Construction Of Pyruvate-producing Escherichia Coli Engineering Strain Based On Redox State Regulation

Pyruvate,as an important chemical raw material,is widely used in materials,food,medicine,cosmetics and other fields.As a way of producing pyruvate,biological fermentation has the advantages of low cost and green.However,there are still some limits in the production of pyruvic acid by microbial fermentation,such as low sugar acid conversion and imbalance of intracellular redox state.This paper intends to construct an E.coli which can accumulate pyruvate by gene deletion,and regulate redox state to improve the production of pyruvate.The main research and results are as follows:?1?An engineering strain for pyruvate production was constructed by using an E.coli HBUT-D which has the ability of producing D-lactate.First,the ldh A and pox B genes were deleted by Red technology to block the conversion of pyruvate to D-lactate and acetic acid.Second,the deletion of succinyl-COA synthetase gene?suc CD?can activate glyoxylate pathway and weaken TCA cycle,suc CD gene was deleted to increase pyruvate accumulation;finally,we try to delete the synthesis of ATP synthetase subunit portion?atp FH?in addition to reduction of ATP synthesis,so as to improve the intensity of glycolysis and pyruvate production.Shake flask fermentation showed that HBUT-P2,which has been deleted ldh A and pox B genes,could accumulate pyruvate to19.56 g/L and yield to 0.79 g/g due to the lack of most of pyruvate decomposition pathways.However,the lack of atp FH and suc CD genes will lead to the decline of the growth ability and pyruvate production,which is not beneficial to the accumulation of pyruvate.?2?The deletion of by-product synthesis gene will increase the concentration of intracellular NADH and inhibit glycolysis pathway,thus affecting the yield of pyruvate.In order to improve the yield of pyruvate,three different methods were used to control the redox state of HBUT-P2.First,at the gene level,the udh A gene from E.coli W3110was overexpressed to catalyze NADH to NAD⁺to regulate the intracellular redox state.The udh A gene was successfully cloned to the original lac Z?gene site of p UC19.Pyruvate fermentation was carried out with 6%glucose as carbon source.After 8 hours of fermentation,1 mmol/L IPTG was added for the expression of udh A gene.Compared with the control HBUT-P2/p UC19,the production intensity of pyruvic acid of the engineering E.coli overexpressing udh A gene increased by about 32.35%,and the conversion rate of glucose increased by 10.80%,but the yield did not increase significantly.?3?At the fermentation level,we tried to use acetoin,Na NO₃,furfural and acetone to regulate the intracellular reducing power.The results of fermentation showed that as an exogenous electron acceptor,Na NO₃ could improve the ability of pyruvate production to a certain extent.Considering oxygen can also act as electron acceptor,which may affect the acceptance of H⁺by Na NO₃,therefor,the effect of adding Na NO₃to the strain on acid production under different dissolved oxygen conditions was compared.The yield of pyruvic acid increased from 3.42 g/L to 5.67 g/L and the glucose conversion rate increased to 90%under the anaerobic condition.Under the microaerobic condition,rotaryshaker at speed of 100 r/min and the addition of 200 m M Na NO₃ can greatly improve the glucose conversion rate,increased from 76%to 95.84%compared with control,but the production of pyruvic acid decreased from 7.98 g/L to6.23 g/L.?4?At the substrate level,sodium gluconate regulates the intracellular reducing power of E.coli.The production of pyruvic acid using sodium gluconate and glucose was compared.It was found that the pyruvic acid production increased from 32.43 g/L to 43.52 g/L,sugar conversion rate and intensity of pyruvate production increased by17.4%and 135.45%respectively.The overall yield of pyruvate increased to 62.42 g/L in the following 5 L fed-batch fermentation.In a word,sodium gluconate can solve the problem of unbalanced reducing power and increase the yield of pyruvate.