Utilisation de souches de levures génétiquement modifiées en œnologie

ENOSAFE (No. 762, Programa AGRO, medida 8).Fundação para a Ciência e a Tecnologia (FCT), l’Instituto Nacional de Investigação Agrária et LeVin - POCI/AGR/56102/2004

A utilização de estirpes de leveduras geneticamente modificadas em enologia

Este estudo foi apoiado pelos projectos ENOSAFE (n.º 762, Programa AGRO, medida 8), pelo Instuituto Nacional de Investigação Agrária e LeVini (POCI/AGR/56102/2004), Fundação para a Ciência e Tecnologia, Portugal

L’utilizzo di ceppi modificati di saccharomyces cerevisiae in enologia

Trata-se de uma tradução do seguinte artigo D. Schuller e M. Casal. 2005. The use of genetically modified yeast in the wine industry. Applied Microbiology and Biotechnology 68, 292-304.Negli ultimi decenni, la scienza e la tecnologia degli alimenti hanno contribuito all’introduzione di nuovi prodotti per soddisfare le richieste nutrizionali, socio-economiche e di qualità. Con la moderna genetica molecolare, l’importanza industriale di Saccharomyces Cerevisiae è accresciuta ulteriormente. La richiesta di ceppi geneticamente modificati (GM) di S. cerevisiae per il biodiesel, l’industria delle bevande e del pane o per la produzione di prodotti biotecnologici (per esempio, enzimi, prodotti farmaceutici) continuerà a crescere in futuro. Negli ultimi anni, sono stati ottenuti numerosi ceppi enologici specializzati di S. cerevisiae , che presentavano diverse e nuove proprietà enologiche, in grado di soddisfare le richieste derivanti dalle moderne pratiche di vinificazione. La comprensione delle complessità del transcrittoma, proteoma e metaboloma darà un contributo decisivo alla conoscenza della composizione genetica dei ceppi commerciali enologici ed influenzerà il miglioramento genetico grazie all’ingegneria genetica. I progressi più importanti riguardanti le implicazioni dell’utilizzo di ceppi GM nell’industria enologica sono discussi in questo articolo. Vengono considerati diversi aspetti, incluse le strategie utilizzate per la costruzione di ceppi nel rispetto degli obblighi legislativi attuali, la valutazione dei rischi ambientali riguardanti i ceppi geneticamente modificati, i metodi per il rilevamento del DNA ricombinante e la proteine e le ragioni dell’atteggiamento negativo dei consumatori nell’utilizzo di tali ceppi.Ringraziamenti Questo studio è stato finanziato dai progetti ENOSAFE (No. 762, Programa AGRO, medida 8) Instituto Nacional de Investigação Agrária and LeVini (POCTI/AGR/56102/2004) Fundação para a Ciência e Tecnologia (FCT

The genetic structure of fermentative vineyard-associated Saccharomyces cerevisiae populations revealed by microsatellite analysis

Prova tipográfica (In Press)From the analysis of six polymorphic microsatellite loci performed in 361 Saccharomyces cerevisiae isolates 93 alleles were identified, being 52 of them described for the first time. All these isolates have a distinct mtDNA RFLP pattern. They are derived from a pool of 1620 isolates obtained from spontaneous fermentations of grapes collected in three vineyards of the Vinho Verde Region in Portugal, during the 2001 – 2003 harvest seasons. For all loci analyzed, observed heterozygosity was three to four times lower than the expected value supposing a Hardy-Weinberg equilibrium (random mating and no evolutionary mechanisms acting), indicating a clonal structure and strong populational substructuring. Genetic differences among S. cerevisiae populations were apparent mainly from gradations in allele frequencies rather than from distinctive “diagnostic” genotypes, and the accumulation of small allele-frequency differences across six loci allowed the identification of population structures. Genetic differentiation in the same vineyard in consecutive years was of the same order of magnitude as the differences verified among the diferent vineyards. Correlation of genetic differentiation with the distance between sampling points within a vineyard suggested a pattern of isolation-by-distance, where genetic divergence in a vineyard increased with size. The continuous use of commercial yeasts has a limited influence on the autochthonous fermentative yeast population collected from grapes and may just slightly change populational structures of strains isolated from sites very close to the winery where they have been used. The present work is the first large-scale approach using microsatellite typing allowing a very fine resolution of indigenous S. cerevisiae populations isolated from vineyards.This study was supported by the project ENOSAFE (Nº 762, Programa AGRO, medida 8), the programme POCI 2010 (project POCI/AGR/56771/2004) and the grant nº 657 C2 from the cooperation agreement between the Portuguese Institute for International Scientific and Technological Cooperation (ICCTI) and the French Embassy in Lisbon. We wish to thank Prof. Célia Pais and for helpful discussions during the preparation of this manuscript. Prof. Paula Sampaio is kindly thanked for assistance with statistical analysis. Magda Silva Graça is gratefully acknowledged for the operation of the DNA sequencer. We also appreciate the kind assistance of the enologists Rui Cunha, Anselmo Mendes, Euclides Rodrigues and José Domingues for facilitating sampling campaigns in the three vineyards

The use of genetically modified Saccharomyces cerevisiae strains in the wine industry

Prova tipográfica (In Press).During the last decades, science and food technology have contributed at an accelerated rate to the introduction of new products to satisfy nutritional, socio-economic and quality requirements. With the emergence of modern molecular genetics, the industrial importance of Saccharomyces cerevisiae, continuously extended. The demand for suitable genetically modified (GM) S. cerevisiae strains for the biofuel, bakery and beverage industries or for the production of biotechnological products (e.g. enzymes, pharmaceutical products) will be continuously growing in the future. Numerous specialized S. cerevisiae wine strains were obtained in the last years, possessing a wide range of optimized or novel oenological properties, capable to satisfy the demanding nature of modern winemaking practice. Unlocking the transcriptome, proteome and metabolome complexities contributes decisively to the knowledge about the genetic make-up of commercial yeast strains and will influence wine strain improvement by genetic engineering. The most relevant advances regarding the importance and implications of the use of genetically modified yeast strains in the wine industry are discussed in this Mini-Review, considering a variety of aspects such as the strategies used for the construction of the strains with respect to current legislation requirements, environmental risk evaluations concerning the deliberate release of genetically modified yeast strains, methods for the detection of recombinant DNA and protein that are currently under evaluation, and the reasons for the critical public perception towards the application of such strains.União Europeia - Fundo Social Europeu (FSE) - III Quadro Comunitário de Apoio (QCA III) - Programa Operacional Agricultura e Desenvolvimento Rural (AGRO) - EnoSafe Nº 762, medida 8.Fundação para a Ciência e a Tecnologia (FCT) - Programa Operacional Ciência, Tecnologia, Inovação (POCTI) - POCTI/AGR/56102/2004.LeVini.Instituto Nacional de Investigação Agrária

Solvent-cast films of an elastin-like polymer fused to an antimicrobial peptide, ABP-CM4, exhibits high antibacterial activity against pseudomonas aeruginosa

The nosocomial infections grew significantly in the last years and became a worldwide problem. Antimicrobial peptides (AMPs) arise as a good treatment to these infections, since traditional antibiotics have become useless against resistant hospital strains. AMPs exhibit a broad range of antimicrobial activity but antitumoral and antiviral activities have also been found. AMPs are usually small, cationic molecules that occur as part of the innate defense mechanism in many organisms, even in microbes and virus. The combination of AMPs with recombinantly produced polymers, such as the Elastin-like Polymers (ELPs), inspired in the mammalian elastin, could improve medical equipment, such as catheters, to overcome infections and biofilms formation. In this work we describe the cloning and recombinant production in Escherichia coli BL21(DE3) of ABP-CM4, a cationic AMP from Bombyx mori, fused to an ELP, consisting of 200 repeats of the pentamer VPAVG (A200). This ELP exhibits thermoresponsive properties, exploitable as a purification method. The morphological characteristics as well as its antibacterial activity of this hybrid polymer were studied as essential for the applicability in medical devices. The ABP-CM4 gene was chemically synthesized, with the inclusion of a formic acid cleavage site, and fused in frame with the N-terminus of the gene coding A200. Production of the recombinant polymer in E. coli BL21(DE3) was achieved and purification was based on the use of the inverse transition cycling method. Formic acid treatment allowed tag removal and obtention of the soluble protein. The hybrid polymer, CM4::A200, and the cleaved ABP-CM4 were tested for its antimicrobial activity in liquid form. Solvent-cast films of CM4::A200, using formic acid as solvent, were tested for the antibacterial activity against Pseudomonas aeruginosa comparing with A200 polymers containing different contents of positively charged aminoacids. The hybrid polymer presented similar morphological and physicochemical features to A200. The cleaved recombinant ABP-CM4 and CM4::A200 showed low levels of inhibition against P. aeruginosa in the liquid form but, in the solvent-cast film form, the inhibition of growth of was almost 100%. This result reveals very good perspectives for the use of these polymers in the medical equipment.Fundação para a Ciência e a Tecnologia (FCT

Cloning, expression and purification of a carbohydrate binding module in Pichia pastoris

The enzymes responsible for carbohydrate degradation are, usually, compose of two distinct modules: catalytic and a substrate binding module. Since these two modules are functionally independent, the CBMs (carbohydrate binding modules) can be fused with bioactive molecules to drive them to starch based biomaterials. In this work, the CBM cloned belongs to human phosphatase laforin, which is involved in metabolism of the glycogen. Aiming at the optimization of large scale expression, CBM peptide production was done by cloning CBM coding sequence in two different systems of Pichia pastoris: pGAPZα C which has a constitutive promoter and pPICZα C which has an inductive promoter. Both expression systems have the secretion signal α- factor. The integration of the CBM coding sequence, in yeast genome and the gene transcription were confirmed by slot-blot and northern-blot, respectively. The fermentation conditions for different P. pastoris clones were optimized and recombinant protein was purified from fermentation medium by affinity chromatography. Purified protein was analysed by western-blot. Fictionalization studies on starch based biomaterials are being performed

Production of the human carbohydrate binding module from laforin protein : a comparative study between bacterial and yeast expression systems

Fundação para a Ciência e a Tecnologia (FCT) - POCTI/BIO/45356/2002 ; (SFRH/BD/18418/2004)

Differential malic acid degradation by indigenous and commercial Saccharomyces cerevisiae wine strains

One thousand six hundred and twenty yeast isolates were obtained from spontaneous fermentations performed with grapes collected in three vineyards of the Vinho Verde Wine Region in northwest Portugal during three subsequent harvest seasons. All isolates were analyzed by mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) and a pattern profile was verified for each isolate, resulting in a total of 294 different profiles, all revealed to belong to the species Saccharomyces cerevisiae. A remarkable heterogeneity of phenotypical traits was found when this collection of indigenous S. cerevisiae strains was screened regarding ethanol tolerance, H2S production, capacity to utilize acetic and malic acid. Malic acid, together with tartaric acid, are the most abundant organic acids in wine contributing to its acidic taste. Excess malic acid removal is of enological interest in this wine region. From the screening of 294 strains, only 3 (318, 319 and 320) showed enhanced malic acid consumption in combination with other desirable phenotypic traits. Their fermentative profiles in a synthetic must medium containing glucose (20%, w/v) and D,L-malic acid (0.6%, w/v) were very similar to the ones observed for the commercial strains QA23 and 71B, but considerable differences were found regarding the activity of key enzymes involved in the metabolism of malic acid (malic enzyme, malate dehydrogenase, fumarase). The best malic acid degrading strain was 71B (40% at the end of fermentation), but from an applied and enological point of view it is still desirable to improve this trait. In order to enhance malic acid consumption, cells were transformed with genetic constructs containing both Kluyveromyves lactis dicarboxylate permease KlJEN2 and the MAE2 malic enzyme from Schizosaccharomyces pombe under constitutive expression. Data will be presented showing how the physiology of malic acid utilization is associated with the distinct yeast genetic backgrounds.Fundação para a Ciência e a Tecnologia (FCT) - POCI 2010 (FEDER/FCT, POCI/AGR/56102/2004), and AGRO (ENOSAFE, Nº 762)