Biosynthesis Of Glutaric Acid In E.coli And Its Fermentation Optimization

Glutaric Acid（Pentanedioic acid）,also known as 1,3-propanedicarboxylic acid or pectic acid,is an important dicarboxylic acid widely used in plastics,chemical engineering,medicine and other fields.In industry,glutaric acid is mainly recovered from the by-products of adipic acid production,but this method has a series of problems such as high environmental pollution,difficulty in separation and purification,and complicated preparation process.Therefore,the biosynthesis of glutaric acid has received extensive attention.Zhao et al.achieved glutaric acid biosynthesis by constructing the adipic acid reverse degradation pathway（RADP）in E.coli.The use of cerulenin inhibits the synthesis of fatty acids to increase the concentration of malonyl-CoA.However,this method has a low glutaric acid titer and cannot meet the industrial needs.On this basis,this subject continues to carry out metabolic engineering and fermentation optimization to further increase the glutaric acid titer.It was found in the early realization that the content of malonyl-CoA in E.coli was low,which limited the increase in glutaric acid titer.In order to solve this problem,the malonate uptake pathway was introduced into E.coli.The expression of the target protein and the transcription of the target gene were verified by SDS-PAGE and RT-qPCR,so that E.coli could use malonate to produce glutaric acid.The results showed that the glutaric acid titer and yield were increased by 2.1times and 10.7%respectively with only 4 g·L^-1 malonate.After a series of optimization of malonic acid concentrations,the glutaric acid titer can be increased by 2.7 times to 0.56 g·L^-1 with 10.4 g·L^-1malonate addition.Based on this research,it was found that the introduction of malonate uptake route increased glutaric acid production,which increased the ATP content in the bacteria.To compensate for the consumption of ATP,engineering strains produced large amounts of acetic acid.In order to enhance the synthesis pathway of glutaric acid and weaken the competition pathway,the fumarate enzyme（fumC）and succinyl-CoA synthetase（sucC）was knocked out in E.coli.It was found that glutaric acid production did not increase after knocking out fumC,while knocking out sucC caused imbalance between cell growth and glutarate production,even non-production of glutaric acid,implying that glutaric acid can not be significantly increased by knocking out the competitive pathways and reducing cofactors.The fermentation optimization was conducted based on the above research.The initial sugar concentration of the fermentation medium was determined to be 8 g·L^-1 through experiments,and0.8 mM IPTG was used to induce the expression of the target protein at OD₆₀₀=2.On the basis of shake-flask fermentation,the optimized conditions of the 5 L fermentor were determined to be 0.5vvm aeration and 400 rpm.At this time,the glutaric acid titer was 3 g·L^-1.And the titer of glutaric acid reached 4.35 g·L^-1 when 10.4 g·L^-1 malonate was added in batch fermentation,which increased by 45%.The optimal feed carbon source glycerin was determined.In order to make the bacteria use glycerin more efficiently,the dissolved oxygen was selected as the parameter to control the feed speed.For the cell growth,the dissolved oxygen parameter was controlled to 0%;When synthesizing the target product,reduce the feed rate to maintain the dissolved oxygen at 50%,and the final glutaric acid titer reached a maximum of 6.3 g·L^-1,achieving a further increase in glutaric acid titer.