Journal
METABOLIC ENGINEERING
Volume 48, Issue -, Pages 138-149Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymben.2018.05.022
Keywords
Bacillus subtilis; Riboflavin; Mutation analysis; Genome sequencing; Transcriptome analysis; Metabolic engineering
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Funding
- National Natural Science Foundation of China [NSFC-21576200, NSFC-21576191, NSFC-21776209, NSFC-21621004]
- Doctoral Scientific Research Foundation of Zhengzhou University of Light Industry [2015BSJJ018]
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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.
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