Microbial production of medium-chain-length oleochemicals have received great research of interest due to their high value in chemical industries. However, medium-chain-length oleochemicals are not naturally abundant in organisms largely because the acyl chain lengths are tightly controlled by endogenous enzymes in the fatty acid synthesis (FAS) and beta-oxidation pathways. Thus, production of medium-chain-length oleochemicals require special enzymes that can catalyze the appropriate intermediates from FAS or beta-oxidation pathways and convert them to desired precursors. In this study, we propose that 3-hydroxyacyl ACP:CoA transferase is a key enzyme in producing medium-chain-length 3-hydroxyacyl-CoA derivatives and characterizing enzyme activities can help control the chain length by converting the medium-chain-length FAS intermediate 3-hydroxyacyl-ACP to 3-hydroxyacyl-CoA, which is the precursor for 3-hydroxy fatty acid or polyhydrixtalkanoates (PHA) synthesis. First of all, we characterized the functionality of PhaG from Pseudomonas putida by heterologous expression in appropriate E. coli hosts. Based on its sequence similarities, 50 homologs (30-95%) were designed to test activities by quantifying intracellular 3-hydroxyacyl-CoA concentration. Synthetic pathways for medium-chain-length PHA and 3-hydroxy fatty acid were constructed, respectively. The optimal 3-hydroxyacyl ACP:CoA transferase candidate was applied to test for medium-chain-length PHA and 3-hydroxy fatty acid production in vivo. Our results showed the 3-hydroxylacyl ACP:CoA transferase can be developed as a key enzyme to control the chain length for 3-hydroxylacyl-CoA derivatives.

Functional characterization of 3-hydroxyacyl ACP:CoA transferase for production of medium-chain-length oleochemicals

2019 Spring National Meeting

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