exhibited a maximum TGase activity of 5.85 U/mL during flask fermentation. Compared to the wild-type strain, the transcription levels of the zymogen TGase genes in the mutants increased significantly as indicated by quantitative real-time PCR, while the gene nucleotide sequences of the mutants did not change at all. It was shown that the overexpression of TGase zymogen gene in the mutants contributes to the increase in TGase production.ARTP is a potentially efficient tool for microbial mutation breeding to bring some significant changes required for the industrial applications.
Fermentative production of γ-aminobutyric acid by the glutamate overproducing Corynebacterium glutamicum from cheap sugar feedstock is generally regarded as one of the most promising methods to reduce the production cost. However, the intracellularly expressed glutamate decarboxylase in C. glutamicum often showed feeble catalysis activity to convert glutamate into γ-aminobutyric acid. Here we tried to secretory express glutamate decarboxylase to achieve efficient extracellular decarboxylation of glutamate, thus improving the γ-aminobutyric acid production by C. glutamicum . We first tested glutamate decarboxylases from different sources, and the mutated glutamate decarboxylase GadBmut from E. coli with better catalytic performance was selected. Then, a signal peptide of the SEC translocation pathway directed the successful secretion of glutamate decarboxylase in C. glutamicum . The extracellular catalysis by secreted glutamate decarboxylase increased the γ-aminobutyric acid generation by three-fold, compared with intracellular catalysis. Enhancing glutamate decarboxylase expression and decreasing γ-aminobutyric acid degradation further increased γ-aminobutyric acid production by 39 %. The fed-batch fermentation of the engineered C. glutamicum strain reached the record high titer (77.6 ± 0.0 g /L), overall yield (0.44 ± 0.00 g/g glucose), and productivity (1.21 ± 0.00 g/L/h). This study demonstrated a unique design of extracellular catalysis for efficient γ-aminobutyric acid production by C. glutamicum .
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