Search

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 SpeBA302V, AtpBS165N and SecYM145V 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.

l-Threonine is an important amino acid for animal feed. Though the industrial fermentation technology of threonine achieved a very high level, there is still significant room to further improve the industrial strains. The biosensor-based high-throughput screening (HTS) technology has demonstrated its powerful applications. Unfortunately, for most of valuable fine chemicals such as threonine, a HTS system has not been established mainly due to the absence of a suitable biosensor. In this study, we developed a HTS method to gain high-yielding threonine-producing strains. Novel threonine sensing promoters including cysJp and cysHp were discovered by proteomic analyses of Escherichia coli in response to extracellular threonine challenges. The HTS method was constructed using a device composed of the fused cysJp and cysHp as a promoter and a linked enhanced green fluorescent protein gene as a reporter. More than 400 strains were selected with fluorescence activated cell sorting technology from a library of 20 million mutants and tested within 1 week. Thirty-four mutants have higher productivities than the starting industrial producer. One mutant produced 17.95 % more threonine in a 5-L jar fermenter. This method should play a functional role for continuous improvement of threonine industry. Additionally, the threonine sensor construction using promoters obtained by proteomics analyses is so convenient that it would be easily extended to develop HTS models for other biochemicals.