Structure-oriented substrate specificity engineering of aldehyde-deformylating oxygenase towards aldehydes carbon chain length

BackgroundAldehyde-deformylating oxygenase (ADO) is an important enzyme involved in the biosynthetic pathway of fatty alk(a/e)nes in cyanobacteria. However, ADO exhibits quite low chain-length specificity with respect to the substrates ranging from C4 to C18 aldehydes, which is not suitable for producing fuels with different properties or different chain lengths.ResultsBased on the crystal structures of cADOs (cyanobacterial ADO) with substrate analogs bound, some amino acids affecting the substrate specificity of cADO were identified, including the amino acids close to the aldehyde group and the hydrophobic tail of the substrate and those along the substrate channel. Using site-directed mutagenesis, selected amino acids were replaced with bulky ones introducing steric hindrance to the binding pocket via large functional groups. All mutants were overexpressed, purified and kinetically characterized. All mutants, except F87Y, displayed dramatically reduced activity towards C14,16,18 aldehydes. Notably, the substrate preferences of some mutants towards different chain-length substrates were enhanced: I24Y for n-heptanal, I27F for n-decanal and n-dodecanal, V28F for n-dodecanal, F87Y for n-decanal, C70F for n-hexanal, A118F for n-butanal, A121F for C4,6,7 aldehydes, V184F for n-dodecanal and n-decanal, M193Y for C6–10 aldehydes and L198F for C7–10 aldehydes. The impact of the engineered cADO mutants on the change of the hydrocarbon profile was demonstrated by co-expressing acyl-ACP thioesterase BTE, fadD and V184F in E. coli, showing that n-undecane was the main fatty alkane.ConclusionsSome amino acids, which can control the chain-length selectivity of substrates of cADO, were identified. The substrate specificities of cADO were successfully changed through structure-guided protein engineering, and some mutants displayed different chain-length preference. The in vivo experiments of V184F in genetically engineered E. coli proved the importance of engineered cADOs on the distribution of the fatty alkane profile. The results would be helpful for the production of fatty alk(a/e)nes in cyanobacteria with different properties.

Related papers:

title: Microbial Biosynthesis of Alkanes
abstract: Toward Alkane Synthesis Alkanes are major components of fossil fuels, and synthesis of alkanes remains a challenge in the conversion of renewable raw materials to fuels. Even though diverse organisms synthesize alkanes, synthesis pathways have remained elusive. Now Schirmer et al. (p. 559) describe an alkane biosynthesis pathway in cyanobacteria that converts intermediates of fatty acid metabolism to alkanes and alkenes. Heterologous expression of the biosynthetic genes resulted in production of alkanes in Escherichia coli. This pathway is likely to be a valuable tool in the production of biofuels. Alkane biosynthesis genes were identified in cyanobacteria and engineered into Escherichia coli for recombinant hydrocarbon production. Alkanes, the major constituents of gasoline, diesel, and jet fuel, are naturally produced by diverse species; however, the genetics and biochemistry behind this biology have remained elusive. Here we describe the discovery of an alkane biosynthesis pathway from cyanobacteria. The pathway consists of an acyl–acyl carrier protein reductase and an aldehyde decarbonylase, which together convert intermediates of fatty acid metabolism to alkanes and alkenes. The aldehyde decarbonylase is related to the broadly functional nonheme diiron enzymes. Heterologous expression of the alkane operon in Escherichia coli leads to the production and secretion of C13 to C17 mixtures of alkanes and alkenes. These genes and enzymes can now be leveraged for the simple and direct conversion of renewable raw materials to fungible hydrocarbon fuels.

title: A perspective: photosynthetic production of fatty acid-based biofuels in genetically engineered cyanobacteria.
abstract: Biofuels are expected to play a key role in the development of a sustainable, economical and environmentally safe source of energy. Microbes offer great potential for applications in technology based biofuel production. Three fundamental questions need to be addressed in order for the development of microbial synthesis of biofuels to be successful. Firstly, what energy resource platform could be used to make biofuels. Secondly, what type of biofuel is the ideal fuel molecule that should be targeted. Finally, what microbial system could be used to transform energy resources into the targeted biofuel molecules. In this perspective, the potential of using photosynthetic microbes (cyanobacteria in particular) in the solar energy driven conversion of carbon dioxide to fatty acid-based biofuels is explored.

title: Metabolic engineering of microbial pathways for advanced biofuels production.
abstract: Production of biofuels from renewable resources such as cellulosic biomass provides a source of liquid transportation fuel to replace petroleum-based fuels. This endeavor requires the conversion of cellulosic biomass into simple sugars, and the conversion of simple sugars into biofuels. Recently, microorganisms have been engineered to convert simple sugars into several types of biofuels, such as alcohols, fatty acid alkyl esters, alkanes, and terpenes, with high titers and yields. Here, we review recently engineered biosynthetic pathways from the well-characterized microorganisms Escherichia coli and Saccharomyces cerevisiae for the production of several advanced biofuels.

title: Next generation biofuel engineering in prokaryotes.
abstract: Next-generation biofuels must be compatible with current transportation infrastructure and be derived from environmentally sustainable resources that do not compete with food crops. Many bacterial species have unique properties advantageous to the production of such next-generation fuels. However, no single species possesses all characteristics necessary to make high quantities of fuels from plant waste or CO2. Species containing a subset of the desired characteristics are used as starting points for engineering organisms with all desired attributes. Metabolic engineering of model organisms has yielded high titer production of advanced fuels, including alcohols, isoprenoids, and fatty acid derivatives. Technical developments now allow engineering of native fuel producers, as well as lignocellulolytic and autotrophic bacteria, for the production of biofuels. Continued research on multiple fronts is required to engineer organisms for truly sustainable and economical biofuel production.

title: Production of advanced biofuels in engineered E. coli.
abstract: Commercial fermentation processes have long taken advantage of the synthetic power of living systems to rapidly and efficiently transform simple carbon sources into complex molecules. In this regard, the ability of yeasts to produce ethanol from glucose at exceptionally high yields has served as a key feature in its use as a fuel, but is also limited by the poor molecular properties of ethanol as a fuel such as high water miscibility and low energy density. Advances in metabolic engineering and synthetic biology allow us to begin constructing new high-flux pathways for production of next generation biofuels that are key to building a sustainable pipeline for liquid transportation fuels.

title: Carboxylic acid reductase is a versatile enzyme for the conversion of fatty acids into fuels and chemical commodities
abstract: Aliphatic hydrocarbons such as fatty alcohols and petroleum-derived alkanes have numerous applications in the chemical industry. In recent years, the renewable synthesis of aliphatic hydrocarbons has been made possible by engineering microbes to overaccumulate fatty acids. However, to generate end products with the desired physicochemical properties (e.g., fatty aldehydes, alkanes, and alcohols), further conversion of the fatty acid is necessary. A carboxylic acid reductase (CAR) from Mycobacterium marinum was found to convert a wide range of aliphatic fatty acids (C6–C18) into corresponding aldehydes. Together with the broad-substrate specificity of an aldehyde reductase or an aldehyde decarbonylase, the catalytic conversion of fatty acids to fatty alcohols (C8–C16) or fatty alkanes (C7–C15) was reconstituted in vitro. This concept was applied in vivo, in combination with a chain-length-specific thioesterase, to engineer Escherichia coli BL21(DE3) strains that were capable of synthesizing fatty alcohols and alkanes. A fatty alcohol titer exceeding 350 mg·L−1 was obtained in minimal media supplemented with glucose. Moreover, by combining the CAR-dependent pathway with an exogenous fatty acid-generating lipase, natural oils (coconut oil, palm oil, and algal oil bodies) were enzymatically converted into fatty alcohols across a broad chain-length range (C8–C18). Together with complementing enzymes, the broad substrate specificity and kinetic characteristics of CAR opens the road for direct and tailored enzyme-catalyzed conversion of lipids into user-ready chemical commodities.

title: Aldehyde Decarbonylases: Enigmatic Enzymes of Hydrocarbon Biosynthesis.
abstract: Long-chain alkane waxes are synthesized by a wide variety of organisms, including plants, animals and microorganisms.1-4 Although chemically alkanes are amongst the most boring of organic molecules, devoid of functionality and thus unreactive, they play essential roles in these organisms’ survival. Plants secrete what are termed “very long chain” alkane waxes (i.e. chain length ~ 30 carbons) onto their leaves and stems.3 These, together with wax esters formed from aliphatic alcohols and carboxylic acids of similar chain length, serve as a waterproof barrier to prevent desiccation. A wide variety of hydrocarbons, including branched and unsaturated molecules, are biosynthesized by insects.2 These are secreted onto the cuticle and play an important role as contact pheromones that mediate all manner of insect:insect recognition and social behaviors; they are also important in preventing desiccation, especially of larval forms.5 Waterfowl secrete alkane-rich oils from their uropygial (preening) glands; these are essential for waterproofing the birds’ feathers, which would otherwise become waterlogged and thus useless for insulation and flight.1 Many algae synthesize large amounts of long-chain alkanes that can comprise up to 30 % of the algae's dry weight.6 These accumulate in the area contained by the inner and outer cell walls known as the trilamellar structure and can be utilized to provide energy when photosynthesis is not possible.7 
 
In recent decades many human beings have become equally dependent on alkanes, in that this class of molecules is the major component of transportation fuels. These fuels are, of course, currently derived from fossil reserves and their combustion is a major source of greenhouse gases. The challenge of producing the next generation of biofuels – those that can effectively function as “drop-in” replacements for gasoline, diesel, and jet fuel – has led to renewed interest, especially in the area of mechanistic enzymology, in how alkanes are biosynthesized.8 
 
The synthesis of completely unfunctionalized molecules presents a challenge for a cell.9 Whereas as a chemist might accomplish this by reducing the corresponding alkene to an alkane using hydrogen and an activated transition metal catalyst, this approach is clearly not biochemically feasible. Historically, an important observation was that naturally-synthesized alkanes invariably comprise an odd number of carbons, suggesting that they are derived from fatty acid biosynthesis through loss of the carboxyl carbon.10 It was subsequently established that long-chain alkanes are synthesized from fatty acids through the intermediacy of the corresponding fatty aldehydes. These are the substrates for a group of enzymes, the aldehyde decarbonylases, which catalyze the removal of the aldehyde carbonyl group to form the alkane. It is the mechanisms of these enzymes that are the focus of this viewpoint. Whereas alkanes might be boring molecules, we hope to show that the mechanisms by which they are formed in Nature, and about which much still remains to be understood, are definitely not! 
 
Although all the various organisms so far investigated appear to synthesize alkanes using fatty aldehydes as the precursor, it has become clear that there are several mechanistically distinct classes of decarbonylases, as illustrated in Figure 1. In insects, the enzyme has been shown to be a cytochrome P450 protein,11,12 whereas in cyanobacteria it is a non-heme di-iron enzyme that is structurally related to enzymes such as ferritin and methane monooxygenase.3,13 In plants, the enzyme is an integral membrane protein that has some sequence similarity to the fatty acid hydroxylase superfamily and stearoyl-CoA desaturase.14 On this basis, it too is presumed to be a metalloenzyme, with iron the most likely metal. The different mechanisms of decarbonylation are reflected in the fate of the aldehyde carbon, which is converted to CO2 in insects,12 HCO2H in cyanobacteria,15,16 and CO in plants and algae.6,17 
 
 
 
Figure 1 
 
The biosynthesis of alkanes proceeds in two steps from fatty acyl-CoA esters. The decarbonylation of the intermediate fatty aldehyde proceeds by one of three different mechanisms depending upon the organism. The insect AD is a membrane-bound P450 type ... 
 
 
 
 
2 Insect decarbonylases 
The biosynthesis of hydrocarbons has been studied in a number of insect species including the domestic house fly, Musca domestica, the fruit fly Drosophila melanogaster, cockroaches Periplaneta americana and Blattella germanica, and the termite Zootermopsis angusticollis.18 Experiments on microsomal preparations of M. domestica demonstrated that molecular oxygen and NADPH were required for the conversion of (Z)-15-tetracosenal to (Z)-9-tricosene and, using 1-14C-material, that the aldehyde carbon was oxidized to CO2 in the process.12 Furthermore, antibodies to either house fly cytochrome P450 reductase or to a cytochrome P450 (CYP6A1) purified from the house fly inhibited (Z)-9-tricosene.19 These initial observations provided support for the involvement of a P450 enzyme in the reaction; this was more recently confirmed with the cloning and heterologous expression of the enzyme, CYP4G1, from Drosophila.11 
 
Further clues to the mechanism of hydrocarbon formation came from analyses of n-tricosane formed by M. domestica microsomal preparations incubated with [2,2-2H2,2-13C]-tetracosanoyl-CoA and [3,3-2H2,3-13C]-tetracosanoyl-CoA which demonstrated that the deuterium atoms on the 2- and 3-positions were retained in the hydrocarbon product.19 Analysis of [l-2H]-tetracosenal incubated with microsomal preparations demonstrated that the aldehyde proton on C-l was transferred to the (Z)-9-tricosene product. Alternative oxidizing agents such as hydrogen peroxide, cumene hydroperoxide, and iodosobenzene were shown to substitute for O2 and NADPH in the reaction. To accommodate these observations, a mechanism has been proposed, shown in Figure 2A, in which the high valent iron-oxo species, resulting from heterolytic cleavage of the O-O bond of the iron-peroxy intermediate, abstracts an electron from the carbonyl group of the aldehyde.19 The reduced iron-oxo species then attacks the carbonyl carbon of the aldehyde to form an iron-hemiacetal diradical. This intermediate is proposed to fragment to form an alkyl radical and an iron-bound formyl radical. In the final step, the alkyl radical then abstracts the formyl hydrogen to produce the hydrocarbon and CO2. 
 
 
 
Figure 2 
 
Comparison of the deformylation reactions catalyzed by insect AD (CYP4G1) and CYP2B4. A – Deformylation of fatty aldehydes by the insect AD is proposed to start with the high-valent iron-oxo species and results in the formation of CO2. The color-coding ... 
 
 
 
P450 enzymes catalyze a notably diverse range of oxidative transformations on a very wide range of substrates.20 Even so, the decarbonylase reaction stands out as being unusual. In most cases P450 enzymes oxidize aldehydes to carboxylic acids through a well understood mechanism involving hydrogen atom abstraction by the high valent iron-oxo intermediate followed by “rebound” of the iron-bound hydroxyl group to give the hydroxylated substrate.21 In some cases decarbonylation of the aldehyde does occur, notably in the aromatization reaction of androst-4-ene-3,17-dione to estrone catalyzed by human aromatase and the deformylation of cyclohexanal catalyzed by CYP2B4

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

title: Conversion of fatty aldehydes to alka(e)nes and formate by a cyanobacterial aldehyde decarbonylase: cryptic redox by an unusual dimetal oxygenase.
abstract: 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.

title: Evidence for only oxygenative cleavage of aldehydes to alk(a/e)nes and formate by cyanobacterial aldehyde decarbonylases.
abstract: Cyanobacterial aldehyde decarbonylases (ADs) catalyze the conversion of C(n) fatty aldehydes to formate (HCO(2)(-)) and the corresponding C(n-1) alk(a/e)nes. Previous studies of the Nostoc punctiforme (Np) AD produced in Escherichia coli (Ec) showed that this apparently hydrolytic reaction is actually a cryptically redox oxygenation process, in which one O-atom is incorporated from O(2) into formate and a protein-based reducing system (NADPH, ferredoxin, and ferredoxin reductase; N/F/FR) provides all four electrons needed for the complete reduction of O(2). Two subsequent publications by Marsh and co-workers [ Das, et al. ( 2011 )  ; Eser, et al. ( 2011 ) Biochemistry 50 , 10743 - 10750 ] reported that their Ec-expressed Np and Prochlorococcus marinus (Pm) AD preparations transform aldehydes to the same products more rapidly by an O(2)-independent, truly hydrolytic process, which they suggested proceeded by transient substrate reduction with obligatory participation by the reducing system (they used a chemical system, NADH and phenazine methosulfate; N/PMS). To resolve this discrepancy, we re-examined our preparations of both AD orthologues by a combination of (i) activity assays in the presence and absence of O(2) and (ii) (18)O(2) and H(2)(18)O isotope-tracer experiments with direct mass-spectrometric detection of the HCO(2)(-) product. For multiple combinations of the AD orthologue (Np and Pm), reducing system (protein-based and chemical), and substrate (n-heptanal and n-octadecanal), our preparations strictly require O(2) for activity and do not support detectable hydrolytic formate production, despite having catalytic activities similar to or greater than those reported by Marsh and co-workers. Our results, especially of the (18)O-tracer experiments, suggest that the activity observed by Marsh and co-workers could have arisen from contaminating O(2) in their assays. The definitive reaffirmation of the oxygenative nature of the reaction implies that the enzyme, initially designated as aldehyde decarbonylase when the C1-derived coproduct was thought to be carbon monoxide rather than formate, should be redesignated as aldehyde-deformylating oxygenase (ADO).

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

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

title: Conversion of fatty aldehydes into alk (a/e)nes by in vitro reconstituted cyanobacterial aldehyde-deformylating oxygenase with the cognate electron transfer system
abstract: BackgroundBiosynthesis of fatty alk(a/e)ne in cyanobacteria has been considered as a potential basis for the sunlight-driven and carbon-neutral bioprocess producing advanced solar biofuels. Aldehyde-deformylating oxygenase (ADO) is a key enzyme involved in that pathway. The heterologous or chemical reducing systems were generally used in in vitro ADO activity assay. The cognate electron transfer system from cyanobacteria to support ADO activity is still unknown.ResultsWe identified the potential endogenous reducing system including ferredoxin (Fd) and ferredoxin-NADP+ reductase (FNR) to support ADO activity in Synechococcus elongatus PCC7942. ADO (Synpcc7942_1593), FNR (SynPcc7942_0978), and Fd (SynPcc7942_1499) from PCC7942 were cloned, overexpressed, purified, and characterized. ADO activity was successfully supported with the endogenous electron transfer system, which worked more effectively than the heterologous and chemical ones. The results of the hybrid Fd/FNR reducing systems demonstrated that ADO was selective against Fd. And it was observed that the cognate reducing system produced less H2O2 than the heterologous one by 33% during ADO-catalyzed reactions. Importantly, kcat value of ADO 1593 using the homologous Fd/FNR electron transfer system is 3.7-fold higher than the chemical one.ConclusionsThe cognate electron transfer system from cyanobacteria to support ADO activity was identified and characterized. For the first time, ADO was functionally in vitro reconstituted with the endogenous reducing system from cyanobacteria, which supported greater activity than the surrogate and chemical ones, and produced less H2O2 than the heterologous one. The identified Fd/FNR electron transfer system will be potentially useful for improving ADO activity and further enhancing the biosynthetic efficiency of hydrocarbon biofuels in cyanobacteria.

title: Engineering self-sufficient aldehyde deformylating oxygenases fused to alternative electron transfer systems for efficient conversion of aldehydes into alkanes.
abstract: Self-sufficient aldehyde deformylating oxygenases (ADOs) from Synechococcus elongatus PCC7942 fused to alternative electron transfer systems were successfully designed, constructed, characterized and used for efficient conversion of aldehydes into alkanes for the first time.

title: Cyanobacterial aldehyde deformylase oxygenation of aldehydes yields n-1 aldehydes and alcohols in addition to alkanes.
abstract: Aldehyde-deformylating oxygenase (ADO) catalyzes O2-dependent release of the terminal carbon of a biological substrate, octadecanal, to yield formate and heptadecane in a reaction that requires external reducing equivalents. We show here that ADO also catalyzes incorporation of an oxygen atom from O2 into the alkane product to yield alcohol and aldehyde products. Oxygenation of the alkane product is much more pronounced with C9-10 aldehyde substrates, so that use of nonanal as the substrate yields similar amounts of octane, octanal, and octanol products. When using doubly-labeled [1,2-13C]-octanal as the substrate, the heptane, heptanal and heptanol products each contained a single 13C-label in the C-1 carbons atoms. The only one-carbon product identified was formate. [18O]-O2 incorporation studies demonstrated formation of [18O]-alcohol product, but rapid solvent exchange prevented similar determination for the aldehyde product. Addition of [1-13C]-nonanol with decanal as the substrate at the outset of the reaction resulted in formation of [1-13C]-nonanal. No 13C-product was formed in the absence of decanal. ADO contains an oxygen-bridged dinuclear iron cluster. The observation of alcohol and aldehyde products derived from the initially formed alkane product suggests a reactive species similar to that formed by methane monooxygenase (MMO) and other members of the bacterial multicomponent monooxygenase family. Accordingly, characterization by EPR and Mössbauer spectroscopies shows that the electronic structure of the ADO cluster is similar, but not identical, to that of MMO hydroxylase component. In particular, the two irons of ADO reside in nearly identical environments in both the oxidized and fully reduced states, whereas those of MMOH show distinct differences. These favorable characteristics of the iron sites allow a comprehensive determination of the spin Hamiltonian parameters describing the electronic state of the diferrous cluster for the first time for any biological system. The nature of the diiron cluster and the newly recognized products from ADO catalysis hold implications for the mechanism of C-C bond cleavage.

title: Structural insights into the catalytic mechanism of aldehyde-deformylating oxygenases
abstract: The fatty alk(a/e)ne biosynthesis pathway found in cyanobacteria gained tremendous attention in recent years as a promising alternative approach for biofuel production. Cyanobacterial aldehyde-deformylating oxygenase (cADO), which catalyzes the conversion of Cn fatty aldehyde to its corresponding Cn-1 alk(a/e)ne, is a key enzyme in that pathway. Due to its low activity, alk(a/e)ne production by cADO is an inefficient process. Previous biochemical and structural investigations of cADO have provided some information on its catalytic reaction. However, the details of its catalytic processes remain unclear. Here we report five crystal structures of cADO from the Synechococcus elongates strain PCC7942 in both its iron-free and iron-bound forms, representing different states during its catalytic process. Structural comparisons and functional enzyme assays indicate that Glu144, one of the iron-coordinating residues, plays a vital role in the catalytic reaction of cADO. Moreover, the helix where Glu144 resides exhibits two distinct conformations that correlates with the different binding states of the di-iron center in cADO structures. Therefore, our results provide a structural explanation for the highly labile feature of cADO di-iron center, which we proposed to be related to its low enzymatic activity. On the basis of our structural and biochemical data, a possible catalytic process of cADO was proposed, which could aid the design of cADO with improved activity.

title: Probing the mechanism of cyanobacterial aldehyde decarbonylase using a cyclopropyl aldehyde.
abstract: 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.

title: Mechanistic insights from reaction of α-oxiranyl-aldehydes with cyanobacterial aldehyde deformylating oxygenase.
abstract: The biosynthesis of long-chain aliphatic hydrocarbons, which are derived from fatty acids, is widespread in  The last step in this pathway involves the decarbonylation of fatty aldehydes to the corresponding alkanes or alkenes. In cyanobacteria, this is catalyzed by an aldehyde deformylating oxygenase. We have investigated the mechanism of this enzyme using substrates bearing an oxirane ring adjacent to the aldehyde carbon. The enzyme catalyzed the deformylation of these substrates to produce the corresponding oxiranes. Performing the reaction in D2O allowed the facial selectivity of proton addition to be examined by (1)H NMR spectroscopy. The proton is delivered with equal probability to either face of the oxirane ring, indicating the formation of an oxiranyl radical intermediate that is free to rotate during the reaction. Unexpectedly, the enzyme also catalyzes a side reaction in which oxiranyl-aldehydes undergo tandem deformylation to furnish alkanes two carbons shorter. We present evidence that this involves the rearrangement of the intermediate oxiranyl radical formed in the first step, resulting in aldehyde that is further deformylated in a second step. These observations provide support for a radical mechanism for deformylation and, furthermore, allow the lifetime of the radical intermediate to be estimated based on prior measurements of rate constants for the rearrangement of oxiranyl radicals.

title: Production of Propane and Other Short-Chain Alkanes by Structure-Based Engineering of Ligand Specificity in Aldehyde-Deformylating Oxygenase
abstract: Biocatalytic propane production: structure-based engineering of aldehyde-deformylating oxygenase improves specificity for short- and medium-chain-length aldehydes and enhances the propane generation in whole-cell biotransformations. This presents new opportunities for developing biocatalytic modules for the production of volatile "drop-in" biofuels.

title: Insights into Substrate and Metal Binding from the Crystal Structure of Cyanobacterial Aldehyde Deformylating Oxygenase with Substrate Bound
abstract: The nonheme diiron enzyme cyanobacterial aldehyde deformylating oxygenase, cADO, catalyzes the highly unusual deformylation of aliphatic aldehydes to alkanes and formate. We have determined crystal structures for the enzyme with a long-chain water-soluble aldehyde and medium-chain carboxylic acid bound to the active site. These structures delineate a hydrophobic channel that connects the solvent with the deeply buried active site and reveal a mode of substrate binding that is different from previously determined structures with long-chain fatty acids bound. The structures also identify a water channel leading to the active site that could facilitate the entry of protons required in the reaction. NMR studies examining 1-[13C]-octanal binding to cADO indicate that the enzyme binds the aldehyde form rather than the hydrated form. Lastly, the fortuitous cocrystallization of the metal-free form of the protein with aldehyde bound has revealed protein conformation changes that are involved in binding iron.

title: Desaturases: Emerging Models for Understanding Functional Diversification of Diiron-containing Enzymes*
abstract: Desaturases and related enzymes perform O2-dependent dehydrogenations initiated at unactivated C-H groups with the use of a diiron active site. Determination of the long-sought oxidized desaturase crystal structure facilitated structural comparison of the active sites of disparate diiron enzymes. Experiments on the castor desaturase are discussed that provide experimental support for a hypothesized ancestral oxidase enzyme in the context of the evolution of the diiron enzyme diverse functionality. We also summarize recent analysis of a castor mutant desaturase that provides valuable insights into the relationship of proposed substrate-binding modes with respect to a range of catalytic outcomes.

title: Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels.
abstract: The ability to generate microorganisms that can produce biofuels similar to petroleum-based transportation fuels would allow the use of existing engines and infrastructure and would save an enormous amount of capital required for replacing the current infrastructure to accommodate biofuels that have properties significantly different from petroleum-based fuels. Several groups have demonstrated the feasibility of manipulating microbes to produce molecules similar to petroleum-derived products, albeit at relatively low productivity (e.g. maximum butanol production is around 20 g/L). For cost-effective production of biofuels, the fuel-producing hosts and pathways must be engineered and optimized. Advances in metabolic engineering and synthetic biology will provide new tools for metabolic engineers to better understand how to rewire the cell in order to create the desired phenotypes for the production of economically viable biofuels.

title: “Designer” Biodiesel: Optimizing Fatty Ester Composition to Improve Fuel Properties†
abstract: Biodiesel is a domestic and renewable alternative with the potential to replace some of the petrodiesel market. It is obtained from vegetable oils, animal fats, or other sources with a significant content of triacylglycerols by means of a transesterification reaction. The fatty acid profile of biodiesel thus corresponds to that of the parent oil or fat and is a major factor influencing fuel properties. Besides being renewable and of domestic origin, advantages of biodiesel compared to petrodiesel include biodegradability, higher flash point, reduction of most regulated exhaust emissions, miscibility in all ratios with petrodiesel, compatibility with the existing fuel distribution infrastructure, and inherent lubricity. Technical problems with biodiesel include oxidative stability, cold flow, and increased NOx exhaust emissions. Solutions to one of these problems often entail increasing the problematic behavior of another property and have included the use of additives or modifying the fatty acid compositi...

title: Overproduction of free fatty acids in E. coli: implications for biodiesel production.
abstract: Whereas microbial fermentation processes for producing ethanol and related alcohol biofuels are well established, biodiesel (methyl esters of fatty acids) is exclusively derived from plant oils. Slow cycle times for engineering oilseed metabolism and the excessive accumulation of glycerol as a byproduct are two major drawbacks of deriving biodiesel from plants. Although most bacteria produce fatty acids as cell envelope precursors, the biosynthesis of fatty acids is tightly regulated at multiple levels. By introducing four distinct genetic changes into the E. coli genome, we have engineered an efficient producer of fatty acids. Under fed-batch, defined media fermentation conditions, 2.5 g/L fatty acids were produced by this metabolically engineered E. coli strain, with a specific productivity of 0.024 g/h/g dry cell mass and a peak conversion efficiency of 4.8% of the carbon source into fatty acid products. At least 50% of the fatty acids produced were present in the free acid form.

title: Engineering acyl carrier protein to enhance production of shortened fatty acids
abstract: BackgroundThe acyl carrier protein (ACP) is an essential and ubiquitous component of microbial synthesis of fatty acids, the natural precursor to biofuels. Natural fatty acids usually contain long chains of 16 or more carbon atoms. Shorter carbon chains, with increased fuel volatility, are desired for internal combustion engines. Engineering the length specificity of key proteins in fatty acid metabolism, such as ACP, may enable microbial synthesis of these shorter chain fatty acids.ResultsWe constructed a homology model of the Synechococcus elongatus ACP, showing a hydrophobic pocket harboring the growing acyl chain. Amino acids within the pocket were mutated to increase steric hindrance to the acyl chain.  ACPs, when over-expressed in Escherichia coli, increased the proportion of shorter chain lipids; I75 W and I75Y showed the strongest effects. Expression of I75 W and I75Y mutant ACPs also increased production of lauric acid in E. coli that expressed the C12-specific acyl-ACP thioesterase from Cuphea palustris.ConclusionsWe engineered the specificity of the ACP, an essential protein of fatty acid metabolism, to alter the E. coli lipid pool and enhance production of medium-chain fatty acids as biofuel precursors. These results indicate that modification of ACP itself could be combined with enzymes affecting length specificity in fatty acid synthesis to enhance production of commodity chemicals based on fatty acids.

title: A process for microbial hydrocarbon synthesis: Overproduction of fatty acids in Escherichia coli and catalytic conversion to alkanes
abstract: The development of renewable alternatives to diesel and jet fuels is highly desirable for the heavy transportation sector, and would offer benefits over the production and use of short‐chain alcohols for personal transportation. Here, we report the development of a metabolically engineered strain of Escherichia coli that overproduces medium‐chain length fatty acids via three basic modifications: elimination of β‐oxidation, overexpression of the four subunits of acetyl‐CoA carboxylase, and expression of a plant acyl–acyl carrier protein (ACP) thioesterase from Umbellularia californica (BTE). The expression level of BTE was optimized by comparing fatty acid production from strains harboring BTE on plasmids with four different copy numbers. Expression of BTE from low copy number plasmids resulted in the highest fatty acid production. Up to a seven‐fold increase in total fatty acid production was observed in engineered strains over a negative control strain (lacking β‐oxidation), with a composition dominated by C12 and C14 saturated and unsaturated fatty acids. Next, a strategy for producing undecane via a combination of biotechnology and heterogeneous catalysis is demonstrated. Fatty acids were extracted from a culture of an overproducing strain into an alkane phase and fed to a Pd/C plug flow reactor, where the extracted fatty acids were decarboxylated into saturated alkanes. The result is an enriched alkane stream that can be recycled for continuous extractions. Complete conversion of C12 fatty acids extracted from culture to alkanes has been demonstrated yielding a concentration of 0.44 g L−1 (culture volume) undecane. Biotechnol. Bioeng. 2010;106: 193–202.

title: Optimization of fatty alcohol biosynthesis pathway for selectively enhanced production of C12/14 and C16/18 fatty alcohols in engineered Escherichia coli
abstract: BackgroundWith the increasing stress from oil price and environmental pollution, aroused attention has been paid to the microbial production of chemicals from renewable sources. The C12/14 and C16/18 alcohols are important feedstocks for the production of surfactants and detergents, which are widely used in the most respected consumer detergents, cleaning products and personal care products worldwide. Though bioproduction of fatty alcohols has been carried out in engineered E. coli, several key problems have not been solved in earlier studies, such as the quite low production of C16/18 alcohol, the lack of optimization of the fatty alcohol biosynthesis pathway, and the uncharacterized performance of the engineered strains in scaled-up system.ResultsWe improved the fatty alcohol production by systematically optimizing the fatty alcohol biosynthesis pathway, mainly targeting three key steps from fatty acyl-acyl carrier proteins (ACPs) to fatty alcohols, which are sequentially catalyzed by thioesterase, acyl-coenzyme A (CoA) synthase and fatty acyl-CoA reductase. By coexpression of thioesterase gene BTE, acyl-CoA synthase gene fadD and fatty acyl-CoA reductase gene acr1, 210.1 mg/L C12/14 alcohol was obtained. A further optimization of expression level of BTE, fadD and acr1 increased the C12/14 alcohol production to 449.2 mg/L, accounting for 75.0% of the total fatty alcohol production (598.6 mg/L). In addition, by coexpression of thioesterase gene ‘tesA, acyl-CoA synthase gene fadD and fatty acyl-CoA reductase gene FAR, 101.5 mg/L C16/18 alcohol was obtained, with C16/18 alcohol accounting for 89.2% of the total fatty alcohol production.ConclusionsTo our knowledge, this is the first report on selective production of C12/14 and C16/18 alcohols by microbial fermentation. This work achieved high-specificity production of both C12/14 and C16/18 alcohols. The encouraging 598.6 mg/L of fatty alcohols represents the highest titer reported so far. In addition, the 101.5 mg/L 89.2% C16/18 alcohol suggests an important breakthrough in C16/18 alcohol production. A more detailed optimization of the expression level of fatty alcohol biosynthesis pathway may contribute to a further improvement of fatty alcohol production.

title: De novo Biosynthesis of Biodiesel by Escherichia coli in Optimized Fed-Batch Cultivation
abstract: Biodiesel is a renewable alternative to petroleum diesel fuel that can contribute to carbon dioxide emission reduction and energy supply. Biodiesel is composed of fatty acid alkyl esters, including fatty acid methyl esters (FAMEs) and fatty acid ethyl esters (FAEEs), and is currently produced through the transesterification reaction of methanol (or ethanol) and triacylglycerols (TAGs). TAGs are mainly obtained from oilseed plants and microalgae. A sustainable supply of TAGs is a major bottleneck for current biodiesel production. Here we report the de novo biosynthesis of FAEEs from glucose, which can be derived from lignocellulosic biomass, in genetically engineered Escherichia coli by introduction of the ethanol-producing pathway from Zymomonas mobilis, genetic manipulation to increase the pool of fatty acyl-CoA, and heterologous expression of acyl-coenzyme A: diacylglycerol acyltransferase from Acinetobacter baylyi. An optimized fed-batch microbial fermentation of the modified E. coli strain yielded a titer of 922 mg L−1 FAEEs that consisted primarily of ethyl palmitate, -oleate, -myristate and -palmitoleate.

title: Fatty alcohol production in engineered E. coli expressing Marinobacter fatty acyl-CoA reductases
abstract: Although successful production of fatty alcohols in metabolically engineered Escherichia coli with heterologous expression of fatty acyl-CoA reductase has been reported, low biosynthetic efficiency is still a hurdle to be overcome. In this study, we examined the characteristics of two fatty acyl-CoA reductases encoded by Maqu_2220 and Maqu_2507 genes from Marinobacter aquaeolei VT8 on fatty alcohol production in E. coli. Fatty alcohols with diversified carbon chain length were obtained by co-expressing Maqu_2220 with different carbon chain length-specific acyl-ACP thioesterases. Both fatty acyl-CoA reductases displayed broad substrate specificities for C12–C18 fatty acyl chains in vivo. The optimized mutant strain of E. coli carrying the modified tesA gene and fadD gene from E. coli and Maqu_2220 gene from Marinobacter aquaeolei VT8 produced fatty alcohols at a remarkable level of 1.725 g/L under the fermentation condition.

title: Molecular Cloning: A Laboratory Manual
abstract: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol

title: Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy
abstract: Circular dichroism (CD) spectroscopy is widely used for protein secondary structure analysis. However, quantitative estimation for β-sheet–containing proteins is problematic due to the huge morphological and spectral diversity of β-structures. We show that parallel/antiparallel orientation and twisting of β-sheets account for the observed spectral diversity. Taking into account the twist of β-structures, our method accurately estimates the secondary structure for a broad range of protein folds, particularly for β-sheet–rich proteins and amyloid fibrils. Moreover, the method can predict the protein fold down to the topology level following the CATH classification. We provide a general tool for a quick and reliable structure analysis using conventional or synchrotron radiation CD spectroscopy, which is especially useful when X-ray or NMR techniques fail. Circular dichroism (CD) spectroscopy is a widely used technique for the study of protein structure. Numerous algorithms have been developed for the estimation of the secondary structure composition from the CD spectra. These methods often fail to provide acceptable results on α/β-mixed or β-structure–rich proteins. The problem arises from the spectral diversity of β-structures, which has hitherto been considered as an intrinsic limitation of the technique. The predictions are less reliable for proteins of unusual β-structures such as membrane proteins, protein aggregates, and amyloid fibrils. Here, we show that the parallel/antiparallel orientation and the twisting of the β-sheets account for the observed spectral diversity. We have developed a method called β-structure selection (BeStSel) for the secondary structure estimation that takes into account the twist of β-structures. This method can reliably distinguish parallel and antiparallel β-sheets and accurately estimates the secondary structure for a broad range of proteins. Moreover, the secondary structure components applied by the method are characteristic to the protein fold, and thus the fold can be predicted to the level of topology in the CATH classification from a single CD spectrum. By constructing a web server, we offer a general tool for a quick and reliable structure analysis using conventional CD or synchrotron radiation CD (SRCD) spectroscopy for the protein science research community. The method is especially useful when X-ray or NMR techniques fail. Using BeStSel on data collected by SRCD spectroscopy, we investigated the structure of amyloid fibrils of various disease-related proteins and peptides.