The Research On Metabolic Engineering Of Escherichia Coli For The Aerobic Production Of Malic Acid

Malic acid also known as 2-hydroxy butyl acid is an important member of the organisms in the tricarboxylic acid cycle,which is widely used in many industries such as food,pharmaceutical and chemical industries.The production methods of malic acid include extraction,enzyme conversion,chemical synthesis and microbial fermentation,which is more and more important because of the increasing of energy and environment degradation.In this paper,Escherichia coli was engineered through metabolic strategy for malic acid production from glycerol and glucose.This paper analyzed the aerobic network model of Escherichia coli for malic acid production from glycerol and glucose respectively.A series of engineered E.Coli mutants were constructed that could efficiently produce malic acid in this study through malic acid consumption pathway elimination,carboxylation flux enhancement,blocking byproducts accumulation and so on.Then we studyed on the ability of the strains to production malic acid under aerobic conditions from glycerol and glucose as carbon source.The results showed that the accumulation of malic acid was affected by different carbon sources.Firstly,in Escherichia coli E2 phosphoenol pyruvate carboxylase gene ppc was overexpressed in E2 yield strains E21,malic acid accumulation from 0.76 to 4.69 g/L from glycerol.Six mutants strains were constructed here through Single knockout and knockdown strategies to knock out the malic dehydrogenase gene mdh,mqo and malic enzyme gene maeA,maeB.Among them,E23(E21,?maeA,?maeB)accumulated 5.14 g / L malic acid from 9.8 g / L glycerol with a yield of 0.36 mol /mol glycerol.The result indicated that blocking the consumption of malic acid is beneficial to the accumulation of malic acid.A study was carried out with E21 through overexpresion the pyruvate carboxykinase pyc gene from Corynebacterium glutamicum and pck gene from Actinobacillus succinogenes to enhanced the carboxylation pathway.The resultes indicated that carboxylation pathway enhancement did not improve the malic acid production from glycerol under aerobic fermentation.With glucose as substrate for aerobic fermentation,Among the mutants,E23(E21,?maeA,?maeB)and E21(pTrcpyc)accumulated up to 0.62 mol / mol and0.79 mol/mol glucose with 10 g/L glucose.The results showed that elimination consumption way of malic acid could improve malic acid production.Meanwhile,due to the concentration of pyruvate from glucose by PTS transport system maintain a high level,pyc gene overexpression could effectively enhance the concersion from pyruvate to oxaloacetate,and further increased the production of malic acid.However,the combination of of malic enzymes deletion and pyc overexpression could not further increase the yield of malic acid.After optimization of fermentation conditions,strain E21(pTrcpyc)produced 12.45 g/L malic acid with a yield of 0.84mol/mol which is 63.2% of the theoretical yield.