Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
6452740 | Metabolic Engineering | 2017 | 11 Pages |
â¢Over-expression of the native groEL and groES led to a better tolerance of C. necator towards exogenous isopropanol.â¢Over-expression of groELS in an engineered isopropanol producing C. necator led to improve the production on fructose.â¢Isopropanol production from CO2 as sole C-source was demonstrated using our engineered C. necator strain in bioreactor.
We previously reported a metabolic engineering strategy to develop an isopropanol producing strain of Cupriavidus necator leading to production of 3.4 g Lâ1 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 15 g Lâ1. 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 9.8 g Lâ1 was achieved in fed-batch culture on fructose as the sole carbon source (equivalent to 16 g Lâ1 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.