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1.
FEMS Yeast Res ; 17(6)2017 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-28873978

RESUMEN

The xylose-fermenting yeasts (CTG clade yeasts, e.g. Scheffersomyces stipitis, Spathaspora passalidarum, Candida amazonensis and Candida jeffriesii) have the potential to be superior platforms for the conversion of lignocellulosic hydrolysate into fuel-grade ethanol and other chemical products. Here, a genetic expression system compatible with the genetic coding characteristics of CTG clade yeasts was constructed for use in xylose-fermenting yeasts. The pRACTH-gfpm plasmid based on an 18S rDNA shuttle vector was capable of stable integration into the genomes of a wide range of heterologous hosts. Green fluorescent protein was transformed and functionally expressed in S. stipitis, S. passalidarum, C. jeffriesii, C. amazonensis and Saccharomyces cerevisiae under control of the SpADH1 promoter and SpCYC1 terminator. Finally, the expression system was useful in multiple yeast hosts for construction of the plasmid pRACTH-ldh. Scheffersomyces stipitis, S. passalidarum, C. jeffriesii, C. amazonensis and S. cerevisiae were enabled to produce lactate from glucose or xylose by pRACTH-based expression of a heterologous lactate dehydrogenase. Among them, C. amazonensis (pRACTH-ldh) exhibited the highest lactate fermentation capacity, which reached a maximum of 44 g L-1 of lactate with a yield of 0.85 g lactate/g xylose.


Asunto(s)
Fermentación , Expresión Génica , Vectores Genéticos , Xilosa/metabolismo , Levaduras/genética , Levaduras/metabolismo , Genes Reporteros , Glucosa/metabolismo , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , L-Lactato Deshidrogenasa/análisis , L-Lactato Deshidrogenasa/genética , Plásmidos , Regiones Promotoras Genéticas , Terminación de la Transcripción Genética
2.
J Ind Microbiol Biotechnol ; 44(1): 75-88, 2017 01.
Artículo en Inglés | MEDLINE | ID: mdl-27844169

RESUMEN

In this study, we constructed an L-methionine-producing recombinant strain from wild-type Escherichia coli W3110 by metabolic engineering. To enhance the carbon flux to methionine and derepression met regulon, thrBC, lysA, and metJ were deleted in turn. Methionine biosynthesis obstacles were overcome by overexpression of metA Fbr (Fbr, Feedback resistance), metB, and malY under control of promoter pN25. Recombinant strain growth and methionine production were further improved by attenuation of metK gene expression through replacing native promoter by metK84p. Blocking the threonine pathway by deletion of thrBC or thrC was compared. Deletion of thrC showed faster growth rate and higher methionine production. Finally, metE, metF, and metH were overexpressed to enhance methylation efficiency. Compared with the original strain E. coli W3110, the finally obtained Me05 (pETMAFbr-B-Y/pKKmetH) improved methionine production from 0 to 0.65 and 5.62 g/L in a flask and a 15-L fermenter, respectively.


Asunto(s)
Escherichia coli/genética , Ingeniería Metabólica/métodos , Metionina/biosíntesis , Carbono/química , Cartilla de ADN , Proteínas de Escherichia coli/metabolismo , Fermentación , Microbiología Industrial , Mutagénesis Sitio-Dirigida , Plásmidos/química , Proteínas Recombinantes/química , Treonina
3.
World J Gastroenterol ; 22(43): 9525-9533, 2016 Nov 21.
Artículo en Inglés | MEDLINE | ID: mdl-27920473

RESUMEN

AIM: To determine the expression and localization of the electrogenic Na+/HCO3- cotransporter (NBC1) in rat pancreas during development. METHODS: The rat pancreas from postnatal and embryos removed from the uterus of pregnant rats that had been sacrificed by CO2 asphyxiation were used. Rat pancreas from embryonic day (E) 15.5 and E18.5 rat embryos was isolated under a stereomicroscope. Rat pancreas from postnatal (P) days 0, 7, 14, 21 and adult was directly isolated by the unaided eye. The RT-PCR analysis of the NBC1 specific region on rat pancreas tissues from different developmental stages. The two antibodies which target the NBC1 common COOH-terminal region and NH2-terminal region detected a clear band of about 145 kDa in the Western blot analysis. The localization of NBC1 was examined by immuno-fluorescence detection. RESULTS: The results revealed the first peak of NBC1 expression at E18.5 and the second peak at P14. Meanwhile, the low NBC1 expression occurred at P7 and adult stages. Our results demonstrated, for the first time, the presence of NBC1 in the plasma membrane of ß and α cells, as well as in the basolateral membrane of acinar cells of the rat pancreas at different stages of development. CONCLUSION: The data strongly suggests that NBC1 is diversely expressed in the pancreas at different developmental stages, where it may exert its functions in pancreatic development especially islet cell growth through HCO3- transport and pH regulation.


Asunto(s)
Células Secretoras de Glucagón/metabolismo , Células Secretoras de Insulina/metabolismo , Páncreas/metabolismo , Simportadores de Sodio-Bicarbonato/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Western Blotting , Técnica del Anticuerpo Fluorescente , Regulación del Desarrollo de la Expresión Génica , Edad Gestacional , Morfogénesis , Páncreas/embriología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Simportadores de Sodio-Bicarbonato/genética
4.
J Biotechnol ; 186: 91-7, 2014 Sep 30.
Artículo en Inglés | MEDLINE | ID: mdl-25011099

RESUMEN

The aproteinogenic amino acid L-phenylglycine (L-Phg) is an important side chain building block for the preparation of several antibiotics and taxol. To biosynthesis L-Phg from glucose, an engineered Escherichia coli containing L-Phg synthetic genes was firstly developed by an L-phenylalanine producing chassis supplying phenylpyruvate. The enzymes HmaS (L-4-hydroxymandelate synthase), Hmo (L-4-hydroxymandelate oxidase) and HpgT (L-4-hydroxyphenylglycine transaminase) from Amycolatopsis orientalis as well as Streptomyces coelicolor were heterologously expressed in E. coli and purified to evaluate their abilities on L-Phg formation. HpgT conversing phenylglyoxylate to L-Phg uses an unusual amino donor L-phenylalanine, which releases another phenylpyruvate as the substrate of HmaS. Thus, a recycle reaction was developed to maximize the utilization of precursor phenylpyruvate. To amplify the accumulation of L-Phg, the effects of attenuating L-phenylalanine transamination was investigated. After deletion of tyrB and aspC, L-Phg yield increased by 12.6-fold. The limiting step in the L-Phg biosynthesis was also studied; the L-Phg yield was further improved by 14.9-fold after enhancing hmaS expression. Finally, by optimizing expression of hmaS, hmo and hpgT and attenuation of L-phenylalanine transamination, the L-Phg yield was increased by 224-fold comparing with the original strain.


Asunto(s)
Escherichia coli/genética , Glicina/análogos & derivados , Proteínas Recombinantes/metabolismo , Transaminasas/metabolismo , Actinomycetales/enzimología , Actinomycetales/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/metabolismo , Glucosa/metabolismo , Glicina/análisis , Glicina/metabolismo , Redes y Vías Metabólicas , Fenilalanina/metabolismo , Proteínas Recombinantes/genética , Streptomyces/enzimología , Streptomyces/genética , Transaminasas/genética
5.
Enzyme Microb Technol ; 49(1): 105-12, 2011 Jun 10.
Artículo en Inglés | MEDLINE | ID: mdl-22112279

RESUMEN

The BGL1 gene, encoding ß-glucosidase in Saccharomycopsis fibuligera, was intracellular, secreted or cell-wall associated expressed in an industrial strain of Saccharomyces cerevisiae. The obtained recombinant strains were studied under aerobic and anaerobic conditions. The results indicated that both the wild type and recombinant strain expressing intracellular ß-glucosidase cannot grow in medium using cellobiose as sole carbon source. As for the recombinant EB1 expressing secreted enzyme and WB1 expressing cell-wall associated enzyme, the maximum specific growth rates (µ(max)) could reach 0.03 and 0.05 h(-1) under anaerobic conditions, respectively. Meanwhile, the surface-engineered S. cerevisiae utilized 5.2 g cellobioseL(-1) and produced 2.3 g ethanol L(-1) in 48 h, while S. cerevisiae secreting ß-glucosidase into culture broth used 3.6 g cellobiose L(-1) and produced 1.5 g ethanolL(-1) over the same period, but no-full depletion of cellobiose were observed for both the used recombinant strains. The results suggest that S. cerevisiae used in industrial ethanol production is deficient in cellobiose transporter. However, when ß-glucoside permease and ß-glucosidase were co-expressed in this strain, it could uptake cellobiose and showed higher growth rate (0.11h(-1)) on cellobiose.


Asunto(s)
Celobiosa/metabolismo , Etanol/metabolismo , Saccharomycopsis/enzimología , beta-Glucosidasa/metabolismo , Anaerobiosis , Secuencia de Bases , Biocombustibles , Transporte Biológico Activo , ADN de Hongos/genética , Estabilidad de Enzimas , Fermentación , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Microbiología Industrial , Cinética , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Saccharomycopsis/genética , Saccharomycopsis/crecimiento & desarrollo , beta-Glucosidasa/genética
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