Biocatalytic production of medium-chain fatty acids and corresponding derivatives

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Marko Kirtz, Konrad B. Otte, Janosch Klebensberger, Bernhard Hauer

University of Stuttgart, Germany

Medium-chain fatty acids (MCFA) and their corresponding -hydroxylated- and dicarboxylic acid derivatives represent important building blocks for the synthesis of a wide variety of industrial relevant products such as biopolymers, lubricants, and plasticizer. The production of MCFA using renewable resources and the de novo fatty acid biosynthesis cycle of the bacterium Escherichia coli offer a sustainable and economical promising alternative to the chemical synthesis of these molecules. In order to achieve this goal, we firstly cloned and expressed the MCFA specific thioesterase FatB2 from the seed of Cuphea hookeriana and the -ketoacyl-ACP-synthase mtKAS from the mitochondria of Arabidopsis thaliana in a -oxidation deficient (fadD) E. coli strain. Interestingly, after optimization of protein solubility, culture conditions, inductor concentrations, and the extraction and analysis methods, octanoic acid levels were only marginally increased in a strain co-expressing both enzymes. In contrast, a strain expressing FatB2 alone showed a 420-fold increase in detectable octanoic acid levels compared to the wildtype, indicating that the maintenance of two plasmids expressing mtKAS and FatB2 most likely increases the metabolic burden of the cell to a level that is detrimental for octanoic acid production. However, the octanoic acid overproducing strain now serves as a platform for our current engineering approaches including i) the production of -hydroxylated octanoic acid by the co-expression of a self-sufficient monooxygenase fusion protein, which has been recently constructed in our lab and demonstrated to exhibit the desired substrate specificity and ii) optimising carbon flux into the fatty acid biosynthesis by the overexpression of the host’s acetyl-CoA carboxylase (accABCD). In addition, we are currently trying to establish a yeast surface display approach, which would allow the screening of a random mutant library of FatB2 for the identification of variants with altered substrate specificity.