Functional Characterization Of Validamycin And Candicidin Biosynthetic Genes

Validamycins, weakly basic C7N-aminocyclitol-containing antibiotics, were isolated from Streptomyces hygroscopicus var. jinggangensis 5008. It is a trehalase inhibitor widely used as a prime control agent against sheath blight disease of rice plants and dumping-off of cucumber seedlings in China and many other eastern Asian countries. The sequence information of the cloned biosynthetic gene cluster helps us to investigate the biosynthesis of this antibiotic.The gene valC, which encodes an enzyme homologous to the 2-epi-5-epi-valiolone kinase (AcbM) of the acarbose biosynthetic pathway, was identified in the validamycin biosynthetic gene cluster. Inactivation of valC resulted in mutants that lack the ability to produce validamycin A. Complementation with full-length valC restored the production of validamycin, thus suggesting a critical function of valC in validamycin biosynthesis. In vitro characterization of ValC revealed a new type of C7-cyclitol kinase, which phosphorylates valienone and validone-but not 2-epi-5-epi-valiolone, 5-epi-valiolone, or glucose-to afford their 7-phosphate derivatives. The results provide new insights into the activity of this enzyme and also confirm the existence of two different pathways leading to the same end-product: the valienamine moiety common to acarbose and validamycin.The gene valD, which encodes a large Vicinal Oxygen Chelate(VOC)superfamily protein, has been identified in the validamycin biosynthetic gene cluster. Inactivation of valD significantly reduced validamycin production, which was fully restored with the full-length valD and partially restored with either N-terminal or C-terminal half by complementation. Heterologously expressed ValD catalyzed the epimerization of 2-epi-5-epi-valiolone to 5-epi-valiolone. Individual and combined site-directed mutations of eight putative active site residues revealed that the N-terminal H44/E107 and the C-terminal H315/E366 are more critical for the activity than the internal H130, E183, H229 and E291.Three biosynthetic genes valB, valK and valO were inactivated respectively. The gene valB encodes a putative nucleotidyl transferase, which may catalyze the adenylation of 1-epi-valienol-1-phosphate. Inactivation of valB abolished validamycin biosynthesis, which was restored by complementation of full-length valB. The gene valK encodes a putative dehydratase, which may catalyze the dehydration of C-5 and C-6 of 5-epi-valiolone to generate valienone. Inactivation of valK abolished validamycin production, which was restored by complementation with cloned valK. These results indicate that valB and valK are essential for validamycin biosynthesis. The gene valO encodes a putative phosphatase/phosphohexo- mutase, which may catalyze the transfer of phosphate group from the C-7 to the C-1 hydroxyl group. However, inactivation of valO did not abolish validamycin production but resulted in significant accumulation of validoxylamine, which could be partially complemented by cloned valO. This result suggests that valO may be involved in conversion of validoxylamine to validamycin.In some antibiotic producers, p-aminobenzoic acid (PABA) or its immediate precursor, 4-amino-4-deoxychorismate (ADC), is involved in primary metabolism and antibiotic biosynthesis. A fused pabAB gene was identified in FR-008/candicidin biosynthetic gene cluster from Streptomyces sp. FR-008. Its deletion abolished FR-008/candicidin biosynthesis, which was restored either by complementation with cloned pabAB or feeding with exogenous PABA. A gene pabC-1 putatively encoding fold-type IV of PLP-dependent enzyme was found in the upstream PKS region, whose inactivation significantly reduced the productivity of antibiotic FR-008 to ca. 20% of the wild-type level. Its specific role for PABA formation was further demonstrated through the successful complementation of an E. coli pabC mutant. Moreover, a free standing gene pabC-2, probably encoding another fold-type IV of PLP-dependent enzyme, was cloned from the same strain. Inactivation of pabC-2 rendered antibiotic FR-008 yield to about 57% of the wild-type level in the mutant, and the complementation of the E. coli pabC mutant established its involvement in PABA biosynthesis. Furthermore, a pabC-1/pabC-2 double mutant only kept about 4% of the wild-type productivity, clearly indicating that pabC-2 also contributed to antibiotic FR-008 biosynthesis. Surprisingly, apparent retarded growth of the double mutant was observed on minimal medium, which suggested that both pabC-1 and pabC-2 were involved in PABA biosynthesis for primary metabolism. Finally, both PabC-1 and PabC-2 were proved to be functional ADC lyases through in vitro enzymatic lysis with the release of pyruvate. To date, pabC-1 and pabC-2 represent the first two functional ADC lyase genes indentified in actinomycetes. The involvement of these two ADC lyase genes in both cell growth and antibiotic FR-008 biosynthesis sets an example for the interplay between primary and secondary metabolisms in bacteria.