A vector carrying the GFP gene (Green fluorescent protein) as a yeast marker for fermentation processes

Abstract

Contaminant yeasts spoil pure culture fermentations and cause great losses in quality and product yields. They can be detected by a variety of methods although none being so efficient for early detection of contaminant yeast cells that appear at low frequency. Pure cultures bearing genetic markers can ease the direct identification of cells and colonies among contaminants. Fast and easy detection are desired and morphological markers would even help the direct visualization of marked pure cultures among contaminants. The GFP gene for green fluorescent protein of Aquorea victoria, proved to be a very efficient marker to visualize transformed cells in mixed populations and tissues. To test this marker in the study of contaminated yeast fermentations, the GFP gene was used to construct a vector under the control of the ADH2 promoter (pYGFP3). Since ADH2 is repressed by glucose the expression of the protein would not interfere in the course of fermentation. The transformed yeasts with the vector pYGFP3 showed high stability and high bioluminescence to permit identification of marked cells among a mixed population of cells. The vector opens the possibility to conduct further studies aiming to develop an efficient method for early detection of spoilage yeasts in industrial fermentative processes.

Obtenção de levedura híbrida fluorescente e resistente a nistatina

“…Com este objetivo, TAVARES (1989) desenvolveu um processo de produção de etanol com o controle de leveduras contaminantes, que utiliza um híbrido M606 de Saccharomyces cerevisiae de alta produtividade e resistente a nistatina, um antifúngico de amplo espectro que pode ser usado para garantir a pureza genética do inóculo industrial. Para facilitar a identificação de leveduras GOMES et al (2000), utilizaram a propriedade de fluorescência expressada pelo gene GFP "Green Fluorescent Protein" sob o controle do promotor da ADH2 em baixas concentrações de glicose. Associar estas duas propriedades em uma levedura industrial permitiria manter a pureza do inóculo e facilitaria a sua identificação.…”

“…Associar estas duas propriedades em uma levedura industrial permitiria manter a pureza do inóculo e facilitaria a sua identificação. Com este objetivo, foram efetuados experimentos de transformação genética do híbrido M606 com o plasmídio pYGFP3 construído por Gomes et al (2000), sem obter o sucesso desejado. Por isto, como passo inicial para obtenção de ambas propriedades em híbrido altamente produtivo, optou-se pela metodologia de cruzamentos entre uma linhagem segregante haplóide resistente a nistatina M606-2c(n) e a linhagem X2904-GFP3 portadora do plasmídio pYGFP3.…”

“…It´s a wide spectrum antifungal that can be used to guarantee the genetic purity of the industrial inoculum. To facilitate the yeasts identification, GOMES et al (2000) used the fluorescence property expressed by the GFP gene "Green Fluorescent Protein", that is controlled by the promoter of ADH2 in a low glucose concentrations. The association of these two properties in an industrial yeast strain would allow to maintain the purity of the inoculum and it would facilitate its identification.…”

“…The association of these two properties in an industrial yeast strain would allow to maintain the purity of the inoculum and it would facilitate its identification. With this aim, an assay of genetic transformation of hybrid M606 with pYGFP3 plasmid built by Gomes et al (2000) was made, but it had not success. Therefore, as initial step for obtaining of both properties in hybrid highly productive, It was opted for the methodology of crossings between a nystatin resistant haploid segregant strain M606-2c(n) and the strain X2904-GFP3(n) harboring of the plasmid pYGFP3.…”

“…Contudo, nem sempre genes marcadores são rapidamente identificados, mas com esta finalidade Gomes et al (2000) obtiveram leveduras cujas células se tornam fluorescentes quando expostas à luz ultravioleta, possibilitando a monitoração rápida e direta na identificação de possíveis contaminantes. Utilizaram o gene GFP "Green Fluorescent Protein" isolado do celenterado Aequorea victoria para a construção do plasmídio pYGFP3, controlado pelo promotor da enzima desidrogenase alcoólica (ADH2) de S. cerevisiae, que apresenta repressão por glicose.…”