(59g) Regulating Malonyl-CoA Metabolism Via Synthetic Antisense RNAs for Enhanced Biosynthesis of Natural Products

Malonyl-CoA is not only a critical precursor for fatty acid biosynthesis, but also serves as an essential building block of natural products such as coumarins, flavonoids, and other polyketides. Its biosynthesis is tightly regulated and its intracellular concentration is usually controlled at low levels, which poses a great challenge to biosynthesize malonyl-CoA derived molecules via metabolic engineering. Previous efforts to increase malonyl-CoA availability were mainly focused on the overexpression of acetyl-CoA carboxylase, the enzyme responsible for the conversion of acetyl-CoA into malonyl-CoA. However, inactivation of its consumption pathway has not been achieved since the disruption of fatty acid biosynthesis would be lethal to the host cells. To overcome this limitation, we report an antisense RNA strategy to down-regulate fatty acid biosynthesis and reduce undesired malonyl-CoA consumption to support heterologous biosynthesis. To increase the stability of the antisense RNAs, we employed an artificial loop-stem structure, which was further optimized in terms of its length and binding position to maximize the interference efficiency. We observed the interference effects at transcriptional, translational, and metabolic levels. Furthermore, we used three heterologous biosynthetic pathways of polyketide compounds to test the applicability of this RNA-mediated down-regulating strategy. The results showed that this strategy greatly improved the biosynthesis of malonyl-CoA derived natural products. This work demonstrates that antisense RNA is an efficient synthetic biology tool to increase microbial production of economically and pharmaceutically valued compounds.