Cloning, Expression And Characterization Of The Sulfur Assimilation Genes, CysE, CysM And CysC From Escherichia Coli And Chondrus Crispus

L-Cysteine and nonproteinaceous amino acid(NPAA) can be widely used in the fields of scientific research and medicine. The production of L-cysteine and nonproteinaceous amino acid(NPAA) using biocatalysis and fermentation has attracted more and more attention, because of its energy-saving, high efficiency and environmental protection. In this study, we reported the molecular cloning of three sulfur assimilation genes, cysE, cysM and cysC, from Escherichia coli and Chondrus crispus. Using recombinant E. coli expressing of serine acetyltransferase(SAT), O-acetylserine sulfhydrylase-B(OASS-B), a large amount of efficient enzymes(biological catalyst) were synthesized. By substitution of uncleophiles, various NPAAs can be synthesize using recombinant enzymes expressed in E. coli, which will provide useful data for the study on the new synthesis method of L-cysteine and NPAAs.In this study, we firstly reported the molecular cloning of E. coli cysE(cysE-ECOLI) cDNA sequences. The open reading frame of cysE-ECOLI was 822 bp, encoding 273 amino acids with a predicted molecular mass of 29.32 kDa and the theoretical isoelectric point was 6.05. The cysE-ECOLI gene was subcloned to expression plasmid pET-22b(+), and the recombinant SAT was existing in the supernatant as a soluble protein. The enzyme was purified to homogeneity with a specific activity of about 180 U/mg. To obtain a better SAT with higher expression, having higher specific activity and easy for mass-produced, we cloned Ch. Crispus cysE(cysE-CHCR) cDNA sequences. The open reading frame of cysE-CHCR was 1143 bp, encoding 380 amino acids with a predicted molecular mass of 40.60 kDa and the theoretical isoelectric point was 6.26. The gene was analyzed by using gene cloning, and bioinformatics methods, which will lay the groundwork for the further study on the protein expression and characterization.Then, the cysM from E. coli was cloned, the open reading frame of cysM was 912 bp, encoding 303 amino acids with a predicted molecular mass of 32.63 kDa and the theoretical isoelectric point was 5.42. The recombinant OASS-B was existed in the supernatant as a soluble protein. The enzyme was purified to homogeneity with a specific activity of about 728 U/mg. Furthermore, sulfur-containing nonproteinaceous amino acids, β-(pyrazol-1-yl)-L-alanine(β-PA), and β-(triazol-1-yl)-L-alanine(β-TA) were synthesized by the recombinant OASS-B using different concentration of OAS. Filtration and evaporation of solvent at reduced pressure gave product as white solid powder. These results indicate that the recombinant OASS-B were useful for the synthesis of sulfur-containing NPAAs by substitution of uncleophiles.Finally, we cloned Ch. Crispus cysC cDNA sequences. The open reading frame of cysC was 699 bp, encoding 232 amino acids with a predicted molecular mass of 25.33 kDa and the theoretical isoelectric point was 8.48. The gene was analyzed by using gene cloning, and bioinformatics methods.