One-Step Biosynthesis of Vitamin C in Saccharomyces cerevisiae

Vitamin C (VC) is comprehensively applied in foods, cosmetics, pharmaceuticals, and especially clinical medicine. Nowadays, the industrial production of VC mainly relies on the classic two-step fermentation route, and researchers have explored the way for one-step fermentation of VC in recent years. In this study, a VC biosynthesis pathway that directly produced VC from glucose was reconstructed in Saccharomyces cerevisiae, and the protein engineering and metabolic engineering strategies were adopted to improve it. First, five exogenous modules from Arabidopsis were introduced into the chassis cells by synthetic biology approaches to obtain the strain YLAA harboring VC biosynthesis. In addition, L-galactose dehydrogenase (L-GalDH) and L-galactono-1,4-lactone dehydrogenase (L-GLDH) were fused and expressed in S. cerevisiae cells for the first time, which increased the intracellular VC accumulation by 2.78-fold, reaching 9.97 ± 0.09 mg/L. Through copy number engineering, it was further confirmed that the last step catalyzed by L-GLDH is the rate-limiting step. GDP-L-galactose phosphorylase (GPP) encoded by vtc2 is another rate-limiting enzyme confirmed by GAL1p overexpression results. Finally, by balancing gene expression and cell growth, the highest production strain with overexpressing vtc2 by multicopy plasmids was constructed. The VC accumulation reached 24.94 ± 1.16 mg/L, which was currently the highest production from glucose in S. cerevisiae. The production of the recombinant strain reached nearly 44 mg/L with the exogenous addition of L-galactose or glutathione. The results further emphasized the importance of the step catalyzed by GPP. The investigation provided experience for the efficient biosynthesis of VC and the determination of rate-limiting steps.

D-galactose catabolism in archaea: operation of the DeLey–Doudoroff pathway in Haloferax volcanii

ABSTRACT The haloarchaeon Haloferax volcanii was found to grow on D-galactose as carbon and energy source. Here we report a comprehensive analysis of D-galactose catabolism in H. volcanii. Genome analyses indicated a cluster of genes encoding putative enzymes of the DeLey–Doudoroff pathway for D-galactose degradation including galactose dehydrogenase, galactonate dehydratase, 2-keto-3-deoxygalactonate kinase and 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolase. The recombinant galactose dehydrogenase and galactonate dehydratase showed high specificity for D-galactose and galactonate, respectively, whereas KDPGal aldolase was promiscuous in utilizing KDPGal and also the C4 epimer 2-keto-3-deoxy-6-phosphogluconate as substrates. Growth studies with knock-out mutants indicated the functional involvement of galactose dehydrogenase, galactonate dehydratase and KDPGal aldolase in D-galactose degradation. Further, the transcriptional regulator GacR was identified, which was characterized as an activator of genes of the DeLey–Doudoroff pathway. Finally, genes were identified encoding components of an ABC transporter and a knock-out mutant of the substrate binding protein indicated the functional involvement of this transporter in D-galactose uptake. This is the first report of D-galactose degradation via the DeLey–Doudoroff pathway in the domain of archaea.

L-Galactosa deshidrogenasa y L-Gulono-1,4-lactona deshidrogenasa influyen en la biosíntesis de vitamina C en Myrciaria dubia (Kunth) McVaugh “camu-camu”

L-Galactosa deshidrogenasa y L-Gulono-1,4-lactona deshidrogenasa influyen en la biosíntesis de vitamina C en Myrciaria dubia (Kunth) McVaugh “camu-camu” L-Galactose dehydrogenase and L-Gulone-1,4-lactone dehydrogenase influence in biosynthesis of vitamin C in Myrciaria dubia (Kunth) McVaugh “camu-camu” Juan C. Castro, Angel Araujo, Freddy Gutiérrez, Sixto A. Imán Laboratorio de Biotecnología. Centro de Investigaciones de Recursos Naturales de la Amazonía (CIRNA),bUniversidad Nacional de la Amazonia Peruana (UNAP). Pasaje Los Paujiles S/N. AAHH Nuevo San Lorenzo. Distrito de San Juan Bautista-Iquitos Instituto Nacional de Innovación Agraria (INIA). San Roque. Iquitos-Perú. DOI: https://doi.org/10.33017/RevECIPeru2012.0024/ Resumen La vitamina C es un nutriente esencial para el hombre y una de sus fuentes naturales son los frutos de Myrciaria dubia. Pero esta planta muestra amplia variación en contenido de vitamina C. Para ayudar a esclarecer las bases bioquímicas de esta variación, nos planteamos como objetivos determinar si las enzimas L-Galactosa deshidrogenasa (L-GalDH) y L-Gulono-1,4-lactona deshidrogenasa (L-GuLDH) están presentes en las hojas y frutos (pulpa y cáscara) del camu-camu y comparar la actividad catalítica de ambas enzimas en la pulpa de frutos cosechados de plantas que producen alto y bajo contenido de vitamina C. Las hojas y frutos fueron obtenidos de seis plantas (tres que producen bajo y tres que producen alto contenido de vitamina C) de la colección de germoplasma del INIA. La vitamina C fue extraída y cuantificada mediante HPLC. Posteriormente, de ambos grupos de plantas, se purificó parcialmente las enzimas y midió por triplicado su actividad catalítica mediante espectrofotometría. Tanto L-GalDH como L-GuLDH fueron detectadas en las hojas, pulpa y cáscara. Ambas enzimas mostraron diferencias en sus actividades catalíticas entre los tejidos analizados (p < 0,05). Al comparar la actividad de estas enzimas entre plantas que producen frutos con alto (2258±217 mg Vit. C/100g pulpa) y bajo (1570±46 mg Vit. C/100g pulpa) contenido de vitamina C se encontró diferencias significativas (p < 0,05) entre ambos grupos. Siendo mayor la actividad catalítica de L-GalDH y L-GuLDH en frutos con alto contenido de vitamina C y mostraron menor actividad catalítica en los frutos con bajo contenido de esta vitamina. Adicionalmente, ensayos cinéticos realizados con la L-GuLDH del camu-camu mostraron que esta enzima tiene una alta afinidad (Km = 2,37 M y Vmax = 9,23 mol.mg prot-1 .min-1) por su sustrato L-Gulono-1,4-lactona. En conclusión, las enzimas L-Galactosa deshidrogenasa y L-Gulono-1,4-lactono deshidrogenasa están presentes en las hojas y frutos del camu-camu, lo que nos indica que las dos vías biosintéticas de vitamina C propuestas para las plantas (vía de Smirnoff-Wheeler y vía de Wolucka), determinan la producción de vitamina C en M. dubia. Asimismo, las evidencias experimentales sugieren que la acumulación de vitamina C en la pulpa de los frutos del camu-camu depende por lo menos de dos procesos: biosíntesis in situ y transporte desde otros tejidos con capacidad biosintética. Además, se evidencia que la actividad catalítica de L-GalDH y L-GulDH influyen en el contenido de vitamina C de la pulpa, de tal modo que una mayor actividad catalítica de ambas enzimas está asociada con más contenido de vitamina C en este tejido. Palabras clave: L-galactosa deshidrogenasa, L-gulono-1,4-lactona deshidrogenasa, biosíntesis de vitamina C, Myrciaria dubia. Abstract Vitamin C is an essential nutrient for humans and one their natural sources are fruits of Myrciaria dubia. But this plant shows wide variation in vitamin C. To help clarify the biochemical basis of this variation, we plan aims to determine whether the enzymes L-Galactose dehydrogenase (L-GalDH) and L-Gulone-1,4-Lactone dehydrogenase (L-GuLDH) are presents in leaves and fruits (pulp and peel) of camu-camu and compare catalytic activity of both enzymes in pulp of fruits harvested from plants that produce high and low content of vitamin C. Leaves and fruits were obtained from six plants (three producing low and three that produce high content of vitamin C) in the germplasm collection of INIA. Vitamin C was extracted and quantified by HPLC. Subsequently, from both groups of plants, was partially purified enzymes and measured by triplicate their catalytic activity by means of spectrophotometry. Both L-GalDH and L-GuLDH were detected in leaves, pulp and peel. Both enzymes showed differences in their catalytic activities among the tissues analyzed (p < 0.05). By comparing the activity of these enzymes between plants that produces fruits with high (2258±217 mg Vit. C/100g pulpa) and low (1570±46 mg Vit. C/100g pulpa) content of vitamin C significant differences were found (p <0.05) between groups. Being higher the catalytic activity of L-GalDH and L-GuLDH in fruits with high content in vitamin C and showed lower catalytic activity in fruits with low content in this vitamin. In addition, kinetic assays performed with the L-GuLDH of camu-camu showed that this enzyme has a high affinity (Km = 2.37 M y Vmax = 9.23 mol.mg prot-1 .min-1) for its substrate L-Gulone-1,4-lactone. In conclusion, the enzymes L-galactose dehydrogenase and L-gulone-1,4-lactone dehydrogenase are present in leaves and fruits of camu-camu, which indicates that the two biosynthetic pathways of vitamin C proposals for plants (pathway of Smirnoff-Wheeler and pathway of Wolucka) determine production of vitamin C in M. dubia. Furthermore, experimental evidences suggest that the accumulation of vitamin C in the pulp of the fruits of camu-camu depends at least of two processes: in situ biosynthesis and transport from other tissues with biosynthetic capacity. Furthermore, it is evident that catalytic activity of L-GalDH and L-GulDH influence in vitamin C content of pulp, so that a higher catalytic activity of both enzymes is associated with more vitamin C content in this tissue. Keywords: L-galactose dehydrogenase, L-gulone-1,4-lactone dehydrogenase, biosynthesis of vitamin C, Myrciaria dubia.

Isoenzyme genotyping and phylogenetic analysis of oxacillin-resistance Staphylococcus aureus isolates

Aim: The propagation of S. aureus in hospital and dental environments is considered an important public health problem since resistant strains can cause serious infections in humans. The genetic variability of 99 oxacillin-resistant S. aureus isolates (ORSA) from the dental patients (oral cavity) and environments (air) was studied by isoenzyme genotyping. Methods: S. aureus isolates were studied using isoenzyme markers (alcohol dehydrogenase, sorbitol dehydrogenase, mannitol-1-phosphate dehydrogenase, malate dehydrogenase, glucose dehydrogenase, D-galactose dehydrogenase, glucose-6-phosphate dehydrogenase, catalase and /-esterase) and genetic (Nei’s statistics) and cluster analysis (UPGMA algorithm). Results: A highly frequent polyclonal pattern was observed in this population of ORSA isolates, suggesting various sources of contamination or microbial dispersion. Genetic relationship analysis showed a high degree of polymorphism between the strains, and it revealed three taxa (A, B and C) distantly genetically related (0.653dij1.432) and fifteen clusters (I to XV) moderately related (0.282dij0.653). These clusters harbored two or more highly related strains (0dij0.282), and the existence of microevolutionary processes in the population of ORSA. Conclusion: This research reinforces the hypothesis of the existence of several sources of contamination and/or dispersal of ORSA of clinical and epidemiologically importance, which could be associated with carriers (patients) and dental environmental (air).