RESUMEN
The acyl-CoA dehydrogenase (FadE) and (R)-specific enoyl-CoA hydratase (PhaJ) are functionally related to the degradation of fatty acids and the synthesis of polyhydroxyalkanoates (PHAs). To verify this, a recombinant Cupriavidus necator H16 harboring the plasmid -pMPJAS03- with fadE from Escherichia coli strain K12 and phaJ1 from Pseudomonas putida strain KT2440 under the arabinose promoter (araC-PBAD) was constructed. The impact of co-expressing fadE and phaJ genes on C. necator H16/pMPJAS03 maintaining the wild-type synthase on short-chain-length/medium-chain-length PHA formation from canola or avocado oil at different arabinose concentrations was investigated. The functional activity of fadEE.c led to obtaining higher biomass and PHA concentrations compared to the cultures without expressing the gene. While high transcriptional levels of phaJ1P.p, at 0.1% of arabinose, aid the wild-type synthase to polymerize larger-side chain monomers, such as 3-Hydroxyoctanoate (3HO) and 3-Hydroxydecanoate (3HD). The presence of even small amounts of 3HO and 3HD in the co-polymers significantly depresses the melting temperature of the polymers, compared to those composed of pure 3-hydroxybutyrate (3HB). Our data presents supporting evidence that the synthesis of larger-side chain monomers by the recombinant strain relies not only upon the affinity of the wild-type synthase but also on the functionality of the intermediate supplying enzymes.
Asunto(s)
Acil-CoA Deshidrogenasa/genética , Cupriavidus necator/genética , Enoil-CoA Hidratasa/genética , Aceites de Plantas/metabolismo , Polihidroxialcanoatos/biosíntesis , Polihidroxialcanoatos/genética , Acil-CoA Deshidrogenasa/metabolismo , Arabinosa/genética , Arabinosa/metabolismo , Caprilatos/metabolismo , Cupriavidus necator/metabolismo , Ácidos Decanoicos/metabolismo , Enoil-CoA Hidratasa/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Grasos/genética , Ácidos Grasos/metabolismo , Hidroxibutiratos/metabolismo , Plásmidos/genética , Polihidroxialcanoatos/metabolismo , Regiones Promotoras Genéticas/genética , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Transcripción Genética/genéticaRESUMEN
Aspergillus niger produces an arsenal of extracellular enzymes that allow synergistic degradation of plant biomass found in its environment. Pectin is a heteropolymer abundantly present in the primary cell wall of plants. The complex structure of pectin requires multiple enzymes to act together. Production of pectinolytic enzymes in A. niger is highly regulated, which allows flexible and efficient capture of nutrients. So far, three transcriptional activators have been linked to regulation of pectin degradation in A. niger. The L-rhamnose-responsive regulator RhaR controls the production of enzymes that degrade rhamnogalacturonan-I. The L-arabinose-responsive regulator AraR controls the production of enzymes that decompose the arabinan and arabinogalactan side chains of rhamnogalacturonan-II. The D-galacturonic acid-responsive regulator GaaR controls the production of enzymes that act on the polygalacturonic acid backbone of pectin. This project aims to better understand how RhaR, AraR and GaaR co-regulate pectin degradation. For that reason, we constructed single, double and triple disruptant strains of these regulators and analyzed their growth phenotype and pectinolytic gene expression in A. niger grown on sugar beet pectin.
Asunto(s)
Aspergillus niger/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Pectinas/metabolismo , Transactivadores/metabolismo , Arabinosa/genética , Arabinosa/metabolismo , Aspergillus niger/metabolismo , Beta vulgaris , Pared Celular/metabolismo , Proteínas Fúngicas/genética , Perfilación de la Expresión Génica , Ácidos Hexurónicos/metabolismo , Ramnosa/genética , Ramnosa/metabolismoRESUMEN
A new assay has been described for mutagenicity testing using an L-arabinose-sensitive strain of Salmonella typhimurium. The test strain SV3 and several L-arabinose-resistant mutants selected therefrom are characterized in the present study by 3 different criteria: inhibition of growth by L-arabinose, accumulation of keto-sugars, and activities of the enzymes involved in L-arabinose catabolism. Strain SV3 (ara-531) shows high levels of inducible L-arabinose isomerase (EC 5.3.1.4) and L-ribulokinase (EC 2.7.1.16) activities, but is deficient in L-ribulose-5-phosphate 4-epimerase (EC 5.1.3.4), the enzyme encoded in Escherichia coli by gene D in the araBAD operon. Addition of L-arabinose to SV3 growing in glycerol or casamino acids stops growth. D-Glucose only partially reverses this inhibition. Reversion of the ara-531 mutation restores different levels of epimerase activity and resistance to L-arabinose. However, the great majority of the L-arabinose-resistant mutants do not utilize L-arabinose. The physiological and enzymatic properties of these L-arabinose non-utilizing mutants suggest that L-arabinose resistance is due to forward mutations in at least 3 other genes, araA, araB and araC, blocking steps prior to L-ribulose 5-phosphate accumulation.