Cloning And Functional Analysis Of Key Genes In Primary Metabolism From Streptomyces Lydicus

Streptolydigin is a novel antibiotic produced by Streptomyces lydicus. It has high antibacteral activity with potential commercial value. But the low productivity limits its application. Enhancing primary metabolism is a feasible approach to improve the antibiotic production. In this study we cloned several key genes of primary metabolism and analyzed their functions.Xylose significantly promoted mycellium growth rate when the media contained low concentration of glucose.The cloned xylB of xylose metabolism gene cluster is 1446 bp in length and encodes 481 amino acids. The results showed S. lydicus can utilize xylose though the efficiency is low.Using the degenerated primers based on the conserved domains, we cloned four key genes (zwf2, glkA, pfkA1, idh) of glucose metabolism from S. lydicus. zwf2 is 1542 bp long and encodes a putative enzyme of 513 amino acids. glkA is 954 bp in length and encode 317 amino acids. pfkA1 is 1026 bp in length and encode 341 amino acids. idh is 2220 bp long and encodes a putative enzyme of 739 amino acids. The active domains of Zwf2 may be AXXXGXGGXA (putative NADP+ binding site) and lysine43 (glucose-6-phosphate binding site). The active domain of three sugar kinases, XylB, GlkA and PfkA1 with the general acid and alkaline catalytic mechanism, may be PYLDGERT, CXCGXGCXEXY and DIXXTDXTXTFGFD while Idh's active domain may be the highly conserved domain HLKATMM. We constructed a constitutive expression plasmid pIB139-zwf2, and then transformed to the S. lydicus.The cloning and function analysis of these key genes of primary metabolism and construction of zwf2-high expression strain lay a foundation of further study on the relationship between primary metabolism and streptolydigin biosynthesis.