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1.
Nat Commun ; 10(1): 4327, 2019 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-31548547

RESUMO

Synthetic RNA-based genetic devices dynamically control a wide range of gene-regulatory processes across diverse cell types. However, the limited throughput of quantitative assays in mammalian cells has hindered fast iteration and interrogation of sequence space needed to identify new RNA devices. Here we report developing a quantitative, rapid and high-throughput mammalian cell-based RNA-Seq assay to efficiently engineer RNA devices. We identify new ribozyme-based RNA devices that respond to theophylline, hypoxanthine, cyclic-di-GMP, and folinic acid from libraries of ~22,700 sequences in total. The small molecule responsive devices exhibit low basal expression and high activation ratios, significantly expanding our toolset of highly functional ribozyme switches. The large datasets obtained further provide conserved sequence and structure motifs that may be used for rationally guided design. The RNA-Seq approach offers a generally applicable strategy for developing broad classes of RNA devices, thereby advancing the engineering of genetic devices for mammalian systems.

2.
Nat Commun ; 10(1): 3634, 2019 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-31406117

RESUMO

Tropane alkaloids (TAs) are a class of phytochemicals produced by plants of the nightshade family used for treating diverse neurological disorders. Here, we demonstrate de novo production of tropine, a key intermediate in the biosynthetic pathway of medicinal TAs such as scopolamine, from simple carbon and nitrogen sources in yeast (Saccharomyces cerevisiae). Our engineered strain incorporates 15 additional genes, including 11 derived from diverse plants and bacteria, and 7 disruptions to yeast regulatory or biosynthetic proteins to produce tropine at titers of 6 mg/L. We also demonstrate the utility of our engineered yeast platform for the discovery of TA derivatives by combining biosynthetic modules from distant plant lineages to achieve de novo production of cinnamoyltropine, a non-canonical TA. Our engineered strain constitutes a starting point for future optimization efforts towards realizing industrial fermentation of medicinal TAs and a platform for the synthesis of TA derivatives with enhanced bioactivities.

3.
Nat Commun ; 10(1): 2673, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31209208

RESUMO

Alternative splicing performs a central role in expanding genomic coding capacity and proteomic diversity. However, programming of splicing patterns in engineered biological systems remains underused. Synthetic approaches thus far have predominantly focused on controlling expression of a single protein through alternative splicing. Here, we describe a modular and extensible platform for regulating four programmable exons that undergo a mutually exclusive alternative splicing event to generate multiple functionally-distinct proteins. We present an intron framework that enforces the mutual exclusivity of two internal exons and demonstrate a graded series of consensus sequence elements of varying strengths that set the ratio of two mutually exclusive isoforms. We apply this framework to program the DNA-binding domains of modular transcription factors to differentially control downstream gene activation. This splicing platform advances an approach for generating diverse isoforms and can ultimately be applied to program modular proteins and increase coding capacity of synthetic biological systems.


Assuntos
Processamento Alternativo/genética , Regulação da Expressão Gênica/genética , Engenharia Genética/métodos , RNA/genética , Fatores de Transcrição/genética , Motivos de Aminoácidos/genética , Animais , Linhagem Celular , Biologia Computacional , Sequência Consenso/genética , Éxons/genética , Biblioteca Gênica , Genes Reporter/genética , Humanos , Íntrons/genética , Mutagênese Sítio-Dirigida/métodos , Domínios Proteicos/genética , Isoformas de Proteínas/genética , RNA/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética
4.
Nat Commun ; 10(1): 2142, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086174

RESUMO

Metabolic engineers endeavor to create a bio-based manufacturing industry using microbes to produce fuels, chemicals, and medicines. Plant natural products (PNPs) are historically challenging to produce and are ubiquitous in medicines, flavors, and fragrances. Engineering PNP pathways into new hosts requires finding or modifying a suitable host to accommodate the pathway, planning and implementing a biosynthetic route to the compound, and discovering or engineering enzymes for missing steps. In this review, we describe recent developments in metabolic engineering at the level of host, pathway, and enzyme, and discuss how the field is approaching ever more complex biosynthetic opportunities.


Assuntos
Produtos Biológicos/metabolismo , Engenharia Metabólica/métodos , Microrganismos Geneticamente Modificados/metabolismo , Plantas/metabolismo , Vias Biossintéticas/genética , Enzimas/genética , Enzimas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Engenharia Metabólica/tendências , Microrganismos Geneticamente Modificados/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Biologia Sintética/métodos , Biologia Sintética/tendências
5.
Artigo em Inglês | MEDLINE | ID: mdl-30602542

RESUMO

In addition to coding for protein sequences, RNA molecules encode a diverse set of gene-regulatory elements. RNA switches are one class of gene-regulatory elements that control protein expression in a manner that is dependent on the concentration of specific ligand molecules. These allosteric gene-regulatory elements have been shown as useful tools in engineering diverse cell types to display novel function. In particular, RNA switches have been used as genetically encoded biosensors and conditional controllers to direct cellular decisions based on the system's changing environment. A significant focus in the field has been the generation of novel RNA switches that are tailored for different biological systems. We review approaches that have been used to generate RNA switches, which leverage the unique physical properties of RNA and the myriad ways in which RNA can modulate gene expression.

6.
Nat Commun ; 10(1): 194, 2019 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-30643127

RESUMO

Repurposed CRISPR-Cas molecules provide a useful tool set for broad applications of genomic editing and regulation of gene expression in prokaryotes and eukaryotes. Recent discovery of phage-derived proteins, anti-CRISPRs, which serve to abrogate natural CRISPR anti-phage activity, potentially expands the ability to build synthetic CRISPR-mediated circuits. Here, we characterize a panel of anti-CRISPR molecules for expanded applications to counteract CRISPR-mediated gene activation and repression of reporter and endogenous genes in various cell types. We demonstrate that cells pre-engineered with anti-CRISPR molecules become resistant to gene editing, thus providing a means to generate "write-protected" cells that prevent future gene editing. We further show that anti-CRISPRs can be used to control CRISPR-based gene regulation circuits, including implementation of a pulse generator circuit in mammalian cells. Our work suggests that anti-CRISPR proteins should serve as widely applicable tools for synthetic systems regulating the behavior of eukaryotic cells.


Assuntos
Bacteriófagos/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Redes Reguladoras de Genes/genética , Técnicas de Cultura de Células , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Células Eucarióticas , Vetores Genéticos/genética , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas , Microscopia Intravital/métodos , Lentivirus/genética , Microscopia de Fluorescência/métodos , Imagem com Lapso de Tempo/métodos , Transdução Genética/métodos , Transfecção/métodos
7.
Nat Prod Rep ; 35(9): 902-920, 2018 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-29897091

RESUMO

Covering: 2006 to 2018 Phytochemicals are important sources for the discovery and development of agricultural and pharmaceutical compounds, such as pesticides and medicines. However, these compounds are typically present in low abundance in nature, and the biosynthetic pathways for most phytochemicals are not fully elucidated. Heterologous production of phytochemicals in plant, bacterial, and yeast hosts has been pursued as a potential approach to address sourcing issues associated with many valuable phytochemicals, and more recently has been utilized as a tool to aid in the elucidation of plant biosynthetic pathways. Due to the structural complexity of certain phytochemicals and the associated biosynthetic pathways, reconstitution of plant pathways in heterologous hosts can encounter numerous challenges. Synthetic biology approaches have been developed to address these challenges in areas such as precise control over heterologous gene expression, improving functional expression of heterologous enzymes, and modifying central metabolism to increase the supply of precursor compounds into the pathway. These strategies have been applied to advance plant pathway reconstitution and phytochemical production in a wide variety of heterologous hosts. Here, we review synthetic biology strategies that have been recently applied to advance complex phytochemical production in heterologous hosts.

8.
Proc Natl Acad Sci U S A ; 115(17): E3922-E3931, 2018 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-29610307

RESUMO

Microbial biosynthesis of plant natural products from simple building blocks is a promising approach toward scalable production and modification of high-value compounds. The pathway for biosynthesis of noscapine, a potential anticancer compound, from canadine was recently elucidated as a 10-gene cluster from opium poppy. Here we demonstrate the de novo production of noscapine in Saccharomyces cerevisiae, through the reconstruction of a biosynthetic pathway comprising over 30 enzymes from plants, bacteria, mammals, and yeast itself, including 7 plant endoplasmic reticulum (ER)-localized enzymes. Optimization directed to tuning expression of pathway enzymes, host endogenous metabolic pathways, and fermentation conditions led to an over 18,000-fold improvement from initial noscapine titers to ∼2.2 mg/L. By feeding modified tyrosine derivatives to the optimized noscapine-producing strain we further demonstrated microbial production of halogenated benzylisoquinoline alkaloids. This work highlights the potential for microbial biosynthetic platforms to support the synthesis of valuable and novel alkaloid compounds, which can advance alkaloid-based drug discovery and development.


Assuntos
Hidrocarbonetos Halogenados/metabolismo , Engenharia Metabólica , Noscapina/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética
9.
Nat Chem ; 10(4): 395-404, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29568052

RESUMO

Phytochemicals are of great pharmaceutical and agricultural importance, but often exhibit low abundance in nature. Recent demonstrations of industrial-scale production of phytochemicals in yeast have shown that microbial production of these high-value chemicals is a promising alternative to sourcing these molecules from native plant hosts. However, a number of challenges remain in the broader application of this approach, including the limited knowledge of plant secondary metabolism and the inefficient reconstitution of plant metabolic pathways in microbial hosts. In this Review, we discuss recent strategies to achieve microbial biosynthesis of complex phytochemicals, including strategies to: (1) reconstruct plant biosynthetic pathways that have not been fully elucidated by mining enzymes from native and non-native hosts or by enzyme engineering; (2) enhance plant enzyme activity, specifically cytochrome P450 activity, by improving efficiency, selectivity, expression or electron transfer; and (3) enhance overall reaction efficiency of multi-enzyme pathways by dynamic control, compartmentalization or optimization with the host's metabolism. We also highlight remaining challenges to - and future opportunities of - this approach.

10.
Nucleic Acids Res ; 46(3): 1541-1552, 2018 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-29244152

RESUMO

As molecular and cellular therapies advance in the clinic, the role of genetic regulation is becoming increasingly important for controlling therapeutic potency and safety. The emerging field of mammalian synthetic biology provides promising tools for the construction of regulatory platforms that can intervene with endogenous pathways and control cell behavior. Recent work has highlighted the development of synthetic biological systems that integrate sensing of molecular signals to regulated therapeutic function in various disease settings. However, the toxicity and limited dosing of currently available molecular inducers have largely inhibited translation to clinical settings. In this work, we developed synthetic microRNA-based genetic systems that are controlled by the pharmaceutical drug leucovorin, which is readily available and safe for prolonged administration in clinical settings. We designed microRNA switches to target endogenous cytokine receptor subunits (IL-2Rß and γc) that mediate various signaling pathways in T cells. We demonstrate the function of these control systems by effectively regulating T cell proliferation with the drug input. Each control system produced unique functional responses, and combinatorial targeting of multiple receptor subunits exhibited greater repression of cell growth. This work highlights the potential use of drug-responsive genetic control systems to improve the management and safety of cellular therapeutics.

11.
J Cell Biol ; 216(1): 73-82, 2017 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-27932576

RESUMO

Synthetic biology is advancing the design of genetic devices that enable the study of cellular and molecular biology in mammalian cells. These genetic devices use diverse regulatory mechanisms to both examine cellular processes and achieve precise and dynamic control of cellular phenotype. Synthetic biology tools provide novel functionality to complement the examination of natural cell systems, including engineered molecules with specific activities and model systems that mimic complex regulatory processes. Continued development of quantitative standards and computational tools will expand capacities to probe cellular mechanisms with genetic devices to achieve a more comprehensive understanding of the cell. In this study, we review synthetic biology tools that are being applied to effectively investigate diverse cellular processes, regulatory networks, and multicellular interactions. We also discuss current challenges and future developments in the field that may transform the types of investigation possible in cell biology.


Assuntos
Biologia Celular , Técnicas Citológicas , Biologia Sintética/métodos , Processamento Alternativo , Comunicação Celular , Epigênese Genética , Redes Reguladoras de Genes , Humanos , Masculino , Interferência de RNA , Transdução de Sinais
12.
Cell Syst ; 3(6): 549-562.e7, 2016 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-27840078

RESUMO

For synthetic biology applications, protein-based transcriptional genetic controllers are limited in terms of orthogonality, modularity, and portability. Although ribozyme-based switches can address these issues, their current two-stage architectures and limited dynamic range hinder their broader incorporation into systems-level genetic controllers. Here, we address these challenges by implementing an RNA-protein hybrid controller with a three-stage architecture that introduces a transcription-based amplifier between an RNA sensor and a protein actuator. To facilitate the construction of these more complex circuits, we use a model-guided strategy to efficiently match the activities of stages. The presence of the amplifier enabled the three-stage controller to have up to 200-fold higher gene expression than its two-stage counterpart and made it possible to implement higher-order controllers, such as multilayer Boolean logic and feedback systems. The modularity inherent in the three-stage architecture along with the sensing flexibility of RNA devices presents a generalizable framework for designing and building sophisticated genetic control systems.

13.
Metab Eng ; 38: 191-203, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27519552

RESUMO

Engineered microbial biosynthesis of plant natural products can support manufacturing of complex bioactive molecules and enable discovery of non-naturally occurring derivatives. Purine alkaloids, including caffeine (coffee), theophylline (antiasthma drug), theobromine (chocolate), and other methylxanthines, play a significant role in pharmacology and food chemistry. Here, we engineered the eukaryotic microbial host Saccharomyces cerevisiae for the de novo biosynthesis of methylxanthines. We constructed a xanthine-to-xanthosine conversion pathway in native yeast central metabolism to increase endogenous purine flux for the production of 7-methylxanthine, a key intermediate in caffeine biosynthesis. Yeast strains were further engineered to produce caffeine through expression of several enzymes from the coffee plant. By expressing combinations of different N-methyltransferases, we were able to demonstrate re-direction of flux to an alternate pathway and develop strains that support the production of diverse methylxanthines. We achieved production of 270µg/L, 61µg/L, and 3700µg/L of caffeine, theophylline, and 3-methylxanthine, respectively, in 0.3-L bench-scale batch fermentations. The constructed strains provide an early platform for de novo production of methylxanthines and with further development will advance the discovery and synthesis of xanthine derivatives.


Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Vias Biossintéticas/genética , Cafeína/biossíntese , Coffea/fisiologia , Engenharia Metabólica/métodos , Saccharomyces cerevisiae/fisiologia , Xantinas/metabolismo , Cafeína/isolamento & purificação , Redes e Vias Metabólicas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Xantinas/isolamento & purificação
14.
Nat Commun ; 7: 12137, 2016 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-27378283

RESUMO

Noscapine is a potential anticancer drug isolated from the opium poppy Papaver somniferum, and genes encoding enzymes responsible for the synthesis of noscapine have been recently discovered to be clustered on the genome of P. somniferum. Here, we reconstitute the noscapine gene cluster in Saccharomyces cerevisiae to achieve the microbial production of noscapine and related pathway intermediates, complementing and extending previous in planta and in vitro investigations. Our work provides structural validation of the secoberberine intermediates and the description of the narcotoline-4'-O-methyltransferase, suggesting this activity is catalysed by a unique heterodimer. We also reconstitute a 14-step biosynthetic pathway of noscapine from the simple alkaloid norlaudanosoline by engineering a yeast strain expressing 16 heterologous plant enzymes, achieving reconstitution of a complex plant pathway in a microbial host. Other engineered yeasts produce previously inaccessible pathway intermediates and a novel derivative, thereby advancing protoberberine and noscapine related drug discovery.


Assuntos
Antineoplásicos/metabolismo , Bioengenharia/métodos , Vias Biossintéticas , Noscapina/metabolismo , Papaver/genética , Saccharomyces cerevisiae , Alcaloides de Berberina , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Descoberta de Drogas/métodos , Metiltransferases/metabolismo , Família Multigênica , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tetra-Hidropapaverolina/metabolismo
15.
Nucleic Acids Res ; 44(7): 2987-99, 2016 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-26969733

RESUMO

The past decade of synthetic biology research has witnessed numerous advances in the development of tools and frameworks for the design and characterization of biological systems. Researchers have focused on the use of RNA for gene expression control due to its versatility in sensing molecular ligands and the relative ease by which RNA can be modeled and designed compared to proteins. We review the recent progress in the field with respect to RNA-based genetic devices that are controlled through small molecule and protein interactions. We discuss new approaches for generating and characterizing these devices and their underlying components. We also highlight immediate challenges, future directions and recent applications of synthetic RNA devices in engineered biological systems.


Assuntos
Regulação da Expressão Gênica , Engenharia Genética/métodos , Sequências Reguladoras de Ácido Ribonucleico , Aptâmeros de Nucleotídeos , Simulação por Computador , Engenharia Metabólica/métodos , Riboswitch , Técnica de Seleção de Aptâmeros
16.
J Biol Eng ; 9: 21, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26594238

RESUMO

BACKGROUND: The cell cycle plays a key role in human health and disease, including development and cancer. The ability to easily and reversibly control the mammalian cell cycle could mean improved cellular reprogramming, better tools for studying cancer, more efficient gene therapy, and improved heterologous protein production for medical or industrial applications. RESULTS: We engineered RNA-based control devices to provide specific and modular control of gene expression in response to exogenous inputs in living cells. Specifically, we identified key regulatory nodes that arrest U2-OS cells in the G0/1 or G2/M phases of the cycle. We then optimized the most promising key regulators and showed that, when these optimized regulators are placed under the control of a ribozyme switch, we can inducibly and reversibly arrest up to ~80 % of a cellular population in a chosen phase of the cell cycle. Characterization of the reliability of the final cell cycle controllers revealed that the G0/1 control device functions reproducibly over multiple experiments over several weeks. CONCLUSIONS: To our knowledge, this is the first time synthetic RNA devices have been used to control the mammalian cell cycle. This RNA platform represents a general class of synthetic biology tools for modular, dynamic, and multi-output control over mammalian cells.

17.
Microb Cell Fact ; 14: 144, 2015 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-26376732

RESUMO

BACKGROUND: Protoberberine alkaloids are bioactive molecules abundant in plant preparations for traditional medicines. Yeast engineered to express biosynthetic pathways for fermentative production of these compounds will further enable investigation of the medicinal properties of these molecules and development of alkaloid-based drugs with improved efficacy and safety. Here, we describe the optimization of a biosynthetic pathway in Saccharomyces cerevisiae for conversion of rac-norlaudanosoline to the protoberberine alkaloid (S)-canadine. RESULTS: This yeast strain is engineered to express seven heterologous enzymes, resulting in protoberberine alkaloid production from a simple benzylisoquinoline alkaloid precursor. The seven enzymes include three membrane-bound enzymes: the flavin-dependent oxidase berberine bridge enzyme, the cytochrome P450 canadine synthase, and a cytochrome P450 reductase. A number of strategies were implemented to improve flux through the pathway, including enzyme variant screening, genetic copy number variation, and culture optimization, that led to an over 70-fold increase in canadine titer up to 1.8 mg/L. Increased canadine titers enable extension of the pathway to produce berberine, a major constituent of several traditional medicines, for the first time in a microbial host. We also demonstrate that this strain is viable at pilot scale. CONCLUSIONS: By applying metabolic engineering and synthetic biology strategies for increased conversion of simple benzylisoquinoline alkaloids to complex protoberberine alkaloids, this work will facilitate chemoenzymatic synthesis or de novo biosynthesis of these and other high-value compounds using a microbial cell factory.


Assuntos
Alcaloides de Berberina/metabolismo , Berberina/análogos & derivados , Engenharia Metabólica/métodos , Saccharomyces cerevisiae/genética , Técnicas de Cultura Celular por Lotes , Berberina/química , Berberina/metabolismo , Alcaloides de Berberina/química , Reatores Biológicos , Vias Biossintéticas , Sistema Enzimático do Citocromo P-450/metabolismo , Fermentação , Dosagem de Genes , Projetos Piloto , Saccharomyces cerevisiae/metabolismo , Tetra-Hidropapaverolina/química , Tetra-Hidropapaverolina/metabolismo
18.
ACS Chem Biol ; 10(11): 2463-2467, 2015 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-26359915

RESUMO

Molecular tools for controlling gene expression are essential for manipulating biological systems. One class of tools includes RNA switches that incorporate RNA-based sensors, known as aptamers. However, most switches reported to date are responsive to toxic molecules or to endogenous metabolites. For effective conditional control, switches must incorporate RNA aptamers that exhibit selectivity against such endogenous metabolites. We report a systematic approach which combines a rapid in vitro assay and an in silico model to support an efficient, streamlined application of aptamers into RNA switches. Model predictions were validated in vivo and demonstrate that the RNA switches enable selective and programmable gene regulation. We demonstrate the method using aptamers that bind the FDA-approved small molecule (6R)-folinic acid, providing access to new molecular targets for gene expression control and much-needed clinically relevant tools for advancing RNA-based therapeutics.


Assuntos
Aptâmeros de Nucleotídeos/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Bioensaio , Leucovorina/química , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/genética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
19.
Nat Methods ; 12(10): 989-94, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26258292

RESUMO

Methods for rapidly assessing sequence-structure-function landscapes and developing conditional gene-regulatory devices are critical to our ability to manipulate and interface with biology. We describe a framework for engineering RNA devices from preexisting aptamers that exhibit ligand-responsive ribozyme tertiary interactions. Our methodology utilizes cell sorting, high-throughput sequencing and statistical data analyses to enable parallel measurements of the activities of hundreds of thousands of sequences from RNA device libraries in the absence and presence of ligands. Our tertiary-interaction RNA devices performed better in terms of gene silencing, activation ratio and ligand sensitivity than optimized RNA devices that rely on secondary-structure changes. We applied our method to build biosensors for diverse ligands and determine consensus sequences that enable ligand-responsive tertiary interactions. These methods advance our ability to develop broadly applicable genetic tools and to elucidate the underlying sequence-structure-function relationships that empower rational design of complex biomolecules.


Assuntos
Aptâmeros de Nucleotídeos/química , Engenharia Genética/métodos , Riboswitch/genética , Aptâmeros de Nucleotídeos/metabolismo , Técnicas Biossensoriais , Interpretação Estatística de Dados , Citometria de Fluxo/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Biblioteca Gênica , Proteínas de Fluorescência Verde/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Ligantes , Nepovirus/genética , RNA Catalítico/química , Ressonância de Plasmônio de Superfície , Teofilina/metabolismo , Teofilina/farmacologia
20.
Science ; 349(6252): 1095-100, 2015 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-26272907

RESUMO

Opioids are the primary drugs used in Western medicine for pain management and palliative care. Farming of opium poppies remains the sole source of these essential medicines, despite diverse market demands and uncertainty in crop yields due to weather, climate change, and pests. We engineered yeast to produce the selected opioid compounds thebaine and hydrocodone starting from sugar. All work was conducted in a laboratory that is permitted and secured for work with controlled substances. We combined enzyme discovery, enzyme engineering, and pathway and strain optimization to realize full opiate biosynthesis in yeast. The resulting opioid biosynthesis strains required the expression of 21 (thebaine) and 23 (hydrocodone) enzyme activities from plants, mammals, bacteria, and yeast itself. This is a proof of principle, and major hurdles remain before optimization and scale-up could be achieved. Open discussions of options for governing this technology are also needed in order to responsibly realize alternative supplies for these medically relevant compounds.


Assuntos
Engenharia Genética/métodos , Hidrocodona/metabolismo , Saccharomyces cerevisiae/enzimologia , Tebaína/metabolismo , Animais , Benzilisoquinolinas/metabolismo , Vias Biossintéticas/genética , Metabolismo dos Carboidratos , Codeína/metabolismo , Hidrocodona/química , Morfinanos/química , Morfinanos/metabolismo , Papaver/enzimologia , Papaver/genética , Tebaína/química
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