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GREACE-assisted adaptive laboratory evolution in endpoint fermentation broth enhances lysine production by Escherichia coli
- Source :
- Microbial Cell Factories, Vol 18, Iss 1, Pp 1-13 (2019), Microbial Cell Factories
- Publication Year :
- 2019
- Publisher :
- BMC, 2019.
-
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 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.
- Subjects :
- 0106 biological sciences
Arabinose
Lysis
Lysine
Mutant
lcsh:QR1-502
Mutagenesis (molecular biology technique)
Bioengineering
medicine.disease_cause
01 natural sciences
Applied Microbiology and Biotechnology
lcsh:Microbiology
dnaQ
03 medical and health sciences
chemistry.chemical_compound
GREACE
010608 biotechnology
Fermentation broth
medicine
Escherichia coli
030304 developmental biology
0303 health sciences
Chemistry
Research
Biochemistry
Metabolic Engineering
Mutagenesis
Fermentation
Directed Molecular Evolution
Lysine production
Adaptive laboratory evolution
Biotechnology
Subjects
Details
- Language :
- English
- ISSN :
- 14752859
- Volume :
- 18
- Issue :
- 1
- Database :
- OpenAIRE
- Journal :
- Microbial Cell Factories
- Accession number :
- edsair.doi.dedup.....a65dee9f21e44d4b03d1e5080f57d538