GREACE-assisted adaptive laboratory evolution in endpoint fermentation broth enhances lysine production by Escherichia coli

Abstract

Background Late-stage fermentation broth contains high concentrations of target chemicals. Additionally, it contains various cellular metabolites which have leaked from lysed cells, which would exert multifactorial stress to industrial hyperproducers and perturb both cellular metabolism and product formation. Although adaptive laboratory evolution (ALE) has been wildly used to improve stress tolerance of microbial cell factories, single-factor stress condition (i.e. target product or sodium chloride at a high concentration) is currently provided. In order to enhance bacterial stress tolerance to actual industrial production conditions, ALE in late-stage fermentation broth is desired. Genome replication engineering assisted continuous evolution (GREACE) employs mutants of the proofreading element of DNA polymerase complex (DnaQ) to facilitate mutagenesis. Application of GREACE coupled-with selection under stress conditions is expected to accelerate the ALE process. Results In this study, GREACE was first modified by expressing a DnaQ mutant KR5-2 using an arabinose inducible promoter on a temperature-sensitive plasmid, which resulted in timed mutagenesis introduction. Using this method, tolerance of a lysine hyperproducer E. coli MU-1 was improved by enriching mutants in a lysine endpoint fermentation broth. Afterwards, the KR5-2 expressing plasmid was cured to stabilize acquired genotypes. By subsequent fermentation evaluation, a mutant RS3 with significantly improved lysine production capacity was selected. The final titer, yield and total amount of lysine produced by RS3 in a 5-L batch fermentation reached 155.0 ± 1.4 g/L, 0.59 ± 0.02 g lysine/g glucose, and 605.6 ± 23.5 g, with improvements of 14.8%, 9.3%, and 16.7%, respectively. Further metabolomics and genomics analyses, coupled with molecular biology studies revealed that mutations SpeB A302V , AtpB S165N and SecY M145V mainly contributed both to improved cell integrity under stress conditions and enhanced metabolic flux into lysine synthesis. Conclusions Our present study indicates that improving a lysine hyperproducer by GREACE-assisted ALE in its stressful living environment is efficient and effective. Accordingly, this is a promising method for improving other valuable chemical hyperproducers. Electronic supplementary material The online version of this article (10.1186/s12934-019-1153-6) contains supplementary material, which is available to authorized users.

Biosensor-Coupled In Vivo Mutagenesis and Omics Analysis Reveals Reduced Lysine and Arginine Synthesis To Improve Malonyl-Coenzyme A Flux in Saccharomyces cerevisiae

“…When combined with omics analysis, a key mechanism can be identified to contribute to specific phenotypes. In vivo mutagenesis has been employed to improve stress tolerance and the production of metabolites such as n -butanol, lysine, tyrosine, and isoprenoid in Escherichia coli ( 17 – 20 ). However, the application of error-prone replication for cell factory construction is relatively rare in Saccharomyces cerevisiae .…”

Advanced strategies and tools to facilitate and streamline microbial adaptive laboratory evolution

“…On the other hand, besides the characterization of obvious phenotypes, like growth rate or production, other omics technologies, including transcriptomic, proteomic, and metabolomic technologies, are being used in molecular level analyses of phenotypes of the evolved or reconstructed microbes [22,35,120,121]. The alterations in the amounts of specific transcripts, proteins, or metabolites could decipher the direct reasons for adaption and reveal the latent targets for further engineering.…”

Intelligent host engineering for metabolic flux optimisation in biotechnology

“…The mutation rate in E. coli has been increased in a principled way through a technique called genome replication engineering assisted continuous evolution (GREACE) [476][477][478]. In this approach, a plasmid carrying a modified DNA proofreading element (the dnaQ gene) is transformed into the initial strain of interest, and then the transformed cells are subject to continuous ALE.…”