RESUMO
Strychnine is a natural product that, through isolation, structural elucidation and synthetic efforts, shaped the field of organic chemistry. Currently, strychnine is used as a pesticide to control rodents1 because of its potent neurotoxicity2,3. The polycyclic architecture of strychnine has inspired chemists to develop new synthetic transformations and strategies to access this molecular scaffold4, yet it is still unknown how plants create this complex structure. Here we report the biosynthetic pathway of strychnine, along with the related molecules brucine and diaboline. Moreover, we successfully recapitulate strychnine, brucine and diaboline biosynthesis in Nicotiana benthamiana from an upstream intermediate, thus demonstrating that this complex, pharmacologically active class of compounds can now be harnessed through metabolic engineering approaches.
Assuntos
Vias Biossintéticas , Engenharia Metabólica , Estricnina , Vias Biossintéticas/genética , Estricnina/análogos & derivados , Estricnina/biossíntese , Estricnina/química , Nicotiana/química , Nicotiana/genética , Nicotiana/metabolismoRESUMO
1-octanol is a valuable molecule in the chemical industry, where it is used as a plasticizer, as a precursor in the production of linear low-density polyethylene (LLDPE), and as a growth inhibitor of tobacco plant suckers. Due to the low availability of eight-carbon acyl chains in natural lipid feedstocks and the selectivity challenges in petrochemical routes to medium-chain fatty alcohols,1-octanol sells for the highest price among the fatty alcohol products. As an alternative, metabolic engineers have pursued sustainable 1-octanol production via engineered microbes. Here, we report demonstration of gram per liter titers in the model bacterium Escherichia coli via the development of a pathway composed of a thioesterase, an acyl-CoA synthetase, and an acyl-CoA reductase. In addition, the impact of deleting fermentative pathways was explored E. coli K12 MG1655 strain for production of octanoic acid, a key octanol precursor. In order to overcome metabolic flux barriers, bioprospecting experiments were performed to identify acyl-CoA synthetases with high activity towards octanoic acid and acyl-CoA reductases with high activity to produce 1-octanol from octanoyl-CoA. Titration of expression of key pathway enzymes was performed and a strain with the full pathway integrated on the chromosome was created. The final strain produced 1-octanol at 1.3â¯g/L titer and a >90% C8 specificity from glycerol. In addition to the metabolic engineering efforts, this work addressed some of the technical challenges that arise when quantifying 1-octanol produced from cultures grown under fully aerobic conditions where evaporation and stripping are prevalent.
Assuntos
1-Octanol/metabolismo , Escherichia coli K12 , Tioléster Hidrolases , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Tioléster Hidrolases/genética , Tioléster Hidrolases/metabolismoRESUMO
Medium-chain length methyl ketones are potential blending fuels due to their cetane numbers and low melting temperatures. Biomanufacturing offers the potential to produce these molecules from renewable resources such as lignocellulosic biomass. In this work, we designed and tested metabolic pathways in Escherichia coli to specifically produce 2-heptanone, 2-nonanone and 2-undecanone. We achieved substantial production of each ketone by introducing chain-length specific acyl-ACP thioesterases, blocking the ß-oxidation cycle at an advantageous reaction, and introducing active ß-ketoacyl-CoA thioesterases. Using a bioprospecting approach, we identified fifteen homologs of E. coli ß-ketoacyl-CoA thioesterase (FadM) and evaluated the in vivo activity of each against various chain length substrates. The FadM variant from Providencia sneebia produced the most 2-heptanone, 2-nonanone, and 2-undecanone, suggesting it has the highest activity on the corresponding ß-ketoacyl-CoA substrates. We tested enzyme variants, including acyl-CoA oxidases, thiolases, and bi-functional 3-hydroxyacyl-CoA dehydratases to maximize conversion of fatty acids to ß-keto acyl-CoAs for 2-heptanone, 2-nonanone, and 2-undecanone production. In order to address the issue of product loss during fermentation, we applied a 20% (v/v) dodecane layer in the bioreactor and built an external water cooling condenser connecting to the bioreactor heat-transferring condenser coupling to the condenser. Using these modifications, we were able to generate up to 4.4 g/L total medium-chain length methyl ketones.
Assuntos
Proteínas de Bactérias , Escherichia coli , Cetonas/metabolismo , Engenharia Metabólica , Providencia/genética , Tioléster Hidrolases , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Redes e Vias Metabólicas , Oxirredução , Providencia/enzimologia , Tioléster Hidrolases/genética , Tioléster Hidrolases/metabolismoRESUMO
The production of valuable chemicals from metabolically engineered microbes can be limited by excretion from the cell. Efflux is often overlooked as a bottleneck in metabolic pathways, despite its impact on alleviating feedback inhibition and product toxicity. In the past, it has been assumed that endogenous efflux pumps and membrane porins can accommodate product efflux rates; however, there are an increasing number of examples wherein overexpressing efflux systems is required to improve metabolite production. In this review, we highlight specific examples from the literature where metabolite export has been studied to identify unknown transporters, increase tolerance to metabolites, and improve the production capabilities of engineered bacteria. The review focuses on the export of a broad spectrum of valuable chemicals including amino acids, sugars, flavins, biofuels, and solvents. The combined set of examples supports the hypothesis that efflux systems can be identified and engineered to confer export capabilities on industrially relevant microbes.
Assuntos
Bactérias/genética , Bactérias/metabolismo , Engenharia Metabólica/métodos , Biocombustíveis , Dinitrocresóis/metabolismo , Escherichia coli/genética , Microbiologia Industrial/métodos , Proteínas de Membrana Transportadoras/metabolismo , Redes e Vias Metabólicas , Biologia de SistemasRESUMO
Thousands of natural products are derived from the fused cyclopentane-pyran molecular scaffold nepetalactol. These natural products are used in an enormous range of applications that span the agricultural and medical industries. For example, nepetalactone, the oxidized derivative of nepetalactol, is known for its cat attractant properties as well as potential as an insect repellent. Most of these naturally occurring nepetalactol-derived compounds arise from only two out of the eight possible stereoisomers, 7S-cis-trans and 7R-cis-cis nepetalactols. Here we use a combination of naturally occurring and engineered enzymes to produce seven of the eight possible nepetalactol or nepetalactone stereoisomers. These enzymes open the possibilities for biocatalytic production of a broader range of iridoids, providing a versatile system for the diversification of this important natural product scaffold.
Assuntos
Produtos Biológicos , Iridoides , Biocatálise , Ciclopentanos , EstereoisomerismoRESUMO
Microbial metabolism is an attractive route for producing medium chain length fatty acids, e.g., octanoic acid, used in the oleochemical industry. One challenge to this strategy is the lack of enzymes that are both highly active in a microbial host and selective toward substrates with desired chain length. Of the many steps in fatty acid biosynthesis, the thioesterase is the most widely used enzyme for controlling chain length. Thioesterases hydrolyze the thioester bond between fatty acids and the acyl-carrier protein (ACP) or coenzyme A (CoA) cofactor. The functional role of thioesterases varies between organisms ( i.e., bacteria vs plant) and therefore so do the substrate specificities. As a result, microbial biocatalysts that utilize a heterologous thioesterase either produce high titers of fatty acids with mixed chain lengths or low titers of products with a narrow chain length distribution. To search for highly active enzymes that selectively hydrolyze octanoyl-ACP, we developed a genetic selection based on the lipoic acid requirement of Escherichia coli. We used the selection to identify variants in a randomly mutagenized library of the C8-specific Cuphea palustris FatB1 thioesterase. After optimizing expression of the thioesterase, E. coli cultures produced 1.7 g/L of octanoic acid with >90% specificity from a single chromosomal copy of this thioesterase. In vitro studies confirmed the mutant thioesterase possessed a 15-fold increase in kcat compared to its native sequence. The high level of specific activity allowed for low levels of expression while maintaining fatty acid titer. The low expression requirement will allow metabolic engineers to use more cellular resources to address other limitations in the pathway and maximize overall productivity.
Assuntos
Engenharia de Proteínas/métodos , Tioléster Hidrolases/metabolismo , Proteína de Transporte de Acila/química , Proteína de Transporte de Acila/metabolismo , Sequência de Aminoácidos , Caprilatos/metabolismo , Coenzima A/química , Coenzima A/metabolismo , Escherichia coli/metabolismo , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Cinética , Mutagênese , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Especificidade por Substrato , Tioléster Hidrolases/química , Tioléster Hidrolases/genéticaRESUMO
Enzyme and metabolic engineering offer the potential to develop biocatalysts for converting natural resources into a wide range of chemicals. To broaden the scope of potential products beyond natural metabolites, methods of engineering enzymes to accept alternative substrates and/or perform novel chemistries must be developed. DNA synthesis can create large libraries of enzyme-coding sequences, but most biochemistries lack a simple assay to screen for promising enzyme variants. Our solution to this challenge is structure-guided mutagenesis in which optimization algorithms select the best sequences from libraries based on specified criteria (i.e. binding selectivity). Here, we demonstrate this approach by identifying medium-chain (C6-C12) acyl-ACP thioesterases through structure-guided mutagenesis. Medium-chain fatty acids, products of thioesterase-catalyzed hydrolysis, are limited in natural abundance compared to long-chain fatty acids; the limited supply leads to high costs of C6-C10 oleochemicals such as fatty alcohols, amines, and esters. Here, we applied computational tools to tune substrate binding to the highly-active 'TesA thioesterase in Escherichia coli. We used the IPRO algorithm to design thioesterase variants with enhanced C12- or C8-specificity while maintaining high activity. After four rounds of structure-guided mutagenesis, we identified three thioesterases with enhanced production of dodecanoic acid (C12) and twenty-seven thioesterases with enhanced production of octanoic acid (C8). The top variants reached up to 49% C12 and 50% C8 while exceeding native levels of total free fatty acids. A comparably sized library created by random mutagenesis failed to identify promising mutants. The chain length-preference of 'TesA and the best mutant were confirmed in vitro using acyl-CoA substrates. Molecular dynamics simulations, confirmed by resolved crystal structures, of 'TesA variants suggest that hydrophobic forces govern 'TesA substrate specificity. We expect that the design rules we uncovered and the thioesterase variants identified will be useful to metabolic engineering projects aimed at sustainable production of medium-chain oleochemicals.