RESUMO
CRISPR technologies have begun to revolutionize T cell therapies; however, conventional CRISPR-Cas9 genome-editing tools are limited in their safety, efficacy, and scope. To address these challenges, we developed multiplexed effector guide arrays (MEGA), a platform for programmable and scalable regulation of the T cell transcriptome using the RNA-guided, RNA-targeting activity of CRISPR-Cas13d. MEGA enables quantitative, reversible, and massively multiplexed gene knockdown in primary human T cells without targeting or cutting genomic DNA. Applying MEGA to a model of CAR T cell exhaustion, we robustly suppressed inhibitory receptor upregulation and uncovered paired regulators of T cell function through combinatorial CRISPR screening. We additionally implemented druggable regulation of MEGA to control CAR activation in a receptor-independent manner. Lastly, MEGA enabled multiplexed disruption of immunoregulatory metabolic pathways to enhance CAR T cell fitness and anti-tumor activity in vitro and in vivo. MEGA offers a versatile synthetic toolkit for applications in cancer immunotherapy and beyond.
Assuntos
Engenharia Metabólica , Linfócitos T , Humanos , Perfilação da Expressão Gênica , Engenharia Metabólica/métodos , RNA , TranscriptomaRESUMO
Synthetic multicellular systems hold promise as models for understanding natural development of biofilms and higher organisms and as tools for engineering complex multi-component metabolic pathways and materials. However, such efforts require tools to adhere cells into defined morphologies and patterns, and these tools are currently lacking. Here, we report a 100% genetically encoded synthetic platform for modular cell-cell adhesion in Escherichia coli, which provides control over multicellular self-assembly. Adhesive selectivity is provided by a library of outer membrane-displayed nanobodies and antigens with orthogonal intra-library specificities, while affinity is controlled by intrinsic adhesin affinity, competitive inhibition, and inducible expression. We demonstrate the resulting capabilities for quantitative rational design of well-defined morphologies and patterns through homophilic and heterophilic interactions, lattice-like self-assembly, phase separation, differential adhesion, and sequential layering. Compatible with synthetic biology standards, this adhesion toolbox will enable construction of high-level multicellular designs and shed light on the evolutionary transition to multicellularity.
Assuntos
Adesão Celular/fisiologia , Engenharia Metabólica/métodos , Biologia Sintética/métodos , Fenômenos Fisiológicos Bacterianos , Evolução Biológica , Adesão Celular/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Escherichia coli/genética , Biblioteca Gênica , Redes e Vias Metabólicas , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/fisiologiaRESUMO
QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans1,2. However, owing to the complex structure of QS-21, its availability is limited. Today, the supply depends on laborious extraction from the Chilean soapbark tree or on low-yielding total chemical synthesis3,4. Here we demonstrate the complete biosynthesis of QS-21 and its precursors, as well as structural derivatives, in engineered yeast strains. The successful biosynthesis in yeast requires fine-tuning of the host's native pathway fluxes, as well as the functional and balanced expression of 38 heterologous enzymes. The required biosynthetic pathway spans seven enzyme families-a terpene synthase, P450s, nucleotide sugar synthases, glycosyltransferases, a coenzyme A ligase, acyl transferases and polyketide synthases-from six organisms, and mimics in yeast the subcellular compartmentalization of plants from the endoplasmic reticulum membrane to the cytosol. Finally, by taking advantage of the promiscuity of certain pathway enzymes, we produced structural analogues of QS-21 using this biosynthetic platform. This microbial production scheme will allow for the future establishment of a structure-activity relationship, and will thus enable the rational design of potent vaccine adjuvants.
Assuntos
Adjuvantes Imunológicos , Engenharia Metabólica , Saccharomyces cerevisiae , Saponinas , Adjuvantes Imunológicos/biossíntese , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/genética , Adjuvantes Imunológicos/metabolismo , Vias Biossintéticas/genética , Desenho de Fármacos , Enzimas/genética , Enzimas/metabolismo , Engenharia Metabólica/métodos , Plantas/enzimologia , Plantas/genética , Plantas/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saponinas/biossíntese , Saponinas/química , Saponinas/genética , Saponinas/metabolismo , Relação Estrutura-AtividadeRESUMO
Monoterpene indole alkaloids (MIAs) are a diverse family of complex plant secondary metabolites with many medicinal properties, including the essential anti-cancer therapeutics vinblastine and vincristine1. As MIAs are difficult to chemically synthesize, the world's supply chain for vinblastine relies on low-yielding extraction and purification of the precursors vindoline and catharanthine from the plant Catharanthus roseus, which is then followed by simple in vitro chemical coupling and reduction to form vinblastine at an industrial scale2,3. Here, we demonstrate the de novo microbial biosynthesis of vindoline and catharanthine using a highly engineered yeast, and in vitro chemical coupling to vinblastine. The study showcases a very long biosynthetic pathway refactored into a microbial cell factory, including 30 enzymatic steps beyond the yeast native metabolites geranyl pyrophosphate and tryptophan to catharanthine and vindoline. In total, 56 genetic edits were performed, including expression of 34 heterologous genes from plants, as well as deletions, knock-downs and overexpression of ten yeast genes to improve precursor supplies towards de novo production of catharanthine and vindoline, from which semisynthesis to vinblastine occurs. As the vinblastine pathway is one of the longest MIA biosynthetic pathways, this study positions yeast as a scalable platform to produce more than 3,000 natural MIAs and a virtually infinite number of new-to-nature analogues.
Assuntos
Antineoplásicos , Reatores Biológicos , Vias Biossintéticas , Engenharia Metabólica , Saccharomyces cerevisiae , Vimblastina , Alcaloides de Vinca , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/provisão & distribuição , Catharanthus/química , Genes Fúngicos , Genes de Plantas , Engenharia Metabólica/métodos , Fosfatos de Poli-Isoprenil , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Triptofano , Vimblastina/biossíntese , Vimblastina/química , Vimblastina/provisão & distribuição , Alcaloides de Vinca/biossíntese , Alcaloides de Vinca/química , Alcaloides de Vinca/provisão & distribuiçãoRESUMO
Orcinol glucoside (OG), mainly found in the rhizome of the traditional Chinese herb Curculigo orchioides Gaertn, is noted for its antidepressant effects. In this study, an efficient screening pipeline was established for identifying the highly active orcinol synthase (ORS) and UDP-dependent glycosyltransferase (UGT) involved in the biosynthesis of OG by combining transcriptome analysis, structure-based virtual screening, and in vitro enzyme activity assays. By enhancing the downstream pathway, metabolic engineering and fermentation optimization, the OG production in Yarrowia lipolytica was improved 100-fold, resulting in a final yield of 43.46 g/L (0.84 g/g DCW), which is almost 6,400-fold higher than the extraction yield from C. orchioides roots. This study provides a reference for rapid identification of functional genes and high-yield production of natural products.
Assuntos
Glucosídeos , Yarrowia , Glucosídeos/metabolismo , Yarrowia/genética , Engenharia Metabólica/métodos , FermentaçãoRESUMO
Tackling climate change challenges requires replacing current chemical industrial processes through the rational and sustainable use of biodiversity resources. To that end, production routes to key bio-based chemicals for the bioeconomy have been identified. However, their production still remains inefficient in terms of titers, rates, and yields; because of the hurdles found when scaling up. In order to make production more efficient, strategies like automated screening and dynamic pathway regulation through biosensors have been applied as part of strain optimization. However, to date, no systematic way exists to design a genetic circuit that is responsive to concentrations of a given target compound. Here, the DetSpace web server provides a set of integrated tools that allows a user to select and design a biological circuit that performs the sensing of a molecule of interest by its enzymatic conversion to a detectable molecule through a transcription factor. In that way, the DetSpace web server allows synthetic biologists to easily design biosensing routes for the dynamic regulation of metabolic pathways in applications ranging from genetic circuits design, screening, production, and bioremediation of bio-based chemicals, to diagnostics and drug delivery.
Assuntos
Internet , Engenharia Metabólica , Software , Engenharia Metabólica/métodos , Biologia Sintética/métodos , Redes e Vias Metabólicas/genética , Técnicas Biossensoriais , Redes Reguladoras de Genes , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
Methanol is an ideal feedstock for chemical and biological manufacturing. Constructing an efficient cell factory is essential for producing complex compounds through methanol biotransformation, in which coordinating methanol use and product synthesis is often necessary. In methylotrophic yeast, methanol utilization mainly occurs in peroxisomes, which creates challenges in driving the metabolic flux toward product biosynthesis. Here, we observed that constructing the cytosolic biosynthesis pathway resulted in compromised fatty alcohol production in the methylotrophic yeast Ogataea polymorpha. Alternatively, peroxisomal coupling of fatty alcohol biosynthesis and methanol utilization significantly improved fatty alcohol production by 3.9-fold. Enhancing the supply of precursor fatty acyl-CoA and cofactor NADPH in the peroxisomes by global metabolic rewiring further improved fatty alcohol production by 2.5-fold and produced 3.6 g/L fatty alcohols from methanol under fed-batch fermentation. We demonstrated that peroxisome compartmentalization is helpful for coupling methanol utilization and product synthesis, and with this approach, constructing efficient microbial cell factories for methanol biotransformation is feasible.
Assuntos
Álcoois Graxos , Metanol , Álcoois Graxos/metabolismo , Metanol/metabolismo , Peroxissomos/metabolismo , Fermentação , Engenharia Metabólica/métodosRESUMO
Cofactor imbalance obstructs the productivities of metabolically engineered cells. Herein, we employed a minimally perturbing system, xylose reductase and lactose (XR/lactose), to increase the levels of a pool of sugar phosphates which are connected to the biosynthesis of NAD(P)H, FAD, FMN, and ATP in Escherichia coli. The XR/lactose system could increase the amounts of the precursors of these cofactors and was tested with three different metabolically engineered cell systems (fatty alcohol biosynthesis, bioluminescence light generation, and alkane biosynthesis) with different cofactor demands. Productivities of these cells were increased 2-4-fold by the XR/lactose system. Untargeted metabolomic analysis revealed different metabolite patterns among these cells, demonstrating that only metabolites involved in relevant cofactor biosynthesis were altered. The results were also confirmed by transcriptomic analysis. Another sugar reducing system (glucose dehydrogenase) could also be used to increase fatty alcohol production but resulted in less yield enhancement than XR. This work demonstrates that the approach of increasing cellular sugar phosphates can be a generic tool to increase in vivo cofactor generation upon cellular demand for synthetic biology.
Assuntos
Engenharia Metabólica , Redes e Vias Metabólicas , Aldeído Redutase/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Álcoois Graxos/metabolismo , Fermentação , Lactose/metabolismo , Engenharia Metabólica/métodos , Fosfatos Açúcares/metabolismo , Xilose/metabolismoRESUMO
The production efficiency of microbial cell factories is sometimes limited by the lack of effective methods to regulate multiple targets in a coordinated manner. Here taking the biosynthesis of glucosamine-6-phosphate (GlcN6P) in Bacillus subtilis as an example, a 'design-build-test-learn' framework was proposed to achieve efficient multiplexed optimization of metabolic pathways. A platform strain was built to carry biosensor signal-amplifying circuits and two genetic regulation circuits. Then, a synthetic CRISPR RNA array blend for boosting and leading (ScrABBLE) device was integrated into the platform strain, which generated 5,184 combinatorial assemblies targeting three genes. The best GlcN6P producer was screened and engineered for the synthesis of valuable pharmaceuticals N-acetylglucosamine and N-acetylmannosamine. The N-acetylglucosamine titer reached 183.9 g liter-1 in a 15-liter bioreactor. In addition, the potential generic application of the ScrABBLE device was also verified using three fluorescent proteins as a case study.
Assuntos
Acetilglucosamina , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Acetilglucosamina/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Redes e Vias Metabólicas , RNA/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Engenharia Metabólica/métodosRESUMO
Synthetic regulation of metabolic fluxes has emerged as a common strategy to improve the performance of microbial cell factories. The present regulatory toolboxes predominantly rely on the control and manipulation of carbon pathways. Nitrogen is an essential nutrient that plays a vital role in growth and metabolism. However, the availability of broadly applicable tools based on nitrogen pathways for metabolic regulation remains limited. In this work, we present a novel regulatory system that harnesses signals associated with nitrogen metabolism to redirect excess carbon flux in Bacillus licheniformis. By engineering the native transcription factor GlnR and incorporating a sorbitol-responsive element, we achieved a remarkable 99% inhibition of the expression of the green fluorescent protein reporter gene. Leveraging this system, we identified the optimal redirection point for the overflow carbon flux, resulting in a substantial 79.5% reduction in acetoin accumulation and a 2.6-fold increase in acetate production. This work highlight the significance of nitrogen metabolism in synthetic biology and its valuable contribution to metabolic engineering. Furthermore, our work paves the way for multidimensional metabolic regulation in future synthetic biology endeavors.
Assuntos
Bacillus licheniformis , Engenharia Metabólica , Sorbitol , Bacillus licheniformis/genética , Bacillus licheniformis/metabolismo , Carbono/metabolismo , Engenharia Metabólica/métodos , Nitrogênio/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sorbitol/metabolismoRESUMO
While there are several genome editing techniques available, few are suitable for dynamic and simultaneous mutagenesis of arbitrary targeted sequences in prokaryotes. Here, to address these limitations, we present a versatile and multiplex retron-mediated genome editing system (REGES). First, through systematic optimization of REGES, we achieve efficiency of â¼100%, 85 ± 3%, 69 ± 14% and 25 ± 14% for single-, double-, triple- and quadruple-locus genome editing, respectively. In addition, we employ REGES to generate pooled and barcoded variant libraries with degenerate RBS sequences to fine-tune the expression level of endogenous and exogenous genes, such as transcriptional factors to improve ethanol tolerance and biotin biosynthesis. Finally, we demonstrate REGES-mediated continuous in vivo protein evolution, by combining retron, polymerase-mediated base editing and error-prone transcription. By these case studies, we demonstrate REGES as a powerful multiplex genome editing and continuous evolution tool with broad applications in synthetic biology and metabolic engineering.
Assuntos
Escherichia coli , Edição de Genes , Edição de Genes/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Mutagênese , Sistemas CRISPR-Cas/genéticaRESUMO
The increase of CO2 emissions due to human activity is one of the preeminent reasons for the present climate crisis. In addition, considering the increasing demand for renewable resources, the upcycling of CO2 as a feedstock gains an extensive importance to establish CO2-neutral or CO2-negative industrial processes independent of agricultural resources. Here we assess whether synthetic autotrophic Komagataella phaffii (Pichia pastoris) can be used as a platform for value-added chemicals using CO2 as a feedstock by integrating the heterologous genes for lactic and itaconic acid synthesis. 13C labeling experiments proved that the resulting strains are able to produce organic acids via the assimilation of CO2 as a sole carbon source. Further engineering attempts to prevent the lactic acid consumption increased the titers to 600 mg L-1, while balancing the expression of key genes and modifying screening conditions led to 2 g L-1 itaconic acid. Bioreactor cultivations suggest that a fine-tuning on CO2 uptake and oxygen demand of the cells is essential to reach a higher productivity. We believe that through further metabolic and process engineering, the resulting engineered strain can become a promising host for the production of value-added bulk chemicals by microbial assimilation of CO2, to support sustainability of industrial bioprocesses.
Assuntos
Engenharia Metabólica , Pichia , Humanos , Pichia/metabolismo , Engenharia Metabólica/métodos , Dióxido de Carbono/metabolismo , Processos AutotróficosRESUMO
Proteins, as essential biomolecules, account for a large fraction of cell mass, and thus the synthesis of the complete set of proteins (i.e., the proteome) represents a substantial part of the cellular resource budget. Therefore, cells might be under selective pressures to optimize the resource costs for protein synthesis, particularly the biosynthesis of the 20 proteinogenic amino acids. Previous studies showed that less energetically costly amino acids are more abundant in the proteomes of bacteria that survive under energy-limited conditions, but the energy cost of synthesizing amino acids was reported to be weakly associated with the amino acid usage in Saccharomyces cerevisiae Here we present a modeling framework to estimate the protein cost of synthesizing each amino acid (i.e., the protein mass required for supporting one unit of amino acid biosynthetic flux) and the glucose cost (i.e., the glucose consumed per amino acid synthesized). We show that the logarithms of the relative abundances of amino acids in S. cerevisiae's proteome correlate well with the protein costs of synthesizing amino acids (Pearson's r = -0.89), which is better than that with the glucose costs (Pearson's r = -0.5). Therefore, we demonstrate that S. cerevisiae tends to minimize protein resource, rather than glucose or energy, for synthesizing amino acids.
Assuntos
Aminoácidos/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Evolução Biológica , Metabolismo Energético/fisiologia , Evolução Molecular , Engenharia Metabólica/métodos , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/fisiologia , Proteoma/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Heme is an oxygen carrier and a cofactor of both industrial enzymes and food additives. The intracellular level of free heme is low, which limits the synthesis of heme proteins. Therefore, increasing heme synthesis allows an increased production of heme proteins. Using the genome-scale metabolic model (GEM) Yeast8 for the yeast Saccharomyces cerevisiae, we identified fluxes potentially important to heme synthesis. With this model, in silico simulations highlighted 84 gene targets for balancing biomass and increasing heme production. Of those identified, 76 genes were individually deleted or overexpressed in experiments. Empirically, 40 genes individually increased heme production (up to threefold). Heme was increased by modifying target genes, which not only included the genes involved in heme biosynthesis, but also those involved in glycolysis, pyruvate, Fe-S clusters, glycine, and succinyl-coenzyme A (CoA) metabolism. Next, we developed an algorithmic method for predicting an optimal combination of these genes by using the enzyme-constrained extension of the Yeast8 model, ecYeast8. The computationally identified combination for enhanced heme production was evaluated using the heme ligand-binding biosensor (Heme-LBB). The positive targets were combined using CRISPR-Cas9 in the yeast strain (IMX581-HEM15-HEM14-HEM3-Δshm1-HEM2-Δhmx1-FET4-Δgcv2-HEM1-Δgcv1-HEM13), which produces 70-fold-higher levels of intracellular heme.
Assuntos
Heme , Engenharia Metabólica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Simulação por Computador , Heme/biossíntese , Heme/genética , Hemeproteínas/biossíntese , Hemeproteínas/genética , Engenharia Metabólica/métodos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Formate has great potential to function as a feedstock for biorefineries because it can be sustainably produced by a variety of processes that don't compete with agricultural production. However, naturally formatotrophic organisms are unsuitable for large-scale cultivation, difficult to engineer, or have inefficient native formate assimilation pathways. Thus, metabolic engineering needs to be developed for model industrial organisms to enable efficient formatotrophic growth. Here, we build a prototype synthetic formate utilizing bacterial microcompartment (sFUT) encapsulating the oxygen-sensitive glycyl radical enzyme pyruvate formate lyase and a phosphate acyltransferase to convert formate and acetyl-phosphate into the central biosynthetic intermediate pyruvate. This metabolic module offers a defined environment with a private cofactor coenzyme A that can cycle efficiently between the encapsulated enzymes. To facilitate initial design-build-test-refine cycles to construct an active metabolic core, we used a "wiffleball" architecture, defined as an icosahedral bacterial microcompartment (BMC) shell with unoccupied pentameric vertices to freely permit substrate and product exchange. The resulting sFUT prototype wiffleball is an active multi enzyme synthetic BMC functioning as platform technology.
Assuntos
Formiatos/metabolismo , Engenharia Metabólica/métodos , Ácido Pirúvico/metabolismo , Acetatos/química , Acetatos/metabolismo , Acetiltransferases , Bactérias/metabolismo , Compartimento Celular/fisiologia , Escherichia coli/genética , Formiatos/química , Ácido Pirúvico/química , Biologia Sintética/métodosRESUMO
SignificanceA gene regulatory system is an important tool for the engineering of biosynthetic pathways of organisms. Here, we report the development of an inducible-ON/OFF regulatory system using a malO operator as a key element. We identified and modulated sequence, position, numbers, and spacing distance of malO operators, generating a series of activating or repressive promoters with tunable strength. The stringency and robustness are both guaranteed in this system, a maximal induction factor of 790-fold was achieved, and nine proteins from different organisms were expressed with high yields. This system can be utilized as a gene switch, promoter enhancer, or metabolic valve in synthetic biology applications. This operator-based engineering strategy can be employed for developing similar regulatory systems in different microorganisms.
Assuntos
Bacillus subtilis , Vias Biossintéticas , Regulação Bacteriana da Expressão Gênica , Maltose , Engenharia Metabólica , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Vias Biossintéticas/genética , Elementos Facilitadores Genéticos , Expressão Gênica , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Maltose/metabolismo , Maltose/farmacologia , Engenharia Metabólica/métodos , Regiões Operadoras Genéticas , Regiões Promotoras Genéticas/genética , Biologia SintéticaRESUMO
Plants are the most sophisticated biofactories and sources of food and biofuels present in nature. By engineering plant metabolism, the production of desired compounds can be increased and the nutritional or commercial value of the plant species can be improved. However, this can be challenging because of the complexity of the regulation of multiple genes and the involvement of different protein interactions. To improve metabolic engineering (ME) capabilities, different tools and strategies for rerouting the metabolic pathways have been developed, including genome editing and transcriptional regulation approaches. In addition, cutting-edge technologies have provided new methods for understanding uncharacterized biosynthetic pathways, protein degradation mechanisms, protein-protein interactions, or allosteric feedback, enabling the design of novel ME approaches.
Assuntos
Sistemas CRISPR-Cas , Plantas , Plantas/genética , Plantas/metabolismo , Edição de Genes/métodos , Engenharia Metabólica/métodos , Redes e Vias Metabólicas , Genoma de PlantaRESUMO
Metabolic engineering reconfigures cellular networks to produce value-added compounds from renewable substrates efficiently. However, identifying strains with desired phenotypes from large libraries through rational or random mutagenesis remains challenging. To overcome this bottleneck, an effective high-throughput screening (HTS) method must be developed to detect and analyze target candidates rapidly. Salidroside is an aromatic compound with broad applications in food, healthcare, medicine, and daily chemicals. However, there currently needs to be HTS methods available to monitor salidroside levels or to screen enzyme variants and strains for high-yield salidroside biosynthesis, which severely limits the development of microbial cell factories capable of efficiently producing salidroside on an industrial scale. This study developed a gene-encoded whole-cell biosensor that is specifically responsive to salidroside. The biosensor was created by screening a site-saturated mutagenic library of uric acid response regulatory protein binding bags. This work demonstrates the feasibility of monitoring metabolic flux with whole-cell biosensors for critical metabolites. It provides a promising tool for building salidroside high-yielding strains for high-throughput screening and metabolic regulation to meet industrial needs.
Assuntos
Técnicas Biossensoriais , Glucosídeos , Ensaios de Triagem em Larga Escala , Engenharia Metabólica , Fenóis , Fenóis/metabolismo , Técnicas Biossensoriais/métodos , Glucosídeos/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Engenharia Metabólica/métodos , Escherichia coli/genética , Escherichia coli/metabolismoRESUMO
Among plant-derived secondary metabolites are benzylisoquinoline alkaloids (BIAs) that play a vital role in medicine. The most conspicuous BIAs frequently found in opium poppy are morphine, codeine, thebaine, papaverine, sanguinarine, and noscapine. BIAs have provided abundant clinically useful drugs used in the treatment of various diseases and ailments With an increasing demand for these herbal remedies, genetic improvement of poppy plants appears to be essential to live up to the expectations of the pharmaceutical industry. With the advent of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated9 (Cas9), the field of metabolic engineering has undergone a paradigm shift in its approach due to its appealing attributes, such as the transgene-free editing capability, precision, selectivity, robustness, and versatility. The potentiality of the CRISPR system for manipulating metabolic pathways in opium poppy was demonstrated, but further investigations regarding the use of CRISPR in BIA pathway engineering should be undertaken to develop opium poppy into a bioreactor synthesizing BIAs at the industrial-scale levels. In this regard, the recruitment of RNA-guided genome editing for knocking out miRNAs, flower responsible genes, genes involved in competitive pathways, and base editing are described. The approaches presented here have never been suggested or applied in opium poppy so far.