RÉSUMÉ
Living systems contain a vast network of metabolic reactions, providing a wealth of enzymes and cells as potential biocatalysts for chemical processes. The properties of protein and cell biocatalysts-high selectivity, the ability to control reaction sequence and operation in environmentally benign conditions-offer approaches to produce molecules at high efficiency while lowering the cost and environmental impact of industrial chemistry. Furthermore, biocatalysis offers the opportunity to generate chemical structures and functions that may be inaccessible to chemical synthesis. Here we consider developments in enzymes, biosynthetic pathways and cellular engineering that enable their use in catalysis for new chemistry and beyond.
Sujet(s)
Biocatalyse , Voies de biosynthèse , Ingénierie cellulaire , Enzymes , Humains , Ingénierie cellulaire/méthodes , Enzymes/métabolisme , Enzymes/composition chimique , Spécificité du substrat , Techniques de chimie synthétiqueRÉSUMÉ
Living organisms carry out a wide range of remarkable functions, including the synthesis of thousands of simple and complex chemical structures for cellular growth and maintenance. The manipulation of this reaction network has allowed for the genetic engineering of cells for targeted chemical synthesis, but it remains challenging to alter the program underlying their fundamental chemical behavior. By taking advantage of the unique ability of living systems to use evolution to find solutions to complex problems, we have achieved yields of up to â¼95% for three C4 commodity chemicals, n-butanol, 1,3-butanediol, and 4-hydroxy-2-butanone. Genomic sequencing of the evolved strains identified pcnB and rpoBC as two gene loci that are able to alter carbon flow by remodeling the transcriptional landscape of the cell, highlighting the potential of synthetic pathways as a tool to identify metabolic control points.
RÉSUMÉ
Living systems provide a promising approach to chemical synthesis, having been optimized by evolution to convert renewable carbon sources, such as glucose, into an enormous range of small molecules. However, a large number of synthetic structures can still be difficult to obtain solely from cells, such as unsubstituted hydrocarbons. In this work, we demonstrate the use of a dual cellular-heterogeneous catalytic strategy to produce olefins from glucose using a selective hydrolase to generate an activated intermediate that is readily deoxygenated. Using a new family of iterative thiolase enzymes, we genetically engineered a microbial strain that produces 4.3 ± 0.4 g l-1 of fatty acid from glucose with 86% captured as 3-hydroxyoctanoic and 3-hydroxydecanoic acids. This 3-hydroxy substituent serves as a leaving group that enables heterogeneous tandem decarboxylation-dehydration routes to olefinic products on Lewis acidic catalysts without the additional redox input required for enzymatic or chemical deoxygenation of simple fatty acids.
Sujet(s)
Alcènes/synthèse chimique , Acides gras/composition chimique , Glucose/métabolisme , Acetyl-coA C-acyltransferase/composition chimique , Acetyl-coA C-acyltransferase/métabolisme , Bactéries/enzymologie , Bactéries/métabolisme , Protéines bactériennes/composition chimique , Protéines bactériennes/métabolisme , Catalyse , Décarboxylation , Énoyl-CoA hydratases/composition chimique , Énoyl-CoA hydratases/métabolisme , Fatty acid desaturases/composition chimique , Fatty acid desaturases/métabolisme , Acides gras/biosynthèse , Acides de Lewis/composition chimique , Oxydoréduction , Palmitoyl-coA hydrolase/composition chimique , Palmitoyl-coA hydrolase/métabolismeRÉSUMÉ
Protein-Observed Fluorine NMR (PrOFâ NMR) spectroscopy is an emerging technique for screening and characterizing small-molecule-protein interactions. The choice of which amino acid to label for PrOFâ NMR can be critical for analysis. Here we report the first use of a protein containing two different fluoroaromatic amino acids for NMR studies. Using the KIX domain of the CBP/p300 as a model system, we examine ligand binding of several small-molecule compounds elaborated from our previous fragment screen and identify a new ligand binding site distinct from those used by native transcription factors. This site was further supported by computational modeling (FTMap and Schrödinger) and 1 H,15 N HSQC/HMQC NMR spectroscopy. Metabolic labeling with multiple fluorinated amino acids provides useful probes for further studying ligand binding and has led to new insight for allosterically regulating transcription-factor protein interactions with small-molecule ligands.
Sujet(s)
Bibliothèques de petites molécules/pharmacologie , Facteurs de transcription CBP-p300/métabolisme , Animaux , Sites de fixation/effets des médicaments et des substances chimiques , Protéine CBP/composition chimique , Protéine CBP/métabolisme , Protéine de liaison à l'élément de réponse à l'AMP cyclique/composition chimique , Protéine de liaison à l'élément de réponse à l'AMP cyclique/métabolisme , Fluor/analyse , Humains , Souris , Modèles moléculaires , Résonance magnétique nucléaire biomoléculaire , Liaison aux protéines/effets des médicaments et des substances chimiques , Domaines protéiques/effets des médicaments et des substances chimiques , Cartes d'interactions protéiques/effets des médicaments et des substances chimiques , Rats , Bibliothèques de petites molécules/composition chimique , Facteurs de transcription CBP-p300/composition chimiqueRÉSUMÉ
(19)Fâ NMR spectroscopy of labeled proteins is a sensitive method for characterizing structure, conformational dynamics, higher-order assembly, and ligand binding. Fluorination of aromatic side chains has been suggested as a labeling strategy for small-molecule ligand discovery for protein-protein interaction interfaces. Using a model transcription factor binding domain of the CREB binding protein (CBP)/p300, KIX, we report the first full small-molecule screen using protein-observed (19)Fâ NMR spectroscopy. Screening of 508 compounds and validation by (1)H-(15)N HSQCâ NMR spectroscopy led to the identification of a minimal pharmacaphore for the MLL-KIX interaction site. Hit rate analysis for the CREB-KIX and MLL-KIX sites provided a metric to assess the ligandability or "druggability" of each interface informing future medicinal chemistry efforts. The structural information from the simplified spectra and data collection speed, affords a new screening tool for analysis of protein interfaces and discovery of small molecules.