The improvement of riboflavin production in <Emphasis Type="Italic">Ashbya gossypii</Emphasis> via disparity mutagenesis and DNA microarray analysis |
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Authors: | Enoch Y Park Yoko Ito Masashi Nariyama Takashi Sugimoto Dwiarti Lies Tatsuya Kato |
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Institution: | (1) Laboratory of Biotechnology, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka 422-8529, Japan;(2) Laboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka 422-8529, Japan |
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Abstract: | We generated a high riboflavin-producing mutant strain of Ashbya gossypii by disparity mutagenesis using mutation of DNA polymerase δ in the lagging strand, resulting in loss of DNA repair function
by the polymerase. Among 1,353 colonies generated in the first screen, 26 mutants produced more than 3 g/L of riboflavin.
By the second screen and single-colony isolation, nine strains that produced more than 5.2 g/L of riboflavin were selected
as high riboflavin-producing strains. These mutants were resistant to oxalic acid and hydrogen peroxide as antimetabolites.
One strain (W122032) produced 13.7 g/L of riboflavin in a 3-L fermentor using an optimized medium. This represents a ninefold
improvement on the production of the wild-type strain. Proteomic analysis revealed that ADE1, RIB1, and RIB5 proteins were
expressed at twofold higher levels in this strain than in the wild type. DNA microarray analysis showed that purine and riboflavin
biosynthetic pathways were upregulated, while pathways related to carbon source assimilation, energy generation, and glycolysis
were downregulated. Genes in the riboflavin biosynthetic pathway were significantly overexpressed during both riboflavin production
and stationary phases, for example, RIB1 and RIB3 were expressed at greater than sixfold higher levels in this strain compared
to the wild type. These results indicate that the improved riboflavin production in this strain is related to a shift in carbon
flux from β-oxidation to the riboflavin biosynthetic pathway. |
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