| With the depletion of fossil resources and increasing environmental pollution,the biorefinery technology that can produce platform chemicals based on renewable resources has attracted more and more attention.Glutarate is an important C5 platform chemical with versatile applications,which can be used for the synthesis of polyesters and polyamides such as nylon-4,5 and nylon-5,5 in chemical industry.Glutarate is obtained via various chemical processes that rely on petrochemical precursors.However,the chemical production method has the disadvantages of serious environmental pollution,high cost and complex production process.Therefore,the bio-based route for glutarate production is now gaining worldwide attention.However,the low yield of glutarate might be the bottleneck restricting its biotechnological production.The in-depth study of the pathways and regulation mechanism of glutarate metabolism can help block the further metabolic pathway of glutarate thoroughly,thereby providing the theoretical and experimental basis for the increase of glutarate production through biotechnological routes in industry.Glutarate is an important metabolite in animals,plants,and microbes.It is distributed in various habitats and can be produced during the biological catabolism of several amino acids(such as L-lysine,L-hydroxylysine,and L-tryptophan)and aromatic compounds(such as nicotinate and benzoate).The only reported glutarate catabolic route is the glutaryl-coenzyme A(CoA)dehydrogenation pathway.In this classical pathway,glutarate is first converted into glutaryl-CoA.Then,glutaryl-CoA dehydrogenase(GcdH)catalyzes the ?,?-dehydrogenation of glutaryl-CoA to glutaconyl-CoA and the decarboxylation of glutaconyl-CoA to crotonyl-CoA and carbon dioxide.Crotonyl-CoA is subsequently converted into acetoacetate-CoA and then into two molecules of acetyl-CoA and finally channeled to the TCA cycle.Although the industrial strain Escherichia coli lacks the GcdH encoding gene,the yield of glutarate from L-lysine by recombinant E.coli is rather low.These intriguing phenomena prompted us to study the other unidentified glutarate catabolic pathway(s)in nature.In this study,the growth of Pseudomonas putida KT2440 on glutarate has been investigated.It was found that the strain with the glutaryl-CoA dehydrogenation pathway disrupted could still grow with glutarate as the sole carbon source,suggesting the presence of other unidentified glutarate metabolism pathway(s)in P.putida KT2440.Based on the comparative genomics and gene expression analysis,we speculated that a new glutarate hydroxylation pathway exists in P.putida KT2440 and the pathway is composed of two key enzymes,CsiD and LhgO.We studied the key enzymes in the glutarate hydroxylation pathway by heterogenous expression and identification of enzymatic properties of CsiD and LhgO.CsiD is a glutarate hydroxylase that belongs to the non-haem Fe2+/2-ketoglutarate(2-KG)-dependent dioxygenase family.CsiD has unspecific activity toward 2-KG to produce succinate and is capable of converting glutarate into L-2-hydroxyglutarate(L-2-HG).CsiD exists as an octamer and the activity of CsiD toward glutarate relies on 2-KG and Fe2+.LhgO is an L-2-HG oxidase that possesses noncovalently bound FAD,capable of converting L-2-HG into 2-KG LhgO seemed to have high substrate specificity.Robust activity was detected only when L-2-HG was used as the substrate.Besides the new glutarate metabolic pathway,a new source of L-2-HG production has also been revealed in this study.Currently,it is generally conceded that L-2-HG is solely produced from the reduction of 2-KG by the promiscuous catalytic activity of L-malate dehydrogenase and L-lactate dehydrogenase under acidic and hypoxic conditions.However,we demonstrated here that L-2-HG could also be produced from the hydroxylation of glutarate and is an intermediate of glutarate catabolism.L-2-HG is a competitive inhibitor of multiple 2-KG-dependent dioxygenases involved in a wide range of biological processes,including prolyl hydroxylases and histone demethylases.Like its mirror-image enantiomer D-2-hydroxyglutrate(D-2-HG),L-2-HG is also viewed as an abnormal metabolite leading to pathogenesis before.With the deepening of the research,L-2-HG was found to have multiple physiological roles,such as helping mitigate cellular reductive stress and playing a physiological role in adaptation to hypoxia.We found that L-2-HG is also an important metabolic intermediate in the catabolism of several organic compounds(such as L-lysine,L-tryptophan,and benzoate).The production and metabolism of L-2-HG are far more complicated than the currently accepted concept.Besides the reported enzymes of L-2-HG production,other unidentified enzymes and components involved in L-2-HG metabolism are still worth studying.We further studied the function of glutarate hydroxylation pathway in the metabolism of glutarate in P.putida KT2440.It was found that both glutaryl-CoA dehydrogenation and glutarate hydroxylation participate in the degradation of glutarate.We demonstrated that the glutarate hydroxylation pathway provides a competitive advantage over the glutaryl-CoA dehydrogenation pathway during glutarate utilization based on the results of growth curves,growth verification on solid plates,competitive fitness assays and modeling of bacterial growth.The competitive advantage of glutarate hydroxylation pathway is not caused by its immediate expression but possibly due to its ability to provide the quickly utilizable metabolic intermediate,succinate.Based on the clarification of glutatare metabolic pathways,we proposed a model for glutarate catabolism in P.putida KT2440.Specifically,glutaryl-CoA is converted to crotonyl-CoA by GcdH,which is then converted into two molecules of acetyl-CoA to supply a C2 compound to the TCA cycle.Glutarate can also be converted into succinate by CsiD and LhgO to supply a C4 compound to the TCA cycle.Lastly,the glyoxylate cycle,which can condense acetyl-CoA into a C4 compound,also participates in the glutarate metabolism.These three pathways(i.e.,the glutarate hydroxylation pathway,glutaryl-CoA dehydrogenation pathway,and the glyoxylate cycle)cooperate to support efficient growth on glutarate.Considering that the glutaryl-Co A dehydrogenation pathway involves ketogenic chemicals,acetoacetate-CoA and acetyl-CoA,and the glutarate hydroxylation pathway involves the glucogenic chemicals,succinate,L-lysine should be viewed as a ketogenic and glucogenic amino acid.Biotechnological production of glutarate can be accomplished through the catabolism of L-lysine.We tried to block the glutarate metabolic pathways to increase glutarate production in P.putida KT2440.Using the P.putida KT2440(?gcdH?csiD?alr)without either the glutarate hydroxylation pathway or the glutaryl-CoA dehydrogenation pathway,the concentration of glutarate was enhanced to 1.94 g L-1.More importantly,the yield of glutarate from L-lysine can be increased to 0.85 mol mol-1.Although GcdH is absent,the homologs of CsiD and LhgO,which are the key enzymes in glutarate hydroxylation pathway,are present in E.coli.That might be the reason for the low yield of glutarate by using recombinant E.coli.Inactivation of CsiD and LhgO in E.coli might be a useful strategy to enhance glutarate production through biotechnological routes.The metabolism of glutarate is tightly regulated by CsiR and GcdR.We studied the regulatory mechanism of CsiR in controlling of the glutarate hydroxylation pathway and GcdR in controlling the glutaryl-CoA dehydrogenation pathway,respectively.The studies were carried out in the aspects of transcriptional analysis of the gene clusters involved in glutarate degradation,transcriptional start sites,promoter activities,binding sites of regulator proteins,and effectors.As for the glutarate hydroxylation pathway,csiD and lhgO are transcribed as a single operon,and csiR and csiD are not co-transcribed.Two transcriptional start sites(TSS)were identified upstream of csiD.The fragment upstream of TSS1 contains a functional glutarate responsive promoter.The regulator protein CsiR exists as an dimer in the active state and specifically binds to the upstream region of the csiD operon.CsiR binds at-10 and-35 regions,which represses the transcription of csiD.The effector molecules that prevent CsiR binding to the csiD promoter region are glutarate and L-2-HG.This study firstly reported a regulator that is involved in the L-2-HG metabolism and can recognize L-2-HG as its effector.As for the glutaryl-CoA dehydrogenation pathway,gcdH and pp0159 are transcribed as a single operon,and gcdR and gcdH are not co-transcribed.The fragment upstream of TSS of gcdH contains a functional glutarate responsive promoter.The regulator protein GcdR can specifically bind to the upstream region of the gcdH operon and activate the transcription of gcdH.The effector molecules of GcdR are glutarate and glutaconate.To investigate the interaction of regulation between the two pathways,we performed promoter activities analysis,growth curve measurement,and electrophoretic mobility shift assays.It was found that the glutarate hydroxylation pathway and the glutaryl-CoA dehydrogenation pathway are independent with each other in the aspect of regulation.There is no cross regulation between CsiR and GcdR.In this study,we discover a novel glutarate hydroxylation metabolic pathway.Based on the biochemical characteristics and physiological function of each key enzyme in the new pathway,a new source of L-2-HG production has been revealed and the role of glutarate hydroxylation pathway in glutarate metabolism has been clarified.The studies on the pathways and regulatory mechanism of glutarate metabolism expand our understanding of glutarate metabolism and regulation,provide valuable reference for searching other sources of L-2-HG production,and establish theoretical basis for glutarate production through biotechnological routes in industry.The study is significant in promoting the application of bio-based routes for glutarate production in industry. |
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