Metabolic Engineering Of Escherichia Coli For Enhanced Production Of Hydroxy Fatty Acids

Polyhydroxy fatty acid (PHA) is a new generation of green biodegradable packaging materials, and hydroxy fatty acid (HFA) is an ideal monomer for PHA. HFA is also a multi-carbon chemical for broad applications. Due to the limitation chemical synthesis method and growing shortage of oil resources, more and more studies are turning to the use of low-cost, renewable carbohydrates for HFA production.Escherichia coli is a model microorganism. Its genetic background is clear and it is easy to be operated by molecular biology tools. E. coli has been widely used for the production of a variety of chemicals. In this study, we used engineered E. coli to prepare HFAs. The cytochrome P450-monooxygenase showed high catalytic activity towards fatty acid ω-carbon position to generate HFA. The P450BM3monooxygenase from Bacillus megaterium was cloned to expression vectors of different copy numbers and heterologously expressed in E. coli. The medium copy number plasmid pACYCDuet-1expression monooxygenase produced the highest levels of hydroxy dodecanoic acid and myristic acid, that is,593.3mg/L and547.3mg/L. The conversion rates of the substrate fatty acids reached59.7%and52.3%, respectively. Quantitative analysis of the hydroxyl products showed that the ratios of ω-1, ω-2, and ω-3were substantially identical, indicating that P450BM3had no hydroxylation site preferences. Through site-directed mutagenesis, a mutant of P450BM3was obtained. The mutant enzyme could convert lauric acid to obtain621.9mg/L hydroxy lauric acid at a conversion rate of62.1%. The ratio of ω-3hydroxy lauric acid was increased from35%to60%.In order to produce HFAs directly from sugars, the metabolic pathway of E. coli was further engineered. Heterologous expression of the thioesterase (BTE) from the California laurel (Umbellularia californica) increased intracellular free fatty acid content and changed free fatty acid compositions. The chain length of the free fatty acids generated were mainly C12and C14, making up more than80%of the total fatty acid profiles. The titer of free fatty acid reached118.9mg/L, and more than95%were present within the cells. Further co-expression of the monooxygenase P450BM3converted free fatty acids to HFAs (72.2mg/L). The proportion of ω-1, ω-2, and ω-3HFAs was substantially similar. By using the mutant P450BM3,79.5mg/L of HFAs were accumulated, and the ratio of ω-3hydroxy lauric acid reached60%.