Stabilisation of the Fatty Acid Decarboxylase from Chlorella variabilis by Caprylic Acid.

The fatty acid photodecarboxylase from Chlorella variabilis NC64 A (CvFAP) catalyses the light-dependent decarboxylation of fatty acids. Photoinactivation of CvFAP still represents one of the major limitations of this interesting enzyme en route to practical application. In this study we demonstrate that the photostability of CvFAP can easily be improved by the administration of medium-chain length carboxylic acids such as caprylic acid indicating that the best way of maintaining CvFAP stability is 'to keep the enzyme busy'.

Radical-based photoinactivation of fatty acid photodecarboxylases.

Fatty acid photodecarboxylases (FAP) are a recently discovered family of FAD-containing, light-activated enzymes, which convert fatty acids to n-alkanes/alkenes with potential applications in the manufacture of fine and speciality chemicals and fuels. Poor catalytic stability of FAPs is however a major limitation. Here, we describe a methodology to purify catalytically stable and homogeneous samples of recombinant Chlorella variabilis NC64A FAP (CvFAP) from Escherichia coli. We demonstrate however that blue light-exposure, which is required for photodecarboxylase activity, also leads to irreversible inactivation of the enzyme, especially in the absence of palmitate substrate. Photoinactivation is attributed to formation of protein based organic radicals, which were observed by EPR spectroscopy. To suppress photoinactivation, we prepared stable and catalytically active FAP in the dark. The steady-state kinetic parameters of CvFAP (kcat: 0.31 ± 0.06 s-1 and KM: 98.8 ± 53.3 µM) for conversion of palmitic acid to pentadecane were determined using gas chromatography. Methods described here should now enable studies of the catalytic mechanism and exploitation of FAPs in biotechnology.

Enzymatic interfacial conversion of acylglycerols in Pickering emulsions stabilized by hydrogel microparticles.

HYPOTHESIS: A critical challenge in the enzymatic conversion of acylglycerols is the limited exposure of the enzyme dissolved in the aqueous solution to the hydrophobic substrate in the oil phase. Positioning the enzyme in a microenvironment with balanced hydrophobicity and hydrophilicity in Pickering emulsion will facilitate the acylglycerol-catalyzing reactions at the interface between the oil and liquid phases. EXPERIMENTS: In this work, to overcome the challenge of biphasic catalysis, we report a method to immobilize enzymes in polyethylene glycol (PEG)-based hydrogel microparticles (HMPs) at the interface between the oil and water phases in Pickering emulsion to promote the enzymatic conversion of acylglycerols. FINDINGS: 3 wt% of HMPs can stabilize the oil-in-water Pickering emulsion for at least 14 days and increase the viscosity of emulsions. Lipase-HMP conjugates showed significantly higher hydrolytic activity in Pickering emulsion; HMP-immobilized lipase SMG1 showed an activity about three times that of free lipase SMG1. Co-immobilization of a lipase and a fatty acid photodecarboxylase from Chlorella variabilis (CvFAP) in Pickering emulsion enables light-driven cascade conversion of triacylglycerols to hydrocarbons, transforming waste oil to renewable biofuels in a green and sustainable approach. HMPs stabilize the Pickering emulsion and promote interfacial biocatalysis in converting acylglycerols to renewable biofuels.

A fatty acid photodecarboxylase-mimicking photonanozyme with defect-induced enzymatic substrate-binding pockets.

Hydrocarbon synthesis hints at the significance of in-depth investigations and detailed explanations of mimicking fatty acid photodecarboxylase (FAP). Considering the importance of photodecarboxylases in hydrocarbon synthesis, we present the potential of defective semiconductor nanomaterials as a novel type of photonanozymes (PNZs) that mimic enzyme-like performance, serving as alternatives to FAP. Ferrum-doped titanium dioxide (Fe-TiO2) was synthesized to introduce appropriate amounts of surface defects including reduced Ti3+ sites and oxygen vacancies, which reduce the band gap of TiO2 and enhance the visible-light absorption, thereby facilitating efficient charge trapping. Notably, the surface defects of Fe-TiO2 PNZs singularly act as enzymatic substrate-binding pockets that enable efficient carboxylic acid adsorption during the dark process, conversely facilitating the formation of more defects and boosting the FAP-like activity for photocatalytic decarboxylation reactions. This work provides a creative strategy for designing substrate-dependent higher-concentration defects as enzyme-like binding sites on promising PNZs that mimic natural photoenzymes.

Time-resolved serial femtosecond crystallography on fatty-acid photodecarboxylase: lessons learned.

Upon absorption of a blue-light photon, fatty-acid photodecarboxylase catalyzes the decarboxylation of free fatty acids to form hydrocarbons (for example alkanes or alkenes). The major components of the catalytic mechanism have recently been elucidated by combining static and time-resolved serial femtosecond crystallography (TR-SFX), time-resolved vibrational and electronic spectroscopies, quantum-chemical calculations and site-directed mutagenesis [Sorigué et al. (2021), Science, 372, eabd5687]. The TR-SFX experiments, which were carried out at four different picosecond to microsecond pump-probe delays, yielded input for the calculation of Fourier difference maps that demonstrated light-induced decarboxylation. Here, some of the difficulties encountered during the experiment as well as during data processing are highlighted, in particular regarding space-group assignment, a pump-laser power titration is described and data analysis is extended by structure-factor extrapolation of the TR-SFX data. Structure refinement against extrapolated structure factors reveals a reorientation of the generated hydrocarbon and the formation of a photoproduct close to Cys432 and Arg451. Identification of its chemical nature, CO2 or bicarbonate, was not possible because of the limited data quality, which was assigned to specificities of the crystalline system. Further TR-SFX experiments on a different crystal form are required to identify the photoproducts and their movements during the catalytic cycle.

Drop-in biofuel production using fatty acid photodecarboxylase from Chlorella variabilis in the oleaginous yeast Yarrowia lipolytica.

BACKGROUND: Oleaginous 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. RESULTS: To 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. CONCLUSIONS: Oleaginous 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.