Study On Validamycin Fermentation Process

Validamycin is an agricultural antibiotic produced by Streptomyces hygroscopicus. It has been developed to an effective bio-pesticide for control of rice sheath blight disease since1970s. It is widely used because of its low toxicity, high efficiency and difficulty to generate drug resistance. In addition, its degradation product valienamine, is an important synthetic precursor of the clinically used antidiabetic agents acarbose and voglibose.It was possible to reveal the fermentation process on molecular level with the cloning of validamycin biosynthetic gene cluster and in-depth study on its biosynthesis and regulation mechanisms. The detailed study of the effects of different strategies on validamycin production on the fermentation kinetics, gene expression and enzyme activity levels will provide useful information for revealing the regulation mechanism of validamycin synthesis. It will be helpful to develop new strategies for validamycin production. But there still lack of information related to fermentation factors on validamycin production based on the fermentation process and molecular mechanism for regulation of validamycin biosynthesis. So it has important values both on the theory and practice to study the fermentation process and regulation mechanism of validamycin.But validamycin production was low by Streptomyces hygroscopicus5008. So it will be important for finding out the significant factors on validamycin production, revealing the regulation mechanism of fermentation conditions, and further enhancing its production by optimization of its fermentation process. Single factor test and Plackett-Burman design were used for screening the major factors that have significant effect on validamycin production in medium components in this study. After statistical analysis, corn powder, soybean powder and yeast extract were found to have significant effect on validamycin production. Their concentrations were further optimized by response surface methodology, and the optimal medium composition was got. The medium was verified by experiment. Validamycin production increased by70%(from the7.5g/L to12.6g/L) in the optimized medium. The composition difference between two medium suggested lower nitrogen level in optimized medium enhanced VAL-A production. The addition of4kinds of nitrogen sources (soybean powder, yeast extract, tryptone and beef extract) all inhibited validamycin production. The results suggested the important effect of nitrogen source on validamycin production. The differences of cell growth, validamycin production, structural gene transcription and enzyme activity related to precursor metabolism between the optimized medium and original medium were analyzed. The results showed that validamycin production and structural gene transcription were largely enhanced in optimized medium, while the cell growth and enzyme activity related to precursor metabolism were not significantly affected. This result suggested that the expression of structural genes was important for validamycin production.In many plant cell cultures, external oxidative stress can activate certain signal transduction pathway, thereby promote the gene expression, and thus improve the secondary metabolites production. But it is unknown whether oxidative stress could enhance antibiotic production by inducing expression of structural genes in Streptomyces. In this study, H2O2was added to study the effect of oxidative stress on validamycin production. By optimizing the addition time and concentration of H2O2, the optimal condition was25μM hydrogen peroxide added at8h, which caused40%enhancement of validmycin production. The addition of reactive oxygen species (ROS) inhibitor diphenyleneiodonium chloride (DPI) and the determination of intracellular ROS level suggested that ROS signal regulated validamycin production. The results of structural gene transcription showed that the induction of reactive oxygen species could enhance transcription of validamycin structural genes. The results of glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and glucose-6-phosphate dehydrogenase (G6PDH) activities suggested that there was a flux redirection from glycolysis pathway to pentose phosphate pathway.Our previous work showed that temperature had a significant effect on validamycin production. High temperature facilitated validamycin production by increasing structural gene transcription, but inhibited cell growth. It should be studied whether temperature shift could enhance validamycin production. In this study, fermentation temperature was controlled at30℃at first, and then changed to42℃at different stages. Validamycin production was enhanced under optimal temperature shift condition, but cell growth was not significantly enhanced. In order to investigate why the temperature shift promoted validamycin production, H2O2and DPI were added before temperature shift to study the role of ROS in this process. The results showed that intracellular ROS level was higher at42℃than30℃, and temperature shift increased ROS level. The addition of DPI delayed the increase of ROS level, and inhibited validamycin production. This result suggested that ROS might be involved in the regulation of temperature on validamyin production. The activity of G6PDH was assayed during temperature shift process. The specific activity of G6PDH was lower at42℃than that of30℃, which suggested that the intracellular concentration of G6PDH was lower at42℃. As the temperature might have a direct affect on the enzyme activity, the activities of the same concentration of crude enzyme were assayed at30℃and 42℃, and its activity was much higher at42℃. The results showed that temperature could regulate G6PDH activity both on expression level and enzyme kinetic level. The transcriptional responses of general stress response sigma factors sigmaB and sigmaH and global regulator phoRP were assayed. The results suggested that they might be involved in regulation of temperature on validamycin A production.In conclusion, the fermentation medium was optimized by statistical methods and validamycin production was effectively enhanced. The multi-level differences on cell growth, validamycin production, gene transcription and enzyme activity were assayed between two media, and the results indicated that the structural gene expression was important for validamycin production. The addition of H2O2and temperature shift methods improved the validamycin production. The regulatory mechanism of the fermentation strategies was studied on signal molecules, gene transcription and enzyme activity levels. The information will be helpful to further understand the regulatory mechanisms of validamyicn synthesis, and it has reference value for other antibiotics fermentation.