RESUMEN
Fungi have historically been the source of numerous important medicinal compounds, but full exploitation of their genetic potential for drug development has been hampered in traditional discovery paradigms. Here we describe a radically different approach, top-down drug discovery (TD3), starting with a massive digital search through a database of over 100,000 fully genomicized fungi to identify loci encoding molecules with a predetermined human target. We exemplify TD3 by the selection of cyclin-dependent kinases (CDKs) as targets and the discovery of two molecules, 1 and 2, which inhibit therapeutically important human CDKs. 1 and 2 exhibit a remarkable mechanism, forming a site-selective covalent bond to the CDK active site Lys. We explored the structure-activity relationship via semi- and total synthesis, generating an analog, 43, with improved kinase selectivity, bioavailability, and efficacy. This work highlights the power of TD3 to identify mechanistically and structurally novel molecules for the development of new medicines.
Asunto(s)
Quinasas Ciclina-Dependientes , Descubrimiento de Drogas , Inhibidores de Proteínas Quinasas , Humanos , Relación Estructura-Actividad , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/síntesis química , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Animales , Genómica/métodos , Modelos MolecularesRESUMEN
Engineering synthetic heterotrophy is a key to the efficient bio-based valorization of renewable and waste substrates. Among these, engineering hemicellulosic pentose utilization has been well-explored in Saccharomyces cerevisiae (yeast) over several decades-yet the answer to what makes their utilization inherently recalcitrant remains elusive. Through implementation of a semi-synthetic regulon, we find that harmonizing cellular and engineering objectives are a key to obtaining highest growth rates and yields with minimal metabolic engineering effort. Concurrently, results indicate that "extrinsic" factors-specifically, upstream genes that direct flux of pentoses into central carbon metabolism-are rate-limiting. We also reveal that yeast metabolism is innately highly adaptable to rapid growth on non-native substrates and that systems metabolic engineering (i.e., functional genomics, network modeling, etc.) is largely unnecessary. Overall, this work provides an alternate, novel, holistic (and yet minimalistic) approach based on integrating non-native metabolic genes with a native regulon system.
Asunto(s)
Pentosas , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Pentosas/metabolismo , Ingeniería Metabólica/métodos , FermentaciónRESUMEN
Engineering the utilization of non-native substrates, or synthetic heterotrophy, in proven industrial microbes such as Saccharomyces cerevisiae represents an opportunity to valorize plentiful and renewable sources of carbon and energy as inputs to bioprocesses. We previously demonstrated that activation of the galactose (GAL) regulon, a regulatory structure used by this yeast to coordinate substrate utilization with biomass formation during growth on galactose, during growth on the non-native substrate xylose results in a vastly altered gene expression profile and faster growth compared with constitutive overexpression of the same heterologous catabolic pathway. However, this effort involved the creation of a xylose-inducible variant of Gal3p (Gal3pSyn4.1), the sensor protein of the GAL regulon, preventing this semi-synthetic regulon approach from being easily adapted to additional non-native substrates. Here, we report the construction of a variant Gal3pMC (metabolic coordinator) that exhibits robust GAL regulon activation in the presence of structurally diverse substrates and recapitulates the dynamics of the native system. Multiple molecular modeling studies suggest that Gal3pMC occupies conformational states corresponding to galactose-bound Gal3p in an inducer-independent manner. Using Gal3pMC to test a regulon approach to the assimilation of the non-native lignocellulosic sugars xylose, arabinose, and cellobiose yields higher growth rates and final cell densities when compared with a constitutive overexpression of the same set of catabolic genes. The subsequent demonstration of rapid and complete co-utilization of all three non-native substrates suggests that Gal3pMC-mediated dynamic global gene expression changes by GAL regulon activation may be universally beneficial for engineering synthetic heterotrophy.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Factores de Transcripción , Factores de Transcripción/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Procesos Heterotróficos , Galactosa/genética , Galactosa/metabolismo , Xilosa/genética , Xilosa/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
Current pathway synthesis tools identify possible pathways that can be added to a host to produce the desired target molecule through the exploration of abstract metabolic and reaction network space. However, not many of these tools explore gene-level information required to physically realize the identified synthesis pathways, and none explore enzyme-host compatibility. Developing tools that address this disconnect between abstract reactions/metabolic design space and physical genetic sequence design space will enable expedited experimental efforts that avoid exploring unprofitable synthesis pathways. This work describes a workflow, termed Probabilistic Pathway Assembly with Solubility Confidence Scores (ProPASS), which links synthesis pathway construction with the exploration of the physical design space as imposed by the availability of enzymes with predicted characterized activities within the host. Predicted protein solubility propensity scores are used as a confidence level to quantify the compatibility of each pathway enzyme with the host Escherichia coli (E. coli). This study also presents a database, termed Protein Solubility Database (ProSol DB), which provides solubility confidence scores in E. coli for 240,016 characterized enzymes obtained from UniProtKB/Swiss-Prot. The utility of ProPASS is demonstrated by generating genetic implementations of heterologous synthesis pathways in E. coli that target several commercially useful biomolecules.
Asunto(s)
Vías Biosintéticas , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Biocatálisis , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Microbiología Industrial , Ingeniería Metabólica , Solubilidad , Flujo de TrabajoRESUMEN
Extending the host substrate range of industrially relevant microbes, such as Saccharomyces cerevisiae, has been a highly-active area of research since the conception of metabolic engineering. Yet, rational strategies that enable non-native substrate utilization in this yeast without the need for combinatorial and/or evolutionary techniques are underdeveloped. Herein, this review focuses on pentose metabolism in S. cerevisiae as a case study to highlight the challenges in this field. In the last three decades, work has focused on expressing exogenous pentose metabolizing enzymes as well as endogenous enzymes for effective pentose assimilation, growth, and biofuel production. The engineering strategies that are employed for pentose assimilation in this yeast are reviewed, and compared with metabolism and regulation of native sugar, galactose. In the case of galactose metabolism, multiple signals regulate and aid growth in the presence of the sugar. However, for pentoses that are non-native, it is unclear if similar growth and regulatory signals are activated. Such a comparative analysis aids in identifying missing links in xylose and arabinose utilization. While research on pentose metabolism have mostly concentrated on pathway level optimization, recent transcriptomics analyses highlight the need to consider more global regulatory, structural, and signaling components.
Asunto(s)
Pentosas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Arabinosa/metabolismo , Galactosa/metabolismo , Ingeniería Metabólica , Biología de SistemasRESUMEN
Nutrient assimilation is the first step that allows biological systems to proliferate and produce value-added products. Yet, implementation of heterologous catabolic pathways has so far relied on constitutive gene expression without consideration for global regulatory systems that may enhance nutrient assimilation and cell growth. In contrast, natural systems prefer nutrient-responsive gene regulation (called regulons) that control multiple cellular functions necessary for cell survival and growth. Here, in Saccharomyces cerevisiae, by partially- and fully uncoupling galactose (GAL)-responsive regulation and metabolism, we demonstrate the significant growth benefits conferred by the GAL regulon. Next, by adapting the various aspects of the GAL regulon for a non-native nutrient, xylose, we build a semi-synthetic regulon that exhibits higher growth rate, better nutrient consumption, and improved growth fitness compared to the traditional and ubiquitous constitutive expression strategy. This work provides an elegant paradigm to integrate non-native nutrient catabolism with native, global cellular responses to support fast growth.