Producing Strain Construction And Fermentation Condition Optimization Of L-tryptophan

L-tryptophan.one of the essential amino acids in human and animal life,is widely used in the pharmaceutical,food and feedstuff industries,and the demand of L-tryptophan in feedstock is maximum.In this study,L-tryptophan producing strain Escherichia coli TRTH was used as original strain under the guidance of metabolic engineering theory and the different gene deletion or over expression of L-tryptophan engineering strains were constructed according to different metabolic problems.In order to decrease the Phosphoenolpyruvate(PEP)consumption through TCA cycle,the L-tryptophan producing strain named as TRTHAptsG was constructed,with the gene ptsG,encoding specific glucose transporter EII CBGlc,knockout.This will increase the L-tryptophan synthesized from PEP.Then the gene glk and glf,which encoding glucokinase in E.coli and glucose transporter in Z.mob,respectively,were overexpressed in TRTHAptsG to construct strain TRTH(pSTV28-KTF)AptsG.The results of 5 L fed-batch fermentation revealed that,the biomass of TRTH(pSTV28-KTF)AptsG and TRTH(pSTV28)AptsG reached 16.24 g DCW/L and 4.58 g DCW/L,which decreased 49.12%and 85.84%compared with TRTH,respectively;L-tryptophan production were 10.25 g/L and 3.1 g/L,which were droped 61.32 g/L and 88.30%compared with TRTH,respectively.The results indicated that the seriously effected the normal growth and metabolism of strains.Although the overexpressions of gene glk and glf could improve the suger transform,the side effects caused by deletion of gene ptsG still couldn’t be solved.Considerated to increase more carbon metabolism into tryptophan snythetic,the strain TRTHAppc was constructed,with the gene ppc,encoding phosphoenolpyruvate carboxylase,knockout.The results of flask fed-batch fermentation revealed that,the biomass,L-tryptophan production and acetic acid of TRTHAppc reached 1.38 g DCW/L,1.5 g/L and 3.4 g/L,which were 18.60%、28.3%and 32.69%of TRTH.The results indicated that the deletion of gene ppc seriously effected the normal growth and L-tryptophan production of strains.The effection was not obvious although adding succinic acid during the fermentation,which indicated that the deletion of gene ppc has a little effect on succinic supply during TRTH fermentation.In order to inhance glyoxylate bypass,the strain TRTHAiclR was constructed,with the gene iclR,the key gene of the glyoxylate shunt,knockout,which will decrease the CO2 loss caused by TCA and make the matebolism more economic.The results of flask fed-batch fermentation revealed that the titer of L-tryptophan and the yield of L-tryptophan from glucose of E.coli TRTHAiclR reached 6.52±0.46 g/L and 13.17%,which were 21.32%and 22.41%higher than TRTH,respectively;6.82 g/L of acetic acid accumulated in the culture of TRTHAiclR.which was 37.63%lower than TRTH.In the 30 L fermentation,the titer and yield of L-tryptophan of TRTHAiclR were 13.01 ± 1.05 g/L and 6.51%,which was 60.42%and 68.31%lower than TRTH,respectively;acetic acid was 18.21 g/L,which was 1.33 times of TRTH.In conclusion,enhancement of glyoxylate shunt resulted in shortage of energy supply and higher acetic acid accumulation,which lead to lower biomass and L-tryptophan titer in 30 L fermentation.After test,energy shortage in recombinational strains was a tough problem.So in order to increase the level of intracellular ATP,the strains TRTHAysaA,TRTHAydaS and TRTHAybiX were constructed,with the gene ysaA,ydaS and ybiX knockout,respectively.Then constructed TRTHAysaAAydaS,with the gene ysaA and ydaS knockout meanwhile.The results of flask fed-batch fermentation revealed that the biomass,L-tryptophan production and yeild on glucose of the recombinational strains increased contrasted with TRTH except TRTHAysaAAydaS;The results of 30 L fed-batch fermentation revealed that the biomass and L-tryptophan production of TRTHAysaA,TRTHAydaS,TRTHAybiX and TRTHAysaAAydaS decreased compared with TRTH;intracellular ATP(mmol/L)concentration was 3.20,7.04,6.04 and 7.13 after 4 h of fermentation,respectively,which were mostly equal to that of the original except TRTHAysaA.However,the intracellular ATP concentration droped sharply later.The results indecated that the way to enhance engery supply should be more careful and systematic.At last,30 L fermentation medium was optimizated with single factor experiment.The optimal medium consists of glucose 7.5 g/L,yeast powder 1.0 g/L,cottonseed Flour 20 g/L,citric acid 2.0 g/L,(NH4)2SO4 1.6 g/L,K2HPO4 7.5 g/L,MgS04.7H20 1.6 g/L and microelement 1 mL/L.E.coli TRTH grow in this medium had a 40.52 g/L titer of L-tryptophan,which was 24.62%higher than that of original.