Identification Of The Enzyme Responsible For The Lactate Utilization In The Late Phase Of Lactococcus Lactis Aerobic Respiration And Clarification Of Its Regulatory Mechanism

Lactococcus lactis (L. lactis) is a dairy microorganism with an important economic value andis widely used as a starter culture for cheeses, buttermilk and butter. It has been found that L. lactiscan undergo respiration when hemin is added to the aerobic culture and the growth and survivalperformances of the respiratory culture are much better than that of the fermentation cultures. Themost distinctive feature in lactococcal respiration is that lactate could be consumed when glucoseis exhausted. Simultaneously, the flavor substances acetoin, diacetyl and acetate are greatlyaccumulated and the biomass is still increased. All these features are very valuable in the foodindustry. No reports have been published regarding the enzyme(s) responding for lactococcallactate utilization and the regulating mechanism of lactate production to consumption. The aim ofthis study was to search out the answers to these questions.The thesis includes four parts: Firstly, potential enzyme candidates responsible for the lactateutilization in the late phase of lactococcal respiration were screened by bioinformatics analysis.Secondly, which enzyme(s) play the dominant role could be preliminarily inferred by measuringdifferent enzyme activities of the crude enzymes prepared from L. lactis respiratory andfermentative cultures. The promising candidates were then over-expressed in E. coli and its lactateoxidation activity and the regulating mechanism of its physical role and lactate oxidation activitywere further studied with the purified enzymes. Finally, the concentrations of key intracellularmetabolites were compared between the cultures before and after lactate utilized in order to verifyif the cellular metabolites changes were benefit for the enzyme catalyzing lactate oxidation.Firstly, potential candidates with lactate oxidation activity were screened by bioinformaticsanalysis. The results showed that the homology of non-flavin, iron-sulfur containing three subunitslactate dehydrogenase LutABC were widely present in L. lactis. As LutABC in Bacillus subtilis168has L-iLDH (NAD-independent L-lactate dehydrogenase) activity, their homologous proteinsin L. lactis may also have the same activity. Flavocytochrome b2, which also has L-iLDH activity,was found to have two homologous proteins in L. lactis. One is L-lactate oxidase and the other is type II IPP isomerase but only the latter could simultaneously meet all the necessary characteristicsand may participate in lactate utilization. Besides the L-iLDH homologous proteins, LDHA, afermentative L-nLDH (NAD-dependent L-LDH), is widely present in L. lacits with lactateoxidation activity and thus it may also participate in lactate utilization.Based on the biomass and metabolites curves measured under respiratory and fermentationconditions, L. lactis cultures were collected at mid-log and stationary phases for crude enzymepreparation. L-nLDH and L-iLDH activities were measured with the crude enzymes. It showed thatthe L-iLDH activity in L. lactis was very weak, independent of culture pattern or growth phase. Incontrast to the faint L-iLDH activity, the L-nLDH activity of L. lactis was always very high, evenin the stationary phase of respiration when lactate could be utilized.A recombinant plasmid carrying fni gene for type II IPP isomerase was constructed with pQE30Xa vector and transformed into E. coli M15for expression. Recombinant FNI was purified usedfor SDS-PAGE analysis, which showed a single band corresponding to41.17kDa. Spectrumscanning showed that FNI was a flavin protein. It was verified that purified recombinant FNI wasbiologically active evidenced by1H NMR analysis that it could effectively catalyze IPPisomerization into DMAPP. However, its nLDH and iLDH activities were extremely low and theactivities would further reduce to be undetectable when the amount of enzyme addition wasreduced or divalent mental ions were added to the reaction mixtures.In this study a new enzyme assay method imitating respiratory cellular condition wasproposed, by which the effect of NADH removal on the NAD-dependent lactate oxidation activitycould be measured. noxE, the gene for a H2O-producing NADH oxidase was expressed withpEASY vector.2,4-Dinitrophenylhydrazine was used to compare the pyruvate production with orwithout NoxE addition. It was verified that the recombinant NoxE was successfully expressed andhad significant biological activity. The lactate oxidation activity of FNI was revalued by the newmethod, however, its lactate oxidation capacity didn’t obviously get higher.To express ldh gene, a recombinant strain E. coli M15(pREP4, pQELDH) was instructed inthe same procedure as for fni gene. The result of SDS-PAGE showed that this strain couldabundantly express recombinant LDHA with the right molecular weight. However, no significantactivity could be detected. It seems necessary to reconstruct a recombinant strain to express ldh.A C-terminal6×His tagged pEASY E2vector was used to express ldh gene. The recombinantE. coli Transetta(DE3)(pEASY-LDH) was constructed in the same procedure as for noxEexpression. SDS-PAGE showed a subunit molecular weight of37.24kDa, which was in goodagreement with the theoretical value. Its pyruvate reduction activity was very weak in the absenceof FBP (fructose1,6-bisphosphate) and was increased more than7000-fold in the presence of1mM FBP. The results showed that the recombinant was successfully constructed and therecombinant LDHA had significant biological activity in a manner of FBP-dependent. Kinetic analysis revealed that the affinities of the enzyme for the forward substrates weremuch higher than those for the reverse ones. Obviously, LDHA was prone to catalyze pyruvatereduction reaction. The result of the effect of FBP and phosphate on both reaction rates showedthat both effectors were found to have significant modulating effect on pyruvate reduction andlactate oxidation rates. However, their activation or inhibitory degree was similar on both reactionsand the equilibrium of the reaction would not be changed. Adenine nucleotides were found to haveinhibitory effect on LDHA. ATP was observed to have a weak and similar inhibitory effect on bothreactions. ADP was a stronger competitive inhibitor and had a comparatively inhibitory effect onthe reverse reaction under the experimental conditions. Nevertheless, its inhibitory degree wouldbe strongly dependent on the concentrations of NADH, NAD and ADP.It was verified that the NADH/NAD ratio had a great effect on the equilibrium of the reaction.Pyruvate reduction activity of LDHA would not be seriously influenced in a wide range ofNADH/NAD ratios. On the other hand, the rate of the reverse reaction was only significant at alow NADH/NAD ratio. Further studies showed that the affinity of LDHA for pyruvate wasdecreased with the decrease of NADH concentration. Lactate was found to be a weak inhibitor ofthe forward reaction while pyruvate was found to have a severe inhibitory effect on the reversereaction.The lactate oxidation activity of LDHA was analyzed using the new method designed in thisstudy. The results showed that the lactate oxidation activity of LDHA was increased2.84foldwhen recombinant NoxE was added to the reaction system. Under respiratory conditions whereNADH could be removed via respiratory chain, the lactate oxidation activity of LDHA would beobviously increased.Cellular metabolites analysis revealed that ADP concentration was always kept at a low levelduring growth. NAD concentration was also constant while cellular NADH and pyruvateconcentrations were significantly decreased when the respiratory culture entered stationary phase.Based on the above results, the cellular metabolites concentrations in the stationary phase culturewere beneficial for LDHA to catalyze lactate oxidation.This study was the first report which verified the fermentative nLDH responsible for lactateutilization in the late phase of L. lactis respiration and proposed the prerequisite of lactateoxidation and the associated transport and metabolism pathways. The results have laid a theoreticalfoundation for modulating lactate metabolism in lactic acid bacteria and provide insight intoenzyme assay under physical conditions.