Dataset Information

Photochemical Mechanism of Light-Driven Fatty Acid Photodecarboxylase.

ABSTRACT: Fatty acid photodecarboxylase (FAP) is a promising target for the production of biofuels and fine chemicals. It contains a flavin adenine dinucleotide cofactor and catalyzes the blue-light-dependent decarboxylation of fatty acids to generate the corresponding alkane. However, little is known about the catalytic mechanism of FAP, or how light is used to drive enzymatic decarboxylation. Here, we have used a combination of time-resolved and cryogenic trapping UV-visible absorption spectroscopy to characterize a red-shifted flavin intermediate observed in the catalytic cycle of FAP. We show that this intermediate can form below the "glass transition" temperature of proteins, whereas the subsequent decay of the species proceeds only at higher temperatures, implying a role for protein motions in the decay of the intermediate. Solvent isotope effect measurements, combined with analyses of selected site-directed variants of FAP, suggest that the formation of the red-shifted flavin species is directly coupled with hydrogen atom transfer from a nearby active site cysteine residue, yielding the final alkane product. Our study suggests that this cysteine residue forms a thiolate-flavin charge-transfer species, which is assigned as the red-shifted flavin intermediate. Taken together, our data provide insights into light-dependent decarboxylase mechanisms catalyzed by FAP and highlight important considerations in the (re)design of flavin-based photoenzymes.

SUBMITTER: Heyes DJ

PROVIDER: S-EPMC7469136 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Photochemical Mechanism of Light-Driven Fatty Acid Photodecarboxylase.

Heyes Derren J DJ Lakavath Balaji B Hardman Samantha J O SJO Sakuma Michiyo M Hedison Tobias M TM Scrutton Nigel S NS

ACS catalysis 20200519 12

Fatty acid photodecarboxylase (FAP) is a promising target for the production of biofuels and fine chemicals. It contains a flavin adenine dinucleotide cofactor and catalyzes the blue-light-dependent decarboxylation of fatty acids to generate the corresponding alkane. However, little is known about the catalytic mechanism of FAP, or how light is used to drive enzymatic decarboxylation. Here, we have used a combination of time-resolved and cryogenic trapping UV-visible absorption spectroscopy to c ...[more]

PMID: 32905273

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Project description:BackgroundOleaginous yeasts are potent hosts for the renewable production of lipids and harbor great potential for derived products, such as biofuels. Several promising processes have been described that produce hydrocarbon drop-in biofuels based on fatty acid decarboxylation and fatty aldehyde decarbonylation. Unfortunately, besides fatty aldehyde toxicity and high reactivity, the most investigated enzyme, aldehyde-deformylating oxygenase, shows unfavorable catalytic properties which hindered high yields in previous metabolic engineering approaches.ResultsTo demonstrate an alternative alkane production pathway for oleaginous yeasts, we describe the production of diesel-like, odd-chain alkanes and alkenes, by heterologously expressing a recently discovered light-driven oxidase from Chlorella variabilis (CvFAP) in Yarrowia lipolytica. Initial experiments showed that only strains engineered to have an increased pool of free fatty acids were susceptible to sufficient decarboxylation. Providing these strains with glucose and light in a synthetic medium resulted in titers of 10.9 mg/L of hydrocarbons. Using custom 3D printed labware for lighting bioreactors, and an automated pulsed glycerol fed-batch strategy, intracellular titers of 58.7 mg/L were achieved. The production of odd-numbered alkanes and alkenes with a length of 17 and 15 carbons shown in previous studies could be confirmed.ConclusionsOleaginous yeasts such as Yarrowia lipolytica can transform renewable resources such as glycerol into fatty acids and lipids. By heterologously expressing a fatty acid photodecarboxylase from the algae Chlorella variabilis hydrocarbons were produced in several scales from microwell plate to 400 mL bioreactors. The lighting turned out to be a crucial factor in terms of growth and hydrocarbon production, therefore, the evaluation of different conditions was an important step towards a tailor-made process. In general, the developed bioprocess shows a route to the renewable production of hydrocarbons for a variety of applications ranging from being substrates for further enzymatic or chemical modification or as a drop-in biofuel blend.

Dataset Information

Photochemical Mechanism of Light-Driven Fatty Acid Photodecarboxylase.

Publications

Photochemical Mechanism of Light-Driven Fatty Acid Photodecarboxylase.

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