RESUMEN
Cell-free protein synthesis (CFPS) is a platform biotechnology that has enabled the on-demand synthesis of proteins for a variety of applications. Numerous advances have improved the productivity of the CFPS platform to result in high-yielding reactions; however, many applications remain limited due to long reaction times. To overcome this limitation, we first established the benchmarks reaction times for CFPS across in-house E. coli extracts and commercial kits. We then set out to fine-tune our in-house extract systems to improve reaction times. Through the optimization of reaction composition and titration of low-cost additives, we have identified formulations that reduce reaction times by 30-50% to obtain high protein titers for biomanufacturing applications, and reduce times by more than 50% to reach the sfGFP detection limit for applications in education and diagnostics. Under optimum conditions, we report the visible observation of sfGFP signal in less than 10 min. Altogether, these advances enhance the utility of CFPS as a rapid, user-defined platform.
Asunto(s)
Escherichia coli/química , Biosíntesis de Proteínas , Sistema Libre de Células/química , Sistema Libre de Células/metabolismo , Escherichia coli/metabolismoRESUMEN
Teaching the processes of transcription and translation is challenging due to the intangibility of these concepts and a lack of instructional, laboratory-based, active learning modules. Harnessing the genetic code in vitro with cell-free protein synthesis (CFPS) provides an open platform that allows for the direct manipulation of reaction conditions and biological machinery to enable inquiry-based learning. Here, we report our efforts to transform the research-based CFPS biotechnology into a hands-on module called the "Genetic Code Kit" for implementation into teaching laboratories. The Genetic Code Kit includes all reagents necessary for CFPS, as well as a laboratory manual, student worksheet, and augmented reality activity. This module allows students to actively explore transcription and translation while gaining exposure to an emerging research technology. In our testing of this module, undergraduate students who used the Genetic Code Kit in a teaching laboratory showed significant score increases on transcription and translation questions in a post-lab questionnaire compared with students who did not participate in the activity. Students also demonstrated an increase in self-reported confidence in laboratory methods and comfort with CFPS, indicating that this module helps prepare students for careers in laboratory research. Importantly, the Genetic Code Kit can accommodate a variety of learning objectives beyond transcription and translation and enables hypothesis-driven science. This opens the possibility of developing Course-Based Undergraduate Research Experiences (CUREs) based on the Genetic Code Kit, as well as supporting next-generation science standards in 8-12th grade science courses.
RESUMEN
Cell-free protein synthesis (CFPS) platforms have undergone numerous workflow improvements to enable diverse applications in research, biomanufacturing, and education. The Escherichia coli cell extract-based platform has been broadly adopted due to its affordability and versatility. The upstream processing of cells to generate crude cell lysate remains time-intensive and technically nuanced, representing one of the largest sources of cost associated with the biotechnology. To overcome these limitations, we have improved the processes by developing a long-lasting autoinduction media formulation for CFPS that obviates human intervention between inoculation and harvest. The cell-free autoinduction (CFAI) media supports the production of robust cell extracts from high cell density cultures nearing the stationary phase of growth. As a result, the total mass of cells and the resulting extract volume obtained increases by 400% while maintaining robust reaction yields of reporter protein, sfGFP (>1 mg/mL). Notably, the CFAI workflow allows users to go from cells on a streak plate to completing CFPS reactions within 24 h. The CFAI workflow uniquely enabled us to elucidate the metabolic limits in CFPS associated with cells grown to stationary phase in the traditional 2× YTPG media. Metabolomics analysis demonstrates that CFAI-based extracts overcome these limits due to improved energy metabolism and redox balance. The advances reported here shed new light on the metabolism associated with highly active CFPS reactions and inform future efforts to tune the metabolism in CFPS systems. Additionally, we anticipate that the improvements in the time and cost-efficiency of CFPS will increase the simplicity and reproducibility, reducing the barriers for new researchers interested in implementing CFPS.
Asunto(s)
Medios de Cultivo/química , Metabolismo Energético , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Ingeniería Metabólica/métodos , Biosíntesis de Proteínas , Productos Biológicos/metabolismo , Tecnología Biomédica/métodos , Sistema Libre de Células/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Metaboloma , Metabolómica/métodos , Oxidación-Reducción , Plásmidos/genética , Reproducibilidad de los ResultadosRESUMEN
Cell-free protein synthesis (CFPS) is a platform biotechnology that enables a breadth of applications. However, field applications remain limited due to the poor shelf-stability of aqueous cell extracts required for CFPS. Lyophilization of E. coli extracts improves shelf life but remains insufficient for extended storage at room temperature. To address this limitation, we mapped the chemical space of ten low-cost additives with four distinct mechanisms of action in a combinatorial manner to identify formulations capable of stabilizing lyophilized cell extract. We report three key findings: (1) unique additive formulations that maintain full productivity of cell extracts stored at 4 °C and 23 °C; (2) additive formulations that enhance extract productivity by nearly 2-fold; (3) a machine learning algorithm that provides predictive capacity for the stabilizing effects of additive formulations that were not tested experimentally. These findings provide a simple and low-cost advance toward making CFPS field-ready and cost-competitive for biomanufacturing.
Asunto(s)
Extractos Celulares , Sistema Libre de Células , Escherichia coli , Biosíntesis de Proteínas/efectos de los fármacos , Biología Sintética/métodos , Biotecnología , Extractos Celulares/química , Extractos Celulares/farmacología , Extractos Celulares/normas , Escherichia coli/química , Escherichia coli/metabolismo , Liofilización/métodosRESUMEN
Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 µg/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per µg of protein produced or $0.019 per µL of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.
Asunto(s)
Sistema Libre de Células , Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/biosíntesis , Biosíntesis de Proteínas , Escherichia coli/genéticaRESUMEN
Malbrancheamide is a dichlorinated fungal indole alkaloid isolated from both Malbranchea aurantiaca and Malbranchea graminicola that belongs to a family of natural products containing a characteristic bicyclo[2.2.2]diazaoctane core. The introduction of chlorine atoms on the indole ring of malbrancheamide differentiates it from other members of this family and contributes significantly to its biological activity. In this study, we characterized the two flavin-dependent halogenases involved in the late-stage halogenation of malbrancheamide in two different fungal strains. MalA and MalA' catalyze the iterative dichlorination and monobromination of the free substrate premalbrancheamide as the final steps in the malbrancheamide biosynthetic pathway. Two unnatural bromo-chloro-malbrancheamide analogues were generated through MalA-mediated chemoenzymatic synthesis. Structural analysis and computational studies of MalA' in complex with three substrates revealed that the enzyme represents a new class of zinc-binding flavin-dependent halogenases and provides new insights into a potentially unique reaction mechanism.
Asunto(s)
Ascomicetos/enzimología , Proteínas Fúngicas/metabolismo , Alcaloides Indólicos/metabolismo , Ascomicetos/química , Ascomicetos/metabolismo , Vías Biosintéticas , Proteínas Fúngicas/química , Halogenación , Alcaloides Indólicos/química , Cinética , Modelos MolecularesRESUMEN
Due to their pivotal role in extender unit selection during polyketide biosynthesis, acyltransferase (AT) domains are important engineering targets. A subset of assembly line polyketide synthases (PKSs) are serviced by discrete, trans-acting ATs. Theoretically, these trans-ATs can complement an inactivated cis-AT, promoting introduction of a noncognate extender unit. This approach requires a better understanding of the substrate specificity and catalytic mechanism of naturally occurring trans-ATs. We kinetically analyzed trans-ATs from the disorazole and kirromycin synthases and compared them to a representative cis-AT from the 6-deoxyerythronolide B synthase (DEBS). During transacylation, the disorazole AT favored malonyl-CoA over methylmalonyl-CoA by >40000-fold, whereas the kirromycin AT favored ethylmalonyl-CoA over methylmalonyl-CoA by 20-fold. Conversely, the disorazole AT had broader specificity than its kirromycin counterpart for acyl carrier protein (ACP) substrates. The presence of the ACP had little effect on the specificity (k(cat)/K(M)) of the cis-AT domain for carboxyacyl-CoA substrates but had a marked influence on the corresponding specificity parameters for the trans-ATs, suggesting that these enzymes do not act strictly by a canonical ping-pong mechanism. To investigate the relevance of the kinetic analysis of isolated ATs in the context of intact PKSs, we complemented an in vitro AT-null DEBS assembly line with either trans-AT. Whereas the disorazole AT efficiently complemented the mutant PKS at substoichiometric protein ratios, the kirromycin AT was considerably less effective. Our findings suggest that knowledge of both carboxyacyl-CoA and ACP specificity is critical to the choice of a trans-AT in combination with a mutant PKS to generate novel polyketides.
Asunto(s)
Acilcoenzima A/metabolismo , Aciltransferasas/metabolismo , Malonil Coenzima A/metabolismo , Modelos Moleculares , Fragmentos de Péptidos/metabolismo , Sintasas Poliquetidas/metabolismo , Aciltransferasas/química , Aciltransferasas/genética , Antibacterianos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biocatálisis , Dominio Catalítico , Macrólidos/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Oxazoles/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Sintasas Poliquetidas/química , Sintasas Poliquetidas/genética , Policétidos/metabolismo , Ingeniería de Proteínas , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Piridonas/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Estereoisomerismo , Especificidad por SustratoRESUMEN
A-74528 is a C30 polyketide natural product that functions as an inhibitor of 2',5'-oligoadenylate phosphodiesterase (2'-PDE), a key regulatory enzyme of the interferon pathway. Modulation of 2'-PDE represents a unique therapeutic approach for regulating viral infections. The gene cluster responsible for biosynthesis of A-74528 yields minute amounts of this natural product together with considerably larger quantities of a structurally dissimilar C30 cytotoxic agent, fredericamycin. Through construction and analysis of a series of knockout mutants, we identified the genes necessary for A-74528 biosynthesis. Remarkably, the formation of six stereocenters and the regiospecific formation of six rings in A-74528 appear to be catalyzed by only two tailoring enzymes, a cyclase and an oxygenase, in addition to the core polyketide synthase. The inferred pathway was genetically refactored in a heterologous host, Streptomyces coelicolor CH999, to produce 3 mg/L A-74528 in the absence of fredericamycin.
Asunto(s)
Adenilil Ciclasas/metabolismo , Oxigenasas/metabolismo , Compuestos Policíclicos/metabolismo , Pironas/metabolismo , Streptomyces coelicolor/metabolismo , Estructura Molecular , Compuestos Policíclicos/química , Pironas/química , Streptomyces coelicolor/químicaRESUMEN
The cyclocinamides possess a unique ß(2)αß(2)α 14-membered tetrapeptide core. The initially reported biological data and intriguing structure, which was without full stereochemical identification, necessitated synthesis of both nominal (all-S) cyclocinamide A and the 11R isomer. The completed synthesis is highlighted by the use of a (cyclo)asparagine-containing dipeptide as a turn inducing fragment. Due to inconsistencies in analytical data between natural and synthetic samples, a re-evaluation of the natural product stereochemistry appears necessary.
Asunto(s)
Productos Biológicos/síntesis química , Péptidos Cíclicos/síntesis química , Productos Biológicos/química , Estructura Molecular , Péptidos Cíclicos/química , EstereoisomerismoRESUMEN
Prenylated indole alkaloids are a diverse group of fungal secondary metabolites and represent an important biosynthetic class. In this study we have identified new halogenated prenyl-indole alkaloids from an invertebrate-derived Malbranchea graminicola strain. Using direct analysis in real time (DART) mass spectrometry, these compounds were initially detected from hyphae of the fungus grown on agar plates, without the need for any organic extraction. Subsequently, the metabolites were isolated from liquid culture in artificial seawater. The structures of two novel chlorinated metabolites, named (-)-spiromalbramide and (+)-isomalbrancheamide B, provide additional insights into the assembly of the malbrancheamide compound family. Remarkably, two new brominated analogues, (+)-malbrancheamide C and (+)-isomalbrancheamide C, were produced by enriching the growth medium with bromine salts.
Asunto(s)
Hongos/química , Alcaloides Indólicos/química , Onygenales/química , Agua de Mar/análisis , Agua de Mar/química , Hongos/metabolismo , Halogenación , Alcaloides Indólicos/aislamiento & purificación , Biología Marina , Espectrometría de Masas , Estructura Molecular , Onygenales/metabolismo , PrenilaciónRESUMEN
The cyclodepsipeptide jasplakinolide (1) (aka jaspamide), isolated previously from the marine sponge Jaspis splendens, is a unique cytotoxin and molecular probe that operates through stabilization of filamentous actin (F-actin). We have recently disclosed that two analogues of 1, jasplakinolides B (3) and E, were referred to the National Cancer Institute's (NCI) Biological Evaluation Committee, and the objective of this study was to reinvestigate a Fijian collection of J. splendens in an effort to find jasplakinolide congeners with similar biological properties. The current efforts have afforded six known jasplakinolide analogues (4-7, 9, 10), two structures requiring revision (8 and 14), and four new congeners of 1 (11-13, 15) including open-chain derivatives and structures with modified ß-tyrosine residues. Compounds were evaluated for biological activity in the NCI's 60 cell line screen and in a microfilament disruption assay in both HCT-116 and HeLa cells. These two phenotypic screens provide evidence that each cytotoxic analogue, including jasplakinolide B (3), operates by modification of microfilaments. The new structure jasplakinolide V (13) has also been selected for study by the NCI's Biological Evaluation Committee. In addition, the results of a clonogenic dose-response study on jasplakinolide are presented.
Asunto(s)
Antineoplásicos/farmacología , Citotoxinas/farmacología , Depsipéptidos/farmacología , Actinas/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Citotoxinas/química , Citotoxinas/aislamiento & purificación , Depsipéptidos/química , Depsipéptidos/aislamiento & purificación , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Fiji , Células HCT116 , Células HeLa , Humanos , Biología Marina , Estructura Molecular , National Cancer Institute (U.S.) , Péptidos Cíclicos , Estereoisomerismo , Estados UnidosRESUMEN
This review focuses on six important parasitic diseases that adversely affect the health and lives of over one billion people worldwide. In light of the global human impact of these neglected tropical diseases (NTDs), several initiatives and campaigns have been mounted to eradicate these infections once and for all. Currently available therapeutics summarized herein are either ineffective and/or have severe and deleterious side effects. Resistant strains continue to emerge and there is an overall unmet and urgent need for new antiparasitic drugs. Marine-derived small molecules (MDSMs) from invertebrates comprise an extremely diverse and promising source of compounds from a wide variety of structural classes. New discoveries of marine natural product privileged structures and compound classes that are being made via natural product library screening using whole cell in vitro assays are highlighted. It is striking to note that for the first time in history the entire genomes of all six parasites have been sequenced and additional transcriptome and proteomic analyses are available. Furthermore, open and shared, publicly available databases of the genome sequences, compounds, screening assays, and druggable molecular targets are being used by the worldwide research community. A combined assessment of all of the above factors, especially of current discoveries in marine natural products, implies a brighter future with more effective, affordable, and benign antiparasitic therapeutics.
Asunto(s)
Antiparasitarios/química , Productos Biológicos/química , Invertebrados/química , Animales , Humanos , Biología MarinaRESUMEN
Human African trypanosomiasis (HAT, commonly known as African sleeping sickness) is categorized as a neglected disease, as it afflicts >50,000 people annually in sub-saharan Africa, and there are few formal programs in the world focused on drug discovery approaches for this disease. In this study, we examined the crude extracts of two fungal strains (Aspergillus fumigatus and Nectria inventa) isolated from deep water sediment which provided >99% growth inhibition at 1microg/mL of Trypanosoma brucei, the causative parasite of HAT. A collection of fifteen natural products was supplemented with six semi-synthetic derivatives and one commercially available compound. Twelve of the compounds, each containing a diketopiperazine core, showed excellent activity against T. brucei (IC(50)=0.002-40microM), with selectivity over mammalian cells as great as 20-fold. The trypanocidal diketopiperazines were also tested against two cysteine protease targets Rhodesain and TbCatB, where five compounds showed inhibition activity at concentrations less than 20microM. A preliminary activity pattern is described and analyzed.