L-cysteine is a ubiquitous and unique sulfur-containing amino acid with important physiological functions. The efficient L-cysteine production via microbial fermentation is interesting and has been paid great attention. In this study, different Escherichia coli K-12 strains (JM109, BW25113, MG1655, W3110) were investigated on their suitability to cysteine-producing plasmid pLH03. The enhancement of precursor synthetic pathway and thiosulfate assimilation pathway resulted in the good performance of BW25113. The expressions of synthetic pathway genes were optimized by two constitutive promoters to assess their effects on L-cysteine production. Main degradation pathway genes were also deleted coordinately for more efficient production of cysteine. The L-cysteine production was further increased through the manipulation of sulfur transcription regulator cysB and sulfur supplement. After the process optimization in a 1.5 L bioreactor, the final engineered strain LH2A1M0B△YTS-pLH03 [BW25113Ptrc2-serA-Ptrc1-cysM- Ptrc-cysB△yhaM△tnaA△sdaA-(pLH03)] accumulated 8.34 g/L of cysteine, laying a certain foundation for cysteine fermentation industry.

The cell free system has been paid more attention due to its potential of facilitating more efficient catalysis of multistep reactions. In this study, an efficient enzymatic cascade of GSH production was developed through the evolution of bifunctional glutathione synthetase (GshF), coupled with polyphosphate kinase (PPK). First, the stability and activity of GshF were enhanced by loop interchange and site-directed mutagenesis. As a result, the GshF half-value period increased 163.3-fold, and its activity raised 18 %. PPK from Jhaorihella thermophile (PPKJT) was characterized and used to regenerate ATP in the GSH synthesis, with hexametaphosphate (PolyP(6)) as the phosphate donor. After the process optimization, 99.9 mM GSH and 7.6 mM oxidized glutathione (GSSG) were produced within 2 h. The molar yield was 95.9 mol/mol based on the amino acid added, while the productivities of GSH achieved 49.95 mM/h, which was the highest yield and productivity ever reported about GSH synthesis.