Genetic Engineering Modification And Metabolic Regulation Research For L-tryptophan Production In Escherichia Coli

L-tryptophan（L-Trp）,one of the essential amino acids for humans and animals,is widely used in food additives,animal feeding stuff and pharmaceutical industries.L-Trp production by microbial fermentation has attracted widespread attention because of the simple operation,cheap raw material,and easy extraction of the product.In recent years,various strains with higher L-Trp production ability have been developed in abroad,using chemical mutagenesis combined with genetic engineering.However,some problems also existed s,such as the accumulation of by-products and the lower conversion rate in the fermentation process,which limited the improvement of industrial production of L-Trp.Based on previous work,L-Trp production strain was further modified by genetic manipulation,using Escherichia coli FB-04 constructed in this laboratory as the original strain.In addition,metabonomics research method was used to analysis the intracellular principal component and explore the influence of genetic modification on intracellular principal component.At the same time,intracellular metabolites were measured to explore the influence of genetic modification on metabolic flow in L-Trp biosynthesis process,providing theoretical basis for further optimization and improvement of strains.（1）Using L-Trp production strain E.coli FB-04 as the original strain,gene fruR,flhD,crp and tyrR which encodes global regulatory protein FruR,Crp,FlhD and TyrR,respectively,was deleted,by employingλRed recombination.Shake flask fermentation results showed that only fruR gene knockout was conducive to the biosynthesis of L-Trp;the final L-Trp content and conversion rate of fruR gene deleting strain E.coli FB-04（ΔfruR）were 1.85 g·L^-1 and 0.032 g·g^-1,respectively,increasing by 59.5%ang 52.4%,compared with E.coli FB-04.Metabonomics analysis indicated that fru R gene deletion had a significant effect on intracellular principal component;the metabolic flow of glycolysis,pentose phosphate pathway and the Krebs cycle（TCA）was dramatically enhanced;the content of L-ser,a terminal substrate in L-Trp branch pathway,was significantly improved.However,L-Trp production was decreased notablely in 3 L fed-batch fermentation process.Thus,we discussed the potential hazard caused by fruR gene deletion.（2）Using L-Trp production strain E.coli FB-04 as the original strain,strain FB-04（ΔptsH） was constructed by deleting ptsH gene（encoding Hpr）;moreover,the native ptsH gene was replaced by different ptsH gene mutants（encouding Hpr mutant N12S,N12A,N38D, S46A,S46N,respectively）in genome,resulting strain FB-04（ptsHN12S）、FB-04（ptsHN12A）、FB-04（ptsHS46A）、FB-04（ptsHS46N）,respectively.Shake flask fermentation results showed that ptsH gene deletion caused the significant restriction of growth and a decresed glucose consumption,however,the conversion rate of ptsH gene deletion strain FB-04（ΔptsH）was two times of that strain FB-04;the final L-Trp content of genomic mutant strains FB-04（ptsHN12S）、FB-04（ptsHN12A）及FB-04（ptsHS46A）was 1.53、1.98、1.82 g·L^-1,respectively,increasing by 31.9、70.7、56.9%,compared to FB-04, moreover,the conversion rates of all mutants were increased in different degree;the fermentation properties of strain FB-04（ptsHS46N）was similar with that of FB-04（ΔptsH）. 3 L fed-batch fermentation results showed that the maximum L-Trp yields of all genomic mutant strains were decreased;among those strains,the conversion rate of strain FB-04（ptsHN12S）was 0.143 g·g^-1,increasing by 33.6%,compared to FB-04.（3）Using L-Trp production strain E.coli FB-04 as the original strain,disruption Pta-AckA pathway by deleting genes pta and ackA,which encodes the Pta and ackA,respectively, forming strain FB-04（Δpta）and FB-04（ΔackA）.Shake flask fermentation results showed that the final acetate content of strain FB-04（Δpta）and FB-04（ΔackA）were 3.6 g·L^-1 and 4.5 g·L^-1,respectively,accounting for 27.9%and 34.9%of the strain FB-04;the final L-Trp content of strain FB-04（Δpta）and FB-04（ΔackA）were 1.40 g·L^-1 and 1.31 g·L^-1, respectively,increasing by 20.7%and 12.9%,compared to strain FB-04;notablely,the growth of strain FB-04（Δpta）was rectricted significantly.Metabolic flux analysis showed that knockout of gene pta led to dramatically decreased metabolic fluxes of glycolysis and TCA,and the significantly enhanced metabolic flux of pentose phosphate pathway.3 L fed-batch fermentation results displayed that even though deleting pta and ackA led to remarkeablely decreased acetate content,L-Trp production of FB-04（ΔackA）had no significantly improved,while L-Trp production and biomass of strain FB-04（Δpta） decreased dramatically.Thus,we discussed the negative impact of the normal physiological metabolism caused by pta deletion.（4）Pta mutant（Pta1）,which contains an amino acid substitution（Pro69Leu）,was found from strain E.coli CCTCC M 2016009,which forms a relatively lower acetate level.Pta and Pta1 were cloned,expressed and purificated,successively,and finally,enzymology properties were studied,using acetyl-coA as the substrate.The results showed that compared to Pta,the k_catat of Pta1 decreased by 37.7%,meanwhile the K_m of Pta1 increased by 190.9%;thus,eventually,value of k_cat/K_m was only 21%of the Pta.The result indicated that Pta1 owned a lower catalytic activity and substrate combination rate,and the application of this mutant in the genome could weaken Pta-Ack A pathway,then reduce the biosynthesis of acetate.（5）Using two-step scarless recombination,the native pta in the FB-04 genome was replaced with the mutant gene pta1 identified from E.coli CCTCC M 2016009,resulting in strain FB-04（pta1）.Shake flask fermentation results showed that compared to FB-04,the final L-Trp content of FB-04（pta1）was 2.09 g·L^-1,increasing by 80.2%;acetate content decreased by 34.9%.Metabolic flux analysis displayed that the native pta replaced by pta1 gene in genome slightly decreased carbon flux through glycolysis and significantly increased carbon fluxes through the pentose phosphate and common aromatic pathways.3 L fed-batch fermentation results showed that the maximum L-Trp yield and conversion rate of FB-04（pta1）were 44.0 g·L^-1 and 0.129 g·g^-1,respectively,increasing by 15.5% and 18.7%,compared with strain FB-04;meanwhile,acetate content of FB-04（pta1）was only 2.1 g·L^-1,which had a significant reduction,compared to FB-04(4.3 g·L^-1).（6）On base of strain FB-04（pta1）,the individual genes pykF and pykA,as well as the combination of pykF and pykA were deleted,forming strainsΔpykF,ΔpykA,and ΔpykF/pykA,respectively.Shake flask fermentation results displayed that compared to FB-04（pta1）,the final L-Trp content ofΔpykF andΔpykA were 2.43 g·L^-1 and 2.25 g·L^-1,respectively,improving by 16%and 8%,while the final L-Trp content ofΔpykF/pyk A was significantly decreased.In addition,the conversion rates of all these mutant strains were increased in different degree,and conversion rate ofΔpykF was highest(0.065 g·g^-1),compared to strain FB-04（pta1）.3 L fed-batch fermentation results showed that the maximum L-Trp yields ofΔpykF andΔpykA were 45.6 g·L^-1 and 44.5 g·L^-1 respectively, increasing by 19.7%and 16.8%,comparing to the original stain FB-04;conversion rates of ΔpykF andΔpykA were 0.14 g·g^-1 and 0.135 g·g^-1,increasing by 30.8%and 26.2%.Moreover,the growth ofΔpykF/pykA was remarkablely rectricted,and the L-Trpproduction as well as conversion rate was significantly lower.