Metabolic Engineering Of Escherichia Coli For The Production Of Medium-chain Omega Position Hydroxy Fatty Acids

Hydroxy fatty acids(HFAs)are fatty acids with one or more hydroxyl groups in their chemical structures.They are widely used in chemical,food,pharmacy and cosmetic industries because of their special properties,such as reactivity,viscosity,stability,solvent miscribility.They can be produced naturally by microbe,plant and animal.However,the commercial application of HFAs is limited due to the low yield,simple category and tedious extraction.The chemical synthesis of HFAs is of large pollution as the massive use of strong acid alkali,and the position of hydroxyl is of poor specificity.In contrast,more and more researches focuse on the production of hydroxy fatty acids by microbial fermentation,especially,utilization of low cost and renewable sugars to produce hydroxyl fatty acids.In this study,metabolic engineering has been applied to successfully yield sub-terminal ω1/2/3-position of HFAs using renewable and cheap glucose and glycerol as substrates in type strain Escherich coli.The complexity of HFAs up to 19 was revealed by gas chromatography-mass spectrometry.Because HFAs were secreted into the culture medium,the cost of extraction and purification was significantly reduced.In this test,first,three plant acyl-acyl carrier protein thioesterases were evaluated for substrate specificity and chosen to precisely control the chain length of fatty acids(C12-C14)by expression in E.coli,providing adequate substrate for the synthesis of medium chain hydroxyl fatty acids.Medium chain fatty acids are also of important physiological function and application values.Second,to improve the production of medium chain hydroxyl fatty acids by metabolic engineering strategies including co-expression of a fatty acid metabolism regulator fadR,knockingout fadD to inhibite the fatty acid degradation,and regulation of gene copy number.The HFAs titer reached 86.1 mg/L,especially ω3-OH-C14:1 was boosted.Third,through addition of cheap and renewable glycerol into medium,HFAs titer was further enhanced to 108 mg/L,although the HFA profile did not change.Fourth,transcriptomic(RNA-Seq)and bioinformatics analysis showed that fec operon was possibly involved in the secret of HFAs in E.coli.The fluorescence quantitative reverse transcription PCR(qRT-PCR)results also verified this conclusion.This study illustrated a metabolic strategy for medium chain HFA production in E.coli by using cheap and renewable glucose and glycerol,without supplement of external medium chain fatty acid.We also first explored key genes involved in HFAs exocytosis.