RESUMEN
Biosynthesis is an environmentally benign and renewable approach that can be used to produce a broad range of natural and, in some cases, new-to-nature products. However, biology lacks many of the reactions that are available to synthetic chemists, resulting in a narrower scope of accessible products when using biosynthesis rather than synthetic chemistry. A prime example of such chemistry is carbene-transfer reactions1. Although it was recently shown that carbene-transfer reactions can be performed in a cell and used for biosynthesis2,3, carbene donors and unnatural cofactors needed to be added exogenously and transported into cells to effect the desired reactions, precluding cost-effective scale-up of the biosynthesis process with these reactions. Here we report the access to a diazo ester carbene precursor by cellular metabolism and a microbial platform for introducing unnatural carbene-transfer reactions into biosynthesis. The α-diazoester azaserine was produced by expressing a biosynthetic gene cluster in Streptomyces albus. The intracellularly produced azaserine was used as a carbene donor to cyclopropanate another intracellularly produced molecule-styrene. The reaction was catalysed by engineered P450 mutants containing a native cofactor with excellent diastereoselectivity and a moderate yield. Our study establishes a scalable, microbial platform for conducting intracellular abiological carbene-transfer reactions to functionalize a range of natural and new-to-nature products and expands the scope of organic products that can be produced by cellular metabolism.
Asunto(s)
Azaserina , Azaserina/biosíntesis , Azaserina/química , Productos Biológicos/química , Productos Biológicos/metabolismo , Familia de Multigenes/genética , Estireno/química , Ciclopropanos/química , Coenzimas/química , Coenzimas/metabolismo , Biocatálisis , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismoRESUMEN
In natural photosynthesis, the light-driven splitting of water into electrons, protons and molecular oxygen forms the first step of the solar-to-chemical energy conversion process. The reaction takes place in photosystem II, where the Mn4CaO5 cluster first stores four oxidizing equivalents, the S0 to S4 intermediate states in the Kok cycle, sequentially generated by photochemical charge separations in the reaction center and then catalyzes the O-O bond formation chemistry1-3. Here, we report room temperature snapshots by serial femtosecond X-ray crystallography to provide structural insights into the final reaction step of Kok's photosynthetic water oxidation cycle, the S3â[S4]âS0 transition where O2 is formed and Kok's water oxidation clock is reset. Our data reveal a complex sequence of events, which occur over micro- to milliseconds, comprising changes at the Mn4CaO5 cluster, its ligands and water pathways as well as controlled proton release through the hydrogen-bonding network of the Cl1 channel. Importantly, the extra O atom Ox, which was introduced as a bridging ligand between Ca and Mn1 during the S2âS3 transition4-6, disappears or relocates in parallel with Yz reduction starting at approximately 700 µs after the third flash. The onset of O2 evolution, as indicated by the shortening of the Mn1-Mn4 distance, occurs at around 1,200 µs, signifying the presence of a reduced intermediate, possibly a bound peroxide.
Asunto(s)
Oxígeno , Fotosíntesis , Complejo de Proteína del Fotosistema II , Oxidación-Reducción , Oxígeno/química , Oxígeno/metabolismo , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/metabolismo , Protones , Agua/química , Agua/metabolismo , Manganeso/química , Manganeso/metabolismo , Calcio/química , Calcio/metabolismo , Peróxidos/metabolismoRESUMEN
Artificial intelligence-based protein structure prediction methods such as AlphaFold have revolutionized structural biology. The accuracies of these predictions vary, however, and they do not take into account ligands, covalent modifications or other environmental factors. Here, we evaluate how well AlphaFold predictions can be expected to describe the structure of a protein by comparing predictions directly with experimental crystallographic maps. In many cases, AlphaFold predictions matched experimental maps remarkably closely. In other cases, even very high-confidence predictions differed from experimental maps on a global scale through distortion and domain orientation, and on a local scale in backbone and side-chain conformation. We suggest considering AlphaFold predictions as exceptionally useful hypotheses. We further suggest that it is important to consider the confidence in prediction when interpreting AlphaFold predictions and to carry out experimental structure determination to verify structural details, particularly those that involve interactions not included in the prediction.
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Inteligencia Artificial , Procesos Mentales , Cristalografía , Conformación ProteicaRESUMEN
Glycosylation is a predominant strategy plants use to fine-tune the properties of small molecule metabolites to affect their bioactivity, transport, and storage. It is also important in biotechnology and medicine as many glycosides are utilized in human health. Small molecule glycosylation is largely carried out by family 1 glycosyltransferases. Here, we report a structural and biochemical investigation of UGT95A1, a family 1 GT enzyme from Pilosella officinarum that exhibits a strong, unusual regiospecificity for the 3'-O position of flavonoid acceptor substrate luteolin. We obtained an apo crystal structure to help drive the analyses of a series of binding site mutants, revealing that while most residues are tolerant to mutations, key residues M145 and D464 are important for overall glycosylation activity. Interestingly, E347 is crucial for maintaining the strong preference for 3'-O glycosylation, while R462 can be mutated to increase regioselectivity. The structural determinants of regioselectivity were further confirmed in homologous enzymes. Our study also suggests that the enzyme contains large, highly dynamic, disordered regions. We showed that while most disordered regions of the protein have little to no implication in catalysis, the disordered regions conserved among investigated homologs are important to both the overall efficiency and regiospecificity of the enzyme. This report represents a comprehensive in-depth analysis of a family 1 GT enzyme with a unique substrate regiospecificity and may provide a basis for enzyme functional prediction and engineering.
RESUMEN
Machine-learning prediction algorithms such as AlphaFold and RoseTTAFold can create remarkably accurate protein models, but these models usually have some regions that are predicted with low confidence or poor accuracy. We hypothesized that by implicitly including new experimental information such as a density map, a greater portion of a model could be predicted accurately, and that this might synergistically improve parts of the model that were not fully addressed by either machine learning or experiment alone. An iterative procedure was developed in which AlphaFold models are automatically rebuilt on the basis of experimental density maps and the rebuilt models are used as templates in new AlphaFold predictions. We show that including experimental information improves prediction beyond the improvement obtained with simple rebuilding guided by the experimental data. This procedure for AlphaFold modeling with density has been incorporated into an automated procedure for interpretation of crystallographic and electron cryo-microscopy maps.
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Algoritmos , Proteínas , Modelos Moleculares , Microscopía por Crioelectrón/métodos , Proteínas/química , Aprendizaje Automático , Conformación ProteicaRESUMEN
This paper describes outcomes of the 2019 Cryo-EM Model Challenge. The goals were to (1) assess the quality of models that can be produced from cryogenic electron microscopy (cryo-EM) maps using current modeling software, (2) evaluate reproducibility of modeling results from different software developers and users and (3) compare performance of current metrics used for model evaluation, particularly Fit-to-Map metrics, with focus on near-atomic resolution. Our findings demonstrate the relatively high accuracy and reproducibility of cryo-EM models derived by 13 participating teams from four benchmark maps, including three forming a resolution series (1.8 to 3.1 Å). The results permit specific recommendations to be made about validating near-atomic cryo-EM structures both in the context of individual experiments and structure data archives such as the Protein Data Bank. We recommend the adoption of multiple scoring parameters to provide full and objective annotation and assessment of the model, reflective of the observed cryo-EM map density.
Asunto(s)
Microscopía por Crioelectrón/métodos , Modelos Moleculares , Cristalografía por Rayos X , Conformación Proteica , Proteínas/químicaRESUMEN
Sunscreen has been used for thousands of years to protect skin from ultraviolet radiation. However, the use of modern commercial sunscreen containing oxybenzone, ZnO, and TiO2 has raised concerns due to their negative effects on human health and the environment. In this study, we aim to establish an efficient microbial platform for production of shinorine, a UV light absorbing compound with anti-aging properties. First, we methodically selected an appropriate host for shinorine production by analyzing central carbon flux distribution data from prior studies alongside predictions from genome-scale metabolic models (GEMs). We enhanced shinorine productivity through CRISPRi-mediated downregulation and utilized shotgun proteomics to pinpoint potential competing pathways. Simultaneously, we improved the shinorine biosynthetic pathway by refining its design, optimizing promoter usage, and altering the strength of ribosome binding sites. Finally, we conducted amino acid feeding experiments under various conditions to identify the key limiting factors in shinorine production. The study combines meta-analysis of 13C-metabolic flux analysis, GEMs, synthetic biology, CRISPRi-mediated gene downregulation, and omics analysis to improve shinorine production, demonstrating the potential of Pseudomonas putida KT2440 as platform for shinorine production.
Asunto(s)
Ingeniería Metabólica , Pseudomonas putida , Protectores Solares , Pseudomonas putida/metabolismo , Pseudomonas putida/genética , Protectores Solares/metabolismoRESUMEN
Sustainable aviation fuel (SAF) will significantly impact global warming in the aviation sector, and important SAF targets are emerging. Isoprenol is a precursor for a promising SAF compound DMCO (1,4-dimethylcyclooctane) and has been produced in several engineered microorganisms. Recently, Pseudomonas putida has gained interest as a future host for isoprenol bioproduction as it can utilize carbon sources from inexpensive plant biomass. Here, we engineer metabolically versatile host P. putida for isoprenol production. We employ two computational modeling approaches (Bilevel optimization and Constrained Minimal Cut Sets) to predict gene knockout targets and optimize the "IPP-bypass" pathway in P. putida to maximize isoprenol production. Altogether, the highest isoprenol production titer from P. putida was achieved at 3.5 g/L under fed-batch conditions. This combination of computational modeling and strain engineering on P. putida for an advanced biofuels production has vital significance in enabling a bioproduction process that can use renewable carbon streams.
Asunto(s)
Pseudomonas putida , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Carbono/metabolismo , Ingeniería MetabólicaRESUMEN
Inspired by the period-four oscillation in flash-induced oxygen evolution of photosystem II discovered by Joliot in 1969, Kok performed additional experiments and proposed a five-state kinetic model for photosynthetic oxygen evolution, known as Kok's S-state clock or cycle1,2. The model comprises four (meta)stable intermediates (S0, S1, S2 and S3) and one transient S4 state, which precedes dioxygen formation occurring in a concerted reaction from two water-derived oxygens bound at an oxo-bridged tetra manganese calcium (Mn4CaO5) cluster in the oxygen-evolving complex3-7. This reaction is coupled to the two-step reduction and protonation of the mobile plastoquinone QB at the acceptor side of PSII. Here, using serial femtosecond X-ray crystallography and simultaneous X-ray emission spectroscopy with multi-flash visible laser excitation at room temperature, we visualize all (meta)stable states of Kok's cycle as high-resolution structures (2.04-2.08 Å). In addition, we report structures of two transient states at 150 and 400 µs, revealing notable structural changes including the binding of one additional 'water', Ox, during the S2âS3 state transition. Our results suggest that one water ligand to calcium (W3) is directly involved in substrate delivery. The binding of the additional oxygen Ox in the S3 state between Ca and Mn1 supports O-O bond formation mechanisms involving O5 as one substrate, where Ox is either the other substrate oxygen or is perfectly positioned to refill the O5 position during O2 release. Thus, our results exclude peroxo-bond formation in the S3 state, and the nucleophilic attack of W3 onto W2 is unlikely.
Asunto(s)
Oxígeno/metabolismo , Fotosíntesis , Agua/química , Agua/metabolismo , Calcio/metabolismo , Cristalografía por Rayos X , Cianobacterias/química , Rayos Láser , Manganeso/metabolismo , Modelos Moleculares , Oxidación-Reducción , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/metabolismo , Plastoquinona/metabolismoRESUMEN
Modular polyketide synthases (PKSs) are polymerases that employ α-carboxyacyl-CoAs as extender substrates. This enzyme family contains several catalytic modules, where each module is responsible for a single round of polyketide chain extension. Although PKS modules typically use malonyl-CoA or methylmalonyl-CoA for chain elongation, many other malonyl-CoA analogues are used to diversify polyketide structures in nature. Previously, we developed a method to alter an extension substrate of a given module by exchanging an acyltransferase (AT) domain while maintaining protein folding. Here, we report in vitro polyketide biosynthesis by 13 PKSs (the wild-type PKS and 12 AT-exchanged PKSs with unusual ATs) and 14 extender substrates. Our â¼200 in vitro reactions resulted in 13 structurally different polyketides, including several polyketides that have not been reported. In some cases, AT-exchanged PKSs produced target polyketides by >100-fold compared to the wild-type PKS. These data also indicate that most unusual AT domains do not incorporate malonyl-CoA and methylmalonyl-CoA but incorporate various rare extender substrates that are equal to in size or slightly larger than natural substrates. We developed a computational workflow to predict the approximate AT substrate range based on active site volumes to support the selection of ATs. These results greatly enhance our understanding of rare AT domains and demonstrate the benefit of using the proposed PKS engineering strategy to produce novel chemicals in vitro.
Asunto(s)
Sintasas Poliquetidas , Policétidos , Sintasas Poliquetidas/metabolismo , Aciltransferasas/química , Dominio Catalítico , Policétidos/metabolismo , Especificidad por SustratoRESUMEN
Photosynthetic light harvesting requires efficient energy transfer within dynamic networks of light-harvesting complexes embedded within phospholipid membranes. Artificial light-harvesting models are valuable tools for understanding the structural features underpinning energy absorption and transfer within chromophore arrays. Here, a method for attaching a protein-based light-harvesting model to a planar, fluid supported lipid bilayer (SLB) is developed. The protein model consists of the tobacco mosaic viral capsid proteins that are gene-doubled to create a tandem dimer (dTMV). Assemblies of dTMV break the facial symmetry of the double disk to allow for differentiation between the disk faces. A single reactive lysine residue is incorporated into the dTMV assemblies for the site-selective attachment of chromophores for light absorption. On the opposing dTMV face, a cysteine residue is incorporated for the bioconjugation of a peptide containing a polyhistidine tag for association with SLBs. The dual-modified dTMV complexes show significant association with SLBs and exhibit mobility on the bilayer. The techniques used herein offer a new method for protein-surface attachment and provide a platform for evaluating excited state energy transfer events in a dynamic, fully synthetic artificial light-harvesting system.
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Fotosíntesis , Proteínas , Transferencia de Energía , Membrana Dobles de Lípidos/químicaRESUMEN
Kelp is an abundant, farmable biomass-containing laminarin and alginate as major polysaccharides, providing an excellent model substrate to study their deconstruction by simple enzyme mixtures. Our previous study showed strong reactivity of the glycoside hydrolase family 55 during hydrolysis of purified laminarin, raising the question of its reactivity with intact kelp. In this study, we determined that a combination of a single glycoside hydrolase family 55 ß-1,3-exoglucanase with a broad-specificity alginate lyase from the polysaccharide lyase family 18 gives efficient hydrolysis of untreated kelp to a mixture of simple sugars, that is, glucose, gentiobiose, mannitol-end glucose, and mannuronic and guluronic acids and their soluble oligomers. Quantitative assignments from nanostructure initiator mass spectrometry (NIMS) and 2D HSQC NMR spectroscopy and analysis of the reaction time-course are provided. The data suggest that binary combinations of enzymes targeted to the unique polysaccharide composition of marine biomass are sufficient to deconstruct kelp into soluble sugars for microbial fermentation.
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Celulasas , Kelp , Kelp/metabolismo , Hidrólisis , Polisacárido Liasas/metabolismo , Polisacáridos , Glucosa , Glicósido Hidrolasas/metabolismo , Especificidad por SustratoRESUMEN
A density-modification procedure for improving maps from single-particle electron cryogenic microscopy (cryo-EM) is presented. The theoretical basis of the method is identical to that of maximum-likelihood density modification, previously used to improve maps from macromolecular X-ray crystallography. Key differences from applications in crystallography are that the errors in Fourier coefficients are largely in the phases in crystallography but in both phases and amplitudes in cryo-EM, and that half-maps with independent errors are available in cryo-EM. These differences lead to a distinct approach for combination of information from starting maps with information obtained in the density-modification process. The density-modification procedure was applied to a set of 104 datasets and improved map-model correlation and increased the visibility of details in many of the maps. The procedure requires two unmasked half-maps and a sequence file or other source of information on the volume of the macromolecule that has been imaged.
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Apoferritinas/química , Microscopía por Crioelectrón/métodos , Programas Informáticos , Procesamiento de Imagen Asistido por Computador , Conformación ProteicaRESUMEN
Pseudomonas putida have emerged as promising biocatalysts for the conversion of sugars and aromatic compounds obtained from lignocellulosic biomass. Understanding the role of carbon catabolite repression (CCR) in these strains is critical to optimize biomass conversion to fuels and chemicals. The CCR functioning in P. putida M2, a strain capable of consuming both hexose and pentose sugars as well as aromatic compounds, was investigated by cultivation experiments, proteomics, and CRISPRi-based gene repression. Strain M2 co-utilized sugars and aromatic compounds simultaneously; however, during cultivation with glucose and aromatic compounds (p-coumarate and ferulate) mixture, intermediates (4-hydroxybenzoate and vanillate) accumulated, and substrate consumption was incomplete. In contrast, xylose-aromatic consumption resulted in transient intermediate accumulation and complete aromatic consumption, while xylose was incompletely consumed. Proteomics analysis revealed that glucose exerted stronger repression than xylose on the aromatic catabolic proteins. Key glucose (Eda) and xylose (XylX) catabolic proteins were also identified at lower abundance during cultivation with aromatic compounds implying simultaneous catabolite repression by sugars and aromatic compounds. Reduction of crc expression via CRISPRi led to faster growth and glucose and p-coumarate uptake in the CRISPRi strains compared to the control, while no difference was observed on xylose+p-coumarate. The increased abundances of Eda and amino acid biosynthesis proteins in the CRISPRi strain further supported these observations. Lastly, small RNAs (sRNAs) sequencing results showed that CrcY and CrcZ homologues levels in M2, previously identified in P. putida strains, were lower under strong CCR (glucose+p-coumarate) condition compared to when repression was absent (p-coumarate or glucose only).IMPORTANCEA newly isolated Pseudomonas putida strain, P. putida M2, can utilize both hexose and pentose sugars as well as aromatic compounds making it a promising host for the valorization of lignocellulosic biomass. Pseudomonads have developed a regulatory strategy, carbon catabolite repression, to control the assimilation of carbon sources in the environment. Carbon catabolite repression may impede the simultaneous and complete metabolism of sugars and aromatic compounds present in lignocellulosic biomass and hinder the development of an efficient industrial biocatalyst. This study provides insight into the cellular physiology and proteome during mixed-substrate utilization in P. putida M2. The phenotypic and proteomics results demonstrated simultaneous catabolite repression in the sugar-aromatic mixtures, while the CRISPRi and sRNA sequencing demonstrated the potential role of the crc gene and small RNAs in carbon catabolite repression.
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Represión Catabólica , Pseudomonas putida , Azúcares/metabolismo , Xilosa/metabolismo , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Glucosa/metabolismo , Hexosas/metabolismo , Pentosas/metabolismo , Carbono/metabolismoRESUMEN
We described a mass spectrometry-based assay to rapidly quantify the production of primary alcohols directly from cell cultures. This novel assay used the combination of TEMPO-based oxidation chemistry and oxime ligation, followed by product analysis based on Nanostructure-Initiator Mass Spectrometry. This assay enables quantitative monitor both C5 to C18 alcohols as well as glucose and gluconate in the growth medium to support strain characterization and optimization. We find that this assay yields similar results to gas chromatography for isoprenol production but required much less acquisition time per sample. We applied this assay to gain new insights into P. Putida's utilization of alcohols and find that this strain largely could not grow on heptanol and octanol.
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Nanoestructuras , Cromatografía de Gases y Espectrometría de Masas , Espectrometría de Masas/métodos , Nanoestructuras/química , Glucosa , EtanolRESUMEN
Human transcription factor IIH (TFIIH) is part of the general transcriptional machinery required by RNA polymerase II for the initiation of eukaryotic gene transcription. Composed of ten subunits that add up to a molecular mass of about 500 kDa, TFIIH is also essential for nucleotide excision repair. The seven-subunit TFIIH core complex formed by XPB, XPD, p62, p52, p44, p34, and p8 is competent for DNA repair, while the CDK-activating kinase subcomplex, which includes the kinase activity of CDK7 as well as the cyclin H and MAT1 subunits, is additionally required for transcription initiation. Mutations in the TFIIH subunits XPB, XPD, and p8 lead to severe premature ageing and cancer propensity in the genetic diseases xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy, highlighting the importance of TFIIH for cellular physiology. Here we present the cryo-electron microscopy structure of human TFIIH at 4.4 Å resolution. The structure reveals the molecular architecture of the TFIIH core complex, the detailed structures of its constituent XPB and XPD ATPases, and how the core and kinase subcomplexes of TFIIH are connected. Additionally, our structure provides insight into the conformational dynamics of TFIIH and the regulation of its activity.
Asunto(s)
Microscopía por Crioelectrón , Factor de Transcripción TFIIH/química , Factor de Transcripción TFIIH/ultraestructura , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/ultraestructura , Adenosina Trifosfato/metabolismo , Humanos , Modelos Moleculares , Mutación , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , ARN Polimerasa II/ultraestructura , Factor de Transcripción TFIIH/genética , Factor de Transcripción TFIIH/metabolismo , Iniciación de la Transcripción GenéticaRESUMEN
In oxygenic photosynthesis, light-driven oxidation of water to molecular oxygen is carried out by the oxygen-evolving complex (OEC) in photosystem II (PS II). Recently, we reported the room-temperature structures of PS II in the four (semi)stable S-states, S1, S2, S3, and S0, showing that a water molecule is inserted during the S2 â S3 transition, as a new bridging O(H)-ligand between Mn1 and Ca. To understand the sequence of events leading to the formation of this last stable intermediate state before O2 formation, we recorded diffraction and Mn X-ray emission spectroscopy (XES) data at several time points during the S2 â S3 transition. At the electron acceptor site, changes due to the two-electron redox chemistry at the quinones, QA and QB, are observed. At the donor site, tyrosine YZ and His190 H-bonded to it move by 50 µs after the second flash, and Glu189 moves away from Ca. This is followed by Mn1 and Mn4 moving apart, and the insertion of OX(H) at the open coordination site of Mn1. This water, possibly a ligand of Ca, could be supplied via a "water wheel"-like arrangement of five waters next to the OEC that is connected by a large channel to the bulk solvent. XES spectra show that Mn oxidation (τ of â¼350 µs) during the S2 â S3 transition mirrors the appearance of OX electron density. This indicates that the oxidation state change and the insertion of water as a bridging atom between Mn1 and Ca are highly correlated.
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Fotosíntesis , Complejo de Proteína del Fotosistema II/metabolismo , Hidrógeno/metabolismo , Magnesio/metabolismo , Oxidación-Reducción , Oxígeno/metabolismo , Fotones , Complejo de Proteína del Fotosistema II/química , Quinonas/metabolismo , Agua/metabolismoRESUMEN
We report a fully automated procedure for the optimization and interpretation of reconstructions from cryo-electron microscopy (cryo-EM) data, available in Phenix as phenix.map_to_model. We applied our approach to 476 datasets with resolution of 4.5 Å or better, including reconstructions of 47 ribosomes and 32 other protein-RNA complexes. The median fraction of residues in the deposited structures reproduced automatically was 71% for reconstructions determined at resolutions of 3 Å or better and 47% for those at resolutions worse than 3 Å.
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Microscopía por Crioelectrón/métodos , Modelos Moleculares , Proteínas de Unión al ARN/química , ARN/química , Ribosomas/química , Microscopía por Crioelectrón/instrumentación , Humanos , Conformación Proteica , ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribosomas/metabolismoRESUMEN
The functionalization of terpenes using cytochrome P450 enzymes is a versatile route to the production of useful derivatives that can be further converted to value-added products. Many terpenes are hydrophobic and volatile making their availability as a substrate for P450 enzymes significantly limited during microbial production. In this study, we developed a strategy to improve the accessibility of terpene molecules for the P450 reaction by linking terpene synthase and P450 together. As a model system, fusion proteins of 1,8-cineole synthase (CS) and P450cin were investigated and it showed an improved hydroxylation of the monoterpenoid 1,8-cineole up to 5.4-fold. Structural analysis of the CS-P450cin fusion proteins by SEC-SAXS indicated a dimer formation with preferred orientations of the active sites of the two domains. We also applied the enzyme fusion strategy to the oxidation of a sesquiterpene epi-isozizaene and the fusion enzymes significantly improved albaflavenol production in engineered E. coli. From the analysis of positive and negative examples of the fusion strategy, we proposed key factors in structure-based prediction and evaluation of fusion enzymes. Developing fusion enzymes for terpene synthase and P450 presents an efficient strategy toward oxidation of hydrophobic terpene compounds. This strategy could be widely applicable to improve the biosynthetic titer of the functionalized products from hydrophobic terpene intermediates.