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Recent Advances in Chemically Modifiable Polyhydroxyalkanoates
Published in Martin Koller, The Handbook of Polyhydroxyalkanoates, 2020
Atahualpa Pinto, Ryan A. Scheel, Christopher T. Nomura
Tappel et al. employed a synthetic/biosynthetic approach to the controlled incorporation of medium-chain-length azidofatty acid monomers into PHA [41]. A shortcoming of previous studies that used fatty acids as substrates for PHA production was the random, uncontrolled mixture of repeating units within the resultant PHA polymers. Since control of repeating unit composition is critical to producing polymers with desirable, reproducible properties, the Nomura lab engineered E. coli to produce PHA polymers with defined repeating unit compositions from fatty acids [41]. The parental strain used was E. coli LS5218 [42], which carries two significant mutations that make it ideal for producing PHA polymers from fatty acids: (1) a fadR mutation for enhanced expression of genes encoding enzymes involved in β-oxidation; and (2) an atoC(Con) mutation which results in the constitutive expression of the Ato short-chain-fatty acid uptake enzymes in E. coli. This strain was further engineered to control the production of enoyl-CoA by deleting the fadB [43] and fadJ [44] genes (E. coli LSBJ), both of which can catalyze the (S)-specific hydration of enoyl-CoA, resulting in a roadblock in β-oxidation. This resulted in a strain where any exogenous fatty acid supplied pooled as its enoyl-CoA intermediate with no loss of carbons from the substrate. For example, supplementation with 4-carbon fatty acid (butyrate) results in a butenoyl-CoA intermediate, and supplementation with 8-carbon fatty acid (octanoate) results in pooling of an octenoyl-CoA intermediate. Thus, specific fatty acid feeding regimens, coupled with expression of the (R)-specific enoyl-CoA hydratase (PhaJ4) and PhaC1(STQK) engineered synthase, results in production of PHA polymers with defined repeating unit compositions [45,46].
Genomics of PHA Synthesizing Bacteria
Published in Martin Koller, The Handbook of Polyhydroxyalkanoates, 2020
Parveen K. Sharma, Jilagamazhi Fu, Nisha Mohanan, David B. Levin
The fatty acid β-oxidation pathway is used for mcl-PHA biosynthesis when fatty acids are used as a sole carbon source. The key precursor for mcl-PHA synthase, (R)-3-hydroxyalkanoate-CoA, is derived from the conversion of trans-2-enoyl-CoA, the intermediate of fatty acid β-oxidation. The key enzyme that carries out this reaction is an (R)-specific enoyl-CoA hydratase coded by phaJ in Pseudomonas species. There are four phaJ homologs identified in P. aeruginosa, and their gene products expressed in recombinant Escherichia coli were demonstrated to provide monomer for mcl-PHA biosynthesis from fatty acids [72]. When expressed in E. coli, PhaJ1 of P. aeruginosa showed substrate specificity toward enoyl-CoAs with acyl chain lengths from C4 to C6 (scl to mcl), while PhaJ2, PhaJ3, and PhaJ4 exhibited substrate specificities toward enoyl-CoAs with acyl chain lengths from C6 to C12 (mcl). Only two phaJ genes (phaJ1 and phaJ4, based on the sequence similarity to those of P. aeruginosa) are present in P. putida, and the product of the phaJ4 ortholog was shown to act as the primary mcl-PHA monomer supplying enzyme in P. putida [73]. Another putative monomer supplying enzyme for mcl-PHA synthesis from fatty acid β-oxidation is a 3-ketoacyl-CoA reductase coded by fabG. This enzyme is an NADPH-dependent 3-ketoacyl reductase and identified in P. aeruginosa as an mcl-PHA monomer supplying protein by reducing mcl-3-ketoacyl-CoAs to mcl-3-hydroxyacyl-CoA from fatty acid β-oxidation [74]. Yet, no evidence has shown that the protein (FadG) played a role as a monomer supplying enzyme for mcl-PHA biosynthesis from fatty acid β-oxidation in P. putida.
Lipids and Amino-Acids as Fuel
Published in Jean-Louis Burgot, Thermodynamics in Bioenergetics, 2019
Concerning, now, the recurrent sequence of degradation, we can say that it evolves in four steps: oxidization by FAD, hydratation, oxidation by NAD+ and thiolysis by CoA. After this sequence, the acyl chain is shortened from two carbon atoms. The sequence is (Figure 116): – Oxidation by FAD. It is the oxidization of the acyl CoAThere is formation of the derivative called the enoyl CoA. It is the trans-∆2-enoyl CoA. This is actually a dehydrogenation under the action of an acyl CoA dehydrogenase. The symbol ∆ shows there is a double bond formed and the superscript indicates its location. Notice that the configuration of the double bond is trans. Steps of oxidation of fatty acids.– the following step is the hydratation of the double bond located between the carbons C2 and C3. The enzyme is the enoyl CoA hydratase. The hydratation is stereospecific. The isomer L is formed alone. It is the L-hydroxyacyl CoA.– the third step is a new step of oxidization. The hydroxyl group in C3 of the L hydroxy derivative is oxidized by NAD+ in a keto derivative with formation of NADH. A ketoacyl CoA is formed.The reaction is catalyzed by the L-3-hydroxyacyl CoA deshydrogenase which is absolutely specific of the preceding L isomer.– the final step is the cleavage of the 3-ketoacyl CoA by the thiol group of a second molecule of CoA. The cleavage is catalyzed by the β-ketothiolase. The reaction is: 3-ketoacylCoA + HS-CoA⇌acetylCoA + acylCoAncarbons(n−2)carbons
Metabolite identification of ibuprofen biodegradation by Patulibacter medicamentivorans under aerobic conditions
Published in Environmental Technology, 2020
Ricado Salgado, Dulce Brito, Joao P. Noronha, Barbara Almeida, Maria R. Bronze, Adrian Oehmen, Gilda Carvalho, Maria T. Barreto Crespo
The oxidation of the IBU molecule with the introduction of hydroxyl groups can be carried out by the enzyme Enoyl-CoA hydratase, which was found to be up-regulated during biodegradation of IBU by P. medicamentovorans [20]. The Enoyl-CoA hydratase (ECH) is known to catalyse a β-oxidation substrate by adding hydroxyl groups and a proton to an unsaturated β-carbon of the molecule. This enzyme is then converted into acyl-CoA and acetyl-CoA generating energy in the form of NADH. The dehydrogenase enzyme oxidizes IBU by reducing an electron acceptor, nicotinamide adenine dinucleotide NAD+ (oxidized)/NADH (reduced) or flavin coenzyme (flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN)). The dehydrogenase enzyme catalyses the oxidation of the alcohols to aldehydes and the transformation of aldehyde to carboxylic acid, corresponding to the passage of the metabolite’s chemical structures from III to V and then XIII, as proposed in mechanism 2 (Figure 2).