RESUMO
Moving cannabinoid production away from the vagaries of plant extraction and into engineered microbes could provide a consistent, purer, cheaper and environmentally benign source of these important therapeutic molecules, but microbial production faces notable challenges. An alternative to microbes and plants is to remove the complexity of cellular systems by employing enzymatic biosynthesis. Here we design and implement a new cell-free system for cannabinoid production with the following features: (1) only low-cost inputs are needed; (2) only 12 enzymes are employed; (3) the system does not require oxygen and (4) we use a nonnatural enzyme system to reduce ATP requirements that is generally applicable to malonyl-CoA-dependent pathways such as polyketide biosynthesis. The system produces ~0.5 g l-1 cannabigerolic acid (CBGA) or cannabigerovarinic acid (CBGVA) from low-cost inputs, nearly two orders of magnitude higher than yeast-based production. Cell-free systems such as this may provide a new route to reliable cannabinoid production.
Assuntos
Canabinoides/biossíntese , Sistema Livre de Células/metabolismo , Malonil Coenzima A/metabolismo , Engenharia Metabólica/métodos , Policetídeos/metabolismo , Terpenos/metabolismo , Trifosfato de Adenosina/biossíntese , Benzoatos/isolamento & purificação , Benzoatos/metabolismo , Canabinoides/isolamento & purificação , Sistema Livre de Células/química , Escherichia coli/enzimologia , Escherichia coli/genética , Expressão Gênica , Humanos , Cinética , Engenharia Metabólica/economia , Organofosfatos/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Policetídeos/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Terpenos/química , TermodinâmicaRESUMO
Cost competitive conversion of biomass-derived sugars into biofuel will require high yields, high volumetric productivities and high titers. Suitable production parameters are hard to achieve in cell-based systems because of the need to maintain life processes. As a result, next-generation biofuel production in engineered microbes has yet to match the stringent cost targets set by petroleum fuels. Removing the constraints imposed by having to maintain cell viability might facilitate improved production metrics. Here, we report a cell-free system in a bioreactor with continuous product removal that produces isobutanol from glucose at a maximum productivity of 4 g L-1 h-1, a titer of 275 g L-1 and 95% yield over the course of nearly 5 days. These production metrics exceed even the highly developed ethanol fermentation process. Our results suggest that moving beyond cells has the potential to expand what is possible for bio-based chemical production.
Assuntos
Bioquímica/métodos , Butanóis/metabolismo , Enzimas/metabolismo , Acetolactato Sintase/química , Acetolactato Sintase/metabolismo , Trifosfato de Adenosina , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Bioquímica/instrumentação , Reatores Biológicos , Sistema Livre de Células , Evolução Molecular Direcionada , Enzimas/química , Enzimas/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glucose/metabolismo , Temperatura , TermodinâmicaRESUMO
Metabolic engineering efforts that harness living organisms to produce natural products and other useful chemicals face inherent difficulties because the maintenance of life processes often runs counter to our desire to maximize important production metrics. These challenges are particularly problematic for commodity chemical manufacturing where cost is critical. A cell-free approach, where biochemical pathways are built by mixing desired enzyme activities outside of cells, can obviate problems associated with cell-based methods. Yet supplanting cell-based methods of chemical production will require the creation of self-sustaining, continuously operating systems where input biomass is converted into desired products at high yields, productivities, and titers. We call the field of designing and implementing reliable and efficient enzyme systems that replace cellular metabolism, synthetic biochemistry.
Assuntos
Bioquímica/tendências , Sistema Livre de Células , Engenharia Metabólica , Biologia Sintética/tendências , BiomassaRESUMO
In the original version of this Article, the genotype of the M30 mutant presented in Fig. 3b was given incorrectly as Y288V/A232S, and the M31 mutant was given incorrectly as M1/A232S. The correct genotype of the M30 mutant is Y288A/A232S and for M31 it is Y288V/A232S. In addition, to keep consistency in genotype formatting, the genotype of the M27 mutant should be Y288V/G286S. The errors have been corrected in both the PDF and HTML versions of the Article.
RESUMO
Prenylation of natural compounds adds structural diversity, alters biological activity, and enhances therapeutic potential. Because prenylated compounds often have a low natural abundance, alternative production methods are needed. Metabolic engineering enables natural product biosynthesis from inexpensive biomass, but is limited by the complexity of secondary metabolite pathways, intermediate and product toxicities, and substrate accessibility. Alternatively, enzyme catalyzed prenyl transfer provides excellent regio- and stereo-specificity, but requires expensive isoprenyl pyrophosphate substrates. Here we develop a flexible cell-free enzymatic prenylating system that generates isoprenyl pyrophosphate substrates from glucose to prenylate an array of natural products. The system provides an efficient route to cannabinoid precursors cannabigerolic acid (CBGA) and cannabigerovarinic acid (CBGVA) at >1 g/L, and a single enzymatic step converts the precursors into cannabidiolic acid (CBDA) and cannabidivarinic acid (CBDVA). Cell-free methods may provide a powerful alternative to metabolic engineering for chemicals that are hard to produce in living organisms.
Assuntos
Produtos Biológicos/metabolismo , Canabinoides/metabolismo , Proteínas Fúngicas/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Engenharia Metabólica/métodos , Estrutura Molecular , Prenilação/fisiologia , Especificidade por SubstratoRESUMO
Synthetic biochemistry seeks to engineer complex metabolic pathways for chemical conversions outside the constraints of the cell. Establishment of effective and flexible cell-free systems requires the development of simple systems to replace the intricate regulatory mechanisms that exist in cells for maintaining high-energy cofactor balance. Here we describe a simple rheostat that regulates ATP levels by controlling the flow down either an ATP-generating or non-ATP-generating pathway according to the free-phosphate concentration. We implemented this concept for the production of isobutanol from glucose. The rheostat maintains adequate ATP concentrations even in the presence of ATPase contamination. The final system including the rheostat produced 24.1 ± 1.8 g/L of isobutanol from glucose in 91% theoretical yield with an initial productivity of 1.3 g/L/h. The molecular rheostat concept can be used in the design of continuously operating, self-sustaining synthetic biochemistry systems.
Assuntos
Trifosfato de Adenosina/metabolismo , Butanóis/metabolismo , Engenharia Metabólica , Sistema Livre de Células , Modelos Moleculares , Transdução de SinaisRESUMO
Cell-free systems designed to perform complex chemical conversions of biomass to biofuels or commodity chemicals are emerging as promising alternatives to the metabolic engineering of living cells. Here we design a system comprises 27 enzymes for the conversion of glucose into monoterpenes that generates both NAD(P)H and ATP in a modified glucose breakdown module and utilizes both cofactors for building terpenes. Different monoterpenes are produced in our system by changing the terpene synthase enzyme. The system is stable for the production of limonene, pinene and sabinene, and can operate continuously for at least 5 days from a single addition of glucose. We obtain conversion yields >95% and titres >15 g l-1. The titres are an order of magnitude over cellular toxicity limits and thus difficult to achieve using cell-based systems. Overall, these results highlight the potential of synthetic biochemistry approaches for producing bio-based chemicals.
Assuntos
Alquil e Aril Transferases/metabolismo , Glucose/metabolismo , Monoterpenos/metabolismo , Bioquímica/métodos , Vias Biossintéticas , Sistema Livre de Células/metabolismo , Biologia Sintética/métodosRESUMO
Extreme acidophiles are capable of growth at pH values near zero. Sustaining life in acidic environments requires extensive adaptations of membranes, proton pumps, and DNA repair mechanisms. Here we describe an adaptation of a core biochemical pathway, the mevalonate pathway, in extreme acidophiles. Two previously known mevalonate pathways involve ATP dependent decarboxylation of either mevalonate 5-phosphate or mevalonate 5-pyrophosphate, in which a single enzyme carries out two essential steps: (1) phosphorylation of the mevalonate moiety at the 3-OH position and (2) subsequent decarboxylation. We now demonstrate that in extreme acidophiles, decarboxylation is carried out by two separate steps: previously identified enzymes generate mevalonate 3,5-bisphosphate and a new decarboxylase we describe here, mevalonate 3,5-bisphosphate decarboxylase, produces isopentenyl phosphate. Why use two enzymes in acidophiles when one enzyme provides both functionalities in all other organisms examined to date? We find that at low pH, the dual function enzyme, mevalonate 5-phosphate decarboxylase is unable to carry out the first phosphorylation step, yet retains its ability to perform decarboxylation. We therefore propose that extreme acidophiles had to replace the dual-purpose enzyme with two specialized enzymes to efficiently produce isoprenoids in extremely acidic environments.
Assuntos
Adaptação Biológica/fisiologia , Ácido Mevalônico/metabolismo , Thermoplasma/metabolismo , Concentração de Íons de Hidrogênio , Thermoplasma/genéticaRESUMO
Synthetic biochemistry, the cell-free production of biologically based chemicals, is a potentially high-yield, flexible alternative to in vivo metabolic engineering. To limit costs, cell-free systems must be designed to operate continuously with minimal addition of feedstock chemicals. We describe a robust, efficient synthetic glucose breakdown pathway and implement it for the production of bioplastic. The system's performance suggests that synthetic biochemistry has the potential to become a viable industrial alternative.
Assuntos
Vias Biossintéticas , Glucose/química , Glucose/metabolismo , Hidroxibutiratos/química , Hidroxibutiratos/metabolismo , Biologia Sintética/métodos , Sistema Livre de CélulasRESUMO
Most biodiesel currently in use consists of fatty acid methyl esters (FAMEs) produced by transesterification of plant oils with methanol. To reduce competition with food supplies, it would be desirable to directly produce biodiesel in microorganisms. To date, the most effective pathway for the production of biodiesel in bacteria yields fatty acid ethyl esters (FAEEs) at up to ~1.5 g/L. A much simpler route to biodiesel produces FAMEs by direct S-adenosyl-L-methionine (SAM) dependent methylation of free fatty acids, but FAME production by this route has been limited to only ~16 mg/L. Here we employ an alternative, broad spectrum methyltransferase, Drosophila melanogaster Juvenile Hormone Acid O-Methyltransferase (DmJHAMT). By introducing DmJHAMT in E. coli engineered to produce medium chain fatty acids and overproduce SAM, we obtain medium chain FAMEs at titers of 0.56 g/L, a 35-fold increase over titers previously achieved. Although considerable improvements will be needed for viable bacterial production of FAMEs and FAEEs for biofuels, it may be easier to optimize and transport the FAME production pathway to other microorganisms because it involves fewer enzymes.
Assuntos
Biocombustíveis , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Escherichia coli/metabolismo , Metiltransferases/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Ensaios Enzimáticos/métodos , Escherichia coli/genética , Ácidos Graxos/metabolismo , Microbiologia Industrial/métodos , Engenharia Metabólica/métodos , Metilação , Metiltransferases/genética , Reprodutibilidade dos Testes , S-Adenosilmetionina/metabolismoRESUMO
In animals, cholesterol is made from 5-carbon building blocks produced by the mevalonate pathway. Drugs that inhibit the mevalonate pathway such as atorvastatin (lipitor) have led to successful treatments for high cholesterol in humans. Another potential target for the inhibition of cholesterol synthesis is mevalonate diphosphate decarboxylase (MDD), which catalyzes the phosphorylation of (R)-mevalonate diphosphate, followed by decarboxylation to yield isopentenyl pyrophosphate. We recently discovered an MDD homolog, mevalonate-3-kinase (M3K) from Thermoplasma acidophilum, which catalyzes the identical phosphorylation of (R)-mevalonate, but without concomitant decarboxylation. Thus, M3K catalyzes half the reaction of the decarboxylase, allowing us to separate features of the active site that are required for decarboxylation from features required for phosphorylation. Here we determine the crystal structure of M3K in the apo form, and with bound substrates, and compare it to MDD structures. Structural and mutagenic analysis reveals modifications that allow M3K to bind mevalonate rather than mevalonate diphosphate. Comparison to homologous MDD structures show that both enzymes employ analogous Arg or Lys residues to catalyze phosphate transfer. However, an invariant active site Asp/Lys pair of MDD previously thought to play a role in phosphorylation is missing in M3K with no functional replacement. Thus, we suggest that the invariant Asp/Lys pair in MDD may be critical for decarboxylation rather than phosphorylation.
Assuntos
Carboxiliases/química , Fosfotransferases (Aceptor do Grupo Álcool)/química , Terpenos/metabolismo , Thermoplasma/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Carboxiliases/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Ácido Mevalônico/análogos & derivados , Ácido Mevalônico/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Alinhamento de Sequência , Thermoplasma/químicaRESUMO
The greatest potential environmental benefit of metabolic engineering would be the production of high-volume commodity chemicals, such as biofuels. Yet, the high yields required for the economic viability of low-value chemicals is particularly hard to achieve in microbes owing to the myriad competing biochemical pathways. An alternative approach, which we call synthetic biochemistry, is to eliminate the organism by constructing biochemical pathways in vitro. Viable synthetic biochemistry, however, will require simple methods to replace the cellular circuitry that maintains cofactor balance. Here we design a simple purge valve module for maintaining NADP(+)/NADPH balance. We test the purge valve in the production of polyhydroxybutyryl bioplastic and isoprene--pathways where cofactor generation and utilization are unbalanced. We find that the regulatory system is highly robust to variations in cofactor levels and readily transportable. The molecular purge valve provides a step towards developing continuously operating, sustainable synthetic biochemistry systems.
Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Butadienos/metabolismo , Genes Sintéticos , Hemiterpenos/metabolismo , Engenharia Metabólica , NADP/genética , NADP/metabolismo , Oxirredução , Pentanos/metabolismo , Biologia SintéticaRESUMO
Microbial fatty acids are an attractive source of precursors for a variety of renewable commodity chemicals such as alkanes, alcohols, and biofuels. Rerouting lipid biosynthesis into free fatty acid production can be toxic, however, due to alterations of membrane lipid composition. Here we find that membrane lipid composition can be altered by the direct incorporation of medium-chain fatty acids into lipids via the Aas pathway in cells expressing the medium-chain thioesterase from Umbellularia californica (BTE). We find that deletion of the aas gene and sequestering exported fatty acids reduces medium-chain fatty acid toxicity, partially restores normal lipid composition, and improves medium-chain fatty acid yields.
Assuntos
Escherichia coli/fisiologia , Ácidos Graxos/biossíntese , Melhoramento Genético/métodos , Engenharia Metabólica/métodos , Palmitoil-CoA Hidrolase/genética , Umbellularia/enzimologia , Clonagem Molecular/métodos , Ácidos Graxos/genética , Deleção de Genes , Palmitoil-CoA Hidrolase/metabolismo , Umbellularia/genéticaRESUMO
Isoprenoids make up a remarkably diverse class of more than 25000 biomolecules that include familiar compounds such as cholesterol, chlorophyll, vitamin A, ubiquinone, and natural rubber. The two essential building blocks of all isoprenoids, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), are ubiquitous in the three domains of life. In most eukaryotes and archaea, IPP and DMAPP are generated through the mevalonate pathway. We have identified two novel enzymes, mevalonate-3-kinase and mevalonate-3-phosphate-5-kinase from Thermoplasma acidophilum, which act sequentially in a putative alternate mevalonate pathway. We propose that a yet unidentified ATP-independent decarboxylase acts upon mevalonate 3,5-bisphosphate, yielding isopentenyl phosphate, which is subsequently phosphorylated by the known isopentenyl phosphate kinase from T. acidophilum to generate the universal isoprenoid precursor, IPP.
Assuntos
Ácido Mevalônico/análogos & derivados , Organofosfatos/metabolismo , Fosfotransferases/metabolismo , Thermoplasma/metabolismo , Ácido Mevalônico/metabolismo , Fosforilação , Fosfotransferases/genéticaRESUMO
The high yields required for the economical production of chemicals and fuels using microbes can be difficult to achieve due to the complexities of cellular metabolism. An alternative to performing biochemical transformations in microbes is to build biochemical pathways in vitro, an approach we call synthetic biochemistry. Here we test whether the full mevalonate pathway can be reconstituted in vitro and used to produce the commodity chemical isoprene. We construct an in vitro synthetic biochemical pathway that uses the carbon and ATP produced from the glycolysis intermediate phosphoenolpyruvate to run the mevalonate pathway. The system involves 12 enzymes to perform the complex transformation, while providing and balancing the ATP, NADPH, and acetyl-CoA cofactors. The optimized system produces isoprene from phosphoenolpyruvate in â¼100% molar yield. Thus, by inserting the isoprene pathway into previously developed glycolysis modules it may be possible to produce isoprene and other acetyl-CoA derived isoprenoids from glucose in vitro.
Assuntos
Vias Biossintéticas , Butadienos/metabolismo , Escherichia coli/metabolismo , Glicólise , Hemiterpenos/metabolismo , Engenharia Metabólica/métodos , Pentanos/metabolismo , Acetilcoenzima A/metabolismo , Trifosfato de Adenosina/metabolismo , Escherichia coli/enzimologia , Química Verde , Ácido Mevalônico/metabolismo , NADP/metabolismo , Fosfoenolpiruvato/metabolismo , Ácido Pirúvico/metabolismoRESUMO
In the actinorhodin type II polyketide synthase, the first polyketide modification is a regiospecific C9-carbonyl reduction, catalyzed by the ketoreductase (actKR). Our previous studies identified the actKR 94-PGG-96 motif as a determinant of stereospecificity. The molecular basis for reduction regiospecificity is, however, not well understood. In this study, we examined the activities of 20 actKR mutants through a combination of kinetic studies, PKS reconstitution, and structural analyses. Residues have been identified that are necessary for substrate interaction, and these observations have suggested a structural model for this reaction. Polyketides dock at the KR surface and are steered into the enzyme pocket where C7-C12 cyclization is mediated by the KR before C9-ketoreduction can occur. These molecular features can potentially serve as engineering targets for the biosynthesis of novel, reduced polyketides.
Assuntos
Oxirredutases do Álcool/química , Oxirredutases do Álcool/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteína de Transporte de Acila/metabolismo , Oxirredutases do Álcool/genética , Antraquinonas/química , Antraquinonas/metabolismo , Proteínas de Bactérias/genética , Domínio Catalítico , Ciclização , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Mutação , NADP/metabolismo , Oxirredução , Policetídeos/metabolismo , Estereoisomerismo , Especificidade por Substrato , Tetralonas/metabolismoRESUMO
BACKGROUND: Biodiesels are methyl esters of fatty acids that are usually produced by base catalyzed transesterification of triacylglyerol with methanol. Some lipase enzymes are effective catalysts for biodiesel synthesis and have many potential advantages over traditional base or acid catalyzed transesterification. Natural lipases are often rapidly inactivated by the high methanol concentrations used for biodiesel synthesis, however, limiting their practical use. The lipase from Proteus mirabilis is a particularly promising catalyst for biodiesel synthesis as it produces high yields of methyl esters even in the presence of large amounts of water and expresses very well in Escherichia coli. However, since the Proteus mirabilis lipase is only moderately stable and methanol tolerant, these properties need to be improved before the enzyme can be used industrially. RESULTS: We employed directed evolution, resulting in a Proteus mirabilis lipase variant with 13 mutations, which we call Dieselzyme 4. Dieselzyme 4 has greatly improved thermal stability, with a 30-fold increase in the half-inactivation time at 50°C relative to the wild-type enzyme. The evolved enzyme also has dramatically increased methanol tolerance, showing a 50-fold longer half-inactivation time in 50% aqueous methanol. The immobilized Dieselzyme 4 enzyme retains the ability to synthesize biodiesel and has improved longevity over wild-type or the industrially used Brukholderia cepacia lipase during many cycles of biodiesel synthesis. A crystal structure of Dieselzyme 4 reveals additional hydrogen bonds and salt bridges in Dieselzyme 4 compared to the wild-type enzyme, suggesting that polar interactions may become particularly stabilizing in the reduced dielectric environment of the oil and methanol mixture used for biodiesel synthesis. CONCLUSIONS: Directed evolution was used to produce a stable lipase, Dieselzyme 4, which could be immobilized and re-used for biodiesel synthesis. Dieselzyme 4 outperforms the industrially used lipase from Burkholderia cepacia and provides a platform for still further evolution of desirable biodiesel production properties.
RESUMO
Bacterial lipases from family I.1 and I.2 catalyze the hydrolysis of triacylglycerol between 25-45°C and are used extensively as biocatalysts. The lipase from Proteus mirabilis belongs to the Proteus/psychrophilic subfamily of lipase family I.1 and is a promising catalyst for biodiesel production because it can tolerate high amounts of water in the reaction. Here we present the crystal structure of the Proteus mirabilis lipase, a member of the Proteus/psychrophilic subfamily of I.1lipases. The structure of the Proteus mirabilis lipase was solved in the absence and presence of a bound phosphonate inhibitor. Unexpectedly, both the apo and inhibitor bound forms of P. mirabilis lipase were found to be in a closed conformation. The structure reveals a unique oxyanion hole and a wide active site that is solvent accessible even in the closed conformation. A distinct mechanism for Ca²âº coordination may explain how these lipases can fold without specific chaperones.
Assuntos
Proteínas de Bactérias/química , Lipase/química , Proteus mirabilis/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/antagonistas & inibidores , Biocatálise , Cálcio/química , Domínio Catalítico , Cristalografia por Raios X , Inibidores Enzimáticos/química , Ligação de Hidrogênio , Lipase/antagonistas & inibidores , Modelos Moleculares , Dados de Sequência Molecular , Organofosfonatos/química , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Solventes/química , Água/químicaRESUMO
This paper describes the X-ray crystallographic structure of a designed cyclic beta-sheet peptide that forms a well-defined hydrogen-bonded dimer that mimics beta-sheet dimers formed by proteins. The 54-membered ring macrocyclic peptide (1a) contains molecular template and turn units that induce beta-sheet structure in a heptapeptide strand that forms the dimerization interface. The X-ray crystallographic structure reveals the structures of the two "Hao" amino acids that help template the beta-sheet structure and the two delta-linked ornithine turn units that link the Hao-containing template to the heptapeptide beta-strand. The Hao amino acids adopt a conformation that resembles a tripeptide in a beta-strand conformation, with one edge of the Hao unit presenting an alternating array of hydrogen-bond donor and acceptor groups in the same pattern as that of a tripeptide beta-strand. The delta-linked ornithines adopt a conformation that resembles a hydrogen-bonded beta-turn, in which the ornithine takes the place of the i+1 and i+2 residues. The dimers formed by macrocyclic beta-sheet 1a resemble the dimers of many proteins, such as defensin HNP-3, the lambda-Cro repressor, interleukin 8, and the ribonuclease H domain of HIV-1 reverse transcriptase. The dimers of 1a self-assemble in the solid state into a barrel-shaped trimer of dimers in which the three dimers are arranged in a triangular fashion. Molecular modeling in which one of the three dimers is removed and the remaining two dimers are aligned face-to-face provides a model of the dimers of dimers of closely related macrocyclic beta-sheet peptides that were observed in solution.
Assuntos
Peptídeos/química , Proteínas/química , Cristalografia por Raios X , Dimerização , Ligação de Hidrogênio , Modelos Moleculares , Conformação ProteicaRESUMO
Polyketides are a class of natural products with diverse structures and biological activities. The structural variability of aromatic products of fungal nonreducing, multidomain iterative polyketide synthases (NR-PKS group of IPKSs) results from regiospecific cyclizations of reactive poly-beta-keto intermediates. How poly-beta-keto species are synthesized and stabilized, how their chain lengths are determined, and, in particular, how specific cyclization patterns are controlled have been largely inaccessible and functionally unknown until recently. A product template (PT) domain is responsible for controlling specific aldol cyclization and aromatization of these mature polyketide precursors, but the mechanistic basis is unknown. Here we present the 1.8 A crystal structure and mutational studies of a dissected PT monodomain from PksA, the NR-PKS that initiates the biosynthesis of the potent hepatocarcinogen aflatoxin B(1) in Aspergillus parasiticus. Despite having minimal sequence similarity to known enzymes, the structure displays a distinct 'double hot dog' (DHD) fold. Co-crystal structures with palmitate or a bicyclic substrate mimic illustrate that PT can bind both linear and bicyclic polyketides. Docking and mutagenesis studies reveal residues important for substrate binding and catalysis, and identify a phosphopantetheine localization channel and a deep two-part interior binding pocket and reaction chamber. Sequence similarity and extensive conservation of active site residues in PT domains suggest that the mechanistic insights gleaned from these studies will prove general for this class of IPKSs, and lay a foundation for defining the molecular rules controlling NR-PKS cyclization specificity.