Abstract

Polyhydroxyalkanoates (PHAs) are a large class of microbial natural biopolymers that accumulated as a kind of carbon and energy reserve in the presence of an excess carbon source.Polyhydroxyalkanoates that contain the medium-chain-length monomers (mcl-PHA) have a wide range of applications owing to their superior physical and mechanical properties. However, plenty of mcl-PHAs synthesized directly from glucose in E. coli are actually obtained from fatty acid -oxidation pathway due to the difficulty in lacking the enzymes that catalyze the transfer of the 3-hydroxyacyl-ACP to 3-hydroxyacyl-CoA. A challenge to synthesize such mcl-PHA from unrelated and renewable source is exploiting the efficient metabolic pathways that lead to the formation of precursor (R)-3-hydroxyacyl-CoA. Here, by engineering the reversed fatty acid β-oxidation cycle, we were able to synthesize mcl-PHA in Escherichia coli directly from glucose. After deletion of the major thioesterases, the engineered E. coli produced 6.6 wt% of cell dry weight mcl-PHA which contains C6-C14 monomers. Deletion of tesB in LZ01 significantly diminished the secretion of fatty acids from 605.25 mg l-1 to 198.48 mg l-1. The total fatty acid secretion in the final strain reduced to 94.44 mg l-1, and C10:0 fatty acid was not detected. Furthermore, when a broad substrate specific PHA synthase from Pseudomonas stutzeri 1317 was employed, recombinant E. coli synthesized 12.1 wt% of cell dry weight scl-mcl PHA copolymers, of which 21.2 mol% is 3-hydroxybutyrate and 78.8 mol% is medium-chain-length monomers. The functional reversal of fatty acid -oxidation cycle provides a promising and encouraging platform to synthesize scl-mcl PHA copolymers with wide range of monomers. The reversed fatty acid β-oxidation cycle offered an efficient metabolic pathway for mcl-PHA biosynthesis in E. coli and can be further optimized.