Laboratory of Biotechnology, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka 422-8529, Japan.
Appl Microbiol Biotechnol. 2011 Sep;91(5):1315-26. doi: 10.1007/s00253-011-3325-0. Epub 2011 May 15.
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.
我们通过使用滞后链 DNA 聚合酶 δ 的突变进行差异诱变，生成了阿氏棉霉高核黄素产生突变株，导致聚合酶丧失 DNA 修复功能。在第一轮筛选中产生的 1353 个菌落中，有 26 个突变株产生的核黄素超过 3 g/L。通过第二轮筛选和单细胞分离，选择了 9 株产生的核黄素超过 5.2 g/L 的菌株作为高核黄素产生菌株。这些突变株对草酸盐和过氧化氢具有抗代谢作用。一株（W122032）在优化培养基的 3-L 发酵罐中产生 13.7 g/L 的核黄素。这比野生型菌株的产量提高了九倍。蛋白质组学分析表明，与野生型相比，该菌株中 ADE1、RIB1 和 RIB5 蛋白的表达水平提高了两倍。DNA 微阵列分析显示，嘌呤和核黄素生物合成途径上调，而与碳源同化、能量产生和糖酵解相关的途径下调。在核黄素生产和静止期，核黄素生物合成途径中的基因均显著过表达，例如，与野生型相比，该菌株中 RIB1 和 RIB3 的表达水平提高了六倍以上。这些结果表明，该菌株核黄素产量的提高与碳通量从β-氧化向核黄素生物合成途径的转移有关。
