Modification Of The Central Metabolism Of E.coli And Its Effect On Tryptophan Yield

Objective:To modify the central metabolism of E. Coli and increase tryptophan yield. Construct a co-expressing plasmid pZA31-ppsA-tktA,put it and pZE12-aroGfbr-trpEDfbr into E.coliMG1655ΔRT-R.And We used this new recombined bacterium to do the elementary fermentation experiment in lab. Compared different culture mediums, cubage, sugar concentration and the altera-tion of pH. And finally picked out the the best condition for further experiments.Methods:PLlacO1-tktA-T1 and PLlacO1-ppsA-T1 fragments were amplified by using pZE12-tktA and pZE12-ppsA as templates, and then ligated to pZA31 plasmid. Afterwards, pZA31-ppsA-tktA recombinant plasmid was constructed by combining the pZA31-tktA recombinant plasmid and PLlacO1-ppsA-T1 fragment and identified by sequencing. Then, the identified pZA31-ppsA-tktA and pZE12-aroGfbr-trpEDfbr plasmid were employed and transferred into E.coli MG1655ΔRT-R (E.coli MG1655ΔRT-R[pZA31-ppsA-tktA/pZE12-aroGfbr-trpEDfbr], engineering strain), while pZE12-aroGfbr-trpEDfbr recombinant plasmid was transferred into E.coli MG1655ΔRT-R (E.coliMG1655ΔRT-R [pZE12-aroGfbr-trpEDfbr], control). IPTG was used to induce the expression of tryptophan.Three hours after the inducement, the activities of ppsA and tktA enzymes were determined. The rest strains were cultured for another 48 hours under stable pH value and glucose concentration, and then collected to measure the tryptophan yield.Results:The size of the amplified fragments were same to that of the expected DNA fragments. Sequencing of the pZA31-ppsA-tktA recombinant plasmid showed that the sequences of the cloned tktA and ppsA were identical with the reported ones.The activities of ppsA and tktA enzymes (U) in the engineering strain were 3- and 3.5-fold higher respectively than those in the control (0.25±0.04 vs. 0.08±0.02, P < 0.05; and 0.21±0.03 vs. 0.06±0.02, P < 0.05, respectively); and tryptophan yield (g/L) in the engineering strain was 1.3 folds of that in the control (1.10±0.05 vs. 0.80±0.05, P < 0.05).Conclusion:We have modified the central metabolism of tryptophan production in E. Coli and increased the tryptophan yield, the method can be developed and used to construct higher-efficient engineering strain for tryptophan production.