Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People's Republic of China; Laboratory of Systems Biology and Biofuels, School of Food and Biological Engineering, Zhengzhou University of Light Industry, Zhengzhou 450000, People's Republic of China; Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, People's Republic of China; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, People's Republic of China.
Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People's Republic of China; Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin 300308, People's Republic of China; Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, People's Republic of China; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, People's Republic of China.
Metab Eng. 2018 Jul;48:138-149. doi: 10.1016/j.ymben.2018.05.022. Epub 2018 Jun 2.
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.
枯草芽孢杆菌商业化核黄素生产已经结合理性和经典的菌株开发近二十年，但如何理性地创造出改良的核黄素生产菌仍然不完全清楚。在这项研究中，我们展示了整合基因组和转录组分析在枯草芽孢杆菌核黄素过量生产遗传基础的联合应用。该方法成功地通过全基因组测序和转录组测序辨别了诱变衍生核黄素生产菌枯草芽孢杆菌 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）和两个无义突变 YhcF（R90*）和 YwaA（Q68*）。将这些特定的突变重新引入野生型菌株中恢复了核黄素过量生产表型，随后的代谢工程极大地提高了核黄素的产量，使核黄素的产量比测序生产菌提高了 3.4 倍。一个新的突变，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活，并增加了细胞内嘌呤代谢物的池，这反过来又增加了核黄素的产量。总之，我们结合基因组和转录组分析，阐明了一个复杂细胞特性的遗传基础，这使得有益突变能够转移到工程参考菌株中，从而成为一个高产菌株。
