Regulation Mechanism On Pyruvate Branch Point Of Rhizopus Oryzae As3.2686during L-lactic Acid Production

At the pyruvate branch of L-lactic acid produced by Rhizopus oryzae, there arepyruvate dehydrogenase (PDH), pyruvate carboxylase (PC), and pyruvatedecarboxylase (PDC) except of lactate dehydrogenase (LDH), these enzymes caninfluence the flux of pyruvate flowing to the L-lactic acid pathway, and producemany byproducts. The study on the distribution and regulation of carbon metabolicflux on pyruvate branch can improve the conversion rate of the target product, andreduce the flux of byproducts; it has important theoretical and practicalsignificance. Therefore, under different aeration conditions of Rhizopus oryzaeAs3.2686in fermenters, by studying the yield of the organic acid and pyruvatelevel of enzyme activities at pyruvate branch point, the metabolic flux analysisequation can be established, and the metabolic flux distribution of differentventilation conditions can be calculated, then the relationship between the theenzyme activities of pyruvate branch and metabolic flux can be studied, and thedegree of control coefficient (Cpi) and flux control coefficient (FCC) can becalculated to find the strongest branch enzyme to control the metabolic productionof L-lactic acid fermentation system. The main findings were as follows:(1) Taking ventilation as a mean to destroy the metabolic balance, study themycelium biomass and specific growth rate first, and determine the total amount ofacid production and specific production rates, as well as the yield of four majororganic acid metabolites (the production of L-lactic acid, malic acid, ethanol,fumaric acid) at0.5L/(L·min)、1.0L/(L·min)、1.5L/(L·min)、2.0L/(L·min)、2.5L/(L·min). The result showed that the production of L-lactic acid were larger at the1.5L/(L·min) and2.0L/(L·min), were39.59g/L and34.60g/L respectively. Malic acid, ethanol,and fumaric acid were the largest at0.5L/(L·min),2.0L/(L·min) and2.0L/(L·min), theproduction of them were6.02g/L,2.05g/L and0.37g/L respectively.(2) On the basis of the constitution of metabolites, building the metabolic networkmodel and metabolic balance equation, then combined with cellular components toestablish of intracellular reactions dosimetry models, calculated the metabolic fluxdistribution of the metabolic network under different ventilation conditions, and summarizethe flux distribution ratio of different pathways at pyruvate branch point. It showed that inthis fermentation system, the conversion rate of glucose-pyruvate first increases and then decreases with the increasing of ventilation, and the sumit reached92.77%at2.0L/(L·min),and the metabolic flux was95.28. At pyruvate branch point, flux distribution of L-lacticacid was60.00%at2.0L/(L·min), larger than the ventilation of1.5L/(L·min), which was53.11%. The largest flux distribution ratios of oxaloacetate, ethanol, acrtyl coenzyme Apathways were30.01%,52.07%and4.02%, under the ventilation2.5L/(L·min),0.5L/(L·min) and1.0L/(L·min) respectively.(3) Through metabolic control analysis (MCA), compared the enzymatic activities ofPDH, PDC, PC, LDH and ADH under aerobic conditions with the fermentation. As theanalysis result, the activity of PDH was lowest at0.5L/(L·min), and highest at2.0L/(L·min), exceeded12U/L between24～60h. From the above, the flow distribution ofacetyl coenzyme A at0.5L/(L·min) reached the maximum; the enzyme activity of PDC at2.0L/(L·min) was very low, but the fact was that actual yield of ethanol was the highestwhich opposite the trend of enzyme activities; the enzyme activities of PC were very low atthe0.5L/(L·min), the fumaric acid production is very low while the actual production ofmalic acid is very high; the trends of LDH enzyme activities and the actual production ofL-lactic acid were consistent, but at the ventilation of2.0L/(L·min) the flux distributionratios of L-lactic acid pathway and the activities of LDH were the highest, though the actualyield of L-lactic acid was slightly lower than the1.5L/(L·min), all of these showed thatthere are other factors limited the transformation of L-lactic acid at2.0L/(L·min); the trendof ADH activities was consistent with the curve of ethanol yield, the enzymatic activity andthe maximum yield were also the highest at the2.0L/(L·min), it described that the fluxdistribution ratio of ethanol pathway was less than1.0L/(L·min), it can be seen that lowventilation restrained conversion in the reaction of catalytzed ethanol.(4) According to the results of correlation analysis coefficient and the degree of controlcoefficient, PC and PDH, LDH and PDC, ADH and PDC, ADH and LDH were greatlysignificant(P<0.01), PDC took a negative control to the flux of pyruvate branch at thesteady state; for L-lactic acid flux, the positive degree of control factors were PDH, PC andLDH successively, and ADH afforded a negative control; for ethanol flux PDH and ADHtook the strongest control roles, and PC afforded a strong negative control role; for acetylcoenzyme A flux, PDH and PC played the main positive control role; for oxaloacetate flux,PDH and PC took the strongest positive role with ADH is negative.(5) Fitting the flux in steady state and the curves of enzyme activity at pyruvate branch,calculating the FCCs of the pyruvate branch enzyme, the results showed that for themetabolic stabilization period, PDC and PC exerted the main control capability to thesystem, synthesizing the results of the degree of control coefficient, PC was the main control enzyme of pyruvate branch to the fermentation.