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1.
Elife ; 102021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34523419

RESUMO

Temporal dynamics of gene expression underpin responses to internal and environmental stimuli. In eukaryotes, regulation of gene induction includes changing chromatin states at target genes and recruiting the transcriptional machinery that includes transcription factors. As one of the most potent defense compounds in Arabidopsis thaliana, camalexin can be rapidly induced by bacterial and fungal infections. Though several transcription factors controlling camalexin biosynthesis genes have been characterized, how the rapid activation of genes in this pathway upon a pathogen signal is enabled remains unknown. By combining publicly available epigenomic data with in vivo chromatin modification mapping, we found that camalexin biosynthesis genes are marked with two epigenetic modifications with opposite effects on gene expression, trimethylation of lysine 27 of histone 3 (H3K27me3) (repression) and acetylation of lysine 18 of histone 3 (H3K18ac) (activation), to form a previously uncharacterized type of bivalent chromatin. Mutants with reduced H3K27me3 or H3K18ac suggested that both modifications were required to determine the timing of gene expression and metabolite accumulation at an early stage of the stress response. Our study indicates that the H3K27me3-H3K18ac bivalent chromatin, which we name as kairostat, plays an important role in controlling the timely induction of gene expression upon stress stimuli in plants.

2.
Proc Natl Acad Sci U S A ; 118(25)2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34140414

RESUMO

Microbial biosynthesis of plant natural products (PNPs) can facilitate access to valuable medicinal compounds and derivatives. Such efforts are challenged by metabolite transport limitations, which arise when complex plant pathways distributed across organelles and tissues are reconstructed in unicellular hosts without concomitant transport machinery. We recently reported an engineered yeast platform for production of the tropane alkaloid (TA) drugs hyoscyamine and scopolamine, in which product accumulation is limited by vacuolar transport. Here, we demonstrate that alleviation of transport limitations at multiple steps in an engineered pathway enables increased production of TAs and screening of useful derivatives. We first show that supervised classifier models trained on a tissue-delineated transcriptome from the TA-producing plant Atropa belladonna can predict TA transporters with greater efficacy than conventional regression- and clustering-based approaches. We demonstrate that two of the identified transporters, AbPUP1 and AbLP1, increase TA production in engineered yeast by facilitating vacuolar export and cellular reuptake of littorine and hyoscyamine. We incorporate four different plant transporters, cofactor regeneration mechanisms, and optimized growth conditions into our yeast platform to achieve improvements in de novo hyoscyamine and scopolamine production of over 100-fold (480 µg/L) and 7-fold (172 µg/L). Finally, we leverage computational tools for biosynthetic pathway prediction to produce two different classes of TA derivatives, nortropane alkaloids and tropane N-oxides, from simple precursors. Our work highlights the importance of cellular transport optimization in recapitulating complex PNP biosyntheses in microbial hosts and illustrates the utility of computational methods for gene discovery and expansion of heterologous biosynthetic diversity.

3.
Elife ; 102021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33860764

RESUMO

Ribozyme switches are a class of RNA-encoded genetic switch that support conditional regulation of gene expression across diverse organisms. An improved elucidation of the relationships between sequence, structure, and activity can improve our capacity for de novo rational design of ribozyme switches. Here, we generated data on the activity of hundreds of thousands of ribozyme sequences. Using automated structural analysis and machine learning, we leveraged these large data sets to develop predictive models that estimate the in vivo gene-regulatory activity of a ribozyme sequence. These models supported the de novo design of ribozyme libraries with low mean basal gene-regulatory activities and new ribozyme switches that exhibit changes in gene-regulatory activity in the presence of a target ligand, producing functional switches for four out of five aptamers. Our work examines how biases in the model and the data set that affect prediction accuracy can arise and demonstrates that machine learning can be applied to RNA sequences to predict gene-regulatory activity, providing the basis for design tools for functional RNAs.


Assuntos
Aptâmeros de Nucleotídeos/genética , Regulação da Expressão Gênica , Aprendizado de Máquina , Modelos Genéticos , Redes Neurais de Computação , RNA Catalítico/genética , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , Desenho Assistido por Computador , Escherichia coli/enzimologia , Escherichia coli/genética , Sequenciamento de Nucleotídeos em Larga Escala , Ligantes , Conformação de Ácido Nucleico , RNA Catalítico/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Análise de Sequência de RNA , Relação Estrutura-Atividade
4.
Nat Commun ; 12(1): 1437, 2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33664255

RESUMO

Biosensors are key components in engineered biological systems, providing a means of measuring and acting upon the large biochemical space in living cells. However, generating small molecule sensing elements and integrating them into in vivo biosensors have been challenging. Here, using aptamer-coupled ribozyme libraries and a ribozyme regeneration method, de novo rapid in vitro evolution of RNA biosensors (DRIVER) enables multiplexed discovery of biosensors. With DRIVER and high-throughput characterization (CleaveSeq) fully automated on liquid-handling systems, we identify and validate biosensors against six small molecules, including five for which no aptamers were previously found. DRIVER-evolved biosensors are applied directly to regulate gene expression in yeast, displaying activation ratios up to 33-fold. DRIVER biosensors are also applied in detecting metabolite production from a multi-enzyme biosynthetic pathway. This work demonstrates DRIVER as a scalable pipeline for engineering de novo biosensors with wide-ranging applications in biomanufacturing, diagnostics, therapeutics, and synthetic biology.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , RNA Catalítico/química , Biologia Sintética/métodos , Expressão Gênica/genética , Proteínas de Fluorescência Verde/metabolismo , Engenharia Metabólica/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
5.
Nat Commun ; 12(1): 1579, 2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33707425

RESUMO

Random mutagenesis is a technique used to generate diversity and engineer biological systems. In vivo random mutagenesis generates diversity directly in a host organism, enabling applications such as lineage tracing, continuous evolution, and protein engineering. Here we describe TRIDENT (TaRgeted In vivo Diversification ENabled by T7 RNAP), a platform for targeted, continual, and inducible diversification at genes of interest at mutation rates one-million fold higher than natural genomic error rates. TRIDENT targets mutagenic enzymes to precise genetic loci by fusion to T7 RNA polymerase, resulting in mutation windows following a mutation targeting T7 promoter. Mutational diversity is tuned by DNA repair factors localized to sites of deaminase-driven mutation, enabling sustained mutation of all four DNA nucleotides at rates greater than 10-4 mutations per bp. We show TRIDENT can be applied to routine in vivo mutagenesis applications by evolving a red-shifted fluorescent protein and drug-resistant mutants of an essential enzyme.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Evolução Molecular Direcionada/métodos , Farmacorresistência Fúngica/genética , Engenharia de Proteínas/métodos , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas Virais/metabolismo , Antifúngicos/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Mutagênese/genética , Regiões Promotoras Genéticas/genética , Pirimetamina/farmacologia
6.
Nat Commun ; 12(1): 1760, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33741955

RESUMO

Plant natural products (PNPs) and their derivatives are important but underexplored sources of pharmaceutical molecules. To access this untapped potential, the reconstitution of heterologous PNP biosynthesis pathways in engineered microbes provides a valuable starting point to explore and produce novel PNP derivatives. Here, we introduce a computational workflow to systematically screen the biochemical vicinity of a biosynthetic pathway for pharmaceutical compounds that could be produced by derivatizing pathway intermediates. We apply our workflow to the biosynthetic pathway of noscapine, a benzylisoquinoline alkaloid (BIA) with a long history of medicinal use. Our workflow identifies pathways and enzyme candidates for the production of (S)-tetrahydropalmatine, a known analgesic and anxiolytic, and three additional derivatives. We then construct pathways for these compounds in yeast, resulting in platforms for de novo biosynthesis of BIA derivatives and demonstrating the value of cheminformatic tools to predict reactions, pathways, and enzymes in synthetic biology and metabolic engineering.


Assuntos
Produtos Biológicos/metabolismo , Vias Biossintéticas/genética , Biologia Computacional/métodos , Engenharia Metabólica/métodos , Noscapina/metabolismo , Saccharomyces cerevisiae/metabolismo , Alcaloides/biossíntese , Benzilisoquinolinas/metabolismo , Noscapina/química , Plantas/genética , Plantas/metabolismo , Saccharomyces cerevisiae/genética , Software
7.
Sci Adv ; 6(44)2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33127687

RESUMO

Chalcone synthase (CHS) canonically catalyzes carbon-carbon bond formation through iterative decarboxylative Claisen condensation. Here, we characterize a previously unidentified biosynthetic capability of SlCHS to catalyze nitrogen-carbon bond formation, leading to the production of a hydroxycinnamic acid amide (HCAA) compound. By expressing a putative tomato (Solanum lycopersicum) gene cluster in yeast (Saccharomyces cerevisiae), we elucidate the activity of a pathway consisting of a carboxyl methyltransferase (SlMT2), which methylates the yeast primary metabolite 3-hydroxyanthranilic acid (3-HAA) to form a methyl ester, and a SlCHS, which catalyzes the condensation of 3-HAA methyl ester and p-coumaroyl-coenzyme A (CoA) through formation of an amide bond. We demonstrate that this aminoacylation activity could be a common secondary activity in plant CHSs by validating the activity in vitro with variants from S. lycopersicum and Arabidopsis thaliana Our work demonstrates yeast as a platform for characterizing putative plant gene clusters with the potential for compound structure and enzymatic activity discovery.

8.
Nature ; 585(7826): 614-619, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32879484

RESUMO

Tropane alkaloids from nightshade plants are neurotransmitter inhibitors that are used for treating neuromuscular disorders and are classified as essential medicines by the World Health Organization1,2. Challenges in global supplies have resulted in frequent shortages of these drugs3,4. Further vulnerabilities in supply chains have been revealed by events such as the Australian wildfires5 and the COVID-19 pandemic6. Rapidly deployable production strategies that are robust to environmental and socioeconomic upheaval7,8 are needed. Here we engineered baker's yeast to produce the medicinal alkaloids hyoscyamine and scopolamine, starting from simple sugars and amino acids. We combined functional genomics to identify a missing pathway enzyme, protein engineering to enable the functional expression of an acyltransferase via trafficking to the vacuole, heterologous transporters to facilitate intracellular routing, and strain optimization to improve titres. Our integrated system positions more than twenty proteins adapted from yeast, bacteria, plants and animals across six sub-cellular locations to recapitulate the spatial organization of tropane alkaloid biosynthesis in plants. Microbial biosynthesis platforms can facilitate the discovery of tropane alkaloid derivatives as new therapeutic agents for neurological disease and, once scaled, enable robust and agile supply of these essential medicines.


Assuntos
Alcaloides/biossíntese , Alcaloides/provisão & distribuição , Hiosciamina/biossíntese , Saccharomyces cerevisiae/metabolismo , Escopolamina/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Animais , Atropa belladonna/enzimologia , Derivados da Atropina/metabolismo , Transporte Biológico , Datura/enzimologia , Glucosídeos/biossíntese , Glucosídeos/metabolismo , Hiosciamina/provisão & distribuição , Lactatos/metabolismo , Ligases/genética , Ligases/metabolismo , Modelos Moleculares , Doenças do Sistema Nervoso/tratamento farmacológico , Oxirredutases/genética , Oxirredutases/metabolismo , Engenharia de Proteínas , Saccharomyces cerevisiae/genética , Escopolamina/provisão & distribuição , Vacúolos/metabolismo
9.
Nat Commun ; 11(1): 2113, 2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32355169

RESUMO

Promoters play a central role in controlling gene regulation; however, a small set of promoters is used for most genetic construct design in the yeast Saccharomyces cerevisiae. Generating and utilizing models that accurately predict protein expression from promoter sequences would enable rapid generation of useful promoters and facilitate synthetic biology efforts in this model organism. We measure the gene expression activity of over 675,000 sequences in a constitutive promoter library and over 327,000 sequences in an inducible promoter library. Training an ensemble of convolutional neural networks jointly on the two data sets enables very high (R2 > 0.79) predictive accuracies on multiple sequence-activity prediction tasks. We describe model-guided design strategies that yield large, sequence-diverse sets of promoters exhibiting activities higher than those represented in training data and similar to current best-in-class sequences. Our results show the value of model-guided design as an approach for generating useful DNA parts.


Assuntos
Regulação Fúngica da Expressão Gênica , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , Separação Celular , Simulação por Computador , Citometria de Fluxo , Proteínas Fúngicas/genética , Biblioteca Gênica , Aprendizado de Máquina , Redes Neurais de Computação , Biologia Sintética/métodos
10.
Nat Commun ; 10(1): 4327, 2019 09 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.


Assuntos
Mamíferos/genética , RNA Catalítico/química , RNA-Seq/métodos , Biologia Sintética/métodos , Animais , Redes Reguladoras de Genes , Engenharia Genética , Células HEK293 , Humanos , Motivos de Nucleotídeos , RNA Catalítico/metabolismo , RNA Catalítico/fisiologia
11.
Nat Commun ; 10(1): 3634, 2019 08 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.


Assuntos
Reatores Biológicos/microbiologia , Engenharia Metabólica/métodos , Compostos Fitoquímicos/biossíntese , Saccharomyces cerevisiae/metabolismo , Tropanos/metabolismo , Saccharomyces cerevisiae/genética , Solanaceae/metabolismo , Alcaloides de Solanáceas/biossíntese
12.
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
13.
Metab Eng Commun ; 9: e00092, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31110942

RESUMO

Cyanogenic glycosides are defense compounds found in a wide range of plant species, including many crops. We demonstrate that the cyanogenic glucoside dhurrin, naturally found in sorghum, can be produced at high titers in Saccharomyces cerevisiae, constituting the first report of cyanogenic glycoside production in a microbe. Genetic modifications to increase the supply of the dhurrin precursor tyrosine enabled dhurrin production in excess of 80 mg/L. The dhurrin-producing yeast strain was used as a chassis to investigate previously uncharacterized enzymes identified close to the biosynthetic gene cluster containing the dhurrin pathway enzymes. This work shows the potential of heterologous expression in yeast to facilitate investigations of plant cyanogenic glycoside pathways.

14.
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
15.
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
16.
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.


Assuntos
RNA/análise , Biologia Sintética/métodos , Sítio Alostérico , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Técnicas Biossensoriais , Escherichia coli/metabolismo , Firmicutes/metabolismo , Expressão Gênica , Biblioteca Gênica , Ligantes , Mycobacterium smegmatis/metabolismo , Conformação Proteica , Engenharia de Proteínas , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RNA/genética , Reprodutibilidade dos Testes
17.
Nat Prod Rep ; 35(9): 902-920, 2018 09 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.


Assuntos
Bactérias/metabolismo , Compostos Fitoquímicos/metabolismo , Plantas/metabolismo , Biologia Sintética/métodos , Leveduras/metabolismo , Bactérias/genética , Técnicas Biossensoriais , Técnicas de Cocultura , Enzimas/genética , Enzimas/metabolismo , Regulação da Expressão Gênica de Plantas , Microrganismos Geneticamente Modificados , Compostos Fitoquímicos/química , Compostos Fitoquímicos/genética , Plantas/genética , Leveduras/genética
18.
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
19.
Nat Chem ; 10(4): 395-404, 2018 04.
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.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Escherichia coli/enzimologia , Compostos Fitoquímicos/biossíntese , Saccharomyces cerevisiae/enzimologia , Sistema Enzimático do Citocromo P-450/genética , Engenharia Metabólica , Compostos Fitoquímicos/química
20.
Nucleic Acids Res ; 46(3): 1541-1552, 2018 02 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.


Assuntos
Proliferação de Células/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Subunidade gama Comum de Receptores de Interleucina/genética , Subunidade beta de Receptor de Interleucina-2/genética , Leucovorina/farmacologia , MicroRNAs/farmacologia , Linfócitos T/efeitos dos fármacos , Animais , Aptâmeros de Nucleotídeos/síntese química , Aptâmeros de Nucleotídeos/metabolismo , Aptâmeros de Nucleotídeos/farmacologia , Pareamento de Bases , Sequência de Bases , Linhagem Celular , Proliferação de Células/genética , Citocinas/genética , Citocinas/imunologia , Relação Dose-Resposta Imunológica , Regulação da Expressão Gênica , Subunidade gama Comum de Receptores de Interleucina/antagonistas & inibidores , Subunidade gama Comum de Receptores de Interleucina/imunologia , Subunidade beta de Receptor de Interleucina-2/antagonistas & inibidores , Subunidade beta de Receptor de Interleucina-2/imunologia , Camundongos , MicroRNAs/síntese química , MicroRNAs/metabolismo , Conformação de Ácido Nucleico , Plasmídeos/química , Plasmídeos/metabolismo , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/imunologia , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/imunologia , Transfecção
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