Integrated whole-genome and transcriptome sequence analysis reveals the genetic characteristics of a riboflavin-overproducing Bacillus subtilis
Integrated whole-genome and transcriptome sequence analysis reveals the genetic characteristics of a riboflavin-overproducing Bacillus subtilis
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综合全基因组和转录组序列分析揭示了过量生产核黄素的枯草芽孢杆菌的遗传特征
DOI：
10.1016/j.ymben.2018.05.022
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发表时间：
2018-07-01
影响因子：
8.4
通讯作者：
Zhao, Xueming
中科院分区：
文献类型：
--
作者：
Wang, Guanglu;Shi, Ting;Zhao, Xueming
通过将理性和经典的菌株开发方法相结合，枯草芽孢杆菌的商业化核黄素生产已经发展了近二十年，但如何理性地创造一种改良的核黄素生产菌株仍未完全被理解。在这项研究中，我们展示了对枯草芽孢杆菌核黄素过量生产的遗传基础进行整合基因组和转录组分析的联合应用。这种方法通过全基因组测序和转录组测序成功地辨别了诱变产生的核黄素生产菌株枯草芽孢杆菌24/pMX45中的阳性突变。这些突变包括RibC（G199D）、ribD(+)(G + 39A)、PurA（P242L）、CcpN（A44S）、YvrH（R222Q）以及两个无义突变YhcF（R90*）和YwaA（Q68*）。将这些特定突变重新引入野生型菌株恢复了核黄素过量生产的表型，并且随后的代谢工程极大地提高了核黄素产量，使核黄素效价相对于测序的生产菌株提高了多达3.4倍。一种新的突变YvrH（R222Q），涉及一个典型的双组分调控系统，使嘌呤从头合成途径失调并增加了细胞内嘌呤代谢物的库，这反过来又增加了核黄素的产量。总之，我们提出了一个结合基因组和转录组分析以阐明复杂细胞特性的遗传基础的案例研究，这使得有益突变能够转移，从而将一个参考菌株改造成一个过量生产菌株。
Commercial riboflavin production with Bacillus subtilis has been developed by combining rational and classical strain development for almost two decades, but how an improved riboflavin producer can be created rationally is still not completely understood. In this study, we demonstrate the combined use of integrated genomic and transcriptomic analysis of the genetic basis for riboflavin over-production in B. subtilis. This methodology succeeded in discerning the positive mutations in the mutagenesis derived riboflavin producer B. subtilis 24/pMX45 through whole-genome sequencing and transcriptome sequencing. These included RibC (G199D), ribD(+)(G + 39A), PurA (P242L), CcpN(A44S), YvrH (R222Q) and two nonsense mutations YhcF (R90*) and YwaA (Q68*). Reintroducing these specific mutations into the wild-type strain recovered the riboflavin overproduction phenotype and subsequent metabolic engineering greatly improved riboflavin production, achieving an up to 3.4-fold increase of the riboflavin titer over the sequenced producer. A novel mutation, YvrH (R222Q), involved in a typical two-component regulatory system deregulated the purine de novo synthesis pathway and increased the pool of intracellular purine metabolites, which in turn increased riboflavin production. Taken together, we present a case study of combining genome and transcriptome analysis to elucidate the genetic underpinnings of a complex cellular property, which enabled the transfer of beneficial mutations to engineer a reference strain into an overproducer.