Over expression of GroESL in Cupriavidus necator for heterotrophic and autotrophic isopropanol production

• Published in: Metabolic engineering 2017-07, Vol.42, p.74-84
• Main Authors: Marc, Jillian, Grousseau, Estelle, Lombard, Eric, Sinskey, Anthony J., Gorret, Nathalie, Guillouet, Stéphane E.
• Format: Article
• Language: English
• Subjects: 2-Propanol - metabolism; Autotrophy; Bacterial Proteins - biosynthesis; Bacterial Proteins - genetics; Biotechnology; Carbon dioxide; Chaperones; Chaperonins - biosynthesis; Chaperonins - genetics; Cupriavidus necator; Cupriavidus necator - genetics; Cupriavidus necator - metabolism; Gas fermentation; Gene Expression; GroEL/S; Hydrogen; Isopropanol; Life Sciences; Metabolic engineering; Ralstonia eutropha
• ISSN: 1096-7176; 1096-7184
• DOI: 10.1016/j.ymben.2017.05.007

We previously reported a metabolic engineering strategy to develop an isopropanol producing strain of Cupriavidus necator leading to production of 3.4gL−1 isopropanol. In order to reach higher titers, isopropanol toxicity to the cells has to be considered. A toxic effect of isopropanol on the growth of C. necator has been indeed observed above a critical value of 15gL−1. GroESL chaperones were first searched and identified in the genome of C. necator. Native groEL and groES genes from C. necator were over-expressed in a strain deleted for PHA synthesis. We demonstrated that over-expressing groESL genes led to a better tolerance of the strain towards exogenous isopropanol. GroESL genes were then over-expressed within the best engineered isopropanol producing strain. A final isopropanol concentration of 9.8gL−1 was achieved in fed-batch culture on fructose as the sole carbon source (equivalent to 16gL−1 after taking into account evaporation). Cell viability was slightly improved by the chaperone over-expression, particularly at the end of the fermentation when the isopropanol concentration was the highest. Moreover, the strain over-expressing the chaperones showed higher enzyme activity levels of the 2 heterologous enzymes (acetoacetate carboxylase and alcohol dehydrogenase) of the isopropanol synthetic operon, translating to a higher specific production rate of isopropanol at the expense of the specific production rate of acetone. Over-expressing the native chaperones led to a 9–18% increase in the isopropanol yield on fructose. •Over-expression of the native groEL and groES led to a better tolerance of C. necator towards exogenous isopropanol.•Over-expression of groELS in an engineered isopropanol producing C. necator led to improve the production on fructose.•Isopropanol production from CO2 as sole C-source was demonstrated using our engineered C. necator strain in bioreactor.