Construction Of Micro-Aerobic Metabolism And The Optimization Of Fermentation Process For Efficient Synthesis Of Medium Chain Fatty Acids By Microbial Biosynthetic

Medium chain fatty acids(MCFA)have broad application prospects in food,medicine and biofuels because of their unique physical and chemical properties.Numerous studies have shown that the reverse fatty acid ?-oxidation pathway(r-BOX)is currently the most efficient medium-chain fatty acid synthesis platform.However,acetyl-CoA and NADH in E.coli native central carbon metabolism cannot ensure efficient reverse ?-oxidation pathway.The acetyl-CoA and NADH synthesis pathways used in previous studies require the addition of sodium acetate and sodium formate,respectively,which restricts the large-scale production of medium-chain fatty acids.In this study,we first constructed a one-step cofactor synthesis pathway with low energy consumption and reduced its feedback inhibition by site-directed mutagenesis.Then we constructed a micro-oxygen fermentation system with glycerol as carbon source and selected it in a micro-oxygen environment.There is still a higher activity of Enterococcus faecalis pyruvate dehydrogenase.After significantly improving the production efficiency of medium-chain fatty acids by using a low-energy cofactor synthesis pathway,we performed amplification experiments on a 5L fermentor.The final yield of medium chain fatty acids was 9.15 g/L through optimization of the fermentation process.The main conclusion of this paper are as follows:1.Devising one-step carbon-and energy-efficient pathway for redox regenerationWe first compare the pyruvate decarboxylation pathway(PDH route)and the acetaldehyde dehydrogenation pathway(ALDH route),select one-step cofactor synthesis with low energy consumption by pyruvate decarboxylation pathway,and then transform it by site-directed mutagenesis.The lpdA gene of the hydrogenase complex(PDHc)was used to reduce the feedback inhibition of NADH,and finally the effect of lpdA(A3 54V)on the production of medium chain fatty acids at different expression intensities was investigated.By using the low-energy acetyl-CoA synthesis pathway instead of the acs-mediated acetyl-CoA synthesis pathway and the formate dehydrogenase-catalyzed NADH production pathway,the medium-chain fatty acid synthesis efficiency is significantly altered,which is significant for the industrial production of microbial synthetic oils.2.Construction of micro-aerobic metabolism for medium chain fatty acid synthesisWe constructed a low-energy one-step synthesis of acetyl-CoA and redox cofactor regeneration pathways and found that the supply of NADH still limits the production of medium-chain fatty acids.In order to increase the supply of NADH,we use glycerol with higher degree of reduction instead of glucose as a carbon source.However,the PDH route does not fully function under micro-oxygen conditions.To further exploit the potential of the PDH route,we sought to find a dihydrolipoamide dehydrogenase that is more active in the slightly dissolved oxygen environment from other microbial genomes..By comparison,we selected EF_lpdA which maintained a high concentration of PDH enzyme activity in a mcro-oxygen environment and a high NADH environment.The EF_lpdA was again subjected to optimization of dissolved oxygen conditions and a medium chain fatty acid yield of 5.1 g/L at a shaking speed of 100 rpm.Given that the natural PDH pathway of bacteria consists of three subunits,we increased the overexpression of the other two subunits to increase the efficiency of the PDH route.Co-expression of pdhA,pdhB,EF_aceF and EF_lpdA resulted in a medium chain fatty acid yield of 6.1 g/L.3.Studies on scale-up of chain fatty acid fermentationThe fermentation of engineering bacteria is divided into the growth stage of the bacteria and the stage of product formation.The main goal in the growth stage is to obtain a large amount of bacteria quickly,and in the production stage after induction,the expression of the target gene is dominant.High-density fermentation of medium-chain fatty acid production strains in fermenters was achieved by optimizing induction time,inducer concentration,and pH parameters.Dissolved oxygen is both a nutrient factor and a parameter of fermentation for E.coli fermentation.Metabolism of micro-oxygen glycerol to produce excess NADH requires additional electron acceptor depletion to achieve redox balance,so we analyzed the dissolved oxygen requirements at different stages of the cell.By controlling the redox balance of the fermentation system in different stages before and after the induction of dissolved oxygen in stages,the yield of the chain fatty acid in the product is improved.The effects of intermittent feeding,constant-speed addition and exponential flow plus glycerol on medium-chain fatty acid fermentation were compared,and then the stages were added in the culture stage.The final medium-chain fatty acid production reached 9.15g/L.