| L-threonine,as one of the eight essential amino acids in human body,is widely used in the fields of food,medicine and feed.With the increasing market demand,it is necessary to develop a strain with high yield of L-threonine.Escherichia coli is often used as a threonine producer due to its excellent growth performance and clear genetic background.In this paper,the laboratory-preserved threonine production strain E.coli THR was used as the starting strain,and it was transformed into the threonine-producing strain E.coli THR8 by metabolic engineering,The shake flask fermentation medium and fermentation conditions were then optimized.Optimized rotation speed and feed in the 5 L fermenter further increased L-threonine production.The main research results are as follows:?1?The carbon flux in the threonine biosynthetic pathway was increased,the intracellular absorption of threonine was inhibited and the feedback inhibition of key enzymes by excess threonine was reduced.The genes encoding Aspartokinase III?lysC?,Phosphofructokinase II?pfkB?and threonine absorption protein?sstT?were deleted by FLP/FRT recombinant enzyme system and the target strains were designed as E.coli THR?lysC?i.e.,E.coli THR1?,E.coli THR?lysC?pfkB?i.e.,E.coli THR2?and E.coli THR?lysC?pfkB?sstT?i.e.,E.coli THR3?.As compared with the original strain,L-threonine yield of strains THR1,THR2 and THR3was increased by 0.55%,5.04%and 9.97%,respectively.The conversion rate of glucose in E.coli THR3 was 10.04%higher than that of the original strain,wherea that in the strains THR1and THR2 were 14.78%and 3.33%lower than that of the original strain.?2?The feedback inhibition of L-threonine on the first key enzyme of the aspartate amino acid branch was relieved,and the extracellular transport ability and cofactor NADPH supply were increased.Aspartatekinase(lysCfbr)and threonine-secreting transporter?thrE?from Corynebacterium glutamicum and NADP+-dependent glyceraldehyde-3-phosphate dehydro-genase?gapC?in Clostridium acetobutylicum were heterologously expressed.Plasmids with exogenous genes lysCfbr,thrE and gapC were constructed and electransfered to strain E.coli THR3,resulted in strains E.coli THR?lysC/pEC-XK99E-lysCfbr?i.e.,E.coli THR4?,E.coli THR?lysC?pfkB/pEC-XK99E-lysCfbrthrE?i.e.,E.coli THR5?and E.coli THR?lysC?pfkB?sstT/pEC-XK99E-lysCfbrthrEgapC?i.e.,E.coli THR6?.The yield of L-threonine of strain THR4,THR5 and THR6 was 17.30%,32.71%and 53.10%higher than that of the original strain,respectively.Moreover,the conversion of glucose of these recombinant strains increased by 29.62%,24.79%and 42.81%,respectively.Among them,the accumulation of L-threonine by E.coli THR6 was more obvious.?3?Increase the supply of the aspartate family amino acid precursor oxaloacetate?OAA?.Pyruvate carboxylase?pycA?in C.glutamicum was inserted into the genome,and NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase encoded by the non-essential gene gapA was knocked out.The strain E.coli THR?lysC?pfkB?sstT?gapA::pycA?ie E.coli THR7?and E.coli THR?lysC?pfkB?sstT?gapA::pycA/pEC-XK99E-lysCfbrthrEgapC?ie E.coli THR8?were constructed.The yield of L-threonine was increased by 17.21%and 60.44%,respectively,and the conversion of glucose was increased by 2.02%and 44.03%,respectively.The yield of threonine accumulated by recombinant strain E.coli THR8 increased to 110.35g·L-1 and the acid production capacity per unit increased to 6.08 g·g-1 DCW,the maximum biomass was 18.12 g DCW·L-1,the growth and threonine-producing adventages of E.coli THR8 were more obvious.?4?Fermentation medium and fermentation conditions were optimized for the success-fully constructed E.coli THR8.Plackeet-burman was used to screen out three factors which had significant influence on L-threonine yield,i.e.,CSL,glucose and MgSO4·7H2O.In addition,the optimal medium formula(g·L-1)for flask fermentation of L-threonine was obtained through Box-Behnkne experiment:glucose 30.5,beet molasses 14 mL·L-1,Betaine1.0,CSL 0.75,H3PO4 0.6 mL·L-1,MgSO4·7H2O 0.80,FeSO4·7H2O 5.0×10-3,KCl 0.80,MnSO4·4H2O 1.0×10-2,CaCO3 20.0.Based on it,single factor optimization was used to optimize the fermentation conditions of shake flask fermentation.The experimental results are as follows:Fermentation temperature 37°C,initial pH 7.0,liquid volume 35 mL/500 mL,inoculum 12.5%,OD56262 0.4,IPTG was added at a final concentration of 0.8 mmol·L-1 after cultivating 8 h.Under this optimized condition in shake flask fermentation,the yield of L-threonine was 11.85 g·L-1,which was 15.61%higher than that before optimization.?5?The rotation speed and feed?glucose?during the fermentation of L-threonine were optimized in a 5 L fermentor.Different stages of cell growth require different levels of dissolved oxygen.In addition,the residual sugar content in the fermentation broth also affects the accumulation of L-threonine.The final L-threonine production reached 118.98 g·L-1 when the residual sugar concentration was maintained at 5-10 g·L-1,which was 7.8%higher than that before optimization. |
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