Alkane biosynthesis by decarbonylation of aldehyde catalyzed by a microsomal preparation from Botryococcus braunii

The final step in the synthesis of n-hydrocarbons in an animal and a higher plant involves enzymatic decarbonylation of aldehydes to the corresponding alkanes by loss of the carbonyl carbon. Whether such a novel reaction is involved in hydrocarbon synthesis in the colonial microalga, Botryococcus braunii, which is known to produce unusually high levels (up to 32% of dry weight) of n-C27, C29, and C31 alka-dienes and -trienes, was investigated. Dithioerythritol severely inhibited the incorporation of [1-14C]acetate into these hydrocarbons with accumulation of the label in the aldehyde fraction in the B. braunii cells. Microsomal preparations of the alga synthesized alkane from fatty acid and aldehyde in the absence of O2. Conversion of fatty acid to alkane required CoA, ATP, and NADH, whereas conversion of aldehyde to alkane did not require the addition of cofactors. That the alkane synthesis involves a decarbonylation was shown by the production of CO and heptadecane from octadecanal. CO was identified by adsorption to RhCl[(C6H6)3P]3. The decarbonylase had a pH optimum at 7.0, an apparent Km of 65 microM, a Vmax of 1.36 nmol/min/mg and was inhibited by the metal chelators EDTA, O-phenanthroline and 8-hydroxyquinoline. It was stimulated nearly threefold by 2 mM ascorbate and inhibited by the presence of O2. A partial (28%) retention of the aldehydic hydrogen of [1-3H]octadecanal in the heptadecane was observed; the remaining 3H was lost to H2O. The microsomal preparation also catalyzed the oxidation of 14CO to 14CO2, with a pH optimum of 7.0. This accounts for the nonstoichiometry of CO to heptadecane observed. In vivo studies with 14CO showed that the label was incorporated into metabolic products. This metabolic conversion of CO, not found in the previously examined hydrocarbon synthesizing systems, may be necessary for organisms that produce large amounts of hydrocarbons such as the present alga. The mechanism of the decarbonylation and the nature of the decarbonylase remain to be elucidated.     

Solubilization, partial purification, and characterization of a fatty aldehyde decarbonylase from a higher plant, Pisum sativum

Enzymatic decarbonylation of fatty aldehydes generates hydrocarbons. The particulate enzyme that catalyzes the decarbonylation has not been solubilized and purified from any organism but a green alga. Here we report the solubilization, purification, and partial characterization of the decarbonylase from a higher plant. Decarbonylase from a particulate preparation from pea (Pisum sativum) leaves, enriched in decarbonylase, was solubilized with beta-octyl glucoside and partially purified. SDS-PAGE showed a major protein band at 67 kDa. Rabbit antibodies raised against this protein specifically cross-reacted with the 67-kDa protein in solubilized microsomal preparations; anti-ribulose bisphosphate carboxylase cross-reacted only with the 49-kDa large subunit of the carboxylase, but not with any protein near 67 kDa, showing the absence of any contamination from cross-linked small-large subunit of the carboxylase found in the green algal enzyme preparation. Anti-67-kDa protein antibodies inhibited decarbonylation catalyzed by the enzyme preparations, showing that this protein represents the decarbonylase. Decarbonylase activity of the purified enzyme required phospholipids for activity; phosphatidylcholine was the preferred lipid although phosphatidylserine and phosphatidylethanolamine could substitute less effectively. Half-maximal activity was observed at 40 microM octadecanal. The purified enzyme produced alkane and CO and was inhibited by O2, NADPH, and DTE. Metal ion chelators severely inhibited the enzyme and Cu2+ fully restored the enzyme activity. Purified enzyme preparations consistently showed the presence of Cu, and copper protoporphyrin IX catalyzed decarbonylation. These results suggest that this higher plant enzyme probably is a Cu enzyme unlike the green algal enzyme that was found to have Co.     

Oxygen-independent alkane formation by non-heme iron-dependent cyanobacterial aldehyde decarbonylase: investigation of kinetics and requirement for an external electron donor

Cyanobacterial aldehyde decarbonylase (cAD) is, structurally, a member of the di-iron carboxylate family of oxygenases. We previously reported that cAD from Prochlorococcus marinus catalyzes the unusual hydrolysis of aldehydes to produce alkanes and formate in a reaction that requires an external reducing system but does not require oxygen [Das et al. (2011) Angew. Chem. 50, 7148-7152]. Here we demonstrate that cADs from divergent cyanobacterial classes, including the enzyme from N. puntiformes that was reported to be oxygen dependent, catalyze aldehyde decarbonylation at a much faster rate under anaerobic conditions and that the oxygen in formate derives from water. The very low activity (<1 turnover/h) of cAD appears to result from inhibition by the ferredoxin reducing system used in the assay and the low solubility of the substrate. Replacing ferredoxin with the electron mediator phenazine methosulfate allowed the enzyme to function with various chemical reductants, with NADH giving the highest activity. NADH is not consumed during turnover, in accord with the proposed catalytic role for the reducing system in the reaction. With octadecanal, a burst phase of product formation, k(prod) = 3.4 ± 0.5 min(-1), is observed, indicating that chemistry is not rate-determining under the conditions of the assay. With the more soluble substrate, heptanal, k(cat) = 0.17 ± 0.01 min(-1) and no burst phase is observed, suggesting that a chemical step is limiting in the reaction of this substrate.     

Biosynthesis of alkanes by particulate and solubilized enzyme preparations from pea leaves (Pisum sativum)

Enzymatic activity responsible for the conversion of fatty acids to alkanes catalyzed by pea leaf homogenate was found to be mainly in the microsomal fraction. This particulate preparation catalyzed alkane formation from n-C18, n-C22, and n-C24 acids at rates comparable to that observed with n-C32 acid with O2 and ascorbate as required cofactors. In each case the major alkane contained two carbon atoms less than the precursor acid. Since the preparation also catalyzed alpha-oxidation, it was suspected that some alpha-oxidation intermediate, with one less carbon atom than the substrate acid, might lose another carbon to generate the alkane. Thin-layer and radio-gas-liquid chromatographic analysis of the products generated from [U-14C]stearic acid by the particulate preparation after different periods of incubation showed that, at all time periods, alpha-hydroxy C18 acid, C17 aldehyde, and C17 acid were the major products. Since C16 alkane was the major product even after short periods of reaction, the C17 aldehyde might have been the immediate precursor of the alkane. Exogenous labeled C18 and C24 aldehyde were converted to alkanes. The alkane-synthesizing activity was solubilized from the microsomal preparation using Triton X-100. The solubilized preparation was retarded in a Sepharose 6-B column, but the hydrocarbon-forming activity was not resolved from alpha-oxidation. The solubilized preparation produced alkane with two carbon atoms less than the parent acid in a time- and protein-dependent manner. The soluble preparation also required O2 and ascorbate and, like the microsomal preparation, was inhibited by dithioerythritol and metal ion chelating agents.     

Modular and selective biosynthesis of gasoline-range alkanes

Typical renewable liquid fuel alternatives to gasoline are not entirely compatible with current infrastructure. We have engineered Escherichia coli to selectively produce alkanes found in gasoline (propane, butane, pentane, heptane, and nonane) from renewable substrates such as glucose or glycerol. Our modular pathway framework achieves carbon-chain extension by two different mechanisms. A fatty acid synthesis route is used to generate longer chains heptane and nonane, while a more energy efficient alternative, reverse-β-oxidation, is used for synthesis of propane, butane, and pentane. We demonstrate that both upstream (thiolase) and intermediate (thioesterase) reactions can act as control points for chain-length specificity. Specific free fatty acids are subsequently converted to alkanes using a broad-specificity carboxylic acid reductase and a cyanobacterial aldehyde decarbonylase (AD). The selectivity obtained by different module pairings provides a foundation for tuning alkane product distribution for desired fuel properties. Alternate ADs that have greater activity on shorter substrates improve observed alkane titer. However, even in an engineered host strain that significantly reduces endogenous conversion of aldehyde intermediates to alcohol byproducts, AD activity is observed to be limiting for all chain lengths. Given these insights, we discuss guiding principles for pathway selection and potential opportunities for pathway improvement.     

Selective hydroxylation of alkanes by an extracellular fungal peroxygenase

Fungal peroxygenases are novel extracellular heme-thiolate biocatalysts that are capable of catalyzing the selective monooxygenation of diverse organic compounds, using only H(2)O(2) as a cosubstrate. Little is known about the physiological role or the catalytic mechanism of these enzymes. We have found that the peroxygenase secreted by Agrocybe aegerita catalyzes the H(2)O(2)-dependent hydroxylation of linear alkanes at the 2-position and 3-position with high efficiency, as well as the regioselective monooxygenation of branched and cyclic alkanes. Experiments with n-heptane and n-octane showed that the hydroxylation proceeded with complete stereoselectivity for the (R)-enantiomer of the corresponding 3-alcohol. Investigations with a number of model substrates provided information about the route of alkane hydroxylation: (a) the hydroxylation of cyclohexane mediated by H(2)(18)(2) resulted in complete incorporation of (18)O into the hydroxyl group of the product cyclohexanol; (b) the hydroxylation of n-hexane-1,1,1,2,2,3,3-D(7) showed a large intramolecular deuterium isotope effect [(k(H)/k(D))(obs)] of 16.0 ± 1.0 for 2-hexanol and 8.9 ± 0.9 for 3-hexanol; and (c) the hydroxylation of the radical clock norcarane led to an estimated radical lifetime of 9.4 ps and an oxygen rebound rate of 1.06 × 10(11) s(-1). These results point to a hydrogen abstraction and oxygen rebound mechanism for alkane hydroxylation. The peroxygenase appeared to lack activity on long-chain alkanes (> C(16)) and highly branched alkanes (e.g. tetramethylpentane), but otherwise exhibited a broad substrate range. It may accordingly have a role in the bioconversion of natural and anthropogenic alkane-containing structures (including alkyl chains of complex biomaterials) in soils, plant litter, and wood.     

Detection of alkanes, alcohols, and aldehydes using bioluminescence

We report a novel method for the rapid, sensitive, and quantitative detection of alkanes, alcohols, and aldehydes that relies on the reaction of bacterial luciferase with an aldehyde, resulting in the emission of light. Primary alcohols with corresponding aldehydes that are within the substrate range of the particular luciferase are detected after conversion to the aldehyde by an alcohol dehydrogenase. In addition, alkanes themselves may be detected by conversion to primary alcohols by an alkane hydroxylase, followed by conversion to the aldehyde by alcohol dehydrogenase. We developed a rapid bioluminescent method by genetically engineering the genes encoding bacterial luciferase, alcohol dehydrogenase, and alkane hydroxylase into a plasmid for simultaneous expression in an E. coli host cell line. Alkanes, alcohols, or aldehydes were detected within seconds, with sensitivity in the micromolar range, by measuring the resulting light emission with a microplate reader. We demonstrate the application of this method for the detection of alkanes, alcohols, and aldehydes and for the detection of alkane hydroxylase and alcohol dehydrogenase activity in vivo. This method is amenable to the high-throughput screening needs required for the identification of novel catalysts.     

Inhibition of CO2 production from aminopyrine or methanol by cyanamide or crotonaldehyde and the role of mitochondrial aldehyde dehydrogenase in formaldehyde oxidation

Previous results have shown that cyanamide or crotonaldehyde are effective inhibitors of the oxidation of formaldehyde by the low-Km mitochondrial aldehyde dehydrogenase, but do not affect the activity of the glutathione-dependent formaldehyde dehydrogenase. These compounds were used to evaluate the enzyme pathways responsible for the oxidation of formaldehyde generated during the metabolism of aminopyrine or methanol by isolated hepatocytes. Both cyanamide and crotonaldehyde inhibited the production of 14CO2 from 14C-labeled aminopyrine by 30-40%. These agents caused an accumulation of formaldehyde which was identical to the loss in CO2 production, indicating that the inhibition of CO2 production reflected an inhibition of formaldehyde oxidation. The oxidation of methanol was stimulated by the addition of glyoxylic acid, which increases the rate of H2O2 generation. Crotonaldehyde inhibited CO2 production from methanol, but caused a corresponding increase in formaldehyde accumulation. The partial sensitivity of CO2 production to inhibition by cyanamide or crotonaldehyde suggests that both the mitochondrial aldehyde dehydrogenase and formaldehyde dehydrogenase contribute towards the metabolism of formaldehyde which is generated from mixed-function oxidase activity or from methanol, just as both enzyme systems contribute towards the metabolism of exogenously added formaldehyde.     

Microbial Assimilation of Hydrocarbons I. Fatty Acids Derived from Normal Alkanes

Fatty acids derived from Micrococcus cerificans growing at the expense of odd- and even-carbon normal alkanes were studied. Results demonstrated that cultures grown with a variety of nonhydrocarbon substrates serving as sole carbon and energy source yielded only even-carbon fatty acids. Even-chain alkanes, dodecane through octadecane serving as sole carbon source, resulted in even-carbon fatty acids with direct correlation between carbon number of the major fatty acid species and carbon number of the alkane substrate. Odd-carbon alkanes, undecane through heptadecane serving as sole carbon source, yielded both odd- and even-carbon fatty acids. A transitional shift from even-carbon fatty acids to odd-carbon fatty acids was observed as the carbon number of the alkane substrate increased. Unsaturated fatty acids were found to comprise a significant percentage of all profiles. Analysis of unsaturated fatty acids showed all odd- and even-carbon acids analyzed were Delta(9) monounsaturated fatty acids.     

Laboratory evolution of a soluble,self-sufficient,highly active alkane hydroxylase

We have converted cytochrome P450 BM-3 from Bacillus megaterium (P450 BM-3), a medium-chain (C12-C18) fatty acid monooxygenase, into a highly efficient catalyst for the conversion of alkanes to alcohols. The evolved P450 BM-3 exhibits higher turnover rates than any reported biocatalyst for the selective oxidation of hydrocarbons of small to medium chain length (C3-C8). Unlike naturally occurring alkane hydroxylases, the best known of which are the large complexes of methane monooxygenase (MMO) and membrane-associated non-heme iron alkane monooxygenase (AlkB), the evolved enzyme is monomeric, soluble, and requires no additional proteins for catalysis. The evolved alkane hydroxylase was found to be even more active on fatty acids than wild-type BM-3, which was already one of the most efficient fatty acid monooxgenases known. A broad range of substrates including the gaseous alkane propane induces the low to high spin shift that activates the enzyme. This catalyst for alkane hydroxylation at room temperature opens new opportunities for clean, selective hydrocarbon activation for chemical synthesis and bioremediation.     

Whole‐cell biocatalytic and de novo production of alkanes from free fatty acids in Saccharomyces cerevisiae

Rapid global industrialization in the past decades has led to extensive utilization of fossil fuels, which resulted in pressing environmental problems due to excessive carbon emission. This prompted increasing interest in developing advanced biofuels with higher energy density to substitute fossil fuels and bio‐alkane has gained attention as an ideal drop‐in fuel candidate. Production of alkanes in bacteria has been widely studied but studies on the utilization of the robust yeast host, Saccharomyces cerevisiae, for alkane biosynthesis have been lacking. In this proof‐of‐principle study, we present the unprecedented engineering of S. cerevisiae for conversion of free fatty acids to alkanes. A fatty acid α‐dioxygenase from Oryza sativa (rice) was expressed in S. cerevisiae to transform C_12–18 free fatty acids to C_11–17 aldehydes. Co‐expression of a cyanobacterial aldehyde deformylating oxygenase converted the aldehydes to the desired alkanes. We demonstrated the versatility of the pathway by performing whole‐cell biocatalytic conversion of exogenous free fatty acid feedstocks into alkanes as well as introducing the pathway into a free fatty acid overproducer for de novo production of alkanes from simple sugar. The results from this work are anticipated to advance the development of yeast hosts for alkane production. Biotechnol. Bioeng. 2017;114: 232–237. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

New pathway for long-chain n-alkane synthesis via 1-alcohol in Vibrio furnissii M1

Alkane biosynthesis in the bacterium Vibrio furnissii M1 involves the synthesis of long-chain alkanes via 1-alcohol. Evidence for this novel pathway are the following. (i) Both even- and odd-carbon-number n-alkanes were produced from glucose, while only even-carbon-number fatty acids were produced in V. furnissii M1. This result cannot be explained by the decarbonylation pathway. (ii) Pentadecane and hexadecane were produced from 1-hexadecanoic acid by membrane fractions of V. furnissii M1, and radioisotope precursor-tracer experiments, in which 1-[1-(14)C]hexadecanoic acid was fed, identified the corresponding alcohol, aldehyde, and alkane derivatives. Since all metabolites maintained the radioisotope label at 1-C, they were produced by a pathway in which the carbon structure was retained, i.e., a reduction pathway. (iii) n-Hexadecane was produced when 1-hexadecanol was fed to membrane preparations.     

Differential expression of the components of the two alkane hydroxylases from Pseudomonas aeruginosa

Oxidation of n-alkanes in bacteria is normally initiated by an enzyme system formed by a membrane-bound alkane hydroxylase and two soluble proteins, rubredoxin and rubredoxin reductase. Pseudomonas aeruginosa strains PAO1 and RR1 contain genes encoding two alkane hydroxylases (alkB1 and alkB2), two rubredoxins (alkG1 and alkG2), and a rubredoxin reductase (alkT). We have localized the promoters for these genes and analyzed their expression under different conditions. The alkB1 and alkB2 genes were preferentially expressed at different moments of the growth phase; expression of alkB2 was highest during the early exponential phase, while alkB1 was induced at the late exponential phase, when the growth rate decreased. Both genes were induced by C(10) to C(22)/C(24) alkanes but not by their oxidation derivatives. However, the alkG1, alkG2, and alkT genes were expressed at constant levels in both the absence and presence of alkanes.     

Purification and characterization of choline oxidase from Arthrobacter globiformis

Choline oxidase was purified from the cells of Arthrobacter globiformis by fractionations with acetone and ammonium sulfate, and column chromatographies on DEAE-cellulose and on Sephadex G-200. The purified enzyme preparation appeared homogeneous on disc gel electrophoresis. The enzyme was a flavoprotein having a molecular weight of approx. 83,000 (gel filtration) or approx. 71,000 (sodium dodecyl sulfate--polyacrylamide disc gel electrophoresis) and an isoelectric point (pI) around pH 4.5. Identification of the reaction products showed that the enzyme catalyzed the following reactions: choline + O2 leads to betaine aldehyde + H2O2, betaine aldehyde + O2 + H2O leads to betaine + H2O2. The enzyme was highly specific for choline and betaine aldehyde (relative reaction velocities: choline, 100%; betaine aldehyde, 46%; N,N-dimethylaminoethanol, 5.2%; triethanolamine, 2.6%; diethanolamine, 0.8%; monoethanolamine, N-methylaminoethanol, methanol, ethanol, propanol, formaldehyde, acetaldehyde, and propionaldehyde, 0%). Its Km values were 1.2 mM for choline and 8.7 mM for betaine aldehyde. The optimum pH for the enzymic reaction was around pH 7.5.     

Metabolic engineering of Cupriavidus necator for heterotrophic and autotrophic alka(e)ne production

Alkanes of defined carbon chain lengths can serve as alternatives to petroleum-based fuels. Recently, microbial pathways of alkane biosynthesis have been identified and enabled the production of alkanes in non-native producing microorganisms using metabolic engineering strategies. The chemoautotrophic bacterium Cupriavidus necator has great potential for producing chemicals from CO₂: it is known to have one of the highest growth rate among natural autotrophic bacteria and under nutrient imbalance it directs most of its carbon flux to the synthesis of the acetyl-CoA derived polymer, polyhydroxybutyrate (PHB), (up to 80% of intracellular content). Alkane synthesis pathway from Synechococcus elongatus (2 genes coding an acyl-ACP reductase and an aldehyde deformylating oxygenase) was heterologously expressed in a C. necator mutant strain deficient in the PHB synthesis pathway. Under heterotrophic condition on fructose we showed that under nitrogen limitation, in presence of an organic phase (decane), the strain produced up to 670 mg/L total hydrocarbons containing 435 mg/l of alkanes consisting of 286 mg/l of pentadecane, 131 mg/l of heptadecene, 18 mg/l of heptadecane, and 236 mg/l of hexadecanal. We report here the highest level of alka(e)nes production by an engineered C. necator to date. We also demonstrated the first reported alka(e)nes production by a non-native alkane producer from CO₂ as the sole carbon source.     

A novel synthesis of branched high-molecular-weight (C40+) long-chain alkanes

Many biological and geochemical questions remain concerning the structures, functions, and properties of naturally occurring high-molecular-weight (C40+) alkanes with various mid-chain alkylation patterns. Above C40, these alkanes are exceedingly difficult to separate and purify, and syntheses can be blocked by the low solubility of intermediates. To overcome these problems, a facile three-step synthesis employing the alkylation of 1,3-dithiane with a suitable alpha,omega-dibromoalkane was developed. Bisalkylation of the bis(dithianyl)alkane intermediate with the appropriate 1-bromoalkane and subsequent desulfurization with Raney nickel furnished the desired long-chain alkane. Long-chain alkanes modified at mid-chain and/or symmetrically near the chain termini (or unmodified, i.e., long-chain n-paraffins) are accessible by the selection of appropriate bromoalkanes. Nine mid-chain methylated (C38H78 to C53H108), one symmetrical terminal-chain dimethylated (C40H82), and four linear (C44H90 to C58H118) long-chain alkanes were synthesized by using this approach. High-temperature gas chromatography (HTGC) was found to have important advantages for evaluating the purity of the synthetic high-molecular-weight alkanes.     

Biochemistry of Short-Chain Alkanes (Tissue-Specific Biosynthesis of n-Heptane in Pinus jeffreyi)

Short-chain (C7-C11) alkanes accumulate as the volatile component of oleoresin (pitch) in several pine species native to western North America. To establish the tissue most amenable for use in detailed studies of short-chain alkane biosynthesis, we examined the tissue specificity of alkane accumulation and biosynthesis in Pinus jeffreyi Grev. & Balf. Short-chain alkane accumulation was highly tissue specific in both 2-year-old saplings and mature trees; heart-wood xylem accumulated alkanes up to 7.1 mg g-1 dry weight, whereas needles and other young green tissue contained oleoresin with monoterpenoid, rather than paraffinic, volatiles. These tissue-specific differences in oleoresin composition appear to be a result of tissue-specific rates of alkane and monoterpene biosynthesis; incubation of xylem tissue with [14C]sucrose resulted in accumulation of radiolabel in alkanes but not monoterpenes, whereas incubation of foliar tissue with 14CO2 resulted in the accumulation of radiolabel in monoterpenes but not alkanes. Furthermore, incubation of xylem sections with [14C]acetate resulted in incorporation of radiolabel into alkanes at rates up to 1.7 nmol h-1 g-1 fresh weight, a rate that exceeds most biosynthetic rates reported with other plant systems for the incorporation of this basic precursor into natural products. This suggests that P. jeffreyi may provide a suitable model for elucidating the enzymology and molecular biology of short-chain alkane biosynthesis.