Elsevier

Metabolic Engineering

Volume 48, July 2018, Pages 138-149
Metabolic Engineering

Integrated whole-genome and transcriptome sequence analysis reveals the genetic characteristics of a riboflavin-overproducing Bacillus subtilis

https://doi.org/10.1016/j.ymben.2018.05.022Get rights and content

Highlights

  • Beneficial mutations including RibC (G199D), ribD+(G+39A), PurA (P242L), CcpN(A44S), YvrH (R222Q), YhcF (R90*) and YwaA (Q68*) for riboflavin productionwere identified.

  • Reintroduction of beneficial mutations recovered the riboflavin overproduction phenotype and subsequent metabolic engineering greatly improved riboflavin production, exhibiting up to 3.4-fold increase.

  • Decreased cell permeability mediated by YvrH (R222Q) or expression of N-acetylmuramoyl-L-alanine amidase enhanced riboflavin production in B. subtilis and 5-aminolevulinic acid in Corynebacterium glutamicum.

Abstract

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.

Introduction

High-performing strains can be created through targeted and nontargeted strain improvement efforts, which have been proved to be powerful tools to construct strains with desired phenotypes. Nontargeted strain improvement allows one to access an enormous number of genotypes if a proper selective pressure is applied. Such nontargeted approaches for strain optimization include: (i) conventional random mutagenesis and followed by screening and genome shuffling (Gong et al., 2009, Peano et al., 2014, Stephanopoulos, 2002, Zhang et al., 2002); (ii) adaptive laboratory evolution (ALE) under cultivation regimes that confer a selective advantage on better performing strains (Brown et al., 2011a, Caspeta et al., 2014, Fletcher et al., 2017, Horinouchi et al., 2017, Lam et al., 2014); (iii) global transcription machinery engineering (gTME) (Alper and Stephanopoulos, 2007), which is particularly effective in introducing phenotypic diversity by reprogramming the cellular transcriptome (Alper et al., 2006, Santos et al., 2012, Zhao et al., 2014). Even in the absence of detailed knowledge on the genetic basis or physiology of the producer strains, the effectiveness of such strategies is beyond dispute. However, due to the uncertainty of mutagenesis, strains developed using nontargeted methods generally have an unknown genetic background, and further improvement typically leads to a slow, incremental increase in performance, especially in the later stages of strain improvement (Dai and Nielsen, 2015). Strains generated through traditional random mutagenesis offer a relatively high ratio of non-beneficial to beneficial mutations, often resulting in ‘sick’ strains that do not perform consistently under the range of conditions required at the industrial scale (Dai and Nielsen, 2015, Warner et al., 2009). What's worse, its ‘black box’ character precludes the rapid transfer of relevant beneficial traits among strains or species.

With the development of systems biology tools, in particular next generation sequencing technology, more and more potentially valuable mutations were identified (Brown et al., 2011b, Garst et al., 2017, Huang et al., 2015, Krober et al., 2016, Lee and Palsson, 2010, Liu et al., 2014, Liu et al., 2017, Wang et al., 2014a, Yang et al., 2010, Zhang et al., 2015), and some of these have been successfully transferred to other host strains (Horinouchi et al., 2017, Lee and Palsson, 2010, Liu et al., 2014). Although these reconstructions could recover a part or most of the performance phenotype of the mutant strains, it is still difficult to identify all the positive mutations due to the large number of mutations and epistatic interactions between the mutations, especially for strains derived from traditional random mutagenesis. For a comprehensive mutation analysis, a broader understanding of the relationship between genomic mutations and transcriptional changes is necessary. Through integrated whole-genome sequencing (WGS) and transcriptome sequencing (RNA-Seq), we were able to obtain comprehensive profiles of genomic and transcriptomic changes such as point mutations, indels and gene fusions for each affected gene to guide the subsequent mutation analysis.

Riboflavin (vitamin B2) is the direct precursor of FMN and FAD, one of the essential components of cellular physiology required by all bacteria, plants and animals. Commercial riboflavin production has been achieved using a combination of classical mutagenesis, genome shuffling and rational metabolic engineering in B. subtilis (Perkins et al., 1999, Revuelta et al., 2017, Schwechheimer et al., 2016, Wang et al., 2011, Wang et al., 2014b). The chemical or physical mutagenesis methods employed to engineer riboflavin production in B. subtilis endowed the strains with high riboflavin production but generated an unclear genetic background, limiting our ability to further explore their tremendous potential. So far, few studies have systematically analyzed the relationship between the genetic characteristics and riboflavin overproduction phenotype of B. subtilis (Paracchini et al., 2017). The objective of the present study was to identify the particular genetic element(s) responsible for the riboflavin overproduction phenotype, to gain important insights into the inner mechanisms through which the nontargeted riboflavin-overproducing mutants were derived.

Section snippets

Bacterial strains, media and chemicals

The bacterial strains used in this work are listed in Supplementary Table S1. All strains were stored at − 80 °C and revived by growing on LB agar slants. B. subtilis 24/pMX45 was derived from B. subtilis 168 by multiple rounds of selection with 8-azaguanine (Azr), decoyinine (Dcr) and roseoflavin (RoFr) for resistance mutations that deregulate the riboflavin biosynthetic pathway and the purine de novo biosynthesis pathway. It contained a low-copy number pSM19035-derived plasmid pMX45 carrying

Identification of genomic changes by whole genome sequencing

High-throughput sequencing technologies are enabling the complete characterization of mutant genomes and facilitating the identification of relevant mutations for different phenotypes. B. subtilis 24/pMX45 showed a specific riboflavin production rate of 42.44 ± 3.35 μmol g−1 CDW h−1 and product yield of 50.48 ± 3.14 mg riboflavin g−1 glucose in LBG medium. To determine the genetic basis for the strain's riboflavin overproduction phenotype, the genome of B. subtilis 24/pMX45 was re-sequenced

Discussion

Comparative genomic analysis of excellent producer strains with their progenitors facilitates the identification of beneficial mutations which can then be combined into a healthy chassis strain or embedded in other strains to generate a robust producer free from the unnecessary mutations (Conrad et al., 2009, Lee and Palsson, 2010, Yang et al., 2010). In the present work, we demonstrated the clear potential of utilizing a combination of multiple “omics” approaches to obtain insights into the

Conclusions

As a case study, we analyzed the genetic characteristics of riboflavin producing strains of B. subtilis using a combined WGS and RNA-Seq approach. Several beneficial mutations were identified and reintroduced into the wild-type strain, successfully recovering most of the high-performance phenotype. It is beneficial to exploit the precious data in genetic databases to confer a high performance on industrial microorganisms. More importantly, some newly identified mutations showed the potential

Acknowledgments

We are grateful to Profs. Chunting Zhang and Feng Gao (Tianjin University) for invaluable assistance and inspiring discussions. We wish to thank the Hebei Pharmaceutical Factory for the kind donation of the strain B. subtilis 24/pMX45. This work was supported by the National Natural Science Foundation of China (NSFC-21576200, NSFC-21576191, NSFC-21776209 and NSFC-21621004) and the Doctoral Scientific Research Foundation of Zhengzhou University of Light Industry (No. 2015BSJJ018).

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