BioChem - DOS

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

Metabolic engineering

Marc, Jillian; Grousseau, Estelle; Lombard, Eric; Sinskey, Anthony J.; Gorret, Nathalie; Guillouet, Sté phane E.

<P><B>Abstract</B></P> <P>We previously reported a metabolic engineering strategy to develop an isopropanol producing strain of <I>Cupriavidus necator</I> leading to production of 3.4gL<SUP>&minus;1</SUP> 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 <I>C. necator</I> has been indeed observed above a critical value of 15gL<SUP>&minus;1</SUP>. GroESL chaperones were first searched and identified in the genome of <I>C. necator</I>. Native <I>groEL</I> and <I>groES</I> genes from <I>C. necator</I> were over-expressed in a strain deleted for PHA synthesis. We demonstrated that over-expressing <I>groESL</I> genes led to a better tolerance of the strain towards exogenous isopropanol. <I>GroESL</I> genes were then over-expressed within the best engineered isopropanol producing strain. A final isopropanol concentration of 9.8gL<SUP>&minus;1</SUP> was achieved in fed-batch culture on fructose as the sole carbon source (equivalent to 16gL<SUP>&minus;1</SUP> 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&ndash;18% increase in the isopropanol yield on fructose.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Over-expression of the native <I>groEL</I> and <I>groES</I> led to a better tolerance of C. necator towards exogenous isopropanol. </LI> <LI> Over-expression of <I>groELS</I> in an engineered isopropanol producing C. necator led to improve the production on fructose. </LI> <LI> Isopropanol production from CO<SUB>2</SUB> as sole C-source was demonstrated using our engineered C. necator strain in bioreactor. </LI> </UL> </P>

2017

Elsevier

1096-7176

1096-7184

42

pp.74-84

10.1016/j.ymben.2017.05.007

http://click.ndsl.kr/servlet/OpenAPIDetailView?keyValue=05787966&target=NART&cn=NART78227196

Ralstonia eutropha; Cupriavidus necator; Isopropanol; Carbon dioxide; Hydrogen; Metabolic engineering; GroEL/S; Chaperones; Gas fermentation; Autotrophy