RESUMEN
Efficient co-utilization of mixed sugar feedstocks remains a biomanufacturing challenge, thus motivating ongoing efforts to engineer microbes for improved conversion of glucose-xylose mixtures. This study focuses on enhancing phenylalanine production by engineering Escherichia coli to efficiently co-utilize glucose and xylose. Flux balance analysis identified E4P flux as a bottleneck which could be alleviated by increasing the xylose-to-glucose flux ratio. A mutant copy of the xylose-specific activator (XylR) was then introduced into the phenylalanine-overproducing E. coli NST74, which relieved carbon catabolite repression and enabled efficient glucose-xylose co-utilization. Carbon contribution analysis through 13 C-fingerprinting showed a higher preference for xylose in the engineered strain (NST74X), suggesting superior catabolism of xylose relative to glucose. As a result, NST74X produced 1.76 g/L phenylalanine from a model glucose-xylose mixture; a threefold increase over NST74. Then, using biomass-derived sugars, NST74X produced 1.2 g/L phenylalanine, representing a 1.9-fold increase over NST74. Notably, and consistent with the carbon contribution analysis, the xylR* mutation resulted in a fourfold greater maximum rate of xylose consumption without significantly impeding the maximum rate of total sugar consumption (0.87 vs. 0.70 g/L-h). This study presents a novel strategy for enhancing phenylalanine production through the co-utilization of glucose and xylose in aerobic E. coli cultures, and highlights the potential synergistic benefits associated with using substrate mixtures over single substrates when targeting specific products.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Azúcares/metabolismo , Xilosa/metabolismo , Biomasa , Fermentación , Glucosa/metabolismo , Aminoácidos Aromáticos/metabolismo , Fenilalanina/metabolismo , Carbono/metabolismo , Factores de Transcripción/genética , Proteínas de Escherichia coli/metabolismoRESUMEN
Anaerobic succinate fermentations can achieve high-titer, high-yield performance while fixing CO2 through the reductive branch of the tricarboxylic acid cycle. To provide the needed CO2, conventional media is supplemented with significant (up to 60 g/L) bicarbonate (HCO3-), and/or carbonate (CO32-) salts. However, producing these salts from CO2 and natural ores is thermodynamically unfavorable and, thus, energetically costly, which reduces the overall sustainability of the process. Here, a series of composite hollow fiber membranes (HFMs) were first fabricated, after which comprehensive CO2 mass transfer measurements were performed under cell-free conditions using a novel, constant-pH method. Lumen pressure and total HFM surface area were found to be linearly correlated with the flux and volumetric rate of CO2 delivery, respectively. Novel HFM bioreactors were then constructed and used to comprehensively investigate the effects of modulating the CO2 delivery rate on succinate fermentations by engineered Escherichia coli. Through appropriate tuning of the design and operating conditions, it was ultimately possible to produce up to 64.5 g/L succinate at a glucose yield of 0.68 g/g; performance approaching that of control fermentations with directly added HCO3-/CO32- salts and on par with prior studies. HFMs were further found to demonstrate a high potential for repeated reuse. Overall, HFM-based CO2 delivery represents a viable alternative to the addition of HCO3-/CO32- salts to succinate fermentations, and likely other 'dark' CO2-fixing fermentations.
Asunto(s)
Dióxido de Carbono , Ácido Succínico , Fermentación , Dióxido de Carbono/farmacología , Sales (Química) , Succinatos , Escherichia coli , Carbonatos/farmacologíaRESUMEN
Robust systematic approaches for the metabolic engineering of cell factories remain elusive. The available models for predicting phenotypical responses and mechanisms are incomplete, particularly within the context of compound toxicity that can be a significant impediment to achieving high yields of a target product. This study describes a Multi-Omic Based Production Strain Improvement (MOBpsi) strategy that is distinguished by integrated time-resolved systems analyses of fed-batch fermentations. As a case study, MOBpsi was applied to improve the performance of an Escherichia coli cell factory producing the commodity chemical styrene. Styrene can be bio-manufactured from phenylalanine via an engineered pathway comprised of the enzymes phenylalanine ammonia lyase and ferulic acid decarboxylase. The toxicity, hydrophobicity, and volatility of styrene combine to make bio-production challenging. Previous attempts to create styrene tolerant E. coli strains by targeted genetic interventions have met with modest success. Application of MOBpsi identified new potential targets for improving performance, resulting in two host strains (E. coli NST74ΔaaeA and NST74ΔaaeA cpxPo) with increased styrene production. The best performing re-engineered chassis, NST74ΔaaeA cpxPo, produced â¼3 × more styrene and exhibited increased viability in fed-batch fermentations. Thus, this case study demonstrates the utility of MOBpsi as a systematic tool for improving the bio-manufacturing of toxic chemicals.
Asunto(s)
Escherichia coli , Ingeniería Metabólica , Escherichia coli/metabolismo , Fermentación , Ingeniería Metabólica/métodos , Fenilalanina/genética , Fenilalanina/metabolismo , Estireno/metabolismoRESUMEN
Carbon loss in the form of CO2 is an intrinsic and persistent challenge faced during conventional and advanced biofuel production from biomass feedstocks. Current mechanisms for increasing carbon conservation typically require the provision of reduced co-substrates as additional reducing equivalents. This need can be circumvented, however, by exploiting the natural heterogeneity of lignocellulosic sugars mixtures and strategically using specific fractions to drive complementary CO2 emitting vs. CO2 fixing pathways. As a demonstration of concept, a coculture-coproduction system was developed by pairing two catabolically orthogonal Escherichia coli strains; one converting glucose to ethanol (G2E) and the other xylose to succinate (X2S). 13C-labeling studies reveled that G2E + X2S cocultures were capable of recycling 24% of all evolved CO2 and achieved a carbon conservation efficiency of 77%; significantly higher than the 64% achieved when all sugars are instead converted to just ethanol. In addition to CO2 exchange, the latent exchange of pyruvate between strains was discovered, along with significant carbon rearrangement within X2S.
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Dióxido de Carbono , Carbono , Técnicas de Cocultivo , Fermentación , Glucosa , XilosaRESUMEN
With the goal of expanding the diversity of tools available for controlling gene expression in cyanobacteria, the T7-RNA polymerase gene expression system from E. coli BL21(DE3) was adapted and systematically engineered for robust function Synechococcus sp. PCC 7002, a fast-growing saltwater strain. Expression of T7-RNA polymerase was controlled via LacI regulation, while functionality was optimized by both further tuning its expression level along with optimizing the translation initiation region of the expressed gene, in this case an enhanced YFP reporter. Under high CO2 conditions, the resulting system displayed a 60-fold dynamic range in expression levels. Furthermore, when maximally induced, T7-RNA polymerase-dependent protein production constituted up to two-thirds of total cellular protein content in Synechococcus sp. PCC 7002. Ultimately, however, this came at the cost of 40% reductions in both biomass and pigmentation levels. Taken together, the developed T7-RNA polymerase gene expression system is effective for controlling and achieving high-level expression of heterologous genes in Synechococcus sp. PCC 7002, making it a valuable tool for cyanobacterial research. KEY POINTS: ⢠Promoter driving T7-RNA polymerase was optimized. ⢠Up to 60-fold dynamic range in expression, depending on CO2 conditions. ⢠Two-thirds of total protein is T7-RNA polymerase dependent.
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Synechococcus , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Fenotipo , Synechococcus/genética , Synechococcus/metabolismoRESUMEN
The high-flux/low-affinity cyanobacterial bicarbonate transporter BicA is a member of sulfate permease/solute carrier 26 (SulP/SLC26) family and plays a major role in cyanobacterial inorganic carbon uptake. In order to study this important membrane protein, robust platforms for over-expression and protocols for purification are required. In this work we have optimized the expression and purification of BicA from strain Synechocystis sp. PCC 6803 (BicA6803) in Escherichia coli. It was determined that expression with C43 (DE3) Rosetta2 at 37 °C produced the highest levels of over-expressed BicA6803 relative to other strains screened, and membrane solubilization with n-dodecyl-ß-d-maltopyranoside facilitated the purification of high levels of stable and homogenous BicA6803. Using these expression and purification strategies, the final yields of purified BicA were 6.5 ± 1.0 mg per liter of culture.
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Proteínas Bacterianas , Expresión Génica , Simportadores de Sodio-Bicarbonato , Synechocystis/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Simportadores de Sodio-Bicarbonato/biosíntesis , Simportadores de Sodio-Bicarbonato/química , Simportadores de Sodio-Bicarbonato/genética , Simportadores de Sodio-Bicarbonato/aislamiento & purificación , Synechocystis/metabolismoRESUMEN
Efficient microbial conversion of lignocellulose into valuable products is often hindered by the presence of furfural, a dehydration product of pentoses in hemicellulose sugar syrups derived from woody biomass. For a cost-effective lignocellulose microbial conversion, robust biocatalysts are needed that can tolerate toxic inhibitors while maintaining optimal metabolic activities. A comprehensive plasmid-based library encoding native multidrug resistance (MDR) efflux pumps, porins, and select exporters from Escherichia coli was screened for furfural tolerance in an ethanologenic E. coli strain. Small multidrug resistance (SMR) pumps, such as SugE and MdtJI, as well as a lactate/glycolate:H+ symporter, LldP, conferred furfural tolerance in liquid culture tests. Expression of the SMR pump potentially increased furfural efflux and cellular viability upon furfural assault, suggesting novel activities for SMR pumps as furfural efflux proteins. Furthermore, induced expression of mdtJI enhanced ethanol fermentative production of LY180 in the presence of furfural or 5-hydroxymethylfurfural, further demonstrating the applications of SMR pumps. This work describes an effective approach to identify useful efflux systems with desired activities for nonnative toxic chemicals and provides a platform to further enhance furfural efflux by protein engineering and mutagenesis.IMPORTANCE Lignocellulosic biomass, especially agricultural residues, represents an important potential feedstock for microbial production of renewable fuels and chemicals. During the deconstruction of hemicellulose by thermochemical processes, side products that inhibit cell growth and production, such as furan aldehydes, are generated, limiting cost-effective lignocellulose conversion. Here, we developed a new approach to increase cellular tolerance by expressing multidrug resistance (MDR) pumps with putative efflux activities for furan aldehydes. The developed plasmid library and screening methods may facilitate new discoveries of MDR pumps for diverse toxic chemicals important for microbial conversion.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Etanol/metabolismo , Furaldehído/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Bioprospección , Proteínas de Escherichia coli/genética , Fermentación , Furaldehído/análogos & derivados , Ingeniería Genética , Lignina/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genéticaRESUMEN
Constructing microbial biocatalysts that produce biorenewables at economically viable yields and titers is often hampered by product toxicity. For production of short chain fatty acids, membrane damage is considered the primary mechanism of toxicity, particularly in regards to membrane integrity. Previous engineering efforts in Escherichia coli to increase membrane integrity, with the goal of increasing fatty acid tolerance and production, have had mixed results. Herein, a novel approach was used to reconstruct the E. coli membrane by enabling production of a novel membrane component. Specifically, trans unsaturated fatty acids (TUFA) were produced and incorporated into the membrane of E. coli MG1655 by expression of cis-trans isomerase (Cti) from Pseudomonas aeruginosa. While the engineered strain was found to have no increase in membrane integrity, a significant decrease in membrane fluidity was observed, meaning that membrane polarization and rigidity were increased by TUFA incorporation. As a result, tolerance to exogenously added octanoic acid and production of octanoic acid were both increased relative to the wild-type strain. This membrane engineering strategy to improve octanoic acid tolerance was found to require fine-tuning of TUFA abundance. Besides improving tolerance and production of carboxylic acids, TUFA production also enabled increased tolerance in E. coli to other bio-products, e.g. alcohols, organic acids, aromatic compounds, a variety of adverse industrial conditions, e.g. low pH, high temperature, and also elevated styrene production, another versatile bio-chemical product. TUFA permitted enhanced growth due to alleviation of bio-product toxicity, demonstrating the general effectiveness of this membrane engineering strategy towards improving strain robustness.
Asunto(s)
Proteínas Bacterianas , Escherichia coli , Ingeniería Metabólica , Pseudomonas aeruginosa/genética , cis-trans-Isomerasas , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Grasos Insaturados , Pseudomonas aeruginosa/enzimología , cis-trans-Isomerasas/biosíntesis , cis-trans-Isomerasas/genéticaRESUMEN
The non-renewable petrochemical phenol is used as a precursor to produce numerous fine and commodity chemicals, including various pharmaceuticals and phenolic resins. Microbial phenol biosynthesis has previously been established, stemming from endogenous tyrosine via tyrosine phenol lyase (TPL). TPL, however, suffers from feedback inhibition and equilibrium limitations, both of which contribute to reduced flux through the overall pathway. To address these limitations, two novel and non-natural phenol biosynthesis pathways, both stemming instead from chorismate, were constructed and comparatively evaluated. The first proceeds to phenol in one heterologous step via the intermediate p-hydroxybenzoic acid, while the second involves two heterologous steps and the associated intermediates isochorismate and salicylate. Maximum phenol titers achieved via these two alternative pathways reached as high as 377 ± 14 and 259 ± 31 mg/L in batch shake flask cultures, respectively. In contrast, under analogous conditions, phenol production via the established TPL-dependent route reached 377 ± 23 mg/L, which approaches the maximum achievable output reported to date under batch conditions. Additional strain development and optimization of relevant culture conditions with respect to each individual pathway is ultimately expected to result in further improved phenol production. Biotechnol. Bioeng. 2016;113: 1745-1754. © 2016 Wiley Periodicals, Inc.
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Bacterias/enzimología , Bacterias/genética , Ingeniería Metabólica/métodos , Fenol/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Técnicas de Cultivo Celular por Lotes , Escherichia coli/genética , Escherichia coli/metabolismo , Glucosa/metabolismo , Redes y Vías Metabólicas , Tirosina/metabolismo , Tirosina Fenol-Liasa/genética , Tirosina Fenol-Liasa/metabolismoRESUMEN
Fermentative production of styrene from glucose has been previously demonstrated in Escherichia coli. Here, we demonstrate the production of styrene from the sugars derived from lignocellulosic biomass depolymerized by fast pyrolysis. A previously engineered styrene-producing strain was further engineered for utilization of the anhydrosugar levoglucosan via expression of levoglucosan kinase. The resulting strain produced 240 ± 3 mg L(-1) styrene from pure levoglucosan, similar to the 251 ± 3 mg L(-1) produced from glucose. When provided at a concentration of 5 g L(-1), pyrolytic sugars supported styrene production at titers similar to those from pure sugars, demonstrating the feasibility of producing this important industrial chemical from biomass-derived sugars. However, the toxicity of contaminant compounds in the biomass-derived sugars and styrene itself limit further gains in production. Styrene toxicity is generally believed to be due to membrane damage. Contrary to this prevailing wisdom, our quantitative assessment during challenge with up to 200 mg L(-1) of exogenously provided styrene showed little change in membrane integrity; membrane disruption was observed only during styrene production. Membrane fluidity was also quantified during styrene production, but no changes were observed relative to the non-producing control strain. This observation that styrene production is much more damaging to the membrane integrity than challenge with exogenously supplied styrene provides insight into the mechanism of styrene toxicity and emphasizes the importance of verifying proposed toxicity mechanisms during production instead of relying upon results obtained during exogenous challenge.
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Biomasa , Metabolismo de los Hidratos de Carbono , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Lignina/metabolismo , Estireno/metabolismo , Estireno/toxicidad , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Escherichia coli/citología , Escherichia coli/genética , Glucosa/análogos & derivados , Glucosa/metabolismo , Lignina/química , Fluidez de la Membrana/efectos de los fármacos , Estireno/farmacologíaRESUMEN
As an important conventional monomer compound, the biological production of styrene carries significant promise with respect to creating novel sustainable materials. Since end-product toxicity presently limits styrene production by previously engineered Escherichia coli, in situ product removal by both solvent extraction and gas stripping were explored as process-based strategies for circumventing its inhibitory effects. In solvent extraction, the addition of bis(2-ethylhexyl)phthalate offered the greatest productivity enhancement, allowing net volumetric production of 836 ± 64 mg/L to be reached, representing a 320 % improvement over single-phase cultures. Gas stripping rates, meanwhile, were controlled by rates of bioreactor agitation and, to a greater extent, aeration. A periodic gas stripping protocol ultimately enabled up to 561 ± 15 mg/L styrene to be attained. Lastly, by relieving the effects of styrene toxicity, new insight was gained regarding subsequent factors limiting its biosynthesis in E. coli and strategies for future strain improvement are discussed.
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Estireno/aislamiento & purificación , Reactores Biológicos , Dietilhexil Ftalato/química , Escherichia coli/genética , Escherichia coli/metabolismo , Estireno/metabolismoRESUMEN
BACKGROUND: Styrene is an important building-block petrochemical and monomer used to produce numerous plastics. Whereas styrene bioproduction by Escherichia coli was previously reported, the long-term potential of this approach will ultimately rely on the use of hosts with improved industrial phenotypes, such as the yeast Saccharomyces cerevisiae. RESULTS: Classical metabolic evolution was first applied to isolate a mutant capable of phenylalanine over-production to 357 mg/L. Transcription analysis revealed up-regulation of several phenylalanine biosynthesis pathway genes including ARO3, encoding the bottleneck enzyme DAHP synthase. To catalyze the first pathway step, phenylalanine ammonia lyase encoded by PAL2 from A. thaliana was constitutively expressed from a high copy plasmid. The final pathway step, phenylacrylate decarboxylase, was catalyzed by the native FDC1. Expression of FDC1 was naturally induced by trans-cinnamate, the pathway intermediate and its substrate, at levels sufficient for ensuring flux through the pathway. Deletion of ARO10 to eliminate the competing Ehrlich pathway and expression of a feedback-resistant DAHP synthase encoded by ARO4K229L preserved and promoted the endogenous availability precursor phenylalanine, leading to improved pathway flux and styrene production. These systematic improvements allowed styrene titers to ultimately reach 29 mg/L at a glucose yield of 1.44 mg/g, a 60% improvement over the initial strain. CONCLUSIONS: The potential of S. cerevisiae as a host for renewable styrene production has been demonstrated. Significant strain improvements, however, will ultimately be needed to achieve economical production levels.
Asunto(s)
Técnicas Químicas Combinatorias/métodos , Ingeniería Metabólica , Saccharomyces cerevisiae/metabolismo , Estireno/metabolismo , Transporte Biológico/efectos de los fármacos , Carboxiliasas/metabolismo , Cinamatos/metabolismo , Glucosa/metabolismo , Mutación/genética , Fenotipo , Fenilalanina/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Transcripción Genética/efectos de los fármacosRESUMEN
Styrene is an important commodity chemical used in polymers and resins, and is typically produced from the petrochemical feedstocks benzene and ethylene. Styrene has recently been produced biosynthetically for the first time using engineered Escherichia coli, and this bio-based route may represent a lower energy and renewable alternative to petroleum-derived styrene. However, the economics of such an approach has not yet been investigated. Using an early-stage technoeconomic evaluation tool, a preliminary economic analysis of bio-based styrene from C(6)-sugar feedstock has been conducted. Owing to styrene's limited water solubility, it was assumed that the resulting fermentation broth would spontaneously form two immiscible liquid phases that could subsequently be decanted. Assuming current C(6) sugar prices and industrially achievable biokinetic parameter values (e.g., product yield, specific growth rate), commercial-scale bio-based styrene has a minimum estimated selling price (MESP) of 1.90 USD kg(-1) which is in the range of current styrene prices. A Monte Carlo analysis revealed a potentially large (0.45 USD kg(-1)) standard deviation in the MESP, while a sensitivity analysis showed feedstock price and overall yield as primary drivers of MESP.
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Reactores Biológicos , Vías Biosintéticas/fisiología , Escherichia coli/fisiología , Microbiología Industrial/economía , Modelos Económicos , Estireno/metabolismo , Carboxiliasas , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentación , Ingeniería Genética , Microbiología Industrial/métodos , Cinética , Método de Montecarlo , Fenilalanina/química , Fenilalanina/metabolismo , Estireno/química , Estireno/economíaRESUMEN
Lignin valorization faces persistent biomanufacturing challenges due to the heterogeneous and toxic carbon substrates derived from lignin depolymerization. To address the heterogeneous nature of aromatic feedstocks, plant cell wall engineering and 'lignin first' pretreatment methods have recently emerged. Next, to convert the resulting aromatic substrates into value-added chemicals, diverse microbial host systems also continue to be developed. This includes microbes that (1) lack aromatic metabolism, (2) metabolize aromatics but not sugars, and (3) co-metabolize both aromatics and sugars, each system presenting unique pros and cons. Considering the intrinsic complexity of lignin-derived substrate mixtures, emerging and non-model microbes with native metabolism for aromatics appear poised to provide the greatest impacts on lignin valorization via biomanufacturing.
RESUMEN
(S)- and (R)-3-hydroxybutyrate (3HB) are precursors to synthesize the biodegradable plastics polyhydroxyalkanoates (PHAs) and many fine chemicals. To date, however, their production has been restricted to petroleum-based chemical industry and sugar-based microbial fermentation, limiting its sustainability and economical feasibility. With the ability to fix CO2 photosynthetically, cyanobacteria have attracted increasing interest as a biosynthesis platform to produce fuels and chemicals from alternative renewable resources. To this end, synthesis metabolic pathways have been constructed and optimized in cyanobacterium Synechocystis sp. PCC 6803 to photosynthetically produce (S)- and (R)-3HB directly from CO2. Both types of 3HB molecules were produced and readily secreted from Synechocystis cells without over-expression of transporters. Additional inactivation of the competing pathway by deleting slr1829 and slr1830 (encoding PHB polymerase) from the Synechocystis genome further promoted the 3HB production. Up to 533.4mg/L 3HB has been produced after photosynthetic cultivation of the engineered cyanobacterium Synechocystis TABd for 21 days. Further analysis indicated that the phosphate consumption during the photoautrophic growth and the concomitant elevated acetyl-CoA pool acted as a key driving force for 3HB biosynthesis in Synechocystis. For the first time, the study has demonstrated the feasibility of photosynthetic production of (S)- and (R)-3HB directly from sunlight and CO2.
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Ácido 3-Hidroxibutírico/biosíntesis , Ingeniería Metabólica/métodos , Fotosíntesis , Synechocystis/metabolismo , Ácido 3-Hidroxibutírico/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Eliminación de Gen , Expresión Génica , Synechocystis/genéticaRESUMEN
Through metabolic pathway engineering, novel microbial biocatalysts can be engineered to convert renewable resources into useful chemicals, including monomer building-blocks for bioplastics production. Here we describe the systematic engineering of Escherichia coli to produce, as individual products, two 5-carbon polyamide building blocks, namely 5-aminovalerate (AMV) and glutarate. The modular pathways were derived using "parts" from the natural lysine degradation pathway of Pseudomonas putida KT2440. Endogenous over-production of the required precursor, lysine, was first achieved through metabolic deregulation of its biosynthesis pathway by introducing feedback resistant mutants of aspartate kinase III and dihydrodipicolinate synthase. Further disruption of native lysine decarboxylase activity (by deleting cadA and ldcC) limited cadaverine by-product formation, enabling lysine production to 2.25 g/L at a glucose yield of 138 mmol/mol (18% of theoretical). Co-expression of lysine monooxygenase and 5-aminovaleramide amidohydrolase (encoded by davBA) then resulted in the production of 0.86 g/L AMV in 48 h. Finally, the additional co-expression of glutaric semialdehyde dehydrogenase and 5-aminovalerate aminotransferase (encoded by davDT) led to the production of 0.82 g/L glutarate under the same conditions. At this output, yields on glucose were 71 and 68 mmol/mol for AMV and glutarate (9.5 and 9.1% of theoretical), respectively. These findings further expand the number and diversity of polyamide monomers that can be derived directly from renewable resources.
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Aminoácidos Neutros/metabolismo , Escherichia coli/metabolismo , Glutaratos/metabolismo , Ingeniería Metabólica/métodos , Aminoácidos Neutros/análisis , Cadaverina/metabolismo , Medios de Cultivo , Escherichia coli/genética , Glutaratos/análisis , Microbiología Industrial , Modelos Lineales , Lisina/metabolismoAsunto(s)
Adenoidectomía , Codeína/efectos adversos , Tonsilectomía , Niño , Preescolar , Codeína/metabolismo , Contraindicaciones , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Quimioterapia/mortalidad , Humanos , Morfina/metabolismo , Dolor Postoperatorio/tratamiento farmacológico , Insuficiencia Respiratoria/inducido químicamenteRESUMEN
Styrene is a large volume, commodity petrochemical with diverse commercial applications, including as a monomer building-block for the synthesis of many useful polymers. Here we demonstrate how, through the de novo design and development of a novel metabolic pathway, styrene can alternatively be synthesized from renewable substrates such as glucose. The conversion of endogenously synthesized l-phenylalanine to styrene was achieved by the co-expression of phenylalanine ammonia lyase and trans-cinnamate decarboxylase. Candidate isoenzymes for each step were screened from bacterial, yeast, and plant genetic sources. Finally, over-expression of PAL2 from Arabidopsis thaliana and FDC1 from Saccharomyces cerevisiae (originally classified as ferulate decarboxylase) in an l-phenylalanine over-producing Escherichia coli host led to the accumulation of up to 260 mg/L in shake flask cultures. Achievable titers already approach the styrene toxicity threshold (determined as ~300 mg/L). To the best of our knowledge, this is the first report of microbial styrene production from sustainable feedstocks.
Asunto(s)
Isomerasas Aldosa-Cetosa/biosíntesis , Carboxiliasas/biosíntesis , Escherichia coli , Organismos Modificados Genéticamente , Estireno/metabolismo , Isomerasas Aldosa-Cetosa/genética , Arabidopsis/enzimología , Arabidopsis/genética , Carboxiliasas/genética , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Expresión Génica , Glucosa/genética , Glucosa/metabolismo , Organismos Modificados Genéticamente/genética , Organismos Modificados Genéticamente/crecimiento & desarrollo , Organismos Modificados Genéticamente/metabolismo , Fenilalanina/genética , Fenilalanina/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genéticaRESUMEN
The American Academy of Dermatology and the American Society of Plastic Surgeons, with the support of other sister societies, conducted the Facial Soft-Tissue Fillers: Assessing the State of the Science conference in December of 2009. The American Academy of Dermatology and the American Society of Plastic Surgeons established a panel of leading experts in the field of soft-tissue fillers-from researchers to clinicians-and other stakeholders for the conference to examine and discuss issues of patient safety, efficacy, and effectiveness in relation to the approved and off-label use of soft-tissue fillers, and other factors, including the training and level of experience of individuals administering fillers. This report summarizes the deliberations and key points made by the panel and presenters to the panel, and includes a summary of the panel's near-term and longer term recommendations for next steps to help guide future efforts to address the safety, efficacy, and effectiveness of facial soft-tissue fillers. This report represents the panel's assessment of the medical knowledge available on facial soft-tissue fillers at the time of the conference.
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Técnicas Cosméticas , Fármacos Dermatológicos/administración & dosificación , Dermatología/métodos , Cara , Cirugía Plástica/métodos , Sistemas de Registro de Reacción Adversa a Medicamentos , Colágeno/administración & dosificación , Colágeno/efectos adversos , Técnicas Cosméticas/efectos adversos , Fármacos Dermatológicos/efectos adversos , Dermatología/educación , Durapatita/administración & dosificación , Durapatita/efectos adversos , Medicina Basada en la Evidencia , Predicción , Humanos , Ácido Hialurónico/administración & dosificación , Ácido Hialurónico/efectos adversos , Ácido Láctico/administración & dosificación , Ácido Láctico/efectos adversos , Poliésteres , Polímeros/administración & dosificación , Polímeros/efectos adversos , Polimetil Metacrilato/administración & dosificación , Polimetil Metacrilato/efectos adversos , Rejuvenecimiento , Investigación , Cirugía Plástica/educación , Resultado del TratamientoRESUMEN
The American Academy of Dermatology and the American Society of Plastic Surgeons, with the support of other sister societies, conducted the Facial Soft-Tissue Fillers: Assessing the State of the Science conference in December of 2009. The American Academy of Dermatology and the American Society of Plastic Surgeons established a panel of leading experts in the field of soft-tissue fillers-from researchers to clinicians-and other stakeholders for the conference to examine and discuss issues of patient safety, efficacy, and effectiveness in relation to the approved and off-label use of soft-tissue fillers, and other factors, including the training and level of experience of individuals administering fillers. This report represents the systematic literature review that examines comprehensively the available evidence and gaps in the evidence related to soft-tissue fillers, to inform and support the work of the state-of-the-science conference panel. This evidence-based medicine review will serve as the foundation for future evidence-based medicine reports in this growing field.