Cyanobacterial Aldehyde Decarbonylase Research Articles

Functional screening of aldehyde decarbonylases for long-chain alkane production by Saccharomyces cerevisiae

BackgroundLow catalytic activities of pathway enzymes are often a limitation when using microbial based chemical production. Recent studies indicated that the enzyme activity of aldehyde decarbonylase (AD) is a critical bottleneck for alkane biosynthesis in Saccharomyces cerevisiae. We therefore performed functional screening to identify efficient ADs that can improve alkane production by S. cerevisiae.ResultsA comparative study of ADs originated from a plant, insects, and cyanobacteria were conducted in S. cerevisiae. As a result, expression of aldehyde deformylating oxygenases (ADOs), which are cyanobacterial ADs, from Synechococcus elongatus and Crocosphaera watsonii converted fatty aldehydes to corresponding Cn−1 alkanes and alkenes. The CwADO showed the highest alkane titer (0.13 mg/L/OD600) and the lowest fatty alcohol production (0.55 mg/L/OD600). However, no measurable alkanes and alkenes were detected in other AD expressed yeast strains. Dynamic expression of SeADO and CwADO under GAL promoters increased alkane production to 0.20 mg/L/OD600 and no fatty alcohols, with even number chain lengths from C8 to C14, were detected in the cells.ConclusionsWe demonstrated in vivo enzyme activities of ADs by displaying profiles of alkanes and fatty alcohols in S. cerevisiae. Among the AD enzymes evaluated, cyanobacteria ADOs were found to be suitable for alkane biosynthesis in S. cerevisiae. This work will be helpful to decide an AD candidate for alkane biosynthesis in S. cerevisiae and it will provide useful information for further investigation of AD enzymes with improved activities.

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.

3P068 アルカンを合成するラン藻由来アルデヒド脱カルボニル化酵素のアラニンスキャン変異解析(01D. 蛋白質:機能,ポスター,第52回日本生物物理学会年会(2014年度))

3P068 アルカンを合成するラン藻由来アルデヒド脱カルボニル化酵素のアラニンスキャン変異解析(01D. 蛋白質:機能,ポスター,第52回日本生物物理学会年会(2014年度))

A high-valent heterobimetallic [Cu(III)(μ-O)2Ni(III)]2+ core with nucleophilic oxo groups.

Cores and effect: Unlike homobimetallic analogues, the heterobimetallic CuNi bis(μ-oxo) diamond core has nucleophilic oxo groups. A similar heterobimetallic core may, therefore, act as a viable intermediate during the deformylation of fatty aldehydes by cyanobacterial aldehyde decarbonylase. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Probing the Mechanism of Cyanobacterial Aldehyde Decarbonylase Using a Cyclopropyl Aldehyde

Cyanobacterial aldehyde decarbonylase (cAD) is a non-heme diiron oxygenase that catalyzes the conversion of fatty aldehydes to alkanes and formate. The mechanism of this chemically unusual reaction is poorly understood. We have investigated the mechanism of C1-C2 bond cleavage by cAD using a fatty aldehyde that incorporates a cyclopropyl group, which can act as a radical clock. When reacted with cAD, the cyclopropyl aldehyde produces 1-octadecene as the rearranged product, providing evidence for a radical mechanism for C-C bond scission. In an alternate pathway, the cyclopropyl aldehyde acts as a mechanism-based irreversible inhibitor of cAD through covalent binding of the alkyl chain to the enzyme.

1P086 アルカンを合成するラン藻由来アルデヒド脱カルボニル化酵素のアラニンスキャン変異解析(01F. 蛋白質:蛋白質工学/進化工学,ポスター,日本生物物理学会年会第51回(2013年度))

1P086 アルカンを合成するラン藻由来アルデヒド脱カルボニル化酵素のアラニンスキャン変異解析(01F. 蛋白質:蛋白質工学/進化工学,ポスター,日本生物物理学会年会第51回(2013年度))

Corrigendum: Oxygen-Independent Decarbonylation of Aldehydes by Cyanobacterial Aldehyde Decarbonylase: A New Reaction of Diiron Enzymes

In this Communication the authors characterized the decarbonylation reaction catalyzed by cyanobacterial aldehyde decarbonylase (cAD) as being independent of oxygen. Further experiments have now led the conclusion that the possibility that the observed activity was due to trace amounts of oxygen in the reaction buffer cannot be excluded. The difficulties in establishing the dependence of the reaction on molecular oxygen stem, in part, from the very low activity of the enzyme under either aerobic or anaerobic conditions. It was found that oxygen scrubbing systems that are routinely employed to remove oxygen from biochemical reactions, such as sodium dithionite, glucose oxidase/glucose and protocatechuate dioxygenase/protocatechuate, are ineffective at decreasing the activity of cAD using the assay conditions described in this Communication, even when included in large excess. Although these observations support the initial assertion that oxygen was not involved, when the assays were performed in an anaerobic chamber capable of maintaining oxygen at very low concentrations, i.e. below 0.5 ppm, (which was not available at the time of the original experiment) very little activity was now observed. Given this discrepancy, the involvement of molecular oxygen in the cAD-catalyzed reaction cannot be unambiguously ruled out. While the data and conclusions in the rest of the paper remain unaltered, the tentative mechanism presented for the enzyme activity will obviously need to be reconsidered.

Correction to Oxygen-Independent Alkane Formation by Non-Heme Iron-Dependent Cyanobacterial Aldehyde Decarbonylase: Investigation of Kinetics and Requirement for an External Electron Donor

Long chain alkanes are produced by a wide variety of organisms including plants (1–2), insects (3–4) and birds (5), in which they function as water-proofing agents, and by green algae where they serve as a store of cellular energy (6). These alkanes, which are typified by an odd number of carbons, are derived from fatty acid biosynthesis through the elongation of the “standard” C16 and C18 fatty acids by specialized fatty acid synthases to “very long chain” fatty acyl-CoA esters of 20 – 34 carbons (2). These acyl-CoA esters are then reduced to the corresponding aldehydes by fatty acyl-CoA reductase (7). In the final, and chemically most unusual step, cleavage of the bond between the aldehyde carbon and the α-carbon produces an odd-chain alkane; this reaction is catalyzed by aldehyde decarbonylase1 (AD) (8–9). The potential utility of this enzyme has not been lost on those seeking to develop new routes to biofuels (10). It appears that there are three distinct AD enzymes that catalyze different chemical reactions to effect removal of the aldehyde carbon. In higher plants and green algae the enzyme is a metal dependent, integral membrane protein that converts the aldehyde carbon to carbon monoxide (9, 11). In insects AD appears to be a cytochrome p450 enzyme that oxidizes the aldehyde carbon to carbon dioxide (4). In cyanobacteria AD (cAD) was recently found to be a small, soluble protein whose structure, shown in Figure 1, is closely related to the non-heme di-iron oxygenases (10). We previously demonstrated that, in the reaction catalyzed by cAD from the cyanobacterium Prochlorococcus marinus MIT9313, the aldehyde carbon is converted to formate (Figure 1) (12). Labeling studies established that the proton in the alkane derives from water and that the aldehyde proton is retained in formate (12), so that the alkane is formally derived by hydrolysis of the aldehyde, albeit in a very unusual reaction. Similar results have been independently demonstrated by Warui et al. (13) for cAD from a related cyanobacterium, Nostoc punctiformes. Figure 1 A: x-ray structure of cAD from P. marinus MIT9313, illustrating the position of the di-iron center and fatty acid bound at the active site. B: detail of the di-iron center and metal coordinating ligands. C: reaction catalyzed by cAD. The color coding ... Our initial characterization of cAD (12) established that it is an iron-dependent enzyme and that an auxiliary reducing system is required for activity. In this respect, the enzyme is similar to other di-iron oxygenases such as methane mono-oxygenase (14–15). However, we determined that oxygen is not a co-substrate in the reaction; the enzyme is in fact more active under anaerobic conditions. Furthermore, the reducing system appears to be absolutely required for alkane formation even under rigorously anaerobic conditions. EPR experiments provided evidence that free radical species are generated in the mechanism. Based on these results we proposed a tentative mechanism, shown in Figure 2, for the enzyme in which the reducing system plays a catalytic role, possibly serving to generate free radicals to facilitate the chemically difficult cleavage of the aldehyde carbon. Figure 2 Mechanistic proposal for the oxygen-independent formation of alkanes by cAD. In this mechanism the external reducing system functions catalytically to generate a reactive ketyl radical anion and facilitate carbon-carbon bond cleavage. For a detailed discussion ... Our findings stand in contrast to a labeling study by Li et al. (16) using cAD from N. punctiformes, which found that cAD activity was dependent upon the presence of oxygen and demonstrated that oxygen was incorporated into formate. To accommodate this finding the authors proposed a “cryptic oxidation” mechanism in which activated oxygen species react with the aldehyde to facilitate C-C bond cleavage. However, the rate of the oxidative reaction was orders of magnitude slower than the rate we measured for the anaerobic reaction. To address the apparent discrepancies noted above, we have directly compared the activities of cAD from representatives of 4 of the major classes of cyanobacteria: Prochlorococcus marinus MIT9313, Nostoc punctiformes PCC73102, Synechococcus sp. RS9917 and Synechocystis sp. PCC6803. These enzymes each exhibit activity under anaerobic conditions. Focusing on the P. marinus enzyme, for which a structure is available, we have undertaken a detailed investigation of the kinetics of the enzyme and the unusual role of the reducing system in the reaction.

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.

Oxygen-independent decarbonylation of aldehydes by cyanobacterial aldehyde decarbonylase: a new reaction of diiron enzymes.

The search for new biofuels has generated increased interest in biochemical pathways that produce hydrocarbons.[1] Although hydrocarbons are simple molecules, the biosynthesis of molecules that lack any chemical functional groups is surprisingly challenging.[2] Biochemical reactions that remove functionality, such as decarboxylations, dehydrations and reduction of double bonds, invariably rely on the presence of adjacent functional groups to stabilize unfavorable transition states. Enzymes involved in hydrocarbon biosynthesis are therefore of interest both for applications in biofuels production and because of the unusual and chemically difficult reactions they catalyze.[3] One enzyme that has attracted particular interest, is aldehyde decarbonylase (AD), which catalyzes the decarbonylation of long-chain fatty aldehydes, to the corresponding alkanes.[4]

Oxygen‐Independent Decarbonylation of Aldehydes by Cyanobacterial Aldehyde Decarbonylase: A New Reaction of Diiron Enzymes

Einfach Wasser zugeben: Mit ihrer Struktur gehören cyanobakterielle Aldehyd-Decarbonylasen zur Enzymfamilie der Nichthäm-Dieisen-Oxygenasen – allerdings katalysiert das Enzym, in einer Sauerstoff-unabhängigen Reaktion, die Hydrolyse von aliphatischen Aldehyden zu Alkanen und Formiat (siehe Schema). Die ungewöhnliche und chemisch schwierige Reaktion läuft höchstwahrscheinlich unter Beteiligung freier Radikale ab. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Conversion of Fatty Aldehydes to Alka(e)nes and Formate by a Cyanobacterial Aldehyde Decarbonylase: Cryptic Redox by an Unusual Dimetal Oxygenase

Cyanobacterial aldehyde decarbonylase (AD) catalyzes conversion of fatty aldehydes (R-CHO) to alka(e)nes (R-H) and formate. Curiously, although this reaction appears to be redox-neutral and formally hydrolytic, AD has a ferritin-like protein architecture and a carboxylate-bridged dimetal cofactor that are both structurally similar to those found in di-iron oxidases and oxygenases. In addition, the in vitro activity of the AD from Nostoc punctiforme (Np) was shown to require a reducing system similar to the systems employed by these O(2)-utilizing di-iron enzymes. Here, we resolve this conundrum by showing that aldehyde cleavage by the Np AD also requires dioxygen and results in incorporation of (18)O from (18)O(2) into the formate product. AD thus oxygenates, without oxidizing, its substrate. We posit that (i) O(2) adds to the reduced cofactor to generate a metal-bound peroxide nucleophile that attacks the substrate carbonyl and initiates a radical scission of the C1-C2 bond, and (ii) the reducing system delivers two electrons during aldehyde cleavage, ensuring a redox-neutral outcome, and two additional electrons to return an oxidized form of the cofactor back to the reduced, O(2)-reactive form.

Detection of formate, rather than carbon monoxide, as the stoichiometric coproduct in conversion of fatty aldehydes to alkanes by a cyanobacterial aldehyde decarbonylase.

The second of two reactions in a recently discovered pathway through which saturated fatty acids are converted to alkanes (and unsaturated fatty acids to alkenes) in cyanobacteria entails scission of the C1-C2 bond of a fatty aldehyde intermediate by the enzyme aldehyde decarbonylase (AD), a ferritin-like protein with a dinuclear metal cofactor of unknown composition. We tested for and failed to detect carbon monoxide (CO), the proposed C1-derived coproduct of alkane synthesis, following the in vitro conversion of octadecanal (R-CHO, where R = n-C(17)H(35)) to heptadecane (R-H) by the Nostoc punctiforme AD isolated following its overproduction in Escherichia coli. Instead, we identified formate (HCO(2)(-)) as the stoichiometric coproduct of the reaction. Results of isotope-tracer experiments indicate that the aldehyde hydrogen is retained in the HCO(2)(-) and the hydrogen in the nascent methyl group of the alkane originates, at least in part, from solvent. With these characteristics, the reaction appears to be formally hydrolytic, but the improbability of a hydrolytic mechanism having the primary carbanion as the leaving group, the structural similarity of the ADs to other O(2)-activating nonheme di-iron proteins, and the dependence of in vitro AD activity on the presence of a reducing system implicate some type of redox mechanism. Two possible resolutions to this conundrum are suggested.