Research On Efficient Biosynthesis Of Medium Chain Fatty Acids Via The Reverse-beta Oxidation Cycle

Compared with the long chain fatty acids,medium chain fatty acids have many important application values due to its characteristics such as fast absorption,reducing fat deposition,regulating energy metabolism.At present,medium chain fatty acids are mainly derived from coconut oil and palm kernel oil.Palm and coconut trees are tropical trees,and they are difficult to plant widely in China.The medium chain fatty acids produced by plants extraction are influenced by seasonal factors and the cost of its purification is relatively high,so the development of high efficient and low cost microbial production of medium chain fatty acids have attracted more attention.In this project,efficient biosynthesis of medium chain fatty acids from Escherichia coli was studied.Based on the preliminary construction of the reverse beta-oxidation cycle,we utilized the CRISPR-cas9 system to analyze the limiting factors on the efficiency of reverse beta-oxidation cycle.And we finally established the effective coupling synthesizing mechanism of the acetyl-coA and NADH cofactors.This research was helpful to analyze the synthesizing mechanism for specific length fatty acids,providing important theoretical references for synthesizing various types of fatty acids and its derivatives.The main results were described as follows:1.Preliminary construction of the reverse beta-oxidation cycleIn order to construct the optimal non natural reverse beta-oxidation cycle,the optimal synthesis pathway gene was screened by literature review,database comparison,codon optimization and so on.The BktB from the Ralstonia eutropha was selected to encode the first step reaction of the sulfur hydrolytic enzyme;the FadB of the Escherichia coli was selected to conduct the 3-Hydroxyacyl-CoA dehydrogenase of the second step and 3-Hydroxyacyl-CoA dehydratase of the third step;the TER from the Euglena gracilis was selected to conduct the Trans-enoyl-CoA reductase of the fourth step reaction;thioesterase encoded by ydiI from Escherichia coli was selected to conduct the termination reaction because of its higher activity against C6,C8,C10 fatty acyl coenzyme A substrate and lower activity of acetyl coenzyme A and butyryl coenzyme A.Reverse beta-oxidation cycle was successfully constructed to obtain an engineering strain for producing MCFAs,demonstrating the successful synthesis in Escherichia coli from substrate glucose.2.The construction of acetyl coenzyme A synthesis pathwayBased on the preliminary construction of MCFAs synthesis pathway,we utilized CRISPR-Cas9 technology to reconstruct the acetyl coenzyme A metabolic pathway related genes of Escherichia coli,and inhibited the metabolic flow of acetyl coenzyme A to lactic acids,succinic acids,acetic acids and ethanol.We excessively expressed acetyl coenzyme A synthesis pathway(ACS)from different strains,toxic by-products acetic acids were recycled to produce intracellular acetyl coenzyme A,and the yield of MCFAs increased to 1.25g/L.By constructing the acetyl coenzyme A synthesis pathway,the passive effects of the accumulation of acetic acid on the growth and metabolism of the bacteria were reduced,and the synthesis efficiency of the reverse beta-oxidation cycle was improved.3.Identification of pathway restrictive cofactor and its coupling synergistic regulation with acetyl coenzyme AThe optimization of the content of intracellular acetyl coenzyme A didn't bring about a continuous increase in the yield of MCFAs,and the synthesis efficiency of the pathway was limited by the level of intracellular redox factors.Based on increasing the expression of acetyl coenzyme A,we coupled the redox balance of the fermentation system by regulating the expression of the reductive cofactor NADH,and relieved the limitation of the intracellular redox synthesis efficiency.After increasing the expression of NADH in the system,the high efficiency acetyl coenzyme A synthesis pathway expressed from the anti feedback inhibition mutant SE_acsL41P from Salmonella enterica enabled product synthesis at maximum titers.By coupling the two cofactors synthesis pathway of acetyl coenzyme A and redox cofactor NADH,the production of MCFAs increased to 4.2g/L.According to the results,redox cofactor NADH and acetyl coenzyme A were identified as the key limiting factors of the reverse beta-oxidation cycle.4.The construction and identification of transport system towards MCFAsIn order to reduce the cytotoxicity and feedback inhibition caused by product accumulation,we chose the cell membrane transport protein pump to accelerate the transport efficiency of MCFAs to the extracellular direction.17 different transporter genes were cloned from the Escherichia coli genome.Overexpression of the transporter gene enhanced the medium chain fatty acids exocytosis pathway.According to the percentage of MCFAs exportation,marA and soxS were identified as transport-in protein,and ompF?acrE and mdtC were identified as transport-out protein.The identification of transport-in protein provided reference for large-scale industrial oil production applying MCFAs as precursors.The MCFAs transport system was constructed to reduce the cytotoxicity caused by the accumulation of products and the cost of products extraction,and to help establish the foundation for the industrial production of fatty acids from microbial fermentation.