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

- 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.

We previously reported a metabolic engineering strategy to develop an isopropanol producing strain of Cupriavidus necator leading to production of 3.4 g L−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 15 g L−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 9.8 g L−1 was achieved in fed-batch culture on fructose as the sole carbon source (equivalent to 16 g L−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.