RESUMO
Engineering polyketide synthases (PKS) to produce new metabolites requires an understanding of catalytic points of failure during substrate processing. Growing evidence indicates the thioesterase (TE) domain as a significant bottleneck within engineered PKS systems. We created a series of hybrid PKS modules bearing exchanged TE domains from heterologous pathways and challenged them with both native and non-native polyketide substrates. Reactions pairing wildtype PKS modules with non-native substrates primarily resulted in poor conversions to anticipated macrolactones. Likewise, product formation with native substrates and hybrid PKS modules bearing non-cognate TE domains was severely reduced. In contrast, non-native substrates were converted by most hybrid modules containing a substrate compatible TE, directly implicating this domain as the major catalytic gatekeeper and highlighting its value as a target for protein engineering to improve analog production in PKS pathways.
Assuntos
Policetídeo Sintases/química , Biocatálise , Macrolídeos/síntese química , Policetídeo Sintases/genética , Domínios Proteicos , Engenharia de Proteínas , Especificidade por SubstratoRESUMO
A potent class of DNA-damaging agents, natural product bis-intercalator depsipeptides (NPBIDs), was evaluated as ultrapotent payloads for use in antibody-drug conjugates (ADCs). Detailed investigation of potency (both in cells and via biophysical characterization of DNA binding), chemical tractability, and in vitro and in vivo stability of the compounds in this class eliminated a number of potential candidates, greatly reducing the complexity and resources required for conjugate preparation and evaluation. This effort yielded a potent, stable, and efficacious ADC, PF-06888667, consisting of the bis-intercalator, SW-163D, conjugated via an N-acetyl-lysine-valine-citrulline- p-aminobenzyl alcohol- N, N-dimethylethylenediamine (AcLysValCit-PABC-DMAE) linker to an engineered variant of the anti-Her2 mAb, trastuzumab, catalyzed by transglutaminase.
Assuntos
Produtos Biológicos/química , Depsipeptídeos/química , Imunoconjugados/química , Substâncias Intercalantes/química , Animais , Antineoplásicos Imunológicos/química , Linhagem Celular Tumoral , DNA/química , Depsipeptídeos/sangue , Depsipeptídeos/farmacocinética , Equinomicina/química , Genes erbB-2 , Meia-Vida , Xenoenxertos , Humanos , Camundongos , Trastuzumab/químicaRESUMO
The human DNA repair enzyme MUTYH excises mispaired adenine residues in oxidized DNA. Homozygous MUTYH mutations underlie the autosomal, recessive cancer syndrome MUTYH-associated polyposis. We report a MUTYH variant, p.C306W (c.918C>G), with a tryptophan residue in place of native cysteine, that ligates the [4Fe4S] cluster in a patient with colonic polyposis and family history of early age colon cancer. In bacterial MutY, the [4Fe4S] cluster is redox active, allowing rapid localization to target lesions by long-range, DNA-mediated signalling. In the current study, using DNA electrochemistry, we determine that wild-type MUTYH is similarly redox-active, but MUTYH C306W undergoes rapid oxidative degradation of its cluster to [3Fe4S]+, with loss of redox signalling. In MUTYH C306W, oxidative cluster degradation leads to decreased DNA binding and enzyme function. This study confirms redox activity in eukaryotic DNA repair proteins and establishes MUTYH C306W as a pathogenic variant, highlighting the essential role of redox signalling by the [4Fe4S] cluster.
Assuntos
Polipose Adenomatosa do Colo/metabolismo , Neoplasias do Colo/metabolismo , DNA Glicosilases/metabolismo , Proteínas Ferro-Enxofre/metabolismo , DNA Glicosilases/genética , Variação Genética/genética , Humanos , Mutação , OxirreduçãoRESUMO
Polyketide synthases (PKSs) represent a powerful catalytic platform capable of effecting multiple carbon-carbon bond forming reactions and oxidation state adjustments. We explored the functionality of two terminal PKS modules that produce the 16-membered tylosin macrocycle, using them as biocatalysts in the chemoenzymatic synthesis of tylactone and its subsequent elaboration to complete the first total synthesis of the juvenimicin, M-4365, and rosamicin classes of macrolide antibiotics via late-stage diversification. Synthetic chemistry was employed to generate the tylactone hexaketide chain elongation intermediate that was accepted by the juvenimicin (Juv) ketosynthase of the penultimate JuvEIV PKS module. The hexaketide is processed through two complete modules (JuvEIV and JuvEV) in vitro, which catalyze elongation and functionalization of two ketide units followed by cyclization of the resulting octaketide into tylactone. After macrolactonization, a combination of in vivo glycosylation, selective in vitro cytochrome P450-mediated oxidation, and chemical oxidation was used to complete the scalable construction of a series of macrolide natural products in as few as 15 linear steps (21 total) with an overall yield of 4.6%.
Assuntos
Antibacterianos/biossíntese , Macrolídeos/metabolismo , Policetídeo Sintases/metabolismo , Policetídeos/metabolismo , Tilosina/análogos & derivados , Antibacterianos/química , Antibacterianos/farmacologia , Biocatálise , Relação Dose-Resposta a Droga , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Macrolídeos/química , Macrolídeos/farmacologia , Testes de Sensibilidade Microbiana , Conformação Molecular , Policetídeo Sintases/química , Policetídeos/química , Policetídeos/farmacologia , Relação Estrutura-Atividade , Tilosina/biossíntese , Tilosina/química , Tilosina/farmacologiaRESUMO
Acyltransferase (AT) domains of polyketide synthases (PKSs) select extender units for incorporation into polyketides and dictate large portions of the structures of clinically relevant natural products. Accordingly, there is significant interest in engineering the substrate specificity of PKS ATs in order to site-selectively manipulate polyketide structure. However, previous attempts to engineer ATs have yielded mutant PKSs with relaxed extender unit specificity, rather than an inversion of selectivity from one substrate to another. Here, by directly screening the extender unit selectivity of mutants from active site saturation libraries of an AT from the prototypical PKS, 6-deoxyerythronolide B synthase, a set of single amino acid substitutions was discovered that dramatically impact the selectivity of the PKS with only modest reductions of product yields. One particular substitution (Tyr189Arg) inverted the selectivity of the wild-type PKS from its natural substrate toward a non-natural alkynyl-modified extender unit while maintaining more than twice the activity of the wild-type PKS with its natural substrate. The strategy and mutations described herein form a platform for combinatorial biosynthesis of site-selectively modified polyketide analogues that are modified with non-natural and non-native chemical functionality.
Assuntos
Aciltransferases/metabolismo , Eritromicina/metabolismo , Mutagênese Sítio-Dirigida , Policetídeo Sintases/metabolismo , Policetídeos/metabolismo , Saccharopolyspora/enzimologia , Aciltransferases/química , Aciltransferases/genética , Eritromicina/química , Macrolídeos/química , Macrolídeos/metabolismo , Mutagênese Sítio-Dirigida/métodos , Mutação Puntual , Policetídeo Sintases/química , Policetídeo Sintases/genética , Policetídeos/química , Domínios Proteicos , Saccharopolyspora/genética , Saccharopolyspora/metabolismo , Especificidade por SubstratoRESUMO
The structural scaffolds of many complex natural products are produced by multifunctional type I polyketide synthase (PKS) enzymes that operate as biosynthetic assembly lines. The modular nature of these mega-enzymes presents an opportunity to construct custom biocatalysts built in a lego-like fashion by inserting, deleting, or exchanging native or foreign domains to produce targeted variants of natural polyketides. However, previously engineered PKS enzymes are often impaired resulting in limited production compared to native systems. Here, we show a versatile method for generating and identifying functional chimeric PKS enzymes for synthesizing custom macrolactones and macrolides. PKS genes from the pikromycin and erythromycin pathways were hybridized in Saccharomyces cerevisiae to generate hybrid libraries. We used a 96-well plate format for plasmid purification, transformations, sequencing, protein expression, in vitro reactions and analysis of metabolite formation. Active chimeric enzymes were identified with new functionality. Streptomyces venezuelae strains that expressed these PKS chimeras were capable of producing engineered macrolactones. Furthermore, a macrolactone generated from selected PKS chimeras was fully functionalized into a novel macrolide analogue. This method permits the engineering of PKS pathways as modular building blocks for the production of new antibiotic-like molecules.
Assuntos
Evolução Molecular , Recombinação Homóloga , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Eritromicina/metabolismo , Escherichia coli/genética , Macrolídeos/metabolismo , Engenharia de Proteínas , Saccharomyces cerevisiae/genética , Streptomyces/metabolismoRESUMO
Polyketide natural products constitute a broad class of compounds with diverse structural features and biological activities. Their biosynthetic machinery, represented by type I polyketide synthases (PKSs), has an architecture in which successive modules catalyse two-carbon linear extensions and keto-group processing reactions on intermediates covalently tethered to carrier domains. Here we used electron cryo-microscopy to determine sub-nanometre-resolution three-dimensional reconstructions of a full-length PKS module from the bacterium Streptomyces venezuelae that revealed an unexpectedly different architecture compared to the homologous dimeric mammalian fatty acid synthase. A single reaction chamber provides access to all catalytic sites for the intramodule carrier domain. In contrast, the carrier from the preceding module uses a separate entrance outside the reaction chamber to deliver the upstream polyketide intermediate for subsequent extension and modification. This study reveals for the first time, to our knowledge, the structural basis for both intramodule and intermodule substrate transfer in polyketide synthases, and establishes a new model for molecular dissection of these multifunctional enzyme systems.
Assuntos
Policetídeo Sintases/química , Policetídeo Sintases/ultraestrutura , Streptomyces/enzimologia , Biocatálise , Domínio Catalítico , Microscopia Crioeletrônica , Ácido Graxo Sintases/química , Macrolídeos/metabolismo , Modelos Moleculares , Policetídeo Sintases/metabolismoRESUMO
The polyketide synthase (PKS) mega-enzyme assembly line uses a modular architecture to synthesize diverse and bioactive natural products that often constitute the core structures or complete chemical entities for many clinically approved therapeutic agents. The architecture of a full-length PKS module from the pikromycin pathway of Streptomyces venezuelae creates a reaction chamber for the intramodule acyl carrier protein (ACP) domain that carries building blocks and intermediates between acyltransferase, ketosynthase and ketoreductase active sites (see accompanying paper). Here we determine electron cryo-microscopy structures of a full-length pikromycin PKS module in three key biochemical states of its catalytic cycle. Each biochemical state was confirmed by bottom-up liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry. The ACP domain is differentially and precisely positioned after polyketide chain substrate loading on the active site of the ketosynthase, after extension to the ß-keto intermediate, and after ß-hydroxy product generation. The structures reveal the ACP dynamics for sequential interactions with catalytic domains within the reaction chamber, and for transferring the elongated and processed polyketide substrate to the next module in the PKS pathway. During the enzymatic cycle the ketoreductase domain undergoes dramatic conformational rearrangements that enable optimal positioning for reductive processing of the ACP-bound polyketide chain elongation intermediate. These findings have crucial implications for the design of functional PKS modules, and for the engineering of pathways to generate pharmacologically relevant molecules.
Assuntos
Biocatálise , Policetídeo Sintases/química , Policetídeo Sintases/metabolismo , Streptomyces/enzimologia , Proteína de Transporte de Acila/química , Proteína de Transporte de Acila/metabolismo , Proteína de Transporte de Acila/ultraestrutura , Aciltransferases/química , Aciltransferases/metabolismo , Aciltransferases/ultraestrutura , Oxirredutases do Álcool/química , Oxirredutases do Álcool/metabolismo , Oxirredutases do Álcool/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Domínio Catalítico , Microscopia Crioeletrônica , Macrolídeos/metabolismo , Modelos Moleculares , Policetídeo Sintases/ultraestrutura , Estrutura Terciária de ProteínaRESUMO
Germicidin synthase (Gcs) from Streptomyces coelicolor is a type III polyketide synthase (PKS) with broad substrate flexibility for acyl groups linked through a thioester bond to either coenzyme A (CoA) or acyl carrier protein (ACP). Germicidin synthesis was reconstituted in vitro by coupling Gcs with fatty acid biosynthesis. Since Gcs has broad substrate flexibility, we directly compared the kinetic properties of Gcs with both acyl-ACP and acyl-CoA. The catalytic efficiency of Gcs for acyl-ACP was 10-fold higher than for acyl-CoA, suggesting a strong preference toward carrier protein starter unit transfer. The 2.9 Å germicidin synthase crystal structure revealed canonical type III PKS architecture along with an unusual helical bundle of unknown function that appears to extend the dimerization interface. A pair of arginine residues adjacent to the active site affect catalytic activity but not ACP binding. This investigation provides new and surprising information about the interactions between type III PKSs and ACPs that will facilitate the construction of engineered systems for production of novel polyketides.
Assuntos
Proteína de Transporte de Acila/metabolismo , Proteínas de Bactérias/metabolismo , Policetídeo Sintases/metabolismo , Pironas/metabolismo , Streptomyces coelicolor/enzimologia , Acil Coenzima A/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cristalografia por Raios X , Modelos Moleculares , Mutação , Policetídeo Sintases/química , Policetídeo Sintases/genética , Ligação Proteica , Transdução de Sinais , Streptomyces coelicolor/química , Streptomyces coelicolor/genética , Especificidade por SubstratoRESUMO
Polyketide natural products generated by type I modular polyketide synthases (PKSs) are vital components in our drug repertoire. To reprogram these biosynthetic assembly lines, we must first understand the steps that occur within the modular "black boxes." Herein, key steps of acyl-CoA extender unit selection are explored by in vitro biochemical analysis of the PikAIV PKS model system. Two complementary approaches are employed: a fluorescent-probe assay for steady-state kinetic analysis, and Fourier Transform Ion Cyclotron Resonance-mass spectrometry (FTICR-MS) to monitor active-site occupancy. Findings from five enzyme variants and four model substrates have enabled a model to be proposed involving catalysis based upon acyl-CoA substrate loading followed by differential rates of hydrolysis. These efforts suggest a strategy for future pathway engineering efforts using unnatural extender units with slow rates of hydrolytic off-loading from the acyltransferase domain.
Assuntos
Acil Coenzima A/química , Antibacterianos/biossíntese , Análise de Fourier , Macrolídeos/metabolismo , Espectrometria de Massas , Policetídeo Sintases/química , Acil Coenzima A/metabolismo , Antibacterianos/química , Domínio Catalítico , Hidrólise , Cinética , Macrolídeos/química , Mutação , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por SubstratoRESUMO
Isoflavonoids possess enormous potential for human health with potential impact on heart disease and cancer, and some display striking affinities for steroid receptors. Synthesized primarily by legumes, isoflavonoids are present in low and variable abundance within complex mixtures, complicating efforts to assess their clinical potential. To satisfy the need for controlled, efficient, and flexible biosynthesis of isoflavonoids, a three-enzyme system has been constructed in yeast that can convert natural and synthetic flavanones into their corresponding isoflavones in practical quantities. Based on the determination of the substrate requirements of isoflavone synthase, a series of natural and nonnatural isoflavones were prepared and their binding affinities for the human estrogen receptors (ER alpha and ER beta) were determined. Structure activity relationships are suggested based on changes to binding affinities related to small variations on the isoflavone structure.
Assuntos
Biotecnologia/métodos , Flavanonas/metabolismo , Flavonoides/metabolismo , Isoflavonas/metabolismo , Fenóis/metabolismo , Receptores de Estrogênio/metabolismo , Saccharomyces cerevisiae/metabolismo , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Humanos , Isoflavonas/química , Estrutura Molecular , Polifenóis , Ligação ProteicaRESUMO
With microbial production becoming the primary choice for natural product synthesis, increasing precursor and cofactor availability has become a chief hurdle for the generation of efficient production platforms. As such, we employed a stoichiometric-based model to identify combinations of gene knockouts for improving NADPH availability in Escherichia coli. Specifically, two different model objectives were used to identify possible genotypes that exhibited either improved overall NADPH production or an improved flux through an artificial reaction coupling NADPH yield to biomass. The top single, double and triple gene deletion candidates were constructed and as a case study evaluated for their ability to produce two polyphenols, leucocyanidin and (+)-catechin. Each is derived from their common precursor dihydroquercetin using two recombinant NADPH-dependent enzymes: dihydroflavonol 4-reductase and leucoanthocyanidin reductase. The best engineered strain carrying Delta pgi, Delta ppc and Delta pldA deletions accumulated up to 817 mg/L of leucocyanidin and 39 mg/L (+)-catechin in batch culture with 10 g/L glucose in modified M9 medium, a 4-fold and 2-fold increase, respectively, compared to the wild-type control.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , NADP/biossíntese , Engenharia de Proteínas , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Modelos BiológicosRESUMO
Plant natural products (NPs) not only serve many functions in an organism's survivability but also demonstrate important pharmacological activities. Isolation of NPs from native sources is frequently limited by low abundance and environmental, seasonal, and regional variation while total chemical synthesis of what are often complex structures is typically commercially infeasible. Reconstruction of biosynthetic pathways in heterologous microorganisms offers significant promise for a scalable means to provide sufficient quantities of a desired NP while using inexpensive renewable resources. To this end, metabolic engineering provides the technological platform for enhancing NP production in these engineered heterologous hosts. Recent advancements in the production of isoprenoids, phenylpropanoids, and alkaloids were made possible by utilizing a variety of techniques including combinatorial biosynthesis, codon optimization, expression of regulatory elements, and protein engineering of P450s.
Assuntos
Produtos Biológicos/biossíntese , Biotecnologia , Plantas/metabolismoRESUMO
Flavan-3-ols, such as green tea catechins represent a major group of phenolic compounds with significant medicinal properties. We describe the construction and optimization of Escherichia coli recombinant strains for the production of mono- and dihydroxylated catechins from their flavanone and phenylpropanoid acid precursors. Use of glucose minimal medium, Fe(II), and control of oxygen availability during shake-flask experiments resulted in production yield increases. Additional production improvement resulted from the use of medium rather than high-copy number plasmids and, in the case of mono-hydroxylated compounds, the addition of extracellular cofactors in the culture medium. The established metabolic engineering approach allowed the biosynthesis of natural catechins at high purity for assessing their possible insulinotropic effects in pancreatic beta-cell cultures. We demonstrated that (+)-afzelechin and (+)-catechin modulated the secretion of insulin by pancreatic beta-cells. These results indicate the potential of applying metabolic engineering approaches for the synthesis of natural and non-natural catechin analogues as drug candidates in diabetes treatments.
Assuntos
Flavonoides/metabolismo , Engenharia Genética , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Benzopiranos/metabolismo , Catequina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glucose , Insulina/biossíntese , Secreção de Insulina , Células Secretoras de Insulina/enzimologia , Fenóis/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
Flavonoids are plant secondary metabolites often used as nutraceutical supplements, but a growing number of unnatural flavonoids are being investigated as therapeutic agents. Cultures of Saccharomyces cerevisiae expressing recombinant flavonoid enzymes, including 4-coumaroyl:CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), and flavanone 3beta-hydroxylase (FHT), produced novel flavanones and dihydroflavonols when fed with a number of aromatic acrylic acids. The flavonoid network also exhibited broad substrate specificity by converting muconic acid into a unique polypropanoid.
Assuntos
Acrilatos/química , Acrilatos/metabolismo , Flavonoides/biossíntese , Flavonoides/química , Saccharomyces cerevisiae/metabolismo , Hidrogênio/química , Estrutura MolecularRESUMO
Industrial biotechnology employs the controlled use of microorganisms for the production of synthetic chemicals or simple biomass that can further be used in a diverse array of applications that span the pharmaceutical, chemical and nutraceutical industries. Recent advances in metagenomics and in the incorporation of entire biosynthetic pathways into Saccharomyces cerevisiae have greatly expanded both the fitness and the repertoire of biochemicals that can be synthesized from this popular microorganism. Further, the availability of the S. cerevisiae entire genome sequence allows the application of systems biology approaches for improving its enormous biosynthetic potential. In this review, we will describe some of the efforts on using S. cerevisiae as a cell factory for the biosynthesis of high-value natural products that belong to the families of isoprenoids, flavonoids and long chain polyunsaturated fatty acids. As natural products are increasingly becoming the center of attention of the pharmaceutical and nutraceutical industries, the use of S. cerevisiae for their production is only expected to expand in the future, further allowing the biosynthesis of novel molecular structures with unique properties.
RESUMO
Flavones are plant secondary metabolites with potent pharmacological properties. We report the functional expression of FSI, a flavonoid 2-oxoglutarate-dependent dioxygenase-encoding flavone synthase from parsley in Escherichia coli. This expression allows the biosynthesis of various flavones from phenylpropanoid acids in recombinant E. coli strains simultaneously expressing five plant-specific flavone biosynthetic genes. The gene ensemble consists of 4CL-2 (4-coumarate:CoA ligase) and FSI (flavone synthase I) from parsley, chsA (chalcone synthase) and chiA (chalcone isomerase) from Petunia hybrida, and OMT1A (7-O-methyltransferase) from peppermint. After a 24-h cultivation, the recombinant E. coli produces significant amounts of apigenin (415 microg/l), luteolin (10 microg/l), and genkwanin (208 microg/l). The majority of the flavone products are excreted in the culture media; however, 25% is contained within the cells. The metabolic engineering strategy presented demonstrates that plant-specific flavones are successfully produced in E. coli for the first time by incorporating a soluble flavone synthase confined only in Apiaceae.
Assuntos
Escherichia coli/metabolismo , Expressão Gênica , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Fitoestrógenos/metabolismo , Escherichia coli/genética , Fermentação , Flavonas/química , Flavonas/metabolismo , Engenharia Genética , Estrutura Molecular , Petroselinum/enzimologia , Petroselinum/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , SolubilidadeRESUMO
Anthocyanins are red, purple, or blue plant pigments that belong to the family of polyphenolic compounds collectively called flavonoids. Their demonstrated antioxidant properties and economic importance to the dye, fruit, and cut-flower industries have driven intensive research into their metabolic biosynthetic pathways. In order to produce stable, glycosylated anthocyanins from colorless flavanones such as naringenin and eriodictyol, a four-step metabolic pathway was constructed that contained plant genes from heterologous origins: flavanone 3beta-hydroxylase from Malus domestica, dihydroflavonol 4-reductase from Anthurium andraeanum, anthocyanidin synthase (ANS) also from M. domestica, and UDP-glucose:flavonoid 3-O-glucosyltransferase from Petunia hybrida. Using two rounds of PCR, each one of the four genes was first placed under the control of the trc promoter and its own bacterial ribosome-binding site and then cloned sequentially into vector pK184. Escherichia coli cells containing the recombinant plant pathway were able to take up either naringenin or eriodictyol and convert it to the corresponding glycosylated anthocyanin, pelargonidin 3-O-glucoside or cyanidin 3-O-glucoside. The produced anthocyanins were present at low concentrations, while most of the metabolites detected corresponded to their dihydroflavonol precursors, as well as the corresponding flavonols. The presence of side product flavonols is at least partly due to an alternate reaction catalyzed by ANS. This is the first time plant-specific anthocyanins have been produced from a microorganism and opens up the possibility of further production improvement by protein and pathway engineering.