Studies On The Mechanisms For Jinggangmycin Biosynthesis In Streptomyces Hygroscopicus 10-22 And 5008

Validamycin, an animoglyciside antibiotics, is used widely against sheath blight disease of rice plants. Based on the gene cluster responsible for the biosynthesis of validamycin identified from Streptomyces hygroscopicus 5008, valA gene encoding a putative 2-epi-5-epi-valiolone synthase is esential for validamycin biosynthesis as confirmed by both bioassay and HPLC analysis.The gene valH in the validamycin biosynthetic gene cluster harbours 12 transmembrane regions, and pertains to a member of the Major Facilitator Superfamily (MFS). The ValH may be a validamycin exporter. An engineered mutant (JXH3) with disrupted valH was obtained, to our surprise; the mutant JXH3 also has the capability of validamycin production, although the yield decreased about 17%. The valH gene placed under the control of ermE* promoter was introduced into the mutant for complementation, and the yield of validamycin increased about 20% compared with the wild type strain.A series of large chromosomal deletions in Streptomyces hygroscopicus 10-22 were aligned on the physical map of the wild-type strain and the mutants were assessed for their ability to produce the aminocyclitol antibiotic 5102-I. Twenty-eight mutants were blocked for jinggangmycin production and all of them were found to lack a 300 kb AseI-F fragment of the wild-type chromosome. One of the cosmids of 300 kb AseI-F fragment conferred 5102-I productivity to Streptomyces lividans ZX1. Three of the overlapping cosmids also hybridized to the valA gene of the validamycin pathway from S. hygroscopicus 5008 as a probe. The valA-homolog (orf1) of S. hygroscopicus 10-22 was shown to be essential for 5102-I. biosynthesis as an engineered mutant with a deletion of orf1 completely abolished 5102-I production. The corresponding knock-out mutant (JXH4) could be complemented by the introduction of an orf1-containing construct. Concurrently, the identities of the genes common to S. hygroscopicus strains 10-22 and 5008 prompted a comparison of the chemical structures of antibiotic 5102-I and validamycin, which led to a clear demonstration that they are identical. And the former is named jinggangmycin.The yield of jinggangmycin produced by S. hygroscopicus 10-22 is very low comparing with the validamycin produced by S. hygroscopicus 5008 in liquid-state fermentation. The most genes in the two gene cluster have high homology, but the regulatory gene between them seem to be different. After disruption of a putative regulatory gene, jin2, the mutant(JXH6) has not obvious effect on yield of jinggangmycin as detected by HPLC analysis, while the yield of jinggangmycin increased about 40% after the regulatory genes valP and valQ were introduced into the S. hygroscopicus 1022.To explore the possible reason that led to different production of jingangmycin between the val cluater and jin cluster. The jin cluster and val gene cluster were integrated into the chromosome of Streptomyces lividans, generating the engineered strain JXH10 and JXH21 respectively. The yield of validamycin produced by the JXH21 is similar to S. hygroscopicus 5008, while the yield of jinggangmycin produced by JXH10 is about 25% comparing with JXH21 and is much higher than the S. hygroscopicus 10-22. For S. hygroscopicus 10-22, the poor ability of jinggangmycin production is predominantly related with the 10-22 strain, instead of the jin cluster. It could be deduced that there are some reason in validamycin gene cluster caused higher yield comparing with the JXH21 and JXH10.With comparison of the three gene cluster responsible for the same antibitic produced by S. hygroscopicus 10-22, S. hygroscopicus 5008 and S. hygroscopicus var. limoneus KCCM 11405, the jinJ gene encoding a putative oxidoreductase may be responsible for hydroxylation of jinggangmycin B/validamyicn B But the engineered strain JXH12 in which the jinJ gene was disruption using JXH10 as start strain also produced the jinggangmycin B. With analysis of the engineered strain XH-9 and XH-6 ( the jinJ gene is absent in both strains), it is seemed that biosynthesis of validamycin B should not need the special oxidoreductase.