Patricia Veiga-Crespo

Construction of a novel Pichia pastoris strain for production of xanthophylls.

In this study, we used the yeast carotenogenic producer Pichia pastoris Pp-EBIL strain, which has been metabolically engineered, by heterologously expressing β-carotene-pathway enzymes to produce β-carotene, as a vessel for recombinant astaxanthin expression. For this purpose, we designed new P. pastoris recombinant-strains harboring astaxanthin-encoding genes from carotenogenic microorganism, and thus capable of producing xanthophyllic compounds. We designed and constructed a plasmid (pGAPZA-WZ) containing both the β-carotene ketolase (crtW) and β-carotene hydroxylase (crtZ) genes from Agrobacterium aurantiacum, under the control of the GAP promoter and containing an AOX-1 terminator. The plasmid was then integrated into the P. pastoris Pp-EBIL strain genomic DNA, producing clone Pp-EBILWZ. The recombinant P. pastoris (Pp-EBILWZ) cells exhibited a strong reddish carotenoid coloration and were confirmed, by HPLC, to produce not only the previous described carotenoids lycopene and β-carotene, but also de novo synthesized astaxanthin.

Cloning and Characterization of the Beer Foaming Gene CFG1from Saccharomyces pastorianus

Foam production is an essential characteristic of beer, generated mainly from the proteins present in the malt and, to a minor extent, from the mannoproteins in brewers yeast cell walls. Here, we describe the isolation and characterization of the novel fermentation gene CFG1 (Carlsbergensis foaming gene) from Saccharomyces pastorianus. CFG1 encodes the cell wall protein Cfg1p, a 105 kDa protein highly homologous to Saccharomyces cerevisiae cell wall mannoproteins, particularly those involved in foam formation, such as Awa1p and Fpg1p. Further characterization of Cfg1p revealed that this novel protein is responsible for beer foam stabilization. This report represents the first time that a brewing yeast foaming gene has been cloned and its action fully characterized.

Role of TolC in Klebsiella oxytoca resistance to antibiotics

OBJECTIVES The Gram-negative human pathogen Klebsiella oxytoca is often resistant to several antibiotics such as fluoroquinolones, erythromycin, tetracycline, chloramphenicol and others. The aim of this study was to look at the mechanisms leading to this resistance and particularly the role of TolC and efflux mechanisms in determining resistance. METHODS Ciprofloxacin accumulation was measured spectrofluorometrically. Growth inhibition assays were performed in the presence or absence of carbonyl cyanide m-chlorophenylhydrazone (10 mg/L, final concentration). The genome of K. oxytoca was analysed for the existence of loci encoding tolC by PCR using primers for the Enterobacter aerogenes tolC gene and subsequently sequenced. A plasmid named pUC18TolC was constructed and inserted into Escherichia coli C600tolC,Tn5, and the function of TolC was analysed. The structure modelling was performed using the Modeller program. RESULTS The existence of the AcrAB efflux mechanism was demonstrated in the species, and a TolC-like protein, a channel-forming protein at the external membrane that allows the extrusion of antibiotics by the AcrAB efflux pump, was cloned, sequenced and a model proposed. CONCLUSIONS K. oxytoca express a functional TolC that lacks a fragment of six amino acids characteristic of the external loops of TolC in E. coli. This makes this species resistant to a few colicins.

FPG1, a gene involved in foam formation in Saccharomyces cerevisiae.

Foam formation in fermentations conducted by Saccharomyces cerevisiae, either at the beginning of the fermentation process or at the end in the case of sparkling wines, is due, to a large extent, to cell wall mannoproteins, which provide hydrophobicity to the yeast cells and favour their floating index as well as stabilization of the foam. The foam may be an undesirable by‐product if it accumulates on top of the fermentation tanks, but its formation is a good property in either beer or sparkling wines. It is therefore important to know the yeast genes involved in foam formation, in order to suppress or potentiate their expression according to the end product to be obtained. The present study identified and characterized, for the first time in an oenological S. cerevisiae strain, a gene involved in foam formation, named FPG1 (foam‐promoting gene). The protein encoded by FPG1 is a mannoprotein precursor present in the cell wall and somewhat homologous to Awa1p, a foaming protein described in a sake S. cerevisiae strain. A foamless strain was prepared by FPG1 deletion, and a foam hyper‐producing strain was also constructed, thus allowing the conclusion that Fpg1p is a mannoprotein involved in yeast frothing. Copyright

Proposal of a method for the genetic transformation of Gordonia jacobaea

Aims:  Gordonia jacobaea is a recently isolated bacterial species with potential industrial application on account of its ability to store large quantities of trans‐canthaxanthin. Its genetic manipulation is, however, difficult and cumbersome owing to the presence of mycolic acids in the cell wall and, especially, because of current lack of knowledge about its basic genetics. The present work describes a method for the genetic transformation of G. jacobaea.

A new disruption vector (pDHO) to obtain heterothallic strains from both Saccharomyces cerevisiae and Saccharomyces pastorianus

Yeasts are responsible for several traits in fermented beverages, including wine and beer, and their genetic manipulation is often necessary to improve the quality of the fermentation product. Improvement of wild-type strains of Saccharomyces cerevisiae and Saccharomyces pastorianus is difficult due to their homothallic character and variable ploidy level. Homothallism is determined by the HO gene in S. cerevisiae and the Sc-HO gene in S. pastorianus. In this work, we describe the construction of an HO disruption vector (pDHO) containing an HO disruption cassette and discuss its use in generating heterothallic yeast strains from homothallic Saccharomyces species.

Combination of cGMP analogue and drug delivery system provides functional protection in hereditary retinal degeneration

Significance Development of treatments for hereditary degeneration of the retina (RD) is hampered by the vast genetic heterogeneity of this group of diseases and by the delivery of the drug to an organ protected by the blood–retina barrier. Here, we present an approach for the treatment of different types of RD, combining an innovative drug therapy with a liposomal system that facilitates drug delivery into the retina. Using different animal models of RD we show that this pharmacological treatment preserved both the viability of cells in the retina as well as retinal function. Thus, our study provides an avenue for the development of therapies for hereditary diseases which cause blindness, an unmet medical need. Inherited retinal degeneration (RD) is a devastating and currently untreatable neurodegenerative condition that leads to loss of photoreceptor cells and blindness. The vast genetic heterogeneity of RD, the lack of “druggable” targets, and the access-limiting blood–retinal barrier (BRB) present major hurdles toward effective therapy development. Here, we address these challenges (i) by targeting cGMP (cyclic guanosine- 3′,5′-monophosphate) signaling, a disease driver common to different types of RD, and (ii) by combining inhibitory cGMP analogs with a nanosized liposomal drug delivery system designed to facilitate transport across the BRB. Based on a screen of several cGMP analogs we identified an inhibitory cGMP analog that interferes with activation of photoreceptor cell death pathways. Moreover, we found liposomal encapsulation of the analog to achieve efficient drug targeting to the neuroretina. This pharmacological treatment markedly preserved in vivo retinal function and counteracted photoreceptor degeneration in three different in vivo RD models. Taken together, we show that a defined class of compounds for RD treatment in combination with an innovative drug delivery method may enable a single type of treatment to address genetically divergent RD-type diseases.

Increased Plasma cGMP in a Family With Autosomal Recessive Retinitis Pigmentosa Due to Homozygous Mutations in the PDE6A Gene

Purpose To describe genotype and phenotype in a family with autosomal recessive retinitis pigmentosa (arRP) carrying homozygous mutations in the gene for the α-subunit of cyclic guanosine monophosphate (cGMP)-hydrolyzing phosphodiesterase 6 (PDE6A). Moreover, to compare their plasma cGMP levels to controls, exploring the possible role for cGMP in RP diagnostics. Methods Seven siblings and their parents were recruited. Microarray, verified by Sanger sequencing, was used for genotyping. Investigations included slit lamp and fundus examination, Goldmann perimetry, full-field and multifocal electroretinography (ERG), and optical coherence tomography (OCT). Cyclic GMP was measured with an immunoassay kit. Results All siblings and their father were homozygous, and the mother heterozygous, for IVS6+1G>A in PDE6A. Seven family members also carried c1532G>A in ABCA4. Visual fields were constricted with mere central remnants in older subjects and additional temporal crescents in younger subjects. Visual acuity ranged from 0.8 to amaurosis. Full-field ERGs showed extinguished rod responses and minimal cone responses. Multifocal ERGs were severely reduced. Optical coherence tomography revealed either general attenuation or central macular edema. Mean plasma cGMP in patients was approximately twice that in controls. Conclusions To our knowledge, this is the first phenotypic description of arRP due to homozygous IVS6+1G>A mutations in PDE6A and these seem here to be associated with severe RP leading to early extinction of rod responses as well as reduced macular function. Additionally, patients showed increased plasma levels of cGMP, indicating a possible role for cGMP measurements as part of the clinical tests for this and, after further investigations, maybe other forms of RP.

Expression of a yeast polygalacturonase gene in Arabidopsis thaliana

Polygalacturonases are enzymes involved in plant cell wall growth and reorganization. Transgenic Arabidopsis thaliana plants with a Saccharomyces cerevisiae endopolygalacturonase gene (PGU1) were obtained. The yeast gene was properly expressed in the plants as it has been shown by RT-PCR as well as by the increase in the endopolygalacturonase activity. The transgenic plants showed conspicuous malformations in early stages of development probably due to a weak cell adhesion. On the other hand, adult plants exhibited almost no phenotypic differences as compared to the wild type plants, this suggesting the appearance of some mechanisms on the plant side to counteract the effect of the overexpressed polygalacturonase.

Fragment of the Ancient RbcL Gene from the Miocene

The development of molecular biology techniques has allowed a new approach to palaeontology and studies on ancient DNA. As a plant fossil resin, amber provided a good matrix for preserving ancient biological material. Some difficulties arise when experimental work is done to extract information concerning these preserved specimens. The major risks in this type of works are the contamination with modern DNA and the degradation of the ancient DNA. A safe method to sterilize amber stones has been designed allowing the amplification of a fragment of the ancient RbcL gene from the Miocene (c.a. 25 million years). Presumably, the gene was from Hymenaea protera, an extinct member of the Leguminoseae family. The phylogenetic tree and divergence rates indicate that since although it is a well-conserved gene, and then should be a good candidate for studying the evolution of plant macrogroups, probably it is not good enough for analyzing divergence among closely related species.