Identification Of Rate-limiting Factors For Validamycin Biosynthesis And Its Engineered Over Production

Jinggangmycin (validamycin), a family of C7N-aminocyclitol antibiotics, are widely used as plant protectants to control rice sheath blight disease in the Far East. Their anti-fungal activity is attributed to validoxylamine A. Validoxylamine A is a potent inhibitor of trehalase due to its structural similarity to trehalose, the primary storage carbohydrate in many fungi. Although in vitro experiments revealed that the aglycone, validoxylamine A, is more active than validamycin A against fungi, in vivo experiments suggested the opposite. It is proposed that the glucose moiety on validamycin A is required for the ef?cient entry into fungal hyphae, in which it is hydrolyzed to yield the more active form validoxylamine A.During the fermentation of S. hygroscopicus TL01 to produce validamycin A, a considerable amount of an intermediate validoxylamine A accumulated. Chemical or enzymatic hydrolysis of validamycin A was not observed during the fermentation process. Over-expression of glucosyltransferase ValG in TL01 did not increase the efficiency of glycosylation. However, increased validamycin A and decreased validoxylamine A production was observed in both the cell-free extract and fermentation broth of TL01 supplemented with a high concentration of UDP-glucose. The enzymatic activity of UDP-glucose pyrophosphorylase (Ugp) in TL01, which catalyzes UDP-glucose formation, was found to be much lower than the activities of other enzymes involved in the biosynthesis of UDP-glucose and the glucosyltransferase ValG. An ugp gene was cloned from S. hygroscopicus 5008 and verified to code for Ugp. In TL01 with an extra copy of ugp, the transcription of ugp was increased for 1.5 times, and Ugp activity was increased by 100%. Moreover, 22 g/L validamycin A and 2.5 g/L validoxylamine A were produced, and the validamycin A/validoxylamine A ratio was increased from 3.15 in TL01 to 5.75. These data prove that validamycin A biosynthesis is limited by the supply of UDP-glucose, which can be relieved by Ugp over-expression.We also investigated the genes from the whole-genome scale that related to validamycin A biosynthesis. A large-scale cultivation and high-throughput screening method was established. For the strong inhibition of trehalase in vitro, an accurate colorimetric assay for quantifying validoxylamine A using trehalose hydrolysis assay and glucose oxidase/peroxidase linking enzymes was developed for a derivative of TL01, which accumulates only validoxylamine A, the biosynthetic precursor of validamycin A.Development of a high throughput method to detect validoxylamine A production using trehalase inhibition assay.Validamycin A was difficult to assay quantitatively because it is colorless. Previous researches used HPLC, but the procedures were time-consuming. Moreover, fungal growth inhibitory assay for validamycin analogs is imprecise and unsuitable for miniaturized plate inhibition assays. So a fast and accurate method was needed to fulfill the high throughput detecting requirements.Through detecting the release of glucose from trehalose hydrolysis, the inhibition ability of each sample could be reflected. Enzymatic activity was measured using an assay in which glucose was detected using glucose oxidase/peroxidase linking enzymes. The glucose (go) assay kit could furnish linear standard curve when the concentration of glucose was 20-80μg/ml. So the concentration of final product of the trehalase hydrolysis assay should be led in that scope. Within the concentration of the inhibitor from 1μg/ml to 9μg/ml, the concentration of released glucose decreased linearly (R2 =0.9961). So the production ability of validoxylamine A for each mutant could be evaluated through the inhibition assay of trehalose hydrolysis.Establishment of large-scale cultivation on 96-square-deep well micro-titer plates.The large-scale cultivation of isolates on 1 ml 96-square-deep well micro-titer plates were developed. This culturing system is reproducible. The standard deviation is 13.78 mg/L with a low variability (±5%) of validoxylamine A production rates between parallel samples, and HPLC analysis showed that less than 5% of validoxylamine A was left in the solid media after extraction, which provided a very suitable system to check the production of validoxylamine A.Modification of Tn5-based transposition using replicative, genetically unstable vectors.The deletion of sti from the Streptomyces plasmid pIJ101 made its derivatives pHZ1358 and pJTU1278 efficient vectors for gene disruption and replacement. The vectors are replicative but genetically unstable in many Streptomyces hosts. Typically, all colonies lost thiostrepton resistance after one round of nonselective growth on mannitol soya flour medium (SFM) plates. So the transposon system was delivered using pJTU1278.All the mutants contained the entire transposon, without the transpotase and the vector. 14 apramycin-resistant mutants were randomly selected for identification of Tn5 insertion sites and each mapped to a unique location on the S. hygroscopicus TL01 chromosome.More than 800 mutants containing random transposon insertions were screened in order to verify the method. Compared with XZ2, 8 mutants showed significant difference in validoxylamine A production (+/-20%, p<0.001) and were localized using the transposon clone strategy. The method could be further used to thoroughly investigate the genes related to validamycin production. The optimized transposon system together with the large-scale cultivation and high throughput screening method could be utilized in other studies.