Engineering adipic acid metabolism in Pseudomonas putida (original) (raw)
Related papers
Initiation of fatty acid biosynthesis in Pseudomonas putida KT2440
Metabolic Engineering
Deciphering the mechanisms of bacterial fatty acid biosynthesis is crucial for both the engineering of bacterial hosts to produce fatty acid-derived molecules and the development of new antibiotics. However, gaps in our understanding of the initiation of fatty acid biosynthesis remain. Here, we demonstrate that the industrially relevant microbe Pseudomonas putida KT2440 contains three distinct pathways to initiate fatty acid biosynthesis. The first two routes employ conventional β-ketoacyl-ACP synthase III enzymes, FabH1 and FabH2, that accept short-and medium-chain-length acyl-CoAs, respectively. The third route utilizes a malonyl-ACP decarboxylase enzyme, MadB. A combination of exhaustive in vivo alanine-scanning mutagenesis, in vitro biochemical characterization, X-ray crystallography, and computational modeling elucidate the presumptive mechanism of malonyl-ACP decarboxylation via MadB. Given that functional homologs of MadB are widespread throughout domain Bacteria, this ubiquitous alternative fatty acid initiation pathway provides new opportunities to target a range of biotechnology and biomedical applications.
One carbon (C1) compounds such as methanol, formate, and CO2 are alternative, sustainable microbial feedstocks for the biobased production of chemicals and fuels. In this study, we engineered the carbon metabolism of the industrially important bacterium Pseudomonas putida to assimilate these three substrates through the reductive glycine pathway. First, we demonstrated the functionality of the C1 assimilation module by coupling the growth of auxotrophic strains to formate assimilation. Next, we extended the module from formate to methanol using both NAD and PQQ dependent methanol dehydrogenases. Finally, we demonstrated CO2-dependent growth through CO2 reduction to formate by the native formate dehydrogenase, which required short-term evolution to rebalance the cellular NADH/NAD+ ratio. This research paves the way to engineer P. putida towards growth on formate, methanol, and CO2 as sole feedstocks, thereby substantially expanding its potential as a sustainable and versatile cell fa...
Engineering glucose metabolism for enhanced muconic acid production in Pseudomonas putida KT2440
Metabolic Engineering, 2020
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
α-Hydroxyglutarate Oxidoreductase of Pseudomonas putida
Journal of Bacteriology, 1969
Oxidation of D-a-hydroxyglutarate to ct-ketoglutarate is catalyzed by D-ca-hydroxyglutarate oxidoreductase, an inducible membrane-bound enzyme of the electron transport particle [ETP; a comminuted cytoplasmic membrane preparation with enzymic properties and chemical composition resembling beef heart mitochondrial ETP (1)] of Pseudomonas putida P2 (P2-ETP). Treatment of P2-ETP with a nonionic detergent yields a preparation with the sedimentation characteristics of a soluble enzyme, but which retains an intact electron transport chain. Oxygen acts solely as a terminal electron acceptor and may be replaced by ferricyanide, 2,6dichlorophenol indophenol, or mammalian cytochrome c. The oxidoreductase is specific for the D-isomer (Km = 4.0 X 10-4 Mfor DL-a-hydroxyglutarate) and is distinct both from L-and D-malate dehydrogenases. Spectral studies suggest that the carrier sequence is substrate -k flavine or nonheme iron -* cyt b --[cyt c] -* oxygen.
Two aerobic pathways for the formation of unsaturated fatty acids in Pseudomonas aeruginosa
Molecular Microbiology, 2006
The double bond in anaerobic unsaturated fatty acid (UFA) biosynthesis is introduced by the FabA dehydratase/isomerase of the bacterial type II fatty acid biosynthetic pathway. A ΔfabA mutant of Pseudomonas aeruginosa grew aerobically, but required a UFA supplement for anaerobic growth. Wild-type cells produced 18:1Δ11 as the principal UFA, whereas the ΔfabA strain produced only 16:1Δ9. The double bond in the 16:1Δ9 was introduced after phospholipid formation and was localized in the sn-2 position. Two predicted membrane proteins, DesA and DesB, possessed the conserved histidine clusters characteristic of fatty acid desaturases. The ΔfabAΔdesA double mutant required exogenous fatty acids for growth but the ΔfabAdesB double mutant did not. Exogenous stearate was converted to 18:1Δ9 and supported the growth of ΔfabAΔdesA double mutant. A ΔfabAΔdesAdesB triple mutant was unable to desaturate exogenous stearate and was an UFA auxotroph. We detected a 2.5-fold increase in desA expression in ΔfabA mutants, whereas desB expression was derepressed by the deletion of the gene encoding a transcriptional repressor DesT. These data add two aerobic desaturases to the enzymes used for fatty acid metabolism in proteobacteria: DesA, a 2-position phospholipid Δ9-desaturase that supplements the anaerobic FabA pathway, and DesB, an inducible acyl-CoA Δ9-desaturase whose expression is repressed by DesT.
Metabolic regulation in Pseudomonas oxalaticus OX1
1978
Metabolic control associated with diauxic growth of Pseudomonas oxalaticus in batch cultures on mixtures of formate and oxalate was investigated by measuring intracellular enzyme and coenzyme concentrations and QO 2 values during transition experiments from oxalate to formate and vice versa. In transition from oxalate to formate oxalyl-CoA reductase concentration declined after the exhaustion of oxalate and ribulose-1, 5-diphosphate carboxylase and 14 CO 2 fixation appeared upon addition of formate.
Canadian Journal of Microbiology, 1993
. Isolation of Brevibacterium sp. R312 mutants potentially useful for the enzymatic production of adipic acid. Can. J. Mutants of Brevibacterium sp. R312 were isolated for the production of adipic acid from adiponitrile. One mutant (Ad) with a modified cell wall showed activity against adipamide 3 times greater than the wild type. Another mutant (ACV2) derived from the Ad strain had 30 times more activity on cyano-5-valeric acid, and 7 times more on adipamide, than the wild type. The presence of an amidase acting on amide intermediates in the hydrolysis of dinitriles to organic acids was demonstrated in these mutants. Key words: Brevibacterium sp., adiponitrile, cyano-5-valeric acid, adipamide, adipic acid, mutant. MOREAU, J. L., BERNET, N., ARNAUD, A., et GALZY, P. 1993. Isolation of Brevibacterium sp. R312 mutants potentially useful for the enzymatic production of adipic acid. Can. J. Microbiol. 39 : 524-528. Des mutants de Brevibacterium sp. R312 ont Ct C isoles en vue de la production d'acide adipique a partir d'adiponitrile. Un mutant a la paroi modifiCe (Ad) prCsente une activitk sur I'adipamide environ 3 fois plus importante que la souche sauvage. Un autre mutant dCrivC de Ad, ACV2, possede une activitC 30 fois plus importante sur l'acide cyano-5-valkrique et 7 fois plus importante sur l'adipamide que la souche sauvage. Une nouvelle amidase responsable de la dCgradation des amides intervenant dans I'hydrolyse des dinitriles en acides organiques a Ct C mise en Cvidence chez ces mutants. Mots clks : Brevibacterium sp., adiponitrile, acide cyano-5-valkrique, adipamide, acide adipique, mutant.