RESUMEN
Retinoids can be produced from E. coli when introduced with the ß-carotene biosynthesis pathway and the BCMO gene. E. coli has no inherent metabolic pathways related to retinoids, therefore only retinal should be produced from the cleavage of ß-carotene by BCMO. However, retinol and retinyl acetate were also produced in significant amounts, by the non-specific activity of inherent promiscuous enzymes or the antibiotic resistance marker of the retinal-producing plasmids. Retinol was produced by the ybbO gene of E. coli which encodes oxidoreductase and retinyl acetate was produced by the chloramphenicol resistance gene, called cat gene which encodes chloramphenicol acetyltransferase, present within the pS-NA plasmid that also contains the mevalonate pathway. The composition of retinoids could be modulated by manipulating the relevant genes. The composition of retinol, a commercially important retinoid, was significantly increased by the overexpression of ybbO gene and the removal of cat gene in the recombinant E. coli, which suggests the possibility of selective retinoid production in the future.
Asunto(s)
Escherichia coli/genética , Escherichia coli/metabolismo , Ingeniería Metabólica/métodos , Vitamina A/metabolismo , Eliminación de Gen , Expresión Génica , Organismos Modificados Genéticamente/genética , Organismos Modificados Genéticamente/metabolismoRESUMEN
To prevent degradation of intracellular retinoids through in situ extraction from the cells, a two-phase culture system was performed. Several organic solvents, including n-alkanes, mineral oils and cosmetic raw materials, were applied as the extraction phase. Of the n-alkanes, n-decane had the highest retinoid production as 134 mg/l after 72 h. For mineral oil, light and heavy mineral oil gave retinoid productions of 158 and 174 mg/l after 96 h, respectively. Of other materials, isopropyl myristate gave the highest retinoid production of 181 mg/l. These results indicate that many types of oils can be applied for retinoid production, and optimization of the in situ extraction process will lead to further improve of economical production for the industrial purpose.
Asunto(s)
Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Retinoides/aislamiento & purificación , Retinoides/metabolismo , Solventes , Biotecnología/métodosRESUMEN
Production of Z-type farnesyl diphosphate (FPP) has not been reported in Escherichia coli. Here we present the fusion enzyme (ILRv) of E. coli E,E-FPP synthase (IspA) and Mycobacterium tuberculosis Z,E-FPP synthase (Rv1086), which can produce primarily Z,E-FPP rather than E,E-FPP, the predominant stereoisomer found in most organisms. Z,E-farnesol (FOH) was produced from E. coli harboring the bottom portion of the MVA pathway and the fusion FPP synthase (ILRv) at a titer of 115.6 mg/L in 2YT medium containing 1% (v/v) glycerol as a carbon source and 5 mM mevalonate. The Z,E-FOH production was improved by 15-fold, compared with 7.7 mg/L obtained from the co-overexpression of separate IspA and Rv1086. The Z,E-FPP was not metabolized in native metabolic pathways of E. coli. It would be of interest to produce Z,E-FPP derived sesquiterpenes from recombinant E. coli due to no loss of Z,E-FPP substrate in endogenous metabolism of the host strain.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Escherichia coli/enzimología , Geraniltranstransferasa/biosíntesis , Mycobacterium tuberculosis/enzimología , Fosfatos de Poliisoprenilo/biosíntesis , Proteínas Bacterianas/genética , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Farnesol/metabolismo , Geraniltranstransferasa/genética , Ácido Mevalónico/metabolismo , Mycobacterium tuberculosis/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , SesquiterpenosRESUMEN
Sesquiterpenes are important materials in pharmaceuticals and industry. Metabolic engineering has been successfully used to produce these valuable compounds in microbial hosts. However, the microbial potential of sesquiterpene production is limited by the poor heterologous expression of plant sesquiterpene synthases and the deficient FPP precursor supply. In this study, we engineered E. coli to produce α-farnesene using a codon-optimized α-farnesene synthase and an exogenous MVA pathway. Codon optimization of α-farnesene synthase improved both the synthase expression and α-farnesene production. Augmentation of the metabolic flux for FPP synthesis conferred a 1.6- to 48.0-fold increase in α-farnesene production. An additional increase in α-farnesene production was achieved by the protein fusion of FPP synthase and α-farnesene synthase. The engineered E. coli strain was able to produce 380.0 mg/L of α-farnesene, which is an approximately 317-fold increase over the initial production of 1.2 mg/L.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Ingeniería Metabólica , Pirofosfatasas/metabolismo , Sesquiterpenos/metabolismo , Codón , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Geraniltranstransferasa/genética , Geraniltranstransferasa/metabolismo , Organismos Modificados Genéticamente , Pirofosfatasas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
BACKGROUND: Retinoids are lipophilic isoprenoids composed of a cyclic group and a linear chain with a hydrophilic end group. These compounds include retinol, retinal, retinoic acid, retinyl esters, and various derivatives of these structures. Retinoids are used as cosmetic agents and effective pharmaceuticals for skin diseases. Retinal, an immediate precursor of retinoids, is derived by ß-carotene 15,15'-mono(di)oxygenase (BCM(D)O) from ß-carotene, which is synthesized from the isoprenoid building blocks isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Retinoids are chemically unstable and biologically degraded via retinoic acid. Although extensive studies have been performed on the microbial production of carotenoids, retinoid production using microbial metabolic engineering has not been reported. Here, we report retinoid production using engineered Escherichia coli that express exogenous BCM(D)O and the mevalonate (MVA) pathway for the building blocks synthesis in combination with a two-phase culture system using a dodecane overlay. RESULTS: Among the BCM(D)O tested in E. coli, the synthetic retinoid synthesis protein (SR), based on bacteriorhodopsin-related protein-like homolog (Blh) of the uncultured marine bacteria 66A03, showed the highest ß-carotene cleavage activity with no residual intracellular ß-carotene. By introducing the exogenous MVA pathway, 8.7 mg/L of retinal was produced, which is 4-fold higher production than that of augmenting the MEP pathway (dxs overexpression). There was a large gap between retinal production and ß-carotene consumption using the exogenous MVA pathway; therefore, the retinal derivatives were analyzed. The derivatives, except for retinoic acid, that formed were identified, and the levels of retinal, retinol, and retinyl acetate were measured. Amounts as high as 95 mg/L retinoids were obtained from engineered E. coli DH5α harboring the synthetic SR gene and the exogenous MVA pathway in addition to dxs overexpression, which were cultured at 29°C for 72 hours with 2YT medium containing 2.0% (w/v) glycerol as the main carbon source. However, a significant level of intracellular degradation of the retinoids was also observed in the culture. To prevent degradation of the intracellular retinoids through in situ extraction from the cells, a two-phase culture system with dodecane was used. The highest level of retinoid production (136 mg/L) was obtained after 72 hours with 5 mL of dodecane overlaid on a 5 mL culture. CONCLUSIONS: In this study, we successfully produced 136 mg/L retinoids, which were composed of 67 mg/L retinal, 54 mg/L retinol, and 15 mg/L retinyl acetate, using a two-phase culture system with dodecane, which produced 68-fold more retinoids than the initial level of production (2.2 mg/L). Our results demonstrate the potential use of E. coli as a promising microbial cell factory for retinoid production.
Asunto(s)
Escherichia coli/metabolismo , Retinoides/biosíntesis , Alcanos/farmacología , Carbono/metabolismo , Diterpenos , Escherichia coli/enzimología , Escherichia coli/crecimiento & desarrollo , Ingeniería Genética , Ácido Mevalónico/metabolismo , Retinaldehído/biosíntesis , Ésteres de Retinilo , Temperatura , Vitamina A/análogos & derivados , Vitamina A/biosíntesis , beta Caroteno/metabolismo , beta-Caroteno 15,15'-Monooxigenasa/genética , beta-Caroteno 15,15'-Monooxigenasa/metabolismoRESUMEN
Farnesol (FOH) production has been carried out in metabolically engineered Escherichia coli. FOH is formed through the depyrophosphorylation of farnesyl pyrophosphate (FPP), which is synthesized from isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) by FPP synthase. In order to increase FPP synthesis, E. coli was metabolically engineered to overexpress ispA and to utilize the foreign mevalonate (MVA) pathway for the efficient synthesis of IPP and DMAPP. Two-phase culture using a decane overlay of the culture broth was applied to reduce volatile loss of FOH produced during culture and to extract FOH from the culture broth. A FOH production of 135.5 mg/L was obtained from the recombinant E. coli harboring the pTispA and pSNA plasmids for ispA overexpression and MVA pathway utilization, respectively. It is interesting to observe that a large amount of FOH could be produced from E. coli without FOH synthase by the augmentation of FPP synthesis. Introduction of the exogenous MVA pathway enabled the dramatic production of FOH by E. coli while no detectable FOH production was observed in the endogenous MEP pathway-only control.
Asunto(s)
Escherichia coli/genética , Escherichia coli/metabolismo , Farnesol/metabolismo , Redes y Vías Metabólicas/genética , Ácido Mevalónico/metabolismo , Medios de Cultivo/química , Dosificación de Gen , Expresión Génica , Geraniltranstransferasa/genética , Hemiterpenos/metabolismo , Compuestos Organofosforados/metabolismo , Plásmidos , Fosfatos de Poliisoprenilo/metabolismo , Sesquiterpenos/metabolismoRESUMEN
Uronate dehydrogenase has been cloned from Pseudomonas syringae pv. tomato strain DC3000, Pseudomonas putida KT2440, and Agrobacterium tumefaciens strain C58. The genes were identified by using a novel complementation assay employing an Escherichia coli mutant incapable of consuming glucuronate as the sole carbon source but capable of growth on glucarate. A shotgun library of P. syringae was screened in the mutant E. coli by growing transformed cells on minimal medium containing glucuronic acid. Colonies that survived were evaluated for uronate dehydrogenase, which is capable of converting glucuronic acid to glucaric acid. In this manner, a 0.8-kb open reading frame was identified and subsequently verified to be udh. Homologous enzymes in P. putida and A. tumefaciens were identified based on a similarity search of the sequenced genomes. Recombinant proteins from each of the three organisms expressed in E. coli were purified and characterized. For all three enzymes, the turnover number (k(cat)) with glucuronate as a substrate was higher than that with galacturonate; however, the Michaelis constant (K(m)) for galacturonate was lower than that for glucuronate. The A. tumefaciens enzyme was found to have the highest rate constant (k(cat) = 1.9 x 10(2) s(-1) on glucuronate), which was more than twofold higher than those of both of the pseudomonad enzymes.
Asunto(s)
Agrobacterium tumefaciens/enzimología , Aldehído Oxidorreductasas , Clonación Molecular , Pseudomonas putida/enzimología , Pseudomonas syringae/enzimología , Proteínas Recombinantes/metabolismo , Agrobacterium tumefaciens/genética , Aldehído Oxidorreductasas/genética , Aldehído Oxidorreductasas/aislamiento & purificación , Aldehído Oxidorreductasas/metabolismo , Medios de Cultivo , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Ácido Glucárico/metabolismo , Ácido Glucurónico/metabolismo , Cinética , Datos de Secuencia Molecular , Mutación , Pseudomonas putida/genética , Pseudomonas syringae/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Análisis de Secuencia de ADNRESUMEN
Alternative microbial hosts have been engineered as biocatalysts for butanol biosynthesis. The butanol synthetic pathway of Clostridium acetobutylicum was first re-constructed in Escherichia coli to establish a baseline for comparison to other hosts. Whereas polycistronic expression of the pathway genes resulted in the production of 34 mg/L butanol, individual expression of pathway genes elevated titers to 200 mg/L. Improved titers were achieved by co-expression of Saccharomyces cerevisiae formate dehydrogenase while overexpression of E. coli glyceraldehyde 3-phosphate dehydrogenase to elevate glycolytic flux improved titers to 580 mg/L. Pseudomonas putida and Bacillus subtilis were also explored as alternative production hosts. Polycistronic expression of butanol biosynthetic genes yielded butanol titers of 120 and 24 mg/L from P. putida and B. subtilis, respectively. Production in the obligate aerobe P. putida was dependent upon expression of bcd-etfAB. These results demonstrate the potential of engineering butanol biosynthesis in a variety of heterologous microorganisms, including those cultivated aerobically.
Asunto(s)
Butanoles/metabolismo , Ingeniería Genética/métodos , Mejoramiento Genético/métodos , Ingeniería de Proteínas/métodos , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Clostridium acetobutylicum/genética , Clostridium acetobutylicum/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Gliceraldehído 3-Fosfato/genéticaRESUMEN
A synthetic pathway has been constructed for the production of glucuronic and glucaric acids from glucose in Escherichia coli. Coexpression of the genes encoding myo-inositol-1-phosphate synthase (Ino1) from Saccharomyces cerevisiae and myo-inositol oxygenase (MIOX) from mice led to production of glucuronic acid through the intermediate myo-inositol. Glucuronic acid concentrations up to 0.3 g/liter were measured in the culture broth. The activity of MIOX was rate limiting, resulting in the accumulation of both myo-inositol and glucuronic acid as final products, in approximately equal concentrations. Inclusion of a third enzyme, uronate dehydrogenase (Udh) from Pseudomonas syringae, facilitated the conversion of glucuronic acid to glucaric acid. The activity of this recombinant enzyme was more than 2 orders of magnitude higher than that of Ino1 and MIOX and increased overall flux through the pathway such that glucaric acid concentrations in excess of 1 g/liter were observed. This represents a novel microbial system for the biological production of glucaric acid, a "top value-added chemical" from biomass.
Asunto(s)
Escherichia coli/metabolismo , Ácido Glucárico/metabolismo , Redes y Vías Metabólicas/genética , Aldehído Oxidorreductasas/genética , Aldehído Oxidorreductasas/metabolismo , Animales , Escherichia coli/genética , Inositol/metabolismo , Inositol-Oxigenasa/genética , Inositol-Oxigenasa/metabolismo , Ratones , Mio-Inositol-1-Fosfato Sintasa/genética , Mio-Inositol-1-Fosfato Sintasa/metabolismo , Pseudomonas syringae/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genéticaRESUMEN
D-Glucuronate is a key metabolite in the process of detoxification of xenobiotics and in a recently constructed synthetic pathway to produce D-glucaric acid, a "top value-added chemical" from biomass. A simple and specific assay of D-glucuronate would be useful for studying these processes, but existing assays are either time-consuming or nonspecific. Using uronate dehydrogenase cloned from Agrobacterium tumefaciens, we developed an assay for D-glucuronate with a detection limit of 5 microM. This method was shown to be more suitable for a system with many interfering compounds than previous methods and was also applied to assays for myo-inositol oxygenase activity.
Asunto(s)
Aldehído Oxidorreductasas/metabolismo , Glucuronatos/análisis , Escherichia coli/enzimología , Glucuronatos/metabolismoRESUMEN
The amplification of gltA gene encoding citrate synthase of TCA cycle was required for the efficient conversion of acetyl-CoA, generated during vanillin production from ferulic acid, to CoA, which is essential for vanillin production. Vanillin of 1.98 g/L was produced from the E. coli DH5alpha (pTAHEF-gltA) with gltA amplification in 48 h of culture at 3.0 g/L of ferulic acid, which was about twofold higher than the vanillin production of 0.91 g/L obtained by the E. coli DH5alpha (pTAHEF) without gltA amplification. The icdA gene encoding isocitrate dehydrogenase of TCA cycle was deleted to make the vanillin producing E. coli utilize glyoxylate bypass which enables more efficient conversion of acetyl-CoA to CoA in comparison with TCA cycle. The production of vanillin by the icdA null mutant of E. coli BW25113 harboring pTAHEF was enhanced by 2.6 times. The gltA amplification of the glyoxylate bypass in the icdA null mutant remarkably increased the production rate of vanillin with a little increase in the amount of vanillin production. The real synergistic effect of gltA amplification and icdA deletion was observed with use of XAD-2 resin reducing the toxicity of vanillin produced during culture. Vanillin of 5.14 g/L was produced in 24 h of the culture with molar conversion yield of 86.6%, which is the highest so far in vanillin production from ferulic acid using recombinant E. coli.
Asunto(s)
Acetilcoenzima A/metabolismo , Benzaldehídos/metabolismo , Ácidos Cumáricos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Adsorción , Benzaldehídos/toxicidad , Biotecnología/métodos , Citrato (si)-Sintasa/genética , Citrato (si)-Sintasa/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Eliminación de Gen , Dosificación de Gen , Glioxilatos/metabolismo , Isocitrato Deshidrogenasa/genética , PoliestirenosRESUMEN
When pT-LYCm4 containing lycopene synthetic genes was co-transformed with pSUcrtY or pSHcrtY containing crtY gene of Pantoea ananatis (P. ananatis) or Pantoea agglomerans (P. agglomerans), beta-carotene productions of 36 and 35 mg/L were obtained, respectively. No lycopene was detected in the beta-carotene production culture. pT-HB, constructed by addition of P. ananatis crtY gene into pT-LYCm4, was used for co-transformation with pSdxs and pSSN12Didi, which increased isopentenyl diphosphate and dimethylallyl diphosphate synthesis. beta-Carotene production significantly increased 1.5-fold (51 mg/L) with the amplification of the dxs gene through pSdxs and 4-fold (135 mg/L) with the mevalonate bottom pathway of pSSN12Didi in the presence of 3.3 mM mevalonate. The pT-DHB, constructed by integrating the dxs gene into pT-HB, was used for cotransformation of Escherichia coli (E. coli) harboring pSSN12Didi, resulting in beta-carotene production of 141 mg/L. Recombinant E. coli harboring pT-DHB and pSSN12Didi was used to maximize beta-carotene production by adjusting the available amounts of glycerol, a carbon source, and mevalonate, the precursor of the mevalonate bottom pathway. When recombinant E. coli was given 16.5 mM mevalonate and 2.5% (w/v) glycerol, beta-carotene production of 503 mg/L in concentration and 49.3 mg/g DCW in content was obtained at 144 h, which was the highest level of carotenoid production in E. coli ever reported in the literature.
Asunto(s)
Escherichia coli/genética , Liasas Intramoleculares/genética , Ácido Mevalónico/farmacología , Terpenos/metabolismo , beta Caroteno/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Ingeniería Genética/métodos , Glicerol/farmacología , Hemiterpenos/metabolismo , Liasas Intramoleculares/metabolismo , Modelos Biológicos , Compuestos Organofosforados/metabolismo , Pantoea/genéticaRESUMEN
Vanillin production was tested with different concentrations of added ferulic acid in E. coli harboring plasmid pTAHEF containing fcs (feruloyl-CoA synthase) and ech (enoyl-CoA hydratase/aldolase) genes cloned from Amycolatopsis sp. strain HR104. The maximum production of vanillin from E. coli DH5alpha harboring pTAHEF was found to be 1.0 g/L at 2.0 g/L of ferulic acid for 48 h of culture. To improve the vanillin production by reducing its toxicity, two approaches were followed: (1) generation of vanillin-resistant mutant of NTG-VR1 through NTG mutagenesis and (2) removal of toxic vanillin from the medium by XAD-2 resin absorption. The vanillin production of NTG-VR1 increased to three times at 5 g/L of ferulic acid when compared with its wild-type strain. When 50% (w/v) of XAD-2 resin was employed in culture with 10 g/L of ferulic acid, the vanillin production of NTG-VR1 was 2.9 g/L, which was 2-fold higher than that obtained with no use of the resin.
Asunto(s)
Resinas Acrílicas/química , Escherichia coli/genética , Escherichia coli/metabolismo , Mejoramiento Genético/métodos , Metilnitronitrosoguanidina/metabolismo , Ultrafiltración/métodos , Absorción , Mutagénesis Sitio-Dirigida , Recombinación Genética/fisiologíaRESUMEN
Geraniol, a monoterpene alcohol, has versatile applications in the fragrance industry, pharmacy and agrochemistry. Moreover, geraniol could be an ideal gasoline alternative. In this study, recombinant overexpression of geranyl diphosphate synthase and the bottom portion of a foreign mevalonate pathway in Escherichia coli MG1655 produced 13.3mg/L of geraniol. Introduction of Ocimum basilicum geraniol synthase increased geraniol production to 105.2mg/L. However, geraniol production encountered a loss from its endogenous dehydrogenization and isomerization into other geranoids (nerol, neral and geranial). Three E. coli enzymes (YjgB, YahK and YddN) were identified with high sequence identity to plant geraniol dehydrogenases. YjgB was demonstrated to be the major one responsible for geraniol dehydrogenization. Deletion of yjgB increased geraniol production to 129.7mg/L. Introduction of the whole mevalonate pathway for enhanced building block synthesis from endogenously synthesized mevalonate improved geraniol production up to 182.5mg/L in the yjgB mutant after 48h of culture, which was a double of that obtained in the wild type control (96.5mg/L). Our strategy for improving geraniol production in engineered E. coli should be generalizable for addressing similar problems during metabolic engineering.
Asunto(s)
Escherichia coli/enzimología , Escherichia coli/genética , Ingeniería de Proteínas , Terpenos/metabolismo , Monoterpenos Acíclicos , Escherichia coli/metabolismo , Farnesiltransferasa/genética , Farnesiltransferasa/metabolismo , Técnicas de Inactivación de Genes , Hidrogenación , Ocimum basilicum/enzimología , Ocimum basilicum/genéticaRESUMEN
Geraniol is an important industrial material and a potential candidate of advanced biofuels. One challenge of microbial geraniol production is the toxicity to hosts. However, the poor understanding on geraniol tolerance mechanism is an obstacle for developing geraniol tolerant host. This study genome-widely screened a shot-gun DNA library of Escherichia coli and found that recA is able to confer geraniol tolerance in E. coli. The recA knockout mutant was found extremely sensitive to geraniol. Based on our data, it was deciphered that recA provided tolerance through SOS response network responding to DNA damage caused by geraniol. RecA-mediated SOS response activates the homologous recombinational repair by RecB and RecN for corrective DNA maintenance. This protection mechanism suggests an effective strategy to combat geraniol toxicity in E. coli.
Asunto(s)
Reparación del ADN , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Rec A Recombinasas/metabolismo , Respuesta SOS en Genética , Terpenos/toxicidad , Monoterpenos Acíclicos , Biocombustibles , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Farmacorresistencia Bacteriana , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Técnicas de Inactivación de Genes , Biblioteca de Genes , Genoma Bacteriano , Viabilidad Microbiana/efectos de los fármacos , Rec A Recombinasas/genética , Reparación del ADN por Recombinación/genéticaRESUMEN
The functional reconstruction of acetoin and meso-2,3-butanediol (meso-2,3-BD) biosynthetic pathways in Escherichia coli have been explored systematically. Pathway construction involved the in vsivo screening of prospective pathway isozymes of yeast and bacterial origin. After substantial engineering of the host background to increase pyruvate availability, E. coli YYC202(DE3) ldhA(() ilvC( expressing ilvBN from E. coli and aldB from L. lactis (encoding acetolactate synthase and acetolactate decarboxylase activities, respectively) was able to produce up to 870 mg/L acetoin, with no coproduction of diacetyl observed. These strains were also found to produce small quantities of meso-2,3-BD, suggesting the existence of endogenous 2,3-BD dehydrogenase activity. Finally, the coexpression of bdh1 from S. cerevisiae, encoding 2,3-BD dehydrogenase, in this strain resulted in the production of up to 1120 mg/L meso-2,3-BD, with glucose a yield of 0.29 g/g. While disruption of the native lactate biosynthesis pathway increased pyruvate precursor availability to this strain, increased availability of NADH for acetoin reduction to meso-2,3-BD was found to be the most important consequence of ldhA deletion.
Asunto(s)
Acetoína/metabolismo , Butileno Glicoles/metabolismo , Técnicas de Cultivo de Célula/métodos , Escherichia coli/metabolismo , Ingeniería Genética/métodos , Anaerobiosis , Medios de Cultivo , Escherichia coli/genética , Redes y Vías Metabólicas , Mutación , Filogenia , Plásmidos/genéticaRESUMEN
Engineered biosynthetic pathways have the potential to produce high-value molecules from inexpensive feedstocks, but a key limitation is engineering enzymes with high activity and specificity for new reactions. Here, we developed a method for combining structure-based computational protein design with library-based enzyme screening, in which inter-residue correlations favored by the design are encoded into a defined-sequence library. We validated this approach by engineering a glucose 6-oxidase enzyme for use in a proposed pathway to convert D-glucose into D-glucaric acid. The most active variant, identified after only one round of diversification and screening of only 10,000 wells, is approximately 400-fold more active on glucose than is the wild-type enzyme. We anticipate that this strategy will be broadly applicable to the discovery of new enzymes for engineered biological pathways.
Asunto(s)
Glucosa Oxidasa/química , Ingeniería de Proteínas , Secuencia de Aminoácidos , Biología Computacional , Biblioteca de Genes , Glucosa Oxidasa/genética , Glucosa Oxidasa/metabolismo , Modelos Moleculares , Especificidad por SustratoRESUMEN
Microbial symbionts are fundamentally important to their host ecology, but microbial communities of invasive marine species remain largely unexplored. Clone libraries and Denaturing gradient gel electrophoresis analyses revealed diverse microbial phylotypes in the invasive marine sponge Mycale armata. Phylotypes were related to eight phyla: Proteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Acidobacteria, Chloroflexi, Crenarchaeota and Firmicutes, with predominant alphaproteobacterial sequences (>58%). Three Bacterial Phylotype Groups (BPG1--associated only with sequence from marine sponges; BPG2--associated with sponges and other marine organisms and BPG3--potential new phylotypes) were identified in M. armata. The operational taxonomic units (OTU) of cluster BPG2-B, belonging to Rhodobacteraceae, are dominant sequences of two clone libraries of M. armata, but constitute only a small fraction of sequences from the non-invasive sponge Dysidea sp. Six OTUs from M. armata were potential new phylotypes because of their low sequence identity with their reference sequences. Our results suggest that M. armata harbors both sponge-specific phylotypes and bacterial phylotypes from other marine organisms.
Asunto(s)
Archaea/clasificación , Archaea/aislamiento & purificación , Bacterias/clasificación , Bacterias/aislamiento & purificación , Poríferos/microbiología , Animales , Archaea/genética , Bacterias/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Electroforesis en Gel de Poliacrilamida , Biblioteca de Genes , Genes de ARNr , Hawaii , Desnaturalización de Ácido Nucleico , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido NucleicoRESUMEN
The increased synthesis of building blocks of IPP (isopentenyl diphosphate) and DMAPP (dimethylallyl diphosphate) through metabolic engineering is a way to enhance the production of carotenoids. Using E. coli as a host, IPP and DMAPP supply can be increased significantly through the introduction of foreign MVA (mevalonate) pathway into it. The MVA pathway is split into two parts with the top and bottom portions supplying mevalonate from acetyl-CoA, and IPP and DMAPP from mevalonate, respectively. The bottom portions of MVA pathway from Streptococcus pneumonia, Enterococcus faecalis, Staphylococcus aureus, Streptococcus pyogenes and Saccharomyces cerevisiae were compared with exogenous mevalonate supplementation for beta-carotene production in recombinant Escherichia coli harboring beta-carotene synthesis genes. The E. coli harboring the bottom MVA pathway of S. pneumoniae produced the highest amount of beta-carotene. The top portions of MVA pathway were also compared and the top MVA pathway of E. faecalis was found out to be the most efficient for mevalonate production in E. coli. The whole MVA pathway was constructed by combining the bottom and top portions of MVA pathway of S. pneumoniae and E. faecalis, respectively. The recombinant E. coli harboring the whole MVA pathway and beta-carotene synthesis genes produced high amount of beta-carotene even without exogenous mevalonate supplementation. When comparing various E. coli strains - MG1655, DH5alpha, S17-1, XL1-Blue and BL21 - the DH5alpha was found to be the best beta-carotene producer. Using glycerol as the carbon source for beta-carotene production was found to be superior to glucose, galactose, xylose and maltose. The recombinant E. coli DH5alpha harboring the whole MVA pathway and beta-carotene synthesis genes produced beta-carotene of 465mg/L at glycerol concentration of 2% (w/v).
Asunto(s)
Escherichia coli/metabolismo , Ácido Mevalónico/metabolismo , Ácido Mevalónico/farmacología , Transducción de Señal/efectos de los fármacos , beta Caroteno/biosíntesis , Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Escherichia coli/genética , Modelos Biológicos , Transducción de Señal/genética , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismoRESUMEN
Carotenoids are ubiquitous pigments synthesized by plants, fungi, algae, and bacteria. Industrially, carotenoids are used in pharmaceuticals, neutraceuticals, and animal feed additives, as well as colorants in cosmetics and foods. Scientific interest in dietary carotenoids has increased in recent years because of their beneficial effects on human health, such as lowering the risk of cancer and enhancement of immune system function, which are attributed to their antioxidant potential. The availability of carotenoid genes from carotenogenic microbes has made possible the synthesis of carotenoids in non-carotenogenic microbes. The increasing interest in microbial sources of carotenoid is related to consumer preferences for natural additives and the potential cost effectiveness of creating carotenoids via microbial biotechnology. In this review, we will describe the recent progress made in metabolic engineering of non-carotenogenic microorganisms with particular focus on the potential of Escherichia coli for improved carotenoid productivity.