| Autor: |
Xiaowei Wang, Qinggang Li, Cunmin Sun, Zhen Cai, Xiaomei Zheng, Xuan Guo, Xiaomeng Ni, Wenjuan Zhou, Yanmei Guo, Ping Zheng, Ning Chen, Jibin Sun, Yin Li, Yanhe Ma |
| Jazyk: |
angličtina |
| Rok vydání: |
2019 |
| Předmět: |
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| Zdroj: |
Microbial Cell Factories, Vol 18, Iss 1, Pp 1-13 (2019) |
| Druh dokumentu: |
article |
| ISSN: |
1475-2859 |
| DOI: |
10.1186/s12934-019-1153-6 |
| Popis: |
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. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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