Engineering Pichia pastoris for the Production of Carotenoids.

Carotenoids are one of the most diverse and widely distributed classes of pigments in the biosphere and exhibit a variety of functions in the nature. Their importance and biotechnological applications are higher and higher, but their sources are not increasing in the same exponential way. Here we describe the process of bioengineering the yeast Pichia pastoris by sequential transformation to get an astaxanthin producer.

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.

Influence of extracellular matrix components on the expression of integrins and regeneration of adult retinal ganglion cells.

Purpose: Retinal ganglion cells (RGCs) are exposed to injury in a variety of optic nerve diseases including glaucoma. However, not all cells respond in the same way to damage and the capacity of individual RGCs to survive or regenerate is variable. In order to elucidate factors that may be important for RGC survival and regeneration we have focussed on the extracellular matrix (ECM) and RGC integrin expression.

Phenotypic map of porcine retinal ganglion cells.

Purpose: Porcine retina is an excellent model for studying diverse retinal processes and diseases. The morphologies of porcine retinal ganglion cells (RGCs) have, however, not yet been described comprehensively. The aim of the present study was to créate a classification of the RGCs using the 1, 1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) tracing method.

Neuroprotective effects of topical CB1 agonist WIN 55212-2 on retinal ganglion cells after acute rise in intraocular pressure induced ischemia in rat.

Neuroprotection in retinal experimental work consists primarily of preventing retinal ganglion cell (RGC) loss after exposure to a hostile event. We have studied the neuroprotective effect on RGCs in an ischemia-reperfusion model by activation of the cannabinoid receptor CB1 using topical application of WIN 55212-2. Intraocular pressure (IOP) was increased by continuous infusion of phosphate buffer saline (PBS) into the anterior chamber of the eye.

Cloning and characterization of the beer foaming gene CFG1 from 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 brewer's 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.

Analysis of canthaxanthin production by Gordonia jacobaea.

Commercial interest in the use of natural pigments isolated from microorganisms has increased in recent years; hence, molecules belonging to the polyisoprenoid group (i.e., β-carotene, astaxanthin, and canthaxanthin) have been the focus of much attention.

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.

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.

Construction of new Pichia pastoris X-33 strains for production of lycopene and β-carotene.

In this study, we used the non-carotenogenic yeast Pichia pastoris X33 as a receptor for β-carotene-encoding genes, in order to obtain new recombinant strains capable of producing different carotenoidic compounds. We designed and constructed two plasmids, pGAPZA-EBI* and pGAPZA-EBI*L*, containing the genes encoding lycopene and β-carotene, respectively. Plasmid pGAPZA-EBI*, expresses three genes, crtE, crtB, and crtI*, that encode three carotenogenic enzymes, geranylgeranyl diphosphate synthase, phytoene synthase, and phytoene desaturase, respectively.

cDNA cloning of a novel gene codifying for the enzyme lycopene β-cyclase from Ficus carica and its expression in Escherichia coli.

Lycopene beta-cyclase (β-LCY) is the key enzyme that modifies the linear lycopene molecule into cyclic β-carotene, an indispensable carotenoid of the photosynthetic apparatus and an important source of vitamin A in human and animal nutrition. Owing to its antioxidant activity, it is commercially used in the cosmetic and pharmaceutical industries, as well as an additive in foodstuffs. Therefore, β-carotene has a large share of the carotenoidic market.

Oily yeasts as oleaginous cell factories.

Oily yeasts have been described to be able to accumulate lipids up to 20% of their cellular dry weight. These yeasts represent a minor proportion of the total yeast population, and only 5% of them have been reported as able to accumulate more than 25% of lipids. The oily yeast genera include Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, and Lipomyces.

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.

Synergism between outer membrane proteins and antimicrobials.

Antibiotic-resistant bacteria are becoming one of the most important problems in health care because of the number of resistant strains and the paucity of new effective antimicrobials. Since antibiotic-resistant bacteria will continue to increase, it is necessary to look for new alternative strategies to fight against them. It is generally accepted that Gram-negative bacteria are intrinsically less susceptible than Gram-positive bacteria to antimicrobials.

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.

Genetic stabilization of Saccharomyces cerevisiae oenological strains by using benomyl.

Wild-type oenological strains of Saccharomyces cerevisiae are usually aneuploid and heterozygotes; thus, when they are used as starters in must fermentation the resulting wine characteristics may vary from year to year. Treatment of a wild-type S. cerevisiae oenological strain with benomyl (methyl-l-butylcarbamoyl-2-benzimidazole carbamate), an antifungal agent shown to cause chromosome loss in yeasts, resulted in a stable starter strain in which the parental oenological traits were unchanged.

Putative ancient microorganisms from amber nuggets.

Evolutionary microbiology studies based on the isolation of ancient DNA and/or microbial samples are scarce due to the difficulty of finding well preserved biological specimens. However, amber is a fossil resin with natural preserving properties for microbial cells and DNA. The visualization by transmission electron microscopy of different microorganism-like specimens found in amber nuggets from both the Miocene and the Cretaceous periods was accompanied by studies of ancient DNA obtained from the nuggets.

Enzybiotics: a look to the future, recalling the past.

The discovery and development of antibiotics was one of the greatest successes of Medicine in the 20th century and allowed the control of many diseases caused by microorganisms. Nevertheless, it is necessary to search constantly for new therapeutic tools in the continuing fight against disease-causing microorganisms and this probably leads us to today's concept of enzybiotics. Although microorganism-degrading enzymes have been known since the beginning of the last century, their use was soon forgotten because of the widespread use of antibiotics.

Cloning and expression of the XPR2 gene from Yarrowia lipolytica in Pichia pastoris.

Yarrowia lipolytica is a dimorphic yeast able to secrete different types of proteases depending on the pH of the environment. At neutral pH, the production of an extracellular alkaline protease (AEP) is induced. This protease could be useful in the leather, detergent, or food industries.

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.

Influence of culture conditions of Gordonia jacobaea MV-26 on canthaxanthin production.

Commercial interest in the use of natural pigments isolated from microorganisms has increased in recent years; hence, molecules belonging to the polyisoprenoid group (i.e. beta-carotene, astaxanthin, and canthaxanthin) have been the focus of much attention.

Ancient genes of Saccharomyces cerevisiae.

Amber is a plant resin mainly produced by coniferous trees that, after entrapping a variety of living beings, was subjected to a process of fossilization until it turned into yellowish, translucent stones. It is also one of the best sources of ancient DNA on which to perform studies on evolution. Here a method for the sterilization of amber that allows reliable ancient DNA extraction with no actual DNA contamination is described.