The Metabolic Pathway Of Acetoin And New Type L-lactate Dehydrogenase In Pseudomonas

2,3-butanediol works as an important primary metabolite in many microbes,it widely exists in different habitat of the nature.There are three enzymes that involve in its anabolism,including ?-acetolactate synthase,?-acetolactate decarboxylase and 2,3-butanediol dehydrogenase.We traditionally thought that the catabolism of 2,3-butanediol mainly includes production of acetoin from 2,3-butanediol and splitting of acetoin.However,2,3-butanediol has three optical isomers:(2R,3R)-2,3-butanediol,(2S,3S)-2,3-butanediol and meso-2,3-butanediol,and acetoin has two optical isomers:(3R)-acetoin and(3S)-acetoin.Only from the angle of chirality to reveal the dehydrogenation of 2,3-butanediol and splitting of acetoin,we can truly illuminate the catabolic mechanism of 2,3-butanediol.In our earlier study,we confirmed that the environmental microorganism standard strain Pseudomonas putida KT2440 can metabolize three optical isomers of 2,3-butanediol,and we identified three kinds of 2,3-butanediol dehydrogenases in production of acetoin from chiral 2,3-butanediol.Thus the first research object of this thesis is to reveal the splitting mechanism of chiral acetoin,meanwhile analyze the regulatory mechanism of the catabolism of 2,3-butanediol.By the analysis of comparative genomics,we found that there is one acetoin metabolic gene cluster in P.putida KT2440,including six genes:acoR,acoX,acoA,acoB,acoC and bdh.Firstly,we knocked out the genes of this gene cluster and examined the physiological function of these mutants.It was the first time that we verified the knock-out of acoX or bdh has no effect in the use of acetoin,the knock-out of other genes made the strains could not use acetoin.Secondly,we expressed and purified AcoAB,the key enzyme of the splitting of acetoin.We confirmed that the subunits of AcoA and AcoB are dispensable for the biochemical function of AcoAB.From chiral catalytic analysis,we firstly ascertain that AcoAB could catalyze the splitting of(3R)-acetoin and(3S)-acetoin in P.putida KT2440.Next,we studied the regulatory mechanism of the catabolism of 2,3-butanediol in P.putida KT2440.We found that all genes in acetoin metabolic gene cluster are inducible expression,gene acoX,acoA,acoB,acoC and bdh were cotranscription and the regulatory gene acoR was transcriptional separately.The 5' RACE method found the transcriptional start site of acoX and acoR.The expression of the key enzyme that involved in catabolism of 2,3-butanediol depended on AcoR and a54.AcoR could activate the transcription of acetoin operon,meanwhile it was negatively autoregulatory.Though all three optical isomers of 2,3-butanediol could induce the expression of acetoin metabolic gene cluster,the true direct effctors of AcoR were(3R)-acetoin and(3S)-acetoin.NAD-independent L-lactate dehydrogenases(L-iLDHs)widely exist in bacteria,yeast and protist,and play significant roles in the utilization of L-lactate of various of organisms.Based on the comparative genomics analysis of different Pseudomonas strains,a novel type L-iLDH was identidied in Pseudomonas stutzeri A1501.In our earlier study,we expressed and purified this protein,and researched the basic properties of this enzyme.We confirmed that this protein is different from FMN-dependent L-iLDHs that have reported.This new type L-iLDH was a dimer and was composed of three subunits,and iron-sulphur cluster may be the co factor of this complex.Thus the second research object of this thesis is to reveal the biological function of the three subunits of this new L-iLDH,and the pivotal role of iron-sulphur cluster in the oxidation of L-lactate.We knocked out three genes that encoded three subunits of LldABC and examined the physiological function of these mutants,and we found that all three subunits are dispensable for the the utilization of L-lactate.The complementation of lldABC in P.stutzeri A1501 and lldD that encodes the FMN dependent L-iLDH in P.stutzeri SDM restored the growth ability of ?lldB on L-lactate.And these two strains'ability of L-lactate utilization was without distinction.We constructed four strains that have site-directed mutagenesis in the binding site of iron-sulphur cluster of LldA and LldB,and confirmed the pivotal role of iron-sulphur cluster in the oxidation of L-lactate.In addition,we also expressed and purified LldA,LldB and LldC these three subunits respectively,and laied foundation of the further study of interaction with three subunits and the revelation of specific catalytic mechanism of LldABC.