Fatty Acid Photodecarboxylase Research Articles

Catalytic Mechanism of Fatty Acid Photodecarboxylase: On the Detection and Stability of the Initial Carbonyloxy Radical Intermediate.

In fatty acid photodecarboxylase (FAP), light-induced formation of the primary radical product RCOO⋅ from fatty acid RCOO- occurs in 300 ps, upon which CO2 is released quasi-immediately. Based on the hypothesis that aliphatic RCOO⋅ (spectroscopically uncharacterized because unstable) absorbs in the red similarly to aromatic carbonyloxy radicals such as 2,6-dichlorobenzoyloxy radical (DCB⋅), much longer-lived linear RCOO⋅ has been suggested recently. We performed quantum chemical reaction pathway and spectral calculations. These calculations are in line with the experimental DCB⋅ decarboxylation dynamics and spectral properties and show that in contrast to DCB⋅, aliphatic RCOO⋅ radicals a) decarboxylate with a very low energetic barrier and on the timescale of a few ps and b) exhibit little red absorption. A time-resolved infrared spectroscopy experiment confirms very rapid, ≪300 ps RCOO⋅ decarboxylation in FAP. We argue that this property is required for the observed high quantum yield of hydrocarbons formation by FAP.

Catalytic Mechanism of Fatty Acid Photodecarboxylase: On the Detection and Stability of the Initial Carbonyloxy Radical Intermediate

AbstractIn fatty acid photodecarboxylase (FAP), light‐induced formation of the primary radical product RCOO⋅ from fatty acid RCOO− occurs in 300 ps, upon which CO2 is released quasi‐immediately. Based on the hypothesis that aliphatic RCOO⋅ (spectroscopically uncharacterized because unstable) absorbs in the red similarly to aromatic carbonyloxy radicals such as 2,6‐dichlorobenzoyloxy radical (DCB⋅), much longer‐lived linear RCOO⋅ has been suggested recently. We performed quantum chemical reaction pathway and spectral calculations. These calculations are in line with the experimental DCB⋅ decarboxylation dynamics and spectral properties and show that in contrast to DCB⋅, aliphatic RCOO⋅ radicals a) decarboxylate with a very low energetic barrier and on the timescale of a few ps and b) exhibit little red absorption. A time‐resolved infrared spectroscopy experiment confirms very rapid, ≪300 ps RCOO⋅ decarboxylation in FAP. We argue that this property is required for the observed high quantum yield of hydrocarbons formation by FAP.

Stereodivergent Protein Engineering of Fatty Acid Photodecarboxylase for Light‐Driven Kinetic Resolution of Sec‐Alcohol Oxalates

AbstractStereodivergent engineering of one enzyme to create stereocomplementary variants for synthesizing optically pure molecules with tailor‐made (R) or (S) configurations on an optional basis is highly desirable and challenging. This study aimed to engineer fatty acid photodecarboxylase from Chlorella variabilis (CvFAP) using the focused rational iterative site‐specific mutagenesis (FRISM) strategy to obtain two highly stereocomplementary variants with excellent selectivity (both giving products with up to 99 % e.e.). These variants were used for the CvFAP‐catalyzed light‐driven kinetic resolution of oxalates or oxamic acids prepared from the corresponding sec‐alcohols or amines, providing a new biotransformation process for preparing chiral sec‐alcohols and amines. Molecular dynamics simulation, kinetic data and transient spectra revealed the source of selectivity. This study represents the first example of the kinetic resolution of sec‐alcohols or amines catalyzed by a pair of stereocomplementary CvFAPs.

Stereodivergent Protein Engineering of Fatty Acid Photodecarboxylase for Light-Driven Kinetic Resolution of Sec-Alcohol Oxalates.

Stereodivergent engineering of one enzyme to create stereocomplementary variants for synthesizing optically pure molecules with tailor-made (R) or (S) configurations on an optional basis is highly desirable and challenging. This study aimed to engineer fatty acid photodecarboxylase from Chlorella variabilis (CvFAP) using the focused rational iterative site-specific mutagenesis (FRISM) strategy to obtain two highly stereocomplementary variants with excellent selectivity (both giving products with up to 99 % e.e.). These variants were used for the CvFAP-catalyzed light-driven kinetic resolution of oxalates or oxamic acids prepared from the corresponding sec-alcohols or amines, providing a new biotransformation process for preparing chiral sec-alcohols and amines. Molecular dynamics simulation, kinetic data and transient spectra revealed the source of selectivity. This study represents the first example of the kinetic resolution of sec-alcohols or amines catalyzed by a pair of stereocomplementary CvFAPs.

Exploring Strategic Approaches for CvFAP Photodecarboxylation through Violet Light Irradiation

In this study, we describe a light-driven photocatalytic decarboxylation of palmitic acid and related fatty acids using Chlorella variabilis fatty acid photodecarboxylase (CvFAP). By utilizing violet light emitting diode (LED) light (50 W; 397 nm), we achieved a remarkable conversion efficiency of 99% within just 4 min, surpassing the previous 79% conversion achieved in 60 min using blue LED light (300 W; 439 nm). Importantly, the use of 50 W violet LED light also resulted in a lower enzyme photoinactivation rate when compared to 300 W blue LED. Comparing the whole-cell biocatalyst with the enzymatic extract, we found that the former demonstrated superior catalytic performance and reduced susceptibility to photoinactivation. Furthermore, whole-cell biocatalyst reuse was demonstrated after five sequential batches. Employing this approach, we successfully synthesized 26 mmol L-1 h-1 of pentadecane, showcasing a promising strategy to improve productivity. These findings represent a significant advancement in CvFAP photodecarboxylation processes compared to the literature, utilizing an alternative light source, with potential implications to the biofuel sector

Continuous Fatty Acid Decarboxylation using an Immobilized Photodecarboxylase in a Membrane Reactor.

The realm of photobiocatalytic alkane biofuel synthesis has burgeoned recently; however, the current dearth of well-established and scalable production methodologies in this domain remains conspicuous. In this investigation, we engineered a modified form of membrane-associated fatty acid photodecarboxylase sourced from Micractinium conductrix (McFAP). This endeavour resulted in creating an innovative assembled photoenzyme-membrane (protein load 5 mg cm-2 ), subsequently integrated into an illuminated flow apparatus to achieve uninterrupted generation of alkane biofuels. Through batch experiments, the photoenzyme-membrane exhibited its prowess in converting fatty acids spanning varying chain lengths (C6-C18). Following this, the membrane-flow mesoscale reactor attained a maximum space-time yield of 1.2 mmol L-1 h-1 (C8) and demonstrated commendable catalytic proficiency across eight consecutive cycles, culminating in a cumulative runtime of eight hours. These findings collectively underscored the photoenzyme-membrane's capability to facilitate the biotransformation of diverse fatty acids, furnishing valuable benchmarks for the conversion of biomass via photobiocatalysis.

Enzymatic Photodecarboxylation on Secondary and Tertiary Carboxylic Acids.

Photoenzymatic decarboxylation of bulky secondary and tertiary carboxylic acids catalyzed by engineered Chlorella variabilis fatty acid photodecarboxylase (CvFAP) is reported. Rational design and directed evolution of wild-type CvFAP are used to improve the reactivity and expand potential applications. Moreover, engineered CvFAP can catalyze light-driven kinetic resolution of α-substituted carboxylic acid. Our work sheds light on the production of chiral building blocks and bioactive molecules from bulky carboxylic acids via the photoenzymatic way.

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.

Continuous hydrocarbon fuels production by photoenzymatic decarboxylation of free fatty acids from waste oils

Fatty acid photodecarboxylase, recently found in Chlorella variabilis NC64A (CvFAP), could efficiently catalyze C16-C18 fatty acids into C1-shortened hydrocarbons under visible light. This provides an alternative approach for hydrocarbon fuels production in mild conditions compared with hydrotreating of oils. In this study, a cascade system integrating lipase hydrolysis and CvFAP decarboxylation was demonstrated for the efficient production of hydrocarbon fuels from waste oils. The continuous CvFAP-catalyzed decarboxylation characteristics and performance of free fatty acids derived from waste oil were investigated for the first time. A hydrocarbon fuel production rate of 18.4 mM/h was obtained at a residence time of 50 min. The regulation of pH value from 6.8 to 8.5 significantly improved the activity of CvFAP, thereby obtaining an optimal conversion of 61.2%. The precipitation of palmitic acid caused a low conversion (37.9%) compared with the other fatty acids, further inhibiting hydrocarbon production. In such a cascade system, various oils can be converted to hydrocarbon fuels with a concentration of 8.97–15.3 mM. A high energy production rate of 204.3 kJ/L/h was achieved, which exhibited the advancement in high-quality biofuel production.

Upper limit efficiency estimates for electromicrobial production of drop-in jet fuels

Microbes which participate in extracellular electron uptake (EEU) or H2 oxidation have the ability to manufacture organic compounds using electricity as the primary source of metabolic energy. So-called electromicrobial production could be valuable to efficiently synthesize drop-in jet fuels using renewable energy. Here, we calculate the upper limit electrical-to-fuel conversion efficiency for a model jet fuel blend containing 85% straight-chain alkanes and 15% terpenoids. When using the Calvin cycle for carbon-fixation, the energy conversion efficiency is 37.8-4.3+1.8% when using EEU for electron delivery and 40.1-4.6+0.7% when using H2 oxidation. The production efficiency can be raised to 44.2-3.7+0.5% when using the Formolase formate-assimilation pathway, and to 49.2-2.1+0.3% with the Wood-Ljungdahl pathway. This efficiency can be further raised by swapping the well-known Aldehyde Deformolating Oxygenase (ADO) termination pathway with the recently discovered Fatty Acid Photodecarboxylase (FAP) pathway. If these systems were supplied with electricity from a maximally-efficient silicon solar photovoltaic, even the least efficient pathway exceeds the maximum solar-to-fuel efficiency of all known forms of photosynthesis.

Photo-driven enzymatic decarboxylation of fatty acids for bio-aviation fuels production in a continuous microfluidic reactor

Fatty acid photodecarboxylase derived from Chlorella variabilis NC64A (CvFAP) is a novel enzyme that can convert fatty acids to corresponding C1-shortened alkanes under blue light illumination, which provides an alternative approach for the production of bio-aviation fuels under mild conditions. To date, the production of bio-aviation fuel using CvFAP is severely limited by an inefficient reactor. Microfluidic reactors have attracted significant attention owing to their high mass-transfer efficiency and low light attenuation in continuous photobiocatalysis. In this study, a microfluidic photobioreactor was proposed for the continuous photoenzymatic decarboxylation of palmitic acid for the first time. The optimal palmitic acid conversion of 96.7% was obtained at a flow rate of 10 μL/min, a blue light intensity of 200 μmol/(m2 s), a reaction temperature of 30 °C, a catalyst concentration of 2.4 mg/mL, a substrate concentration of 12 mM, and a cosolvent volume ratio of 30%. The maximum pentadecane production rate of 59.8 mM/h was achieved. The highest turnover frequency of CvFAP up to 19,186/h, as ever reported, was obtained at a flow rate of 40 μL/min. Meanwhile, the energy yield of 33.6 kJ/g and energy production rate of 533.5 kJ/L/h were achieved in continuous bio-aviation fuels production. Taken together, the continuous photoenzymatic decarboxylation of fatty acids in a microfluidic photobioreactor is a promising approach for bio-aviation fuels production.

Immobilization of fatty acid photodecarboxylase in magnetic nickel ferrite nanoparticle

Fatty acid photodecarboxylase in Chlorella variabilis NC64A (CvFAP) performed excellent ability to exclusively decarboxylate renewable fatty acids for C1-shortened hydrocarbons fuel production under visible light. However, the large-scale application by such an approach is limited by the free state of CvFAP catalyst, which is unstable for efficient biofuel production. In this study, CvFAP was immobilized in magnetic nickel ferrite (NiFe2O4) nanoparticles for facile recovery by a simple procedure. The shift of Ni 2p in electron binding energy was detected to clarify the interaction between Ni2+ and histidine of CvFAP. The coordination of NiFe2O4 and CvFAP contributed to an efficient affinity binding with an immobilization capacity of 98 mg/g carrier. Hydrocarbon fuel concentration of 3.7 mM was obtained by NiFe2O4@CvFAP-induced photoenzymatic decarboxylation. The high stability of CvFAP in terms of residual enzyme activity of 79.7% at pH 9.0 and that of 68% at organic solvent ratio of 60%, respectively, were observed.

Photodecarboxylase Displayed on Microbial Cell‐Surface for Enhanced Biocatalytic Performance

AbstractSurface display technology has emerged as a powerful tool for enhancing the stability, accessibility, and reusability of enzymes. Fatty acid photodecarboxylase from Chlorella variabilis NC64A (CvFAP) is capable of catalyzing decarboxylation reactions from free fatty acids to alkanes under blue light, however, its stability and reusability in catalytic cycles remain to be improved. Herein, we successfully displayed CvFAP on the surface of Escherichia coli cells utilizing full‐length ice nucleation protein as the anchor protein. By comparing the performance of the surface displayed CvFAP with whole‐cells harboring CvFAP, it was found that surface displaying of CvFAP significantly reduces the reaction time and enhances substrate accessibility, resulting in a turnover number of 9972. Also, compared to soluble enzymes, the surface displayed CvFAP exhibits enhanced stability and reusability in the decarboxylation reaction. Furthermore, the broad substrate scope of surface displayed CvFAP suggests its potential for large‐scale hydrocarbon and fatty chemicals production. Overall, our results highlight the value of surface display approach in broadening the application of photodecarboxylase.

Autocatalytic effect boosts the production of medium-chain hydrocarbons by fatty acid photodecarboxylase.

Ongoing climate change is driving the search for renewable and carbon-neutral alternatives to fossil fuels. Photocatalytic conversion of fatty acids to hydrocarbons by fatty acid photodecarboxylase (FAP) represents a promising route to green fuels. However, the alleged low activity of FAP on C2 to C12 fatty acids seemed to preclude the use for synthesis of gasoline-range hydrocarbons. Here, we reveal that Chlorella variabilis FAP (CvFAP) can convert n-octanoic acid in vitro four times faster than n-hexadecanoic acid, its best substrate reported to date. In vivo, this translates into a CvFAP-based production rate over 10-fold higher for n-heptane than for n-pentadecane. Time-resolved spectroscopy and molecular modeling demonstrate that CvFAP's high catalytic activity on n-octanoic acid is, in part, due to an autocatalytic effect of its n-heptane product, which fills the rest of the binding pocket. These results represent an important step toward a bio-based and light-driven production of gasoline-like hydrocarbons.

Moving towards the Application of Biocatalysis in Food Waste Biorefinery

Waste valorization is an important strategy to reduce environmental pollution and dependency on petroleum-based fuels. In this regard, utilization of food waste as a versatile and low-cost resource is important. Several advanced catalytic methods for the valorization of food waste have been widely investigated for the production of liquid biofuels. Along this line, chemical catalysts have been explored for the synthesis of liquid biofuels. Chemo-catalysis is mainly metal based, which requires harsh process conditions. Alternatively, biocatalysts are currently being investigated as a result of several advantages such as mild reaction conditions, recyclability, selectivity and biodegradability. In this work, recent biocatalytic technologies for the preparation of liquid biofuels through food waste valorization are discussed thoroughly. Lipases are employed for the synthesis of biodiesel and the upgradation of bio-oil, whereas methane mono-oxygenases could be explored for the production of methanol via the oxidation of methane generated from food wastes. Industrial production of ethanol from food waste using bioconversion technologies is a success story. To date, there has been no specific report on the use of food waste for propanol preparation using enzymes. The ABE process (Acetone–Butanol–Ethanol) (using suitable microorganisms) is used for butanol preparation, where the vacuum stripping system is integrated to remove butanol from the broth and circumvent inhibition. The synthesis of hydrocarbon fuels from fatty acids and triglycerides can be carried out using enzymes, such as carboxylic acid reductase and fatty acid photodecarboxylase (an algal photoenzyme). Both carboxylic acid reductase and fatty acid photodecarboxylase have not yet been applied in the direct valorization of food wastes. Furthermore, limitations of the reported methods, societal and economic aspects and a fresh perspective on the subject, along with important examples, are described.

Green light enhanced the photostability and catalytic performance of fatty acid photodecarboxylase.

Green light was documented to improve the photostability of fatty acid photodecarboxylase from Chlorella variabilis (CvFAP). Compared to blue light, green light increased the pentadecane yield by 27.6% and improved the residual activity of CvFAP to 5.9-fold after the preillumination. Kinetics and thermodynamics indicated that blue light facilitated a high CvFAP activity.

Cell-free metabolic engineering enables selective biotransformation of fatty acids to value-added chemicals

Fatty acid-derived products such as alkanes, fatty aldehydes, and fatty alcohols have many applications in the chemical industry. These products are predominately produced from fossil resources, but their production processes are often not environmentally friendly. While microbes like Escherichia coli have been engineered to convert fatty acids to corresponding products, the design and optimization of metabolic pathways in cells for high productivity is challenging due to low mass transfer, heavy metabolic burden, and intermediate/product toxicity. Here, we describe an E. coli-based cell-free protein synthesis (CFPS) platform for in vitro conversion of long-chain fatty acids to value-added chemicals with product selectivity, which can also avoid the above issues when using microbial production systems. We achieve the selective biotransformation by cell-free expression of different enzymes and the use of different conditions (e.g., light and heating) to drive the biocatalysis toward different final products. Specifically, in response to blue light, cell-free expressed fatty acid photodecarboxylase (CvFAP, a photoenzyme) was able to convert fatty acids to alkanes with approximately 90% conversion. When the expressed enzyme was switched to carboxylic acid reductase (CAR), fatty acids were reduced to corresponding fatty aldehydes, which, however, could be further reduced to fatty alcohols by endogenous reductases in the cell-free system. By using a thermostable CAR and a heating treatment, the endogenous reductases were deactivated and fatty aldehydes could be selectively accumulated (>97% in the product mixture) without over-reduction to alcohols. Overall, our cell-free platform provides a new strategy to convert fatty acids to valuable chemicals with notable properties of operation flexibility, reaction controllability, and product selectivity.

Ultrafast Dynamics of Fatty Acid Photodecarboxylase in Anionic Semiquinone State.

Fatty acid photodecarboxylase is a newly identified blue-light driven photoenzyme that catalyzes decarboxylation of fatty acids. The catalytic reaction involves a transient anionic semiquinone of flavin cofactor (FAD•-) as an intermediate, but photochemical properties of this anionic radical are largely unknown. Here, we have anaerobically produced the wild-type FAP in the FAD•- state and conducted femtosecond-resolved fluorescence and absorption measurements. We have observed the multiphasic deactivation dynamics of excited states on multiple time scales from a few picoseconds even to a few nanoseconds through conical intersections between various electronic states. Interestingly, the nanosecond components can only be observed from higher electronic excited states. Our results show the complexity of the energy landscapes of various excited states and rule out the occurrence of electron or proton transfer with nearby residue(s) in the active site.

Ultrafast Dynamics and Catalytic Mechanism of Fatty Acid Photodecarboxylase.

Fatty acid photodecarboxylase is a newly discovered flavin photoenzyme that converts a carboxylic acid into a hydrocarbon and a carbon dioxide molecule through decarboxylation. The enzymatic reactions are poorly understood. In this study, we carefully characterized its dynamic evolution with femtosecond spectroscopy. We observed initial electron transfer from the substrate to the flavin cofactor in 347 ps with a stretched dynamic behavior and subsequently captured the critical carbonyloxy radical. The dominant process following this step was decarboxylation in 5.8 ns to form an alkyl radical and a carbon dioxide molecule. We further identified the absorption bands of two carbonyloxy and alkyl radical intermediates. The overall enzymatic quantum efficiency determined by our obtained timescales is 0.81, consistent with the steady-state value. The results are essential to the elucidation of the enzyme mechanism and catalytic photocycle, providing a molecular basis for potential design of flavin-based artificial photoenzymes.

Immobilization and Application of Fatty Acid Photodecarboxylase in Deep Eutectic Solvents.

Since its discovery in 2017, the fatty acid decarboxylase (FAP) photoenzyme has been the focus of extensive research, given its ability to convert fatty acids into alka(e)nes using merely visible blue light. Unfortunately, there are still some drawbacks that limit the applicability of this biocatalyst, such as poor solubility of the substrates in aqueous media, poor photostability, and the impossibility of reusing the catalyst for several cycles. In this work, we demonstrate the use of FAP in non-conventional media as a free enzyme and an immobilized preparation. Namely, its applicability in deep eutectic solvents (DESs) and a proof-of-concept immobilization using a commercial His-tag selective carrier, a thorough study of reaction and immobilization conditions in each case, as well as reusability studies are shown. We observed an almost complete selectivity of the enzyme towards C18 decarboxylation over C16 when used in a DES, with a product analytical yield up to 81 % when using whole cells. Furthermore, when applying the immobilized enzyme in DES, we obtained yields >10-fold higher than the ones obtained in aqueous media.