D-2-Hydroxyglutarate Metabolic Mechanism In Pseudomonas And 2-oxobutyrate Production Via Biocatalysis

Glycolysis and TCA cycle are two core metabolic pathways in organisms.Pyruvate,oxaloacetate,and 2-ketoglutarate?2-KG?are 3 key intermediates that belong to 2-keto carboxylic acids in glycolysis and TCA.In the presence of reducing agents and reductases,these 2-keto carboxylic acids can be reduced into lactate,malate,and 2-hydroxyglutarate?2-HG?,respectively.Malate is a key metabolite that takes part in TCA cycle.The catabolism and anabolism of 2-HG and lactate have been validated to exist in animals,plants and microorganisms via metabolomics methods.The mechanism of lactate metabolism and its physiological roles have been well-studied in different organisms,while there is relatively little research about D-2-HG.In recent years,studies have found that many kinds of cancers are accompanied by D-2-HG accumulation and D-2-HG could serve as a toxic metabolic product to promote oncogenesis.The purpose of this thesis is to elucidate the physiological significance of the toxic D-2-HG production through research on the mechanism of D-2-HG anabolism and catabolism,which may be beneficial for the study on the occurrence of diseases related to D-2-HG metabolism.D-2-Hydroxyglutarate dehydrogenase?D2HGDH?has been reported to act as the catabolic enzyme in both animals and plants.In this study,the distribution and evolution of D2HGDH in microorganisms were analyzed by bioinformatics method at first and it was found that the homologous proteins of D2HGDH are widespread in eukaryotic and prokaryotic microorganisms,including bacteria and yeast.Then,P.stutzeri A1501 was taken as the research subject and its D2HGDH homologue was overexpressed and purified.The obtained protein product was proved to possess the dehydrogenase activities using D-isomers of 2-HG and malate as substrates,and catalyze D-2-HG to 2-KG via in vitro experiments.The enzymatic properties of this first microbial D2HGDH were characterized.It was showed that the protein with the molecular mass of 103.9-kDa is a homodimeric protein that binds one FAD per subunit.The enzyme showed Michaelis-Menten kinetics with D-2-HG as the substrate.The K_m is 0.17 mM,the Vmax is 4.56 U mg^-1 protein and the Kcat/K_m is 2723.55 min^-1 mM^-1.The effects of pH,temperature and metal ions on D2HGDH activity were also determined.The D2HGDH activity in P.stutzeri A1501 could be detected at similar levels through activity staining in various mediums and all growth periods.And intracellular D-2-HG concentration was found with a range from 20?M to 60 ?M at various growth periods,which were determined by the constructed extraction and quantitative method for intracellular D-2-HG in this study.These results suggested that the D-2-HG homeostasis might be due to the continuous occurrence of both the D-2-HG anabolism and catabolism.To determine the in vivo function of D2HGDH,the chromosomal d2hgdh gene encoding D2HGDH in P.stutzeri A1501?WT?was deleted to construct P.stutzeri WT??dhg?and the latter was complemented with d2hgdh gene to result another strain,designated as P.stutzeri WT??dhg:dhg+?.The strains were cultured in medium with D-2-HG as the single carbon source and it was proved that the d2hgdh gene is crucial to the assimilation of D-2-HG.When the strains grew in medium with glucose as the single carbon source,P.stutzeri WT??dhg?showed measurable high intracellular D-2-HG concentration?about 100 times greater than that of P.stutzeri A1501?.In comparison,the intracellular D-2-HG concentration of P.stutzeri WT??dhg:dhg+?returned to normal level.In addition,the d2hgdh gene disruption would lower biomass yield and growth rate of the P.stutzeri WT??dhg?strain growing in various kinds of mediums and cause the great accumulation of D-2-HG(up to 0.425 g l^-1,about 2800 ?M)in medium.D-2-HG,as an end metabolic product,could not be catabolized directly by cells.It was speculated that the d2hgdh gene disruption led to the produced D-2-HG unable to be assimilated,and the D-2-HG accumulation would decrease the amount of carbon source that could be assimilated and finally affect the growth of P.stutzeri WT??dhg?.For P.stutzeri A1501?WT?and P.stutzeri WT??dhg:dhg+?,D-2-HG catabolism that play important roles in cell total metabolism,is active to convert the produced large amounts of D-2-HG within cells into 2-KG that can be assimilated by cells,and fulfill the full use of carbon and energy.D2HGDH belonging to flavin dependent dehydrogenase is not a membrane protein and could not directly transfer electrons into respiratory chain.However,D-2-HG catabolism means the continuous electron generation in D-2-HG dehydrogenation reaction and the efficient electron transfer of the flavins that gain electrons in D2HGDH.In the study,electron transfer flavoprotein?ETF?in P.stutzeri A1501 was expressed and purified.In vitro experiments showed that ETF addition could mediate the electron transfer between D2HGDH and dichlorophenol-indophenol?DCIP?,and improve the rate of D2HGDH-catalyzed reaction.When ETF was considered as substrate of D2HGDH,the K_m,Vmax and Vcat/K_m values were 7.7?M,7.50 U mg^-1 and 1.03 × 105 min^-1 mM^-1,respectively.Moreover,ETF addition significantly increased the Vmax value of D2HGDH towards D-2-HG,while the K_m value remained less affected.This indicated that ETF may serve as the true electron acceptor of D2HGDH within the cell.To investigate the electron transfer function of ETF in vivo,the chromosomal etf gene encoding ETF in P.stutzeri A1501?WT?was deleted to construct P.stutzeri WT??etf?and the latter was complemented with etf gene to result another strain,designated as P.stutzeri WT P.stutzeri WT??etf:etf+?.The etf gene was validated to be crucial to the intracellular D-2-HG homeostasis and D-2-HG assimilation.Meanwhile,D-2-HG could also be in great accumulation in culture.Combined the in vivo and in vitro results,ETF was proved to play key roles in D-2-HG catabolism.It was speculated that ETF serves as the direct electron acceptor of D2HGDH and coordinates with D2HGDH to form an efficient mechanism of D-2-HG catabolism.As mentioned,D-2-HG catabolism and anabolism in P.stutzeri A1501 are balanced,and D-2-HG concentration remains steady in normal physiological conditions.The continuous D-2-HG catabolism indicates the existence of continuous D-2-HG production.Comparative genome analysis of the adjacent gene of d2hgdh was performed and the D-3-phosphoglycerate dehydrogenase?SerA?encoding gene serA was found in the genomic neighborhood of the d2hgdh gene in many microorganisms,including P.stutzeri A1501.This indicates that there may be functional relationship between SerA and D2HGDH.SerA in P.stutzeri A1501 was expressed and purified.It was proved that SerA could reduce 2-KG with perfect stereoselectivity to produce D-2-HG in the presence of NADH.Gene of serA was disrupted in P.stutzeri WT??dhg?to obtain the double mutant of serA and d2hgdh gene,designated as P.stutzeri WT??dhg?serA?,which showed little accumulation of intracellular D-2-HG.From above in vitro and in vivo results,SerA was proved to function as the key enzyme of D-2-HG anabolism in P.stutzeri A1501.SerA catalyzes the glycolytic D-3-phosphoglycerate?D-3-PG?oxidation into 3-phosphohydroxypyruvate?3-PHP?,which is the first step of L-serine biosynthesis.The function relationship between SerA and D2HGDH suggests the roles of D-2-HG production may be related to L-serine synthesis.In the study,it was found that SerA-catalyzed D-3-PG oxidation was difficult to proceed while SerA-catalyzed 2-KG reduction was relatively easy to proceed.2-KG addition could improve the rate of SerA catalyzed D-3-PG oxidation.D-2-HG production could couple and facilitate the D-3-PG oxidation reaction as follows.On the one hand,SerA catalyzed 2-KG reduction is accompanied by NADH oxidation,which prevents SerA and NADH from forming tight complex and is in favor of the SerA catalyzed reaction between D-3-PG and NAD.On the other hand,D-2-HG production reaction could couple D-3-PG oxidation to effectively reduce the free energy of SerA-catalyzed reaction.In addition,D-2-HG could inhibit SerA-catalyzed 2-KG reduction and D-3-PG oxidation and thus D-2-HG may be an allosteric inhibitor of SerA.Thus,D-2-HG catabolism in vivo aims to convert inactive D-2-HG to active 2-KG that flows into cell metabolism to fulfill the carbon and energy reproduction,and maintain the low concentration of D-2-HG to prevent the inhibition effect of D-2-HG on SerA.In conclusion,D2HGDH,SerA,and ETF coordinate to form a D-2-HG metabolic module with the physiological role in guaranteeing multiple and efficient turnovers of SerA catalyzed D-3-PG oxidation.D-3-PG is the key node to connect glycolysis and L-serine synthesis pathway.Experiments confirmed that a large amount of glycolytic carbon flux and the coupled flux of 2-KG,an intermediate from TCA cycle,flow through the D-2-HG metabolic module into L-serine synthesis pathway.Due to the high flux,D-2-HG should be considered as an important primary metabolite but not a waste.Comparative genome and phylogenetic analysis indicate the D-2-HG metabolic module is widespread in different organisms and plays important roles in connecting the core metabolic pathways of glycolysis,TCA cycle,and amino acids synthesis.Another part work of the dissertation involves 2-oxobutyrate?2-OBA?production via biocatalysis.2-OBA is an important intermediate with many applications in the drug and chemical industries.2-OBA could also be transformed from L-threonine through an L-threonine dehydratase?L-TD?catalyzed deamination and dehydration process.Considering that the fermentation of L-threonine could be easily carried out and reach a rather high concentration,L-threonine might be a suitable starting material for the 2-OBA production.In this study,P.stutzeri SDM possessing high activity of biosynthetic L-TD was obtained through screening.Using the whole cells of strain SDM as biocatalyst,an efficient biocatalytic system to produce 2-OBA from L-threonine was constructed.The optimal catalytic conditions were identified as follows:pH,8.0;temperature,50?;biocatalyst concentration,9.2 g dry cell weight l^-1;L-threonine,30 g l^-1.Under the optimum conditions,the biocatalytic process produced 2-OBA at a high concentration(25.6 g l^-1)with a highest molar conversion rate?99.6%?and a highest productivity(41.8 mM h^-1)at 6 h,in comparison with other biological production processes.In addition,2-hydroxybutyric acid?2-HBA?could be used to produce 2-OBA through dehydrogenation and synthesize L-isoleucine and some medicines.2-HBA is also an important industrial intermediate.However,there was no available and inexpensive process to prepare 2-HBA.In this study,G.oxydans DSM 2003 and a low-cost substrate,1,2-butanediol?1,2-BDO?,were used to construct a new approach to produce D-2-hydroxybutyric acid?D-2-HBA?.Using 60 g l^-1 wet cells of G.oxydans DSM 2003 as biocatalyst and 40 g l^-1 of racemic 1,2-BDO as substrate,the biocatalytic process?pH was adjusted to 6-7;37?;EDTA,20 mM?produced D-2-HBA at a high concentration(19.7 g l^-1)with a high enantiomer excess value?99%?after 21 h conversion.