Strain Construction And Metabolic Regulation For L-tryptophan Production

L-tryptophan （L-Trp） is widely used in food, animal feed and pharmaceutical industries as an essential amino acid for humans. The production of L-Trp by microbial fermentation has the virtues of cheap sugar and simple extraction technology. Accompanied by the application of metabolic engineering in strain improvement, some L-Trp hyper-production strains was developed, however, there have existed some problems such as the obvious decrease of L-Trp production rate at later-stage of fermentation, the accumulation of byproducts etc.. In addition, the development of these strains is all involved in multiple rounds of random mutagenesis. Previous studies showed that random mutagenesis often produces unexpected mutations at some locations in genome together with desirable ones. Since it is difficult to ascertain the influence of these unidentified mutations, further strain improvement would be affected. In China, the relevant research of metabolic engineering of L-Trp has been only carried out since 2000, and the L-Trp production of constructed strains was much less than world’s advanced level, which hindered the development of domestic industry of L-Trp.In the present study, an L-Trp production strain E.coli FB-T1/pSV05 was developed by a series of defined genetic manipulations based on known regulatory and metabolic information. In addition, metabolic flux of L-Trp synthesis pathway was analyzed by determining the concentrations of multiple intracellular metabolites, which provided the basis for further improvement of L-Trp production. The main contents and results of this thesis are as following:（1） The feedback resistant 3-deoxy-D-arabinoheptulosonate 7-phosphate （DAHP） synthase encoded by aroF was achieved by deleting its residue Ile11. The feedback resistant anthranilate （ANTA） synthase encoded by trpED was achieved by replacing the residue Ser40 of TrpE with Phe. The trpR gene, encoding trp repressor, was knocked out from the genome of E. coli W3110 and the trpR mutant was named as FB-01. Both aroF^fbr and trpE^fbrD were cloned into the expression vector pSV next to the PR and PL promoter, resulting in the plasmids pSV04, and the recombinant plasmid was transformed into FB-01. The Fermentation results showed that the production of L-Trp of FB-01/pSV04 was 2.8 g/L. while, the concentrations of byproducts of L-Phe, L-Tyr, and ANTA in the culture medium were 1.8 g/L, 1.4 g/L, and 2.3 g/L, respectively（2） The degradation pathway of L-Trp was cut off by knocking out its critical gene tnaA based onΔtrpR background, resulting in the strain FB-02. Strain FB-02/pSV04 produced 7.8 g/L L-Trp. However, more byproducts were accumulated in the culture and the concentrations of L-Phe, L-Tyr, and ANTA reached 5.3 g/L, 3.6 g/L, and 5.4 g/L, respectively. In order to prevent the accumulation of L-Phe and L-Tyr, L-Phe and L-Tyr synthesis pathways were blocked. Based onΔtrpR.tnaA background, pheA, which encodes a bifunctional enzyme catalyzing the first two reaction steps of the L-Phe branch pathway in E. coli, was further knocked out and the resulting strain was named as FB-03. The fermentation results of FB-03/pSV04 showed that less than 1 g/L L-Phe was accumulated, but more L-Tyr （5.1 g/L） was observed in the culture media. Based onΔtrpR.tnaA.pheA background, tyrA was further knocked out on genome and resulted in FB-04. When pheA and tyrA were both knocked out, FB-04/pSV04 grew normally only by adding appropriate amount of L-Phe （2 g/L） and L-Tyr （3 g/L） into the medium. The fermentation results indicated that L-Trp production was further increased to 13.3 g/L, and no obvious change in the concentration of ANTA was observed.（3） The three genes of mtr, tnaB, and aroP of L-Trp uptake system were respectively knocked out from the genome of FB-03, resulting in the strains FB-T1, FB-T2 and FB-T3. The results showed that the L-Trp uptake activities of mtr, tnaB, and aroP knockout mutants was decreased by 48%, 34% and 17% when compared to that of the parent FB-04 （2.9 nmol min-1 （mg dry weight） -1）. The fermentation results of FB-T1/pSV04, FB-T2/pSV04 and FB-T3/pSV04 indicated that the L-Trp production of the mtr, tnaB, and aroP knockout mutants was 14.7, 13.1, and 12.3 g/L, respectively, which were increased by 34%, 19%, and 12% when compared to that of FB-03/pSV04. Furthermore, The L-Tyr production of mtr, tnaB, and aroP mutants was decreased by 40%, 21%, and, 15% as compared to the parent strain. The ANTA production of mtr, tnaB, and aroP mutants was decreased by 25%, 11%, and 6% as compared to the parent strain. In addition, it was found that the increase rate of intracellular concentration of L-Trp in mtr mutant was much lower than that in the parent in the process of fermentation. At the culture time of 16 h and 24 h, which represented the middle-stage of fermentation, the intracellular concentration of L-Trp of mtr mutant was 27.4 and 50.9μmol/L, respectively, which was decreased by 42% and 35% when compared to that of the parent. Based on these characteristics, the mechanism of improvement of L-Trp production caused by gene knockouts of L-Trp uptake system was analyzed.（4） The prediction of RNA secondary structure of E. coli trp leader of trp operon was performed, the resulted indicated that the free energy of trp leader （trpL） was -36.5 kcal/mol and was decreased by 32% when the sequences from 10 to 118 of trpL was knockout by using the Red recombination system. However, the fermentation results indicated that the accumulations of L-Trp, L-Tyr, and ANTA have no changes occurred after the 10-118th sequences were knocked out. The restriction endonucleases analysis of trpEDCBA was analyzed by using DNAMAN software. The sequences of trpDCBA was divided into three DNA fragments, trpD*C*, trpC*B*,trpB*A by using its only three restriction sites of Eag I、Sca I and Hpa I. The three DNA fragments were cloned into pSV04 step by step. Finally, the plasmid pSV-aroF^fbr-trpE^fbrDCBA （pSV05） was constructed and transformed into strain FB-T1. The fermentation results indicated that the L-Trp production of FB-T1/pSV05 was 17.1 g/L with 13% conversion ratio from glucose, which were increased by 21% when compared to that of FB-T1/pSV04. In addition, The ANTA production of FB-T1/pSV05 was 1.9 g/L, which was decreased by 51%, as compared to strain FB-T1/pSV04. （5） Metabolic network of L-Trp biosynthesis from glucose as carbon source in E. coli was constructed based on known metabolic information from the KEGG database resource （http://www.genome.jp/kegg/）. More than twenty kinds of intracellular metabolites concentrations were respectively determined by high performance liquid chromatography-tandem mass spectrometry （LC-MS/MS） when the cells of E. coli FB-02/pSV04, FB-03/pSV04, and FB-T1/pSV04 were grown up to the mid-exponential phase. Then the Flux distribution of metabolic network of L-Trp biosynthesis was computed. Finally, the characteristics of metabolic flux of L-Trp biosynthesis pathway, including uptake rate of glucose, main pathways, and critical products, were analyzed and the improvement of synthesis of erythrose 4-phosphate （E4P） and L-serine （L-Ser） was identified as the limited step of L-Trp sysnthesis pathway in FB-T1/pSV05.