RESUMEN
The canonical glycolysis pathway is responsible for converting glucose into 2 molecules of acetyl-coenzyme A (acetyl-CoA) through a cascade of 11 biochemical reactions. Here, we have designed and constructed an artificial phosphoketolase (APK) pathway, which consists of only 3 types of biochemical reactions. The core enzyme in this pathway is phosphoketolase, while phosphatase and isomerase act as auxiliary enzymes. The APK pathway has the potential to achieve a 100% carbon yield to acetyl-CoA from any monosaccharide by integrating a one-carbon condensation reaction. We tested the APK pathway in vitro, demonstrating that it could efficiently catabolize typical C1-C6 carbohydrates to acetyl-CoA with yields ranging from 83% to 95%. Furthermore, we engineered Escherichia coli stain capable of growth utilizing APK pathway when glycerol act as a carbon source. This novel catabolic pathway holds promising route for future biomanufacturing and offering a stoichiometric production platform using multiple carbon sources.
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Aldehído-Liasas , Carbono , Acetilcoenzima A , Carbono/metabolismo , Aldehído-Liasas/genética , Aldehído-Liasas/metabolismo , Glucosa/metabolismo , Ingeniería MetabólicaRESUMEN
Optical spatial differentiation is a typical operation of optical analog computing and can single out the edge to accelerate the subsequent image processing, but in some cases, overall information about the object needs to be presented synchronously. Here, we propose a multifunctional optical device based on structured chiral photonic crystals for the simultaneous realization of real-time dual-mode imaging. This optical differentiator is realized by self-organized large-birefringence cholesteric liquid crystals, which are photopatterned to encode with a special integrated geometric phase. Two highly spin-selective modes of second-order spatial differentiation and bright-field imaging are exhibited in the reflected and transmitted directions, respectively. Two-dimensional edges of both amplitude and phase objects have been efficiently enhanced in high contrast and the broadband spectrum. This work extends the ingenious building of hierarchical chiral nanostructures, enriches their applications in the emerging frontiers of optical computing, and boasts considerable potential in machine vision and microscopy.
RESUMEN
N2 adsorption is a prerequisite for activation and transformation. Time-of-flight mass spectrometry experiments show that the Nb2C6H4+ cation, resulting from the gas-phase reaction of Nb2+ with C6H6, is more favorable for N2 adsorption than Nb+ and Nb2+ cations. Density functional theory calculations reveal the effect of the ortho-C6H4 ligand on N2 adsorption. In Nb2C6H4+, interactions between the Nb-4d and C-2p orbitals enable the Nb2+ cation to form coordination bonds with the ortho-C6H4 ligand. Although the ortho-C6H4 ligand in Nb2C6H4+ is not directly involved in the reaction, its presence increases the polarity of the cluster and brings the highest occupied molecular orbital (HOMO) closer to the lowest occupied molecular orbital (LUMO) of N2, thereby increasing the N2 adsorption energy, which effectively facilitates N2 adsorption and activation. This study provides fundamental insights into the mechanisms of N2 adsorption in "transition metal-organic ligand" systems.
RESUMEN
Studying the cleavage of the CâO bond during CO2 activation at room temperature is highly significant for comprehending the CO2 conversion processes. Herein, mass spectrometry experiments and density functional theory calculations indicate that the niobium carbide anions Nb3C4- can continuously convert five CO2 molecules to CO under thermal collision conditions, while the other clusters with less carbon ligands Nb3C1-3- reduce fewer CO2 molecules. Size-dependent reactivity of Nb3C1-4- cluster anions toward CO2 is observed. Interestingly, the carbon atoms in Nb3C4- not only act as highly active adsorption sites for CO2 but also serve as electron donors to reduce CO2. The stored electrons are released through a carbon-carbon coupling process. Our findings on the role of carbon ligands in enhancing transition metal carbide reactivity can offer new insights for designing active sites on catalysts with both high activity and selectivity.
RESUMEN
Vortices are hallmarks of a wide range of nonequilibrium phenomena in fluids at multiple length scales. In this work, we numerically study the whirling motion of self-propelled soft point particles confined in circular domain, and aim at addressing the stability issue of the coherent vortex structure. By the combination of dynamical and statistical analysis at the individual particle level, we reveal the persistence of the whirling motion resulting from the subtle competition of activity and geometric confinement. In the stable whirling motion, the scenario of the coexistence of the irregular microscopic motions of individual particles and the regular global whirling motion is fundamentally different from the motion of a vortex in passive fluid. Possible orientational order coexisting with the whirling are further explored. This work shows the stability mechanism of vortical dynamics in active media under the alignment rule in confined space and may have implications in creating and harnessing macroscale coherent dynamical states by tuning the confining geometry.
Asunto(s)
Movimiento (Física)RESUMEN
Febrifugine is a kind of quinazolinone compound with high biological activity from a Chinese herb called Chang Shan (Dichroa febrifuga). Febrifugine and its derivatives possess extensive biological activities, some of which exhibited anti-tumor activities as FAK inhibitors. However, they are not very effective at inhibiting tumor metastasis, perhaps because tumors gain energy through compensatory activation of other signaling pathways that promote cell migration and invasion. Therefore, seventeen novel febrifugine derivatives with quinazolinone skeleton were designed, synthesized and acted as potential FAK/PLK1 dual inhibitors. These compounds were determined by 1 H-NMR, 13 C-NMR and MS. Most of the compounds exhibited good inhibitory activity against cancer cell lines by computer-assisted screening, antitumor activity test and FAK/PLK1 inhibitory activity test, wherein compound 3b was screened as a high-efficiency lead compound.
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Antineoplásicos , Inhibidores de Proteínas Quinasas , Quinazolinonas , Antineoplásicos/química , Línea Celular , Proliferación Celular , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Espectroscopía de Resonancia Magnética , Simulación del Acoplamiento Molecular , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinonas/química , Quinazolinonas/farmacología , Esqueleto , Relación Estructura-Actividad , Quinasa 1 de Adhesión Focal/antagonistas & inhibidores , Quinasa Tipo Polo 1RESUMEN
Enzymatic electrosynthesis has gained more and more interest as an emerging green synthesis platform, particularly for the fixation of CO2 . However, the simultaneous utilization of CO2 and a nitrogenous molecule for the enzymatic electrosynthesis of value-added products has never been reported. In this study, we constructed an in vitro multienzymatic cascade based on the reductive glycine pathway and demonstrated an enzymatic electrocatalytic system that allowed the simultaneous conversion of CO2 and NH3 as the sole carbon and nitrogen sources to synthesize glycine. Through effective coupling and the optimization of electrochemical cofactor regeneration and the multienzymatic cascade reaction, 0.81â mM glycine was yielded with a highest reaction rate of 8.69â mg L-1 h-1 and faradaic efficiency of 96.8 %. These results imply a promising alternative for enzymatic CO2 electroreduction and expand its products to nitrogenous chemicals.
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Dióxido de Carbono , Carbono , Glicina , NitrógenoRESUMEN
Dihydroxy-acid dehydratase (DHAD) plays an important role in the utilization of glycerol or glucose for the production of value-added chemicals in the in vitro synthetic enzymatic biosystem. The low activity of DHAD in the dehydration of glycerate to pyruvate hampers its applications in biosystems. Protein engineering of a thermophilic DHAD from Sulfolobus solfataricus (SsDHAD) was performed to increase its dehydration activity. A triple mutant (I161M/Y145S/G205K) with a 10-fold higher activity on glycerate dehydration was obtained after three rounds of iterative saturation mutagenesis (ISM) based on computational analysis. The shrunken substrate-binding pocket and newly formed hydrogen bonds were the reason for the activity improvement of the mutant. For the in vitro synthetic enzymatic biosystems of converting glucose or glycerol to L-lactate, the biosystems with the mutant SsDHAD showed 3.32- and 2.34-fold higher reaction rates than the wild type, respectively. This study demonstrates the potential of protein engineering to improve the efficiency of in vitro synthetic enzymatic biosystems by enhancing the enzyme activity of rate-limited enzymes. KEY POINTS: ⢠A screening method was established for the protein engineering of SsDHAD. ⢠A R3 mutant of SsDHAD with 10-fold higher activity was obtained. ⢠The R3 mutant exhibits higher productivity in the in vitro biosystems.
Asunto(s)
Glicerol , Sulfolobus solfataricus , Deshidratación , Glucosa , Humanos , Hidroliasas/metabolismo , Sulfolobus solfataricus/genéticaRESUMEN
A series of novel quinazolinone hydrazide derivatives were designed and synthesized as EGFR inhibitors. The results indicated that most of the aimed compounds had potential anti-tumor cell proliferation and EGFR inhibitory activities. In the comprehensive analysis of all the tested compounds, the target compound 9c showed the best anti-tumor cell proliferation activity, (IC50 =1.31â µM for MCF-7, IC50 =1.89â µM for HepG2, IC50 =2.10â µM for SGC), and IC50 =0.59â µM for the EGFR inhibitory activity. Docking results showed that compound 9c could ideally insert the active site and interact with the critical amino acid residues (Val702, Lys721, Met769, Asp831) in the active site.
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Antineoplásicos , Neoplasias , Antineoplásicos/química , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Receptores ErbB , Humanos , Hidrazinas/farmacología , Simulación del Acoplamiento Molecular , Estructura Molecular , Inhibidores de Proteínas Quinasas/química , Quinazolinonas/química , Relación Estructura-ActividadRESUMEN
Adenosine triphosphate (ATP), the universal energy currency of life, has a central role in numerous biochemical reactions with potential for the synthesis of numerous high-value products. ATP can be regenerated by three types of mechanisms: substrate level phosphorylation, oxidative phosphorylation, and photophosphorylation. Current ATP regeneration methods are mainly based on substrate level phosphorylation catalyzed by one enzyme, several cascade enzymes, or in vitro synthetic enzymatic pathways. Among them, polyphosphate kinases and acetate kinase, along with their respective phosphate donors, are the most popular approaches for in vitro ATP regeneration. For in vitro artificial pathways, either ATP-free or ATP-balancing strategies can be implemented via smart pathway design by choosing ATP-independent enzymes. Also, we discuss some remaining challenges and suggest perspectives, especially for industrial biomanufacturing. Development of ATP regeneration systems featuring low cost, high volumetric productivity, long lifetime, flexible compatibility, and great robustness could be one of the bottom-up strategies for cascade biocatalysis and in vitro synthetic biology.
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Adenosina Trifosfato , Enzimas , Biología Sintética , Adenosina Trifosfato/metabolismo , Biocatálisis , Enzimas/metabolismo , Regeneración , Biología Sintética/métodos , Biología Sintética/tendenciasRESUMEN
Thiazolidinedione (TZD) is a novel peroxides proliferator activated receptor γ (PPARγ) agonist with many side effects. Herein, we developed a series of novel TZD analogues as partial agonists targeting PPARγ. The study of anti-hyperglycemic activity and anti-inflammatory activity enabled us to identify a novel compound, 4 g, which quickly recover the blood glucose of mice at the concentration of 100 mg/kg, and show similar anti-inflammatory activity to ibuprofen at the concentration of 20 mg/kg. The competitive binding assay confirmed direct binding of 4 g to the LBD of PPARγ with IC50 being 1790 nM, and dose-dependently increased the transcriptional activity of PPARγ. Besides, through computer-aided drug design software simulation docking, it was found that compound 4 g showed the best binding ability to target protein PPARγ. Furthermore, because of the introduction of benzene containing group at N3 position, the stability of H12 in the active pocket is reduced and the stability of H3 and ß-fold is increased, showing the characteristics of some PPARγ agonists, based on the docking model analysis. Together, these results suggest that 4 g is a promising PPARγ agonist that deserves further investigation.
Asunto(s)
Diseño de Fármacos , PPAR gamma/agonistas , Tiazolidinedionas/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Programas Informáticos , Relación Estructura-Actividad , Tiazolidinedionas/síntesis química , Tiazolidinedionas/químicaRESUMEN
Secretory production of recombinant proteins provides a simple approach to the production and purification of target proteins in the enzyme industry. We developed a combined strategy for the secretory production of three large-size heterologous enzymes with a special focus on 83-kDa isoamylase (IA) from an archaeon Sulfolobus tokodaii in a bacterium Bacillus subtilis. First, a secretory protein of the B. subtilis family 5 glycoside hydrolase endoglucanase (Cel5) was used as a fusion partner, along with the NprB signal peptide, to facilitate secretory production of IA. This secretory partner strategy was effective for the secretion of two other large enzymes: family 9 glycoside hydrolase from Clostridium phytofermentas and cellodextrin phosphorylase from Clostridium thermocellum. Second, the secretion of Cel5-IA was improved by directed evolution with two novel double-layer Petri-dish-based high-throughput screening (HTS) methods. The high-sensitivity HTS relied on the detection of high-activity Cel5 on the carboxymethylcellulose/Congo-red assay. The second modest-sensitivity HTS focused on the detection of low-activity IA on the amylodextrin-I2 assay. After six rounds of HTS, a secretory Cel5-IA level was increased to 234 mg/L, 155 times the wild-type IA with the NprB signal peptide only. This combinatory strategy could be useful to enhance the secretory production of large-size heterologous proteins in B. subtilis.
Asunto(s)
Bacillus subtilis/enzimología , Evolución Molecular Dirigida/métodos , Glucosiltransferasas/metabolismo , Glicósido Hidrolasas/metabolismo , Isoamilasa/metabolismo , Sistemas de Translocación de Proteínas/metabolismo , Proteínas Recombinantes de Fusión/aislamiento & purificación , Bacillus subtilis/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Celulasa/metabolismo , Clostridium thermocellum/metabolismo , Metaloendopeptidasas/metabolismo , Señales de Clasificación de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Sulfolobus/metabolismoRESUMEN
The upgrade of D-xylose, the most abundant pentose, to value-added biochemicals is economically important to next-generation biorefineries. myo-Inositol, as vitamin B8, has a six-carbon carbon-carbon ring. Here we designed an in vitro artificial NAD(P)-free 12-enzyme pathway that can effectively convert the five-carbon xylose to inositol involving xylose phosphorylation, carbon-carbon (C-C) rearrangement, C-C bond circulation, and dephosphorylation. The reaction conditions catalyzed by all thermostable enzymes from hyperthermophilic microorganisms Thermus thermophiles, Thermotoga maritima, and Archaeoglobus fulgidus were optimized in reaction temperature, buffer type and concentration, enzyme composition, Mg2+ concentration, and fed-batch addition of ATP. The 11-enzyme cocktail, whereas a fructose 1,6-bisphosphatase from T. maritima has another function of inositol monophosphatase, converted 20â¯mM xylose to 16.1â¯mM inositol with a conversion efficiency of 96.6% at 70⯰C. Polyphosphate was found to replace ATP for xylulose phosphorylation due to broad substrate promiscuity of the T. maritima xylulokinase. The Tris-HCl buffer effectively mitigated the Maillard reaction at 70⯰C or higher temperature. The co-production of value-added biochemicals, such as inositol, from wood sugar could greatly improve economics of new biorefineries, similar to oil refineries that make value-added plastic precursors to subsidize gasoline/diesel production.
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Suplementos Dietéticos/análisis , Ingeniería Metabólica/métodos , Azúcares/química , Madera/química , Xilosa/química , Adenosina Trifosfato/metabolismo , Archaeoglobus/enzimología , Archaeoglobus/metabolismo , Catálisis , Inositol/metabolismo , Magnesio/metabolismo , Redes y Vías Metabólicas , NAD/metabolismo , Fosforilación , Thermotoga maritima/enzimología , Thermus/enzimología , Thermus/metabolismoRESUMEN
Carbon dioxide (CO2) is an appealing carbon feedstock for the sustainable production of biocommodities. Here we designed three in vitro artificial enzymatic pathways featuring the ATP-excess, ATP-deficit, and ATP-balanced pathways for the biotransformation of starch and CO2 to malate. This ATP-balanced pathway without exogenous ATP donors can auto-regulate its carbon fluxes from glyceraldehyde 3-phosphate to 3-phosphoglycerate via either the ATP-generating pathway (a part of glycolysis) or no-ATP-generating pathway from a hyperthermophilic archaeon Thermococcus kodakarensis. The ATP-balanced pathway enabled to produce up to 52.4â¯mM malate with 95.3% of the theoretical yield, that is, 2â¯mol of malate synthesized from 1â¯mol of glucose of starch and 2â¯mol of CO2. This pathway also enabled to produce high-yield malate regardless of ATP/ADP ratios. Anaerobic reaction conditions and/or the addition of a reducing agent dithiothreitol were of importance for creating an anoxic environment for biocatalysis of enzyme cocktails and for mitigating the deactivation of enzymes and degradation of intermediates. This new pathway could provide a green route for direct conversion of CO2 to many building blocks, a promising alternative of petrochemical-based production of biocommodities.
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Proteínas Arqueales/química , Dióxido de Carbono/química , Malatos/síntesis química , Almidón/química , Thermococcus/enzimología , Proteínas Arqueales/genética , Dióxido de Carbono/metabolismo , Sistema Libre de Células/química , Sistema Libre de Células/metabolismo , Malatos/química , Malatos/metabolismo , Ingeniería Metabólica , Almidón/metabolismo , Thermococcus/genéticaRESUMEN
Thermostability and specific activity of enzymes are two of the most important properties for industrial biocatalysts. Here, we developed a petri dish-based double-layer high-throughput screening (HTS) strategy for rapid identification of desired mutants of polyphosphate glucokinase (PPGK) from a thermophilic actinobacterium, Thermobifida fusca YX, with both enhanced thermostability and activity. Escherichia coli colonies representing a PPGK mutant library were grown on the first-layer Phytagel-based plates, which can remain solid for 1 h, even at heat treatment temperatures of more than 100°C. The second layer that was poured on the first layer contained agarose, substrates, glucose 6-phosphate dehydrogenase (G6PDH), the redox dye tetranitroblue tetrazolium (TNBT), and phenazine methosulfate. G6PDH was able to oxidize the product from the PPGK-catalyzed reaction and generate NADH, which can be easily examined by a TNBT-based colorimetric assay. The best mutant obtained after four rounds of directed evolution had a 7,200-fold longer half-life at 55°C, 19.8°C higher midpoint of unfolding temperature (Tm ), and a nearly 3-fold enhancement in specific activities compared to those of the wild-type PPGK. The best mutant was used to produce 9.98 g/liter myo-inositol from 10 g/liter glucose, with a theoretical yield of 99.8%, along with two other hyperthermophilic enzymes at 70°C. This PPGK mutant featuring both great thermostability and high activity would be useful for ATP-free production of glucose 6-phosphate or its derived products.IMPORTANCE Polyphosphate glucokinase (PPGK) is an enzyme that transfers a terminal phosphate group from polyphosphate to glucose, producing glucose 6-phosphate. A petri dish-based double-layer high-throughput screening strategy was developed by using ultrathermostable Phytagel as the first layer instead of agar or agarose, followed by a redox dye-based assay for rapid identification of ultrathermostable PPGK mutants. The best mutant featuring both great thermostability and high activity could produce glucose 6-phosphate from glucose and polyphosphate without in vitro ATP regeneration.
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Actinomycetales/enzimología , Coevolución Biológica , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Actinomycetales/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Estabilidad de Enzimas , Enzimas , Escherichia coli/genética , Biblioteca de Genes , Glucosa/metabolismo , Glucosafosfato Deshidrogenasa/metabolismo , Ensayos Analíticos de Alto Rendimiento , Cinética , Mutación , Polifosfatos/metabolismo , Estabilidad Proteica , TemperaturaRESUMEN
Cofactor-dependent oxidoreduction and electron transfer play an important role in in vitro bioelectricity generation and many other enzyme biocatalysis reactions. To facilitate such electron generation and transfer, several approaches based on the coimmobilization of cofactors and oxidoreductases have been demonstrated. Herein, a convenient and immobilization-free approach of constructing enzyme-cofactor and enzyme-mediator conjugates was developed. The in vitro bioelectricity generation reactions via enzymatic fuel cells were evaluated. The cells equipped by the conjugates exhibited significantly improved power output and stability in contrast to those mediated by unconjugated enzymes. These results may bring a new avenue in constructing efficient in vitro electron transfer chains for various biocatalysis applications.
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Fuentes de Energía Bioeléctrica , Coenzimas/química , Enzimas/química , Biocatálisis , Transporte de Electrón , Técnicas In Vitro , Oxidación-ReducciónRESUMEN
Fructose 1,6-diphosphate (FDP) is a widely used medicine and is also a precursor of two important three-carbon phosphates - glyceraldehyde 3-phosphate (GA3P) and dihydroxyacetone phosphate (DHAP) for the biosynthesis of numerous fine chemicals. An in vitro synthetic cofactor-free enzymatic pathway comprised of four hyperthermophilic enzymes was designed to produce FDP from starch and pyrophosphate. All of four hyperthermophilic enzymes (i.e., alpha-glucan phosphorylase from Thermotaga maritima, phosphoglucomutase from Thermococcus kodakarensis, glucose 6-phosphate isomerase from Thermus thermophilus, and pyrophosphate phosphofructokinase from T. maritima) were overexpressed in E. coli BL21(DE3) and purified by simple heat precipitation. The optimal pH and temperature of one-pot biosynthesis were 7.2 and 70°C, respectively. The optimal enzyme ratios of αGP, PGM, PGI and PFK were 2:2:1:2 in terms of units. Via step-wise addition of new substrates, up to 125 ± 4.6mM FDP was synthesized after 7-h reaction. This de novo ATP-free enzymatic pathway comprised of all hyperthermophilic enzymes could drastically decrease the manufacturing costs of FDP and its derivatives GA3P and DHAP, better than those catalyzed by ATP-regeneration cascade biocatalysis, the use of mesophilic enzymes, whole cell lysates, and microbial cell factories.
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Escherichia coli , Fructosadifosfatos/biosíntesis , Ingeniería Metabólica , Proteínas Arqueales/biosíntesis , Proteínas Arqueales/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Fructosadifosfatos/genética , Thermococcus/enzimología , Thermococcus/genética , Thermotoga maritima/enzimología , Thermotoga maritima/genética , Thermus thermophilusRESUMEN
Rare codon in a heterologous gene may cause premature termination of protein synthesis, misincorporation of amino acids, and/or slow translation of mRNA, decreasing the heterologous protein expression. However, its hypothetical function pertaining to functional protein folding has been barely reported. Here, we investigated the effects of selective introduction of synonymous rare codons (SRCs) to two codon-optimized (i.e., rare codon-free) genes sucrose phosphorylase (SP) gene from Thermoanaerobacterium thermosaccharolyticum and amidohydrolase gene from Streptomyces caatingaensis on their expression levels in Escherichia coli BL21(DE3). We investigated the introduction of a single SRC to the coding regions of alpha-helix, beta-strand, or linker in the first half of rare codon-free sp and ah gene. The introduction of a single SRC in the beginning of the coding regions of beta-strand greatly enhanced their soluble expression levels as compared to the other regions. Also, we applied directed evolution to test multi-SRC-containing sp gene mutants for enhanced soluble SP expression levels. To easily identify the soluble SP expression level of colonies growing on Petri dishes, mCherry fluorescent protein was used as a SP-folding reporter when it was fused to the 3' end of the sp gene mutant libraries. After three rounds of screening, the best sp gene mutant containing nine SRCs exhibited an approximately six-fold enhancement in soluble protein expression level as compared to the wild-type and rare codon-free sp control. This study suggests that the selective introduction of SRCs can attenuate translation at specific points and such discontinuous attenuation can temporally separate the translation of segments of the peptide chains and actively coordinates their co-translational folding, resulting in enhanced functional protein expression. Biotechnol. Bioeng. 2017;114: 1054-1064. © 2016 Wiley Periodicals, Inc.
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Codón/genética , Evolución Molecular Dirigida/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Glucosiltransferasas/genética , Mutación Silenciosa/genética , Clonación Molecular , Glucosiltransferasas/química , Glucosiltransferasas/metabolismo , Modelos Moleculares , Biosíntesis de Proteínas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Thermoanaerobacterium/enzimología , Thermoanaerobacterium/genéticaRESUMEN
Myo-Inositol (vitamin B8) is widely used in the drug, cosmetic, and food & feed industries. Here, we present an in vitro non-fermentative enzymatic pathway that converts starch to inositol in one vessel. This in vitro pathway is comprised of four enzymes that operate without ATP or NAD+ supplementation. All enzyme BioBricks are carefully selected from hyperthermophilic microorganisms, that is, alpha-glucan phosphorylase from Thermotoga maritima, phosphoglucomutase from Thermococcus kodakarensis, inositol 1-phosphate synthase from Archaeoglobus fulgidus, and inositol monophosphatase from T. maritima. They were expressed efficiently in high-density fermentation of Escherichia coli BL21(DE3) and easily purified by heat treatment. The four-enzyme pathway supplemented with two other hyperthermophilic enzymes (i.e., 4-α-glucanotransferase from Thermococcus litoralis and isoamylase from Sulfolobus tokodaii) converts branched or linear starch to inositol, accomplishing a very high product yield of 98.9 ± 1.8% wt./wt. This in vitro (aeration-free) biomanufacturing has been successfully operated on 20,000-L reactors. Less costly inositol would be widely added in heath food, low-end soft drink, and animal feed, and may be converted to other value-added biochemicals (e.g., glucarate). This biochemical is the first product manufactured by the in vitro synthetic biology platform on an industrial scale. Biotechnol. Bioeng. 2017;114: 1855-1864. © 2017 Wiley Periodicals, Inc.
Asunto(s)
Reactores Biológicos/microbiología , Escherichia coli/fisiología , Inositol/metabolismo , Complejos Multienzimáticos/fisiología , Ingeniería de Proteínas/métodos , Almidón/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Vías Biosintéticas/fisiología , Industria Farmacéutica/métodos , Inositol/genética , Inositol/aislamiento & purificación , Monoéster Fosfórico Hidrolasas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Biología Sintética/métodosRESUMEN
Biomanufacturing is a type of manufacturing that utilizes biological systems (e.g., living microorganisms, resting cells, animal cells, plant cells, tissues, enzymes, or in vitro synthetic (enzymatic) systems) to produce commercially important biomolecules for use in the agricultural, food, material, energy, and pharmaceutical industries. History of biomanufacturing could be classified into the three revolutions in terms of respective product types (mainly), production platforms, and research technologies. Biomanufacturing 1.0 focuses on the production of primary metabolites (e.g., butanol, acetone, ethanol, citric acid) by using mono-culture fermentation; biomanufacturing 2.0 focuses on the production of secondary metabolites (e.g., penicillin, streptomycin) by using a dedicated mutant and aerobic submerged liquid fermentation; and biomanufacturing 3.0 focuses on the production of large-size biomolecules-proteins and enzymes (e.g., erythropoietin, insulin, growth hormone, amylase, DNA polymerase) by using recombinant DNA technology and advanced cell culture. Biomanufacturing 4.0 could focus on new products, for example, human tissues or cells made by regenerative medicine, artificial starch made by in vitro synthetic biosystems, isobutanol fermented by metabolic engineering, and synthetic biology-driven microorganisms, as well as exiting products produced by far better approaches. Biomanufacturing 4.0 would help address some of the most important challenges of humankind, such as food security, energy security and sustainability, water crisis, climate change, health issues, and conflict related to the energy, food, and water nexus.