Systematic Metabolic Engineering Of Escherichia Coli For L-methionine Production

Methionine is a sulfur-containing amino acid essential for humans and animals.It is widely used in pharmacy,the food industry and feed additives,with a worldwide production of ~800 000 tons in 2013(>600 000 tons were used as feed additives).To date,all the commercial production of methionine relies on acrolein chemical synthesis,in which the petrified material methyl mercaptan,acrolein,and hydrogen cyanide are used to synthesize a racemic mixture of DL-methionine.Since pharmaceutical and medical applications often need chiral pure methionine,enzymatic processes were used to convert the DL-racemate into the pure isomers.Although animals can metabolize both stereoisomers of methionine,L-methionine is easier to be utilized than D-methionine when fed at low levels.Low-cost,environmentally friendly bacterial production of L-methionine based on natural renewable resources is becoming more attractive,compared with the chemical synthesis process that are limited by environmental constraints(e.g.poisonous substrates,intermediates and waste)and high energy consumption.Over the past few decades,L-methionine-producing strains were obtained by screening or inducing mutants.However,no industrial strain for L-methionine production has been developed so far.With the deep understanding of the mechanisms of L-methionine biosynthesis pathway in Escherichia coli,rational metabolic engineering strategies to develop L-methionine producing strain are becoming more attractive.In this thesis,feedback inhibition,synthetic bottleneck and growth repression in the L-methionine pathway were investigated.A systematic metabolic engineering of E.coli for L-methionine production was carried out by functional analysis of important genes,optimizing the biosynthesis pathway,modifying the transporter and exporter of L-methionine.The L-methionine productivity was improved step by step.Additionally,we have explored some new regulation mechanisms of L-methionine biosynthesis pathway,which lays a foundation for further metabolic modification of E.coli.L-homoserine is the precursor and carbon skeleton of L-methionine.A high-L-homoserine-producing strain was firstly constructed from wild type E.coli W3110 using metabolic engineering.The lysA,metA,and thrBC genes were inactivated to block the competing and degradation pathways of L-homoserine.To enhance the carbon flux to L-homoserine,the effects of the lysC,thrA,and metL genes on L-homoserine production and the impact of L-homoserine on the aspartokinases(AKs)AKI,AKII,and AKIII were studied.The results demonstrated that,the bottleneck of the L-homoserine biosynthetic pathway was overcome and carbon flux to L-homoserine was enhanced by overexpressing the metL gene in E.coli W3110.The rhtA gene was also overexpressed under the control of the pN25 promoter to improve the survivability under the stress of high concentration as well as the productivity of L-homoserine.Moreover,we verified that TdcC is responsible for the import of L-homoserine and the inactivation of the TdcC transporter further increased L-homoserine production.These strategies improved the recombinant strain HM5(pBRmetL–pNrhtA)production of L-homoserine from 0 to 39.54 g·L-1 in fed-batch fermentation.To enhance the carbon flux to L-methionine and derepression of met regulon,thrBC,lysA,and metJ were deleted in turn.L-methionine biosynthesis obstacles were overcome by overexpression of metAFbr(Fbr,Feedback resistance),and malY under control of promoter pN25.Recombinant strain growth and L-methionine production were further improved by attenuation of metK gene expression through replacing native promoter by metK84 p,and the L-methionine biosynthesis obstacles were also overcome by overexpression of metAFbr.Blocking the threonine pathway by deletion of thrBC or thrC was compared.Deletion of thrC showed faster growth rate and higher L-methionine production.We constructed an L-methionine-producing recombinant strain from wild-type E.coli W3110 by the above metabolic engineering strategies,which improved L-methionine production from 0 to 0.4 g·L-1 and 3.5 g·L-1 in a 500 m L flask and a 15-L fermenter,respectively.metE,metF and metH were overexpressed to enhance methylation efficiency.Enhancing transfer of the methyl groups by deregulation of metE or metH to overexpress MetE or MetH significantly improved L-methionine production.Compared with MetE,MetH exhibited higher methylation efficiency.The effect of different expression level of metH on L-methionine production were also studied.Fermentation results demonstrated that much high or low expression of metH will decrease the production of L-methionine.Replacing native metH promoter of Me06 by pN25,the regulation control of metH was deregulated,and the L-methionine production of Me08(pETMAFbr-B-Y)reached to 5.43 g·L-1 in a 15-L fermenter.The MetD mutants were constructed to study the effect of inactivating MetD transporter system of E.coli W3110 on the production of L-methionine.metNIQ genes expression ratio and the uptake capacity of L-methionine were significantly increased by the deregulation of MetJ repression.By deleting the metNIQ genes cluster of E.coli W3110 and Me05,the MetD transport capacity were inactivated which resulted in the reduced uptake capacity of L-methionine.Besides,we deleted the metNIQ genes cluster,metN,metI,and metQ of L-methionine-producing chassis strain Me06.Growth curves and flask batch fermentation showed that,the growth and production of L-methionine were improved by the deletion of metI.The L-methionine production improved from 0.39 g·L-1 to 0.45 g·L-1,increased by 15.4%.The L-methionine yield on biomass improved from 0.14 to 0.15 g·g-1 DCW.Expression of yjeH was deregulated under control of tac promoter,through constitutive expression of YjeH at low level.This resulted in low concentration of intracellular L-methionine,and improved L-methionine production from 0.45 to 0.58 g·L-1.By modification of MetD transporter system,enhancing L-methionine exporter and methylation efficiency,the fed-batch fermentation production of L-methionine of Me15(pETMAFbr-B-Y/pKK-tacyjeH)reached to 7.19 g·L-1,which increased by 32.4% compared with Me08(pETMAFbr-B-Y).To enhance the supplement of cysteine for L-methionine,cys regulon were depressed under control of tac promoter.The fermentation results demonstrated that L-methionine production were improved by overexpressing cysC,cysE,cysH and cysK genes.Coexpression of cysE and cysC,the carbon flux to cysteine and sulfate reduction efficiency were enhanced,which resulted further improvement of L-methionine production.With enhancing L-methionine export rate,the L-methionine production of recombinant strain Me15(pETMAFbr-B-Y/pKK tacyjeH-cysE-C)increased to 1.16 g·L-1 and 10.10 g·L-1 in a 500 m L flask and a 15-L fermenter,respectively.