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1.
Proc Natl Acad Sci U S A ; 116(28): 13964-13969, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31243147

RESUMO

Carboxylases are biocatalysts that capture and convert carbon dioxide (CO2) under mild conditions and atmospheric concentrations at a scale of more than 400 Gt annually. However, how these enzymes bind and control the gaseous CO2 molecule during catalysis is only poorly understood. One of the most efficient classes of carboxylating enzymes are enoyl-CoA carboxylases/reductases (Ecrs), which outcompete the plant enzyme RuBisCO in catalytic efficiency and fidelity by more than an order of magnitude. Here we investigated the interactions of CO2 within the active site of Ecr from Kitasatospora setae Combining experimental biochemistry, protein crystallography, and advanced computer simulations we show that 4 amino acids, N81, F170, E171, and H365, are required to create a highly efficient CO2-fixing enzyme. Together, these 4 residues anchor and position the CO2 molecule for the attack by a reactive enolate created during the catalytic cycle. Notably, a highly ordered water molecule plays an important role in an active site that is otherwise carefully shielded from water, which is detrimental to CO2 fixation. Altogether, our study reveals unprecedented molecular details of selective CO2 binding and C-C-bond formation during the catalytic cycle of nature's most efficient CO2-fixing enzyme. This knowledge provides the basis for the future development of catalytic frameworks for the capture and conversion of CO2 in biology and chemistry.


Assuntos
Aminoácidos/química , Dióxido de Carbono/química , Ácidos Graxos Dessaturases/química , Modelos Moleculares , Aminoácidos/genética , Aminoácidos/metabolismo , Dióxido de Carbono/metabolismo , Proteínas de Transporte/química , Catálise , Domínio Catalítico/genética , Enzimas/química , Ácidos Graxos Dessaturases/metabolismo , Streptomycetaceae/química , Streptomycetaceae/enzimologia
2.
Chembiochem ; 22(1): 84-91, 2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-32783358

RESUMO

Natural products and secondary metabolites comprise an indispensable resource from living organisms that have transformed areas of medicine, agriculture, and biotechnology. Recent advances in high-throughput DNA sequencing and computational analysis suggest that the vast majority of natural products remain undiscovered. To accelerate the natural product discovery pipeline, cell-free metabolic engineering approaches used to develop robust catalytic networks are being repurposed to access new chemical scaffolds, and new enzymes capable of performing diverse chemistries. Such enzymes could serve as flexible biocatalytic tools to further expand the unique chemical space of natural products and secondary metabolites, and provide a more sustainable route to manufacture these molecules. Herein, we highlight select examples of natural product biosynthesis using cell-free systems and propose how cell-free technologies could facilitate our ability to access and modify these structures to transform synthetic and chemical biology.


Assuntos
Produtos Biológicos/metabolismo , Produtos Biológicos/química , Sequenciamento de Nucleotídeos em Larga Escala , Engenharia Metabólica , Estrutura Molecular
3.
Arch Microbiol ; 203(7): 4149-4159, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34059946

RESUMO

A novel acyl-CoA dehydrogenase involved in degradation of the auxin indoleacetate by Aromatoleum aromaticum was identified as a decarboxylating benzylmalonyl-CoA dehydrogenase (IaaF). It is encoded within the iaa operon coding for enzymes of indoleacetate catabolism. Using enzymatically produced benzylmalonyl-CoA, the reaction was characterized as simultaneous oxidation and decarboxylation of benzylmalonyl-CoA to cinnamoyl-CoA and CO2. Oxygen served as electron acceptor and was reduced to H2O2, whereas electron transfer flavoprotein or artificial dyes serving as electron acceptors for other acyl-CoA dehydrogenases were not used. The enzyme is homotetrameric, contains an FAD cofactor and is enantiospecific in benzylmalonyl-CoA turnover. It shows high catalytic efficiency and strong substrate inhibition with benzylmalonyl-CoA, but otherwise accepts only a few medium-chain alkylmalonyl-CoA compounds as alternative substrates with low activities. Its reactivity of oxidizing 2-carboxyacyl-CoA with simultaneous decarboxylation is unprecedented and indicates a modified reaction mechanism for acyl-CoA dehydrogenases, where elimination of the 2-carboxy group replaces proton abstraction from C2.


Assuntos
Proteínas de Bactérias , Ácidos Indolacéticos , Oxirredutases , Rhodocyclaceae , Proteínas de Bactérias/metabolismo , Peróxido de Hidrogênio/metabolismo , Ácidos Indolacéticos/metabolismo , Cinética , Oxirredutases/genética , Oxirredutases/metabolismo , Rhodocyclaceae/enzimologia
4.
Proc Natl Acad Sci U S A ; 115(13): 3380-3385, 2018 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-29531083

RESUMO

Many reactions within a cell are thermodynamically unfavorable. To efficiently run some of those endergonic reactions, nature evolved intermediate-channeling enzyme complexes, in which the products of the first endergonic reactions are immediately consumed by the second exergonic reactions. Based on this concept, we studied how archaea overcome the unfavorable first reaction of isoprenoid biosynthesis-the condensation of two molecules of acetyl-CoA to acetoacetyl-CoA catalyzed by acetoacetyl-CoA thiolases (thiolases). We natively isolated an enzyme complex comprising the thiolase and 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGCS) from a fast-growing methanogenic archaeon, Methanothermococcus thermolithotrophicus HMGCS catalyzes the second reaction in the mevalonate pathway-the exergonic condensation of acetoacetyl-CoA and acetyl-CoA to HMG-CoA. The 380-kDa crystal structure revealed that both enzymes are held together by a third protein (DUF35) with so-far-unknown function. The active-site clefts of thiolase and HMGCS form a fused CoA-binding site, which allows for efficient coupling of the endergonic thiolase reaction with the exergonic HMGCS reaction. The tripartite complex is found in almost all archaeal genomes and in some bacterial ones. In addition, the DUF35 proteins are also important for polyhydroxyalkanoate (PHA) biosynthesis, most probably by functioning as a scaffold protein that connects thiolase with 3-ketoacyl-CoA reductase. This natural and highly conserved enzyme complex offers great potential to improve isoprenoid and PHA biosynthesis in biotechnologically relevant organisms.


Assuntos
Acetilcoenzima A/metabolismo , Acetil-CoA C-Acetiltransferase/química , Acetil-CoA C-Acetiltransferase/metabolismo , Acil Coenzima A/metabolismo , Archaea/enzimologia , Hidroximetilglutaril-CoA Sintase/química , Hidroximetilglutaril-CoA Sintase/metabolismo , Sítios de Ligação , Catálise , Domínio Catalítico , Cristalografia por Raios X , Conformação Proteica
5.
Nat Chem Biol ; 14(12): 1127-1132, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30374166

RESUMO

Cells must cope with toxic or reactive intermediates formed during metabolism. One coping strategy is to sequester reactions that produce such intermediates within specialized compartments or tunnels connecting different active sites. Here, we show that propionyl-CoA synthase (PCS), an ∼ 400-kDa homodimer, three-domain fusion protein and the key enzyme of the 3-hydroxypropionate bi-cycle for CO2 fixation, sequesters its reactive intermediate acrylyl-CoA. Structural analysis showed that PCS forms a multicatalytic reaction chamber. Kinetic analysis suggested that access to the reaction chamber and catalysis are synchronized by interdomain communication. The reaction chamber of PCS features three active sites and has a volume of only 33 nm3. As one of the smallest multireaction chambers described in biology, PCS may inspire the engineering of a new class of dynamically regulated nanoreactors.


Assuntos
Acil Coenzima A/metabolismo , Coenzima A Ligases/química , Coenzima A Ligases/metabolismo , Catálise , Coenzima A Ligases/genética , Cristalografia por Raios X , Cinética , Domínios Proteicos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Espalhamento a Baixo Ângulo , Sphingomonadaceae/enzimologia , Sphingomonadaceae/genética , Difração de Raios X
6.
Microb Cell Fact ; 19(1): 160, 2020 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-32778124

RESUMO

BACKGROUND: Thioesters of coenzyme A participate in 5% of all enzymatic reactions. In microbial cell factories, they function as building blocks for products of recognized commercial value, including natural products such as polyketides, polyunsaturated fatty acids, biofuels, and biopolymers. A core spectrum of approximately 5-10 short chain thioesters is present in many microbes, as inferred from their genomic repertoire. The relevance of these metabolites explains the high interest to trace and quantify them in microbial cells. RESULTS: Here, we describe a common workflow for extraction and absolute quantification of short chain CoA thioesters in different gram-positive and gram-negative bacteria and eukaryotic yeast, i.e. Corynebacterium glutamicum, Streptomyces albus, Pseudomonas putida, and Yarrowia lipolytica. The approach assessed intracellular CoA thioesters down to the picomolar level and exhibited high precision and reproducibility for all microbes, as shown by principal component analysis. Furthermore, it provided interesting insights into microbial CoA metabolism. A succinyl-CoA synthase defective mutant of C. glutamicum exhibited an unaffected level of succinyl-CoA that indicated a complete compensation by the L-lysine pathway to bypass the disrupted TCA cycle. Methylmalonyl-CoA, an important building block of high-value polyketides, was identified as dominant CoA thioester in the actinomycete S. albus. The microbe revealed a more than 10,000-fold difference in the abundance of intracellular CoA thioesters. A recombinant strain of S. albus, which produced different derivatives of the antituberculosis polyketide pamamycin, revealed a significant depletion of CoA thioesters of the ethylmalonyl CoA pathway, influencing product level and spectrum. CONCLUSIONS: The high relevance of short chain CoA thioesters to synthetize industrial products and the interesting insights gained from the examples shown in this work, suggest analyzing these metabolites in microbial cell factories more routinely than done so far. Due to its broad application range, the developed approach appears useful to be applied this purpose. Hereby, the possibility to use one single protocol promises to facilitate automatized efforts, which rely on standardized workflows.


Assuntos
Coenzima A/análise , Ésteres/análise , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/metabolismo , Engenharia Metabólica/métodos , Yarrowia/metabolismo , Técnicas de Cultura Celular por Lotes , Bactérias Gram-Negativas/genética , Bactérias Gram-Positivas/genética , Espaço Intracelular/química , Reprodutibilidade dos Testes , Yarrowia/genética
7.
J Biol Chem ; 293(44): 17200-17207, 2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30217823

RESUMO

The enoyl-thioester reductase InhA catalyzes an essential step in fatty acid biosynthesis of Mycobacterium tuberculosis and is a key target of antituberculosis drugs to combat multidrug-resistant M. tuberculosis strains. This has prompted intense interest in the mechanism and intermediates of the InhA reaction. Here, using enzyme mutagenesis, NMR, stopped-flow spectroscopy, and LC-MS, we found that the NADH cofactor and the CoA thioester substrate form a covalent adduct during the InhA catalytic cycle. We used the isolated adduct as a molecular probe to directly access the second half-reaction of the catalytic cycle of InhA (i.e. the proton transfer), independently of the first half-reaction (i.e. the initial hydride transfer) and to assign functions to two conserved active-site residues, Tyr-158 and Thr-196. We found that Tyr-158 is required for the stereospecificity of protonation and that Thr-196 is partially involved in hydride transfer and protonation. The natural tendency of InhA to form a covalent C2-ene adduct calls for a careful reconsideration of the enzyme's reaction mechanism. It also provides the basis for the development of effective tools to study, manipulate, and inhibit the catalytic cycle of InhA and related enzymes of the short-chain dehydrogenase/reductase (SDR) superfamily. In summary, our work has uncovered the formation of a covalent adduct during the InhA catalytic cycle and identified critical residues required for catalysis, providing further insights into the InhA reaction mechanism important for the development of antituberculosis drugs.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/enzimologia , Oxirredutases/química , Oxirredutases/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Biocatálise , Domínio Catalítico , Modelos Moleculares , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Oxirredutases/genética , Conformação Proteica
8.
Nat Chem Biol ; 13(7): 745-749, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28504678

RESUMO

Enzymes are highly specific biocatalysts, yet they can promote unwanted side reactions. Here we investigated the factors that direct catalysis in the enoyl-thioester reductase Etr1p. We show that a single conserved threonine is essential to suppress the formation of a side product that would otherwise act as a high-affinity inhibitor of the enzyme. Substitution of this threonine with isosteric valine increases side-product formation by more than six orders of magnitude, while decreasing turnover frequency by only one order of magnitude. Our results show that the promotion of wanted reactions and the suppression of unwanted side reactions operate independently at the active site of Etr1p, and that the active suppression of side reactions is highly conserved in the family of medium-chain dehydrogenases/reductases (MDRs). Our discovery emphasizes the fact that the active destabilization of competing transition states is an important factor during catalysis that has implications for the understanding and the de novo design of enzymes.


Assuntos
Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Treonina/farmacologia , Biocatálise , Candida tropicalis/enzimologia , Relação Dose-Resposta a Droga , Mitocôndrias/enzimologia , Estrutura Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Relação Estrutura-Atividade , Treonina/química
9.
Nat Chem Biol ; 11(6): 398-400, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25867044

RESUMO

An improved understanding of enzymes' catalytic proficiency and stereoselectivity would further enable applications in chemistry, biocatalysis and industrial biotechnology. We use a chemical probe to dissect individual catalytic steps of enoyl-thioester reductases (Etrs), validating an active site tyrosine as the cryptic proton donor and explaining how it had eluded definitive identification. This information enabled the rational redesign of Etr, yielding mutants that create products with inverted stereochemistry at wild type-like turnover frequency.


Assuntos
Biotecnologia/métodos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Engenharia de Proteínas/métodos , Sítios de Ligação , Catálise , Modelos Moleculares , Conformação Proteica , Prótons , Estereoisomerismo , Especificidade por Substrato , Tirosina/química , Tirosina/genética
10.
Molecules ; 21(4): 517, 2016 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-27104508

RESUMO

Coenzyme A (CoA) is a ubiquitous cofactor present in every known organism. The thioesters of CoA are core intermediates in many metabolic processes, such as the citric acid cycle, fatty acid biosynthesis and secondary metabolism, including polyketide biosynthesis. Synthesis of CoA-thioesters is vital for the study of CoA-dependent enzymes and pathways, but also as standards for metabolomics studies. In this work we systematically tested five chemo-enzymatic methods for the synthesis of the three most abundant acyl-CoA thioester classes in biology; saturated acyl-CoAs, α,ß-unsaturated acyl-CoAs (i.e., enoyl-CoA derivatives), and α-carboxylated acyl-CoAs (i.e., malonyl-CoA derivatives). Additionally we report on the substrate promiscuity of three newly described acyl-CoA dehydrogenases that allow the simple conversion of acyl-CoAs into enoyl-CoAs. With these five methods, we synthesized 26 different CoA-thioesters with a yield of 40% or higher. The CoA esters produced range from short- to long-chain, include branched and α,ß-unsaturated representatives as well as other functional groups. Based on our results we provide a general guideline to the optimal synthesis method of a given CoA-thioester in respect to its functional group(s) and the commercial availability of the precursor molecule. The proposed synthetic routes can be performed in small scale and do not require special chemical equipment, making them convenient also for biological laboratories.


Assuntos
Coenzima A/química , Ésteres/síntese química , Acil Coenzima A/síntese química , Acil Coenzima A/química , Ácidos Carboxílicos , Ésteres/química , Especificidade por Substrato
11.
bioRxiv ; 2024 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-39149402

RESUMO

Biotechnological processes hold tremendous potential for the efficient and sustainable conversion of one-carbon (C1) substrates into complex multi-carbon products. However, the development of robust and versatile biocatalytic systems for this purpose remains a significant challenge. In this study, we report a hybrid electrochemical-biochemical cell-free system for the conversion of C1 substrates into the universal biological building block acetyl-CoA. The synthetic reductive formate pathway (ReForm) consists of five core enzymes catalyzing non-natural reactions that were established through a cell-free enzyme engineering platform. We demonstrate that ReForm works in a plug-and-play manner to accept diverse C1 substrates including CO2 equivalents. We anticipate that ReForm will facilitate efforts to build and improve synthetic C1 utilization pathways for a formate-based bioeconomy.

12.
Nat Commun ; 14(1): 3897, 2023 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-37400446

RESUMO

Antibody discovery is bottlenecked by the individual expression and evaluation of antigen-specific hits. Here, we address this bottleneck by developing a workflow combining cell-free DNA template generation, cell-free protein synthesis, and binding measurements of antibody fragments in a process that takes hours rather than weeks. We apply this workflow to evaluate 135 previously published antibodies targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including all 8 antibodies previously granted emergency use authorization for coronavirus disease 2019 (COVID-19), and demonstrate identification of the most potent antibodies. We also evaluate 119 anti-SARS-CoV-2 antibodies from a mouse immunized with the SARS-CoV-2 spike protein and identify neutralizing antibody candidates, including the antibody SC2-3, which binds the SARS-CoV-2 spike protein of all tested variants of concern. We expect that our cell-free workflow will accelerate the discovery and characterization of antibodies for future pandemics and for research, diagnostic, and therapeutic applications more broadly.


Assuntos
COVID-19 , Animais , Humanos , Camundongos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Anticorpos Neutralizantes , Anticorpos Antivirais
13.
Methods Mol Biol ; 2433: 199-215, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34985746

RESUMO

Biological systems provide a sustainable and complimentary approach to synthesizing useful chemical products. Metabolic engineers seeking to establish economically viable biosynthesis platforms strive to increase product titers, rates, and yields. Despite continued advances in genetic tools and metabolic engineering techniques, cellular workflows remain limited in throughput. It may take months to test dozens of unique pathway designs even in a robust model organism, such as Escherichia coli. In contrast, cell-free protein synthesis enables the rapid generation of enzyme libraries that can be combined to reconstitute metabolic pathways in vitro for biochemical synthesis in days rather than weeks. Cell-free reactions thereby enable comparison of hundreds to thousands of unique combinations of enzyme homologs and concentrations, which can quickly identify the most productive pathway variants to test in vivo or further characterize in vitro. This cell-free pathway prototyping strategy provides a complementary approach to accelerate cellular metabolic engineering efforts toward highly productive strains for metabolite production.


Assuntos
Vias Biossintéticas , Engenharia Metabólica , Sistema Livre de Células/metabolismo , Engenharia Metabólica/métodos , Redes e Vias Metabólicas , Biossíntese de Proteínas
14.
Nat Commun ; 13(1): 3058, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35650184

RESUMO

Carbon-negative synthesis of biochemical products has the potential to mitigate global CO2 emissions. An attractive route to do this is the reverse ß-oxidation (r-BOX) pathway coupled to the Wood-Ljungdahl pathway. Here, we optimize and implement r-BOX for the synthesis of C4-C6 acids and alcohols. With a high-throughput in vitro prototyping workflow, we screen 762 unique pathway combinations using cell-free extracts tailored for r-BOX to identify enzyme sets for enhanced product selectivity. Implementation of these pathways into Escherichia coli generates designer strains for the selective production of butanoic acid (4.9 ± 0.1 gL-1), as well as hexanoic acid (3.06 ± 0.03 gL-1) and 1-hexanol (1.0 ± 0.1 gL-1) at the best performance reported to date in this bacterium. We also generate Clostridium autoethanogenum strains able to produce 1-hexanol from syngas, achieving a titer of 0.26 gL-1 in a 1.5 L continuous fermentation. Our strategy enables optimization of r-BOX derived products for biomanufacturing and industrial biotechnology.


Assuntos
Ciclo do Carbono , Escherichia coli , Processos Autotróficos , Escherichia coli/metabolismo , Fermentação , Oxirredução
15.
ACS Cent Sci ; 8(8): 1091-1101, 2022 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-36032767

RESUMO

Enoyl-CoA carboxylases/reductases (ECRs) are some of the most efficient CO2-fixing enzymes described to date. However, the molecular mechanisms underlying the extraordinary catalytic activity of ECRs on the level of the protein assembly remain elusive. Here we used a combination of ambient-temperature X-ray free electron laser (XFEL) and cryogenic synchrotron experiments to study the structural organization of the ECR from Kitasatospora setae. The K. setae ECR is a homotetramer that differentiates into a pair of dimers of open- and closed-form subunits in the catalytically active state. Using molecular dynamics simulations and structure-based mutagenesis, we show that catalysis is synchronized in the K. setae ECR across the pair of dimers. This conformational coupling of catalytic domains is conferred by individual amino acids to achieve high CO2-fixation rates. Our results provide unprecedented insights into the dynamic organization and synchronized inter- and intrasubunit communications of this remarkably efficient CO2-fixing enzyme during catalysis.

16.
Sci Transl Med ; 14(646): eabn1252, 2022 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-35412328

RESUMO

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to arise and prolong the coronavirus disease 2019 (COVID-19) pandemic. Here, we used a cell-free expression workflow to rapidly screen and optimize constructs containing multiple computationally designed miniprotein inhibitors of SARS-CoV-2. We found the broadest efficacy was achieved with a homotrimeric version of the 75-residue angiotensin-converting enzyme 2 (ACE2) mimic AHB2 (TRI2-2) designed to geometrically match the trimeric spike architecture. Consistent with the design model, in the cryo-electron microscopy structure TRI2-2 forms a tripod at the apex of the spike protein that engaged all three receptor binding domains simultaneously. TRI2-2 neutralized Omicron (B.1.1.529), Delta (B.1.617.2), and all other variants tested with greater potency than the monoclonal antibodies used clinically for the treatment of COVID-19. TRI2-2 also conferred prophylactic and therapeutic protection against SARS-CoV-2 challenge when administered intranasally in mice. Designed miniprotein receptor mimics geometrically arrayed to match pathogen receptor binding sites could be a widely applicable antiviral therapeutic strategy with advantages over antibodies in greater resistance to viral escape and antigenic drift, and advantages over native receptor traps in lower chances of autoimmune responses.


Assuntos
COVID-19 , SARS-CoV-2 , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Microscopia Crioeletrônica , Humanos , Camundongos , Glicoproteína da Espícula de Coronavírus
17.
Curr Opin Biotechnol ; 69: 136-144, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33453438

RESUMO

Industrial biotechnology is an attractive approach to address the need for low-cost fuels and products from sustainable resources. Unfortunately, cells impose inherent limitations on the effective synthesis and release of target products. One key constraint is that cellular survival objectives often work against the production objectives of biochemical engineers. Additionally, industrial strains release CO2 and struggle to utilize sustainable, potentially profitable feedstocks. Cell-free biotechnology, which uses biological machinery harvested from cells, can address these challenges with advantages including: (i) shorter development times, (ii) higher volumetric production rates, and (iii) tolerance to otherwise toxic molecules. In this review, we highlight recent advances in cell-free technologies toward the production of non-protein products beyond lab-scale demonstrations and describe guiding principles for designing cell-free systems. Specifically, we discuss carbon and energy sources, reaction homeostasis, and scale-up. Expanding the scope of cell-free biomanufacturing practice could enable innovative approaches for the industrial production of green chemicals.


Assuntos
Biotecnologia , Sistema Livre de Células
18.
Front Bioeng Biotechnol ; 8: 590341, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33195157

RESUMO

The modern cell-free protein synthesis (CFPS) system is expanding the opportunity of cell-free biomanufacturing as a versatile platform for synthesizing various therapeutic proteins. However, synthesizing human protein in the bacterial CFPS system remains challenging due to the low expression level, protein misfolding, inactivity, and more. These challenges limit the use of a bacterial CFPS system for human therapeutic protein synthesis. In this study, we demonstrated the improved performance of a customized CFPS platform for human therapeutic protein production by investigating the factors that limit cell-free transcription-translation. The improvement of the CFPS platform has been made in three ways. First, the cell extract was prepared from the rare tRNA expressed host strain, and CFPS was performed with a codon-optimized gene for Escherichia coli codon usage bias. The soluble protein yield was 15.2 times greater with the rare tRNA overexpressing host strain as cell extract and codon-optimized gene in the CFPS system. Next, we identify and prioritize the critical biomanufacturing factors for highly active crude cell lysate for human protein synthesis. Lastly, we engineer the CFPS reaction conditions to enhance protein yield. In this model, the therapeutic protein filaggrin expression was significantly improved by up to 23-fold, presenting 28 ± 5 µM of soluble protein yield. The customized CFPS system for filaggrin biomanufacturing described here demonstrates the potential of the CFPS system to be adapted for studying therapeutic proteins.

19.
Nat Commun ; 11(1): 5812, 2020 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-33199707

RESUMO

Carbon fixation is one of the most important biochemical processes. Most natural carbon fixation pathways are thought to have emerged from enzymes that originally performed other metabolic tasks. Can we recreate the emergence of a carbon fixation pathway in a heterotrophic host by recruiting only endogenous enzymes? In this study, we address this question by systematically analyzing possible carbon fixation pathways composed only of Escherichia coli native enzymes. We identify the GED (Gnd-Entner-Doudoroff) cycle as the simplest pathway that can operate with high thermodynamic driving force. This autocatalytic route is based on reductive carboxylation of ribulose 5-phosphate (Ru5P) by 6-phosphogluconate dehydrogenase (Gnd), followed by reactions of the Entner-Doudoroff pathway, gluconeogenesis, and the pentose phosphate pathway. We demonstrate the in vivo feasibility of this new-to-nature pathway by constructing E. coli gene deletion strains whose growth on pentose sugars depends on the GED shunt, a linear variant of the GED cycle which does not require the regeneration of Ru5P. Several metabolic adaptations, most importantly the increased production of NADPH, assist in establishing sufficiently high flux to sustain this growth. Our study exemplifies a trajectory for the emergence of carbon fixation in a heterotrophic organism and demonstrates a synthetic pathway of biotechnological interest.


Assuntos
Ciclo do Carbono , Escherichia coli/metabolismo , Adaptação Fisiológica , Simulação por Computador , Escherichia coli/enzimologia , Fermentação , Cinética , Mutação/genética , Oxirredução , Açúcares/metabolismo
20.
Synth Biol (Oxf) ; 5(1): ysaa019, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33344777

RESUMO

Industrial biotechnology aims to produce high-value products from renewable resources. This can be challenging because model microorganisms-organisms that are easy to use like Escherichia coli-often lack the machinery required to utilize desired feedstocks like lignocellulosic biomass or syngas. Non-model organisms, such as Clostridium, are industrially proven and have desirable metabolic features but have several hurdles to mainstream use. Namely, these species grow more slowly than conventional laboratory microbes, and genetic tools for engineering them are far less prevalent. To address these hurdles for accelerating cellular design, cell-free synthetic biology has matured as an approach for characterizing non-model organisms and rapidly testing metabolic pathways in vitro. Unfortunately, cell-free systems can require specialized DNA architectures with minimal regulation that are not compatible with cellular expression. In this work, we develop a modular vector system that allows for T7 expression of desired enzymes for cell-free expression and direct Golden Gate assembly into Clostridium expression vectors. Utilizing the Joint Genome Institute's DNA Synthesis Community Science Program, we designed and synthesized these plasmids and genes required for our projects allowing us to shuttle DNA easily between our in vitro and in vivo experiments. We next validated that these vectors were sufficient for cell-free expression of functional enzymes, performing on par with the previous state-of-the-art. Lastly, we demonstrated automated six-part DNA assemblies for Clostridium autoethanogenum expression with efficiencies ranging from 68% to 90%. We anticipate this system of plasmids will enable a framework for facile testing of biosynthetic pathways in vitro and in vivo by shortening development cycles.

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