Abstract:Renewable production of hydrocarbons is being pursued as a petroleum-independent source of commodity chemicals and replacement for biofuels. The bacterial biosynthesis of long-chain olefins represents one such platform. The process is initiated by OleA catalyzing the condensation of two fatty acyl-coenzyme A substrates to form a β-keto acid. Here, the mechanistic role of the conserved His285 is investigated through mutagenesis, activity assays, and X-ray crystallography. Our data demonstrate that His285 is req… Show more
“…The roles of active site residues H285, E117, and C143 of OleA have been studied using site-directed mutagenesis and X-ray crystal structures. All of these residues are important for the first step of olefin biosynthesis. − OleD is an NAD(P)H-dependent reductase and a member of the short-chain dehydrogenase superfamily which catalyzes reduction of β-keto acid to generate β-hydroxy acid . OleC is a β-lactone synthetase found in X. campestris , S. maltophilia, Micrococcus luteus , Lysobacter dokdonensis , and Arenimonas malthae which catalyzes β-lactone synthesis from β-hydroxy acid .…”
Section: Production Of Triglyceride-
Fatty Acid- and
Glycerol-derived...mentioning
Since the industrial revolution, the rapid growth and development of global industries have depended largely upon the utilization of coal-derived chemicals, and more recently, the utilization of petroleum-based chemicals. These developments have followed a linear economy model (produce, consume, and dispose). As the world is facing a serious threat from the climate change crisis, a more sustainable solution for manufacturing, i.e., circular economy in which waste from the same or different industries can be used as feedstocks or resources for production offers an attractive industrial/business model. In nature, biological systems, i.e., microorganisms routinely use their enzymes and metabolic pathways to convert organic and inorganic wastes to synthesize biochemicals and energy required for their growth. Therefore, an understanding of how selected enzymes convert biobased feedstocks into special (bio)chemicals serves as an important basis from which to build on for applications in biocatalysis, metabolic engineering, and synthetic biology to enable biobased processes that are greener and cleaner for the environment. This review article highlights the current state of knowledge regarding the enzymatic reactions used in converting biobased wastes (lignocellulosic biomass, sugar, phenolic acid, triglyceride, fatty acid, and glycerol) and greenhouse gases (CO 2 and CH 4 ) into value-added products and discusses the current progress made in their metabolic engineering. The commercial aspects and life cycle assessment of products from enzymatic and metabolic engineering are also discussed. Continued development in the field of metabolic engineering would offer diversified solutions which are sustainable and renewable for manufacturing valuable chemicals.
“…The roles of active site residues H285, E117, and C143 of OleA have been studied using site-directed mutagenesis and X-ray crystal structures. All of these residues are important for the first step of olefin biosynthesis. − OleD is an NAD(P)H-dependent reductase and a member of the short-chain dehydrogenase superfamily which catalyzes reduction of β-keto acid to generate β-hydroxy acid . OleC is a β-lactone synthetase found in X. campestris , S. maltophilia, Micrococcus luteus , Lysobacter dokdonensis , and Arenimonas malthae which catalyzes β-lactone synthesis from β-hydroxy acid .…”
Section: Production Of Triglyceride-
Fatty Acid- and
Glycerol-derived...mentioning
Since the industrial revolution, the rapid growth and development of global industries have depended largely upon the utilization of coal-derived chemicals, and more recently, the utilization of petroleum-based chemicals. These developments have followed a linear economy model (produce, consume, and dispose). As the world is facing a serious threat from the climate change crisis, a more sustainable solution for manufacturing, i.e., circular economy in which waste from the same or different industries can be used as feedstocks or resources for production offers an attractive industrial/business model. In nature, biological systems, i.e., microorganisms routinely use their enzymes and metabolic pathways to convert organic and inorganic wastes to synthesize biochemicals and energy required for their growth. Therefore, an understanding of how selected enzymes convert biobased feedstocks into special (bio)chemicals serves as an important basis from which to build on for applications in biocatalysis, metabolic engineering, and synthetic biology to enable biobased processes that are greener and cleaner for the environment. This review article highlights the current state of knowledge regarding the enzymatic reactions used in converting biobased wastes (lignocellulosic biomass, sugar, phenolic acid, triglyceride, fatty acid, and glycerol) and greenhouse gases (CO 2 and CH 4 ) into value-added products and discusses the current progress made in their metabolic engineering. The commercial aspects and life cycle assessment of products from enzymatic and metabolic engineering are also discussed. Continued development in the field of metabolic engineering would offer diversified solutions which are sustainable and renewable for manufacturing valuable chemicals.
“…Another homologous protein pair, LstAB, is a heterodimer made by Streptomyces toxytricini that produces lipstatin, which is hydrogenated to make the antiobesity drug tetrahydrolipstatin, known commercially as Orlistat or Xenical [16] , [24] . The best studied enzyme catalyzing these condensation reactions with long chain substrates is the OleA from the plant pathogen Xanthomonas campestris [17] , [25] , [26] , [27] , [28] . Fig.…”
Section: Introductionmentioning
confidence: 99%
“…X-ray structures of the X. campestris OleA enzyme have been solved to 1.8 Å resolution, helping to reveal features of the Claisen condensation reaction [25] , [26] , [27] , [28] . Initial studies with native enzyme and inhibitors revealed the reactivity of Cys143 and the occupation of a hydrophobic tunnel denoted as Channel A ( Fig.…”
Graphical abstract
The thiolase enzyme OleA initiates the biosynthesis of β-lactone natural products and membrane hydrocarbons via acyl-Coenzyme A (CoA) substrates, but this study showed that
p
-nitrophenyl alkanoates can substitute, yielding new insights into the enzyme mechanism and providing a cheaper and more available starting substrate.
“…As such, there is significant interest in identifying and reengineering these enzymes for biotechnological purposes (14). Our understanding thus far is derived almost exclusively from mechanistic and X-ray crystallographic studies with OleA from X. campestris (15)(16)(17)(18) and a study on hydrocarbon biosynthesis in Micrococcus luteus (19). X. campestris OleA catalyzes the condensation of acyl-CoA substrates with C 10 -C 16 acyl chains and produces long-chain hydrocarbons via deoxygenation reactions catalyzed by OleC and OleB proteins.…”
OleA, a member of the thiolase superfamily, is known to catalyze the Claisen condensation of long-chain acyl coenzyme A (acyl-CoA) substrates, initiating metabolic pathways in bacteria for the production of membrane lipids and β-lactone natural products. OleA homologs are found in diverse bacterial phyla, but to date, only one homodimeric OleA has been successfully purified to homogeneity and characterized in vitro. A major impediment for the identification of new OleA enzymes has been protein instability and time-consuming in vitro assays. Here, we developed a bioinformatic pipeline to identify OleA homologs and a new rapid assay to screen OleA enzyme activity in vivo and map their taxonomic diversity. The screen is based on the discovery that OleA displayed surprisingly high rates of p-nitrophenyl ester hydrolysis, an activity not shared by other thiolases, including FabH. The high rates allowed activity to be determined in vitro and with heterologously expressed OleA in vivo via the release of the yellow p-nitrophenol product. Seventy-four putative oleA genes identified in the genomes of diverse bacteria were heterologously expressed in Escherichia coli, and 25 showed activity with p-nitrophenyl esters. The OleA proteins tested were encoded in variable genomic contexts from seven different phyla and are predicted to function in distinct membrane lipid and β-lactone natural product metabolic pathways. This study highlights the diversity of unstudied OleA proteins and presents a rapid method for their identification and characterization.
IMPORTANCE Microbially produced β-lactones are found in antibiotic, antitumor, and antiobesity drugs. Long-chain olefinic membrane hydrocarbons have potential utility as fuels and specialty chemicals. The metabolic pathway to both end products share bacterial enzymes denoted as OleA, OleC, and OleD that transform acyl-CoA cellular intermediates into β-lactones. Bacteria producing membrane hydrocarbons via the Ole pathway additionally express a β-lactone decarboxylase, OleB. Both β-lactone and olefin biosynthesis pathways are initiated by OleA enzymes that define the overall structure of the final product. There is currently very limited information on OleA enzymes apart from the single representative from Xanthomonas campestris. In this study, bioinformatic analysis identified hundreds of new, putative OleA proteins, 74 proteins were screened via a rapid whole-cell method, leading to the identification of 25 stably expressed OleA proteins representing seven bacteria phyla.
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