A comparative transcriptome profiling between a riboflavin-producing Bacillus subtilis strain RH33 and the wild-type strain B. subtilis 168 was performed, complemented with metabolite pool and nucleotide sequence analysis, to rationally identify new targets for improving riboflavin production. The pur operon (purEKBCSQLFMNHD) together with other PurR-regulated genes (glyA, guaC, pbuG, xpt-pbuX, yqhZ-folD, and pbuO) was all down-regulated in RH33, which consequently limited the supply of the riboflavin precursors. As 5-phospho-ribosyl-1(a)-pyrophosphate (PRPP) strongly inhibits the binding of PurR to its targets, it was inferred that the reduced expression of PurR-regulated genes might be caused by a low PRPP pool, which was subsequently confirmed by metabolite analysis. Thus, we selected and co-overexpressed prs and ywlF genes in RH33, which are involved in the biosynthetic pathway of PRPP from ribulose-5-phosphate. This co-amplification led to an elevated PRPP pool and thus the increased transcript abundances of PurR-regulated genes participated in riboflavin precursor biosynthesis. The riboflavin titer was increased by 25% (up to 15 g l(-1)) in fed-batch fermentation.
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机译:在产生核黄素的枯草芽孢杆菌菌株RH33和野生型枯草芽孢杆菌168之间进行了比较转录组分析,并辅以代谢物库和核苷酸序列分析,以合理地确定新的靶标,以改善核黄素的生产。嘌呤操纵子(purEKBCSQLFMNHD)以及其他PurR调控的基因(glyA,guaC,pbuG,xpt-pbuX,yqhZ-folD和pbuO)在RH33中均被下调,因此限制了核黄素前体的供应。由于5-磷酸-核糖基-1(a)-焦磷酸(PRPP)强烈抑制PurR与其靶标的结合,因此推断PurR调节基因的表达降低可能是由于低PPPP池引起的,随后它经代谢物分析证实。因此,我们选择并共表达了RH33中的prs和ywlF基因,这些基因参与了5核糖磷酸PRPP的生物合成途径。这种共扩增导致PRPP库增加,因此PurR调控基因的转录丰度增加,参与了核黄素前体的生物合成。分批补料发酵的核黄素滴度提高了25%(最高15 g l(-1))。
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