Construntion Of L-Isoleucine Producing Strain And Optimization Of Fermentation Technology

L-isoleucine, one of the eight essential amino acids for human, plays an important part in the metabolism of our body. During the process of L-isoleucine synthesis, the enzyme acetohydroxy acid synthase catalyzes α-ketobutyrate and pyruvate to generate L-isoleucine. Acetohydroxy acid synthase (AHAS) shows higher affinity to α-ketobutyrate than to pyruvate. Therefore, increasing the supply of intracellular α-ketobutyrate may lead to preferred synthesis of L-isoleucine catalyzed by acetohydroxy acid synthase. What’s more, if the AHAS, one of the limited enzymes for L-isoleucine synthesis, is strengthened, and as a result, the limited reactions will be accelerated with more L-isoleucine accumulation.To investigate effects of increasing supplies of intracellular α-ketobutyrate and strengthened AHAS on L-isoleucine fermentation, L-isoleucine producer C. glutamicum YILW was used as the original strain for molecular construction. C. glutamicum YILWpXMJ19cimA was constructed through the overexpression of cimA, C. glutamicum YILWPtac was constructed by the insertion of Ptac, just in the upstream of ilvB in genome. C. glutamicum YILWPtacpXMJ19cimA was constructed by the overexpression of plasmid based on C. glutamicum YILWPtac. The real-time PCR showed the relative transcription of cimA and ilv B was increased by 920.0 and 12.5 fold, respectively. The feasibility of cimA gene overexpression in C. glutamicum YILWpXMJ19cimA was determined by SDS-PAGE.Fed-batch fermentation experiment was performed to compare the L-isoleucine. production of original strain to recombinant strains. The result discovered that the L-isoleucine production of YILWpXMJ19cimA and YILWPtac increased by 8.9% and 14.8% with the yield increased by 17.8% and 18.6%, respectively, compared to original strain YILW. The unit cell production was 15.1% and 20.3% higher than that of YILW. YILWpXMJ19cimA and YILWPtac excreted 25.0% and 29.9% lower L-alanine than that from original strain. Compared to YILW, YILWPtacpXMJ19cimA exhibited 14.5% higher L-isoleucine production with 42.4% and 17.6% higher L-isoleucine yield and unit cell production, respectively. The accumulation of L-alanine, one of the byproducts, was 8.6% lower than that of the parent strain. The L-isoleucine yield of YILWPtacpXMJ19cimA was 20.8% and 20.0% higher than that of YILWpXMJ19cimA and YILWPtac, respectively. These results illustrated Ptac promoter replacement combined with the expression of cimA to increase the supplies of α-ketobutyrate and strengthenning the limited AHAS led to increase in L-isoleucine production with relative obvious increase of yield. Metabolism analysis showed metabolic flux for L-isoleucine synthesis increased from 1.09% to 2.33%,7.69% and 4.26%, respectively.The 5 L-fermentor experiment came across problem that strains went into stationary phase too early with growth retardation and low glucose and oxygen consumption. In order to solve the problem, a variety of nutrients including soybean protein hydrolysate, core steep liquor, amino acid mixture, VH, purine nucleoside and microelement were fed into the fermentor in turns. As a result, the problem of low biomass was still existed. The feeding experiment exluded the reason that the nutrient contents of what we had fed was the key factor to limit the strains growth. Maybe, it is a certain or multiple nutrtion in core steep liquor that account for the growth and L-isoleucine production.