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Effect of Oxygen Carrier on Validamycin a Biosynthesis and Exploration on Its Mechanism

Author: FengJinSong
Tutor: ZhengXiaoDong
School: Zhejiang University
Course: Fermentation Engineering
Keywords: validamycin A oxygen carrier amino acid rapid determination method gene expression
CLC: S482.2
Type: Master's thesis
Year: 2013
Downloads: 6
Quote: 0
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Abstract


Validamycin A (Val-A) is a widely used agro-antibiotic, as one kind of secondary metabolites produced by aerobic bacteria, dissolved oxygen concentration could heavily affect its production during fermentation process. Oxygen carrier is one kind of compounds which could significantly improve oxygen transfer rate in the enviornment. Its function has been tested and verified by many fermentation experiments. The value of Val-A in agriculture has been fullly reflected and demand for its output increases year by year. So how to effectively improve its fermantation production becomes a hot-spot issue. Reports have proved that oxidative stress can increase production of Val-A, but practical strategy on how to improve dissolved oxygen concentration in fermentation process is limited, and mechanism of how oxygen carrier influence intracellular metabolism is not clear. In this study, by adding oxygen carrier, a novel strategy to enhance Val-A production in Streptomyces hygroscopicus5008was introduced. By screening the optimal condition,0.01%(v/v) liquid paraffin was proved to stimulate Val-A production most. Further research on cell growth and sugar utilization indicated intracellular metabolism regulation was the reason leading to increase of Val-A production. According to character of oxygen carrier, reactive oxygen species (ROS) level were detected to reflected oxidative stress change. We found ROS induced by liquid paraffin was factor which activated the metabolism regulation. Responding to enhances oxidative stress, global regulators and Val-A structure related genes got up-regulation. Besides, significant redirection of carbon metabolism from glycolysis pathway to pentose phosphated pathway (PPP) contributed to more precursor production. These results provided comprehensive understanding about the effect of liquid paraffin on Val-A biosynthesis.During the fermentation, we found a stable relationship between pigment secretion and Val-A production. Using spectral absorption (SA) value as interior label, a rapid determination method for determining Val-A production was established. Compared with traditional high-performance liquid chromatography assay, spectral absorption assay were not only convenient and cost-effective but also stable and precise. It was a new method for quantification in industrial fermentation process of Val-A.Besides, there is a close relationship between amino acid metabolism and aminoglycoside antibiotic biosynthesis. Nine kinds of amino acids were added during the fermentation of Streptomyces hygroscopicus5008to investigate effects of amino acids on biosynthesis of validamycin A (Val-A). The results revealed addition of amino acids could affect activities of key enzymes in carbon metabolism and redirect their flux which benefits to accumulation of Val-A precursor and stimulates biosynthesis of Val-A.In the end, liquid paraffin’s effect on Val-A was conducted on larger scale fermentation. The result in a5L fermentation showed that liquid paraffin had advantage on stimulating Val-A production. The information will provide a useful strategy for improvement of industrial fermentation.

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