Development of industrial platforms: domestication of yeast for improved alcoholic fermentation

Author(s)

Felipe Galzerani

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2010-09-30

Examining board members

Gonçalo Amarante Guimarães Pereira; Anderson Ferreira da Cunha; Luiz Humberto Gomes

• Advisor: Juan Lucas Argueso; Gonçalo Amarante Guimarães Pereira

Abstract

The energy crisis and the environmental concern have made ethanol an attractive renewable energy source. Brazil and the United States have been, in the last years, the largest producers of this important energy resource. Brazilian ethanol is produced through fermentation of sucrose from sugar cane by Saccharomyces cerevisiae yeasts. Laboratory strains of S. cerevisiae are among the most well characterized organisms in modern biology. For decades they have undergone a series of genetic changes with the creation of mutations in markers genes that now enable current procedures for genetic manipulation. In contrast, the industrial fermentation process is carried out by wild strains that remain as of yet genetically unexplored hindering the application of genetic engineering techniques. The objective of the present study was to domesticate the JAY270 diploid strain, which is an isolate of the industrial yeast Pedra-2, simplifying its genetic manipulation into a biological platform for the introduction of characteristics that improve its performance in industries. The genetic manipulation strategy was based on the Delitto Perfetto procedure to preserve the highly heterogeneous genome of the JAY270 strain including its genetics properties related to characteristics of industrial interest. Two integrative cassettes containing different genetic markers were constructed from plasmids to be used in deletion of both copies of the URA3 gene of the JAY270 strain. A repair homologous fragment was employed in the removal of the selective markers used during the manipulation steps, avoiding any remaining transgenic traces. The auxotrophy to uracil was achieved after complete deletion of the URA3 genes from the JAY270 diploid genome. The heterogeneous genomic architecture of the JAY270 was maintained as well as its hybrid vigor related to fermentative characteristics of industrial interest. The auxotrophic yeast generated in this study will facilitate the application of genetic engineering techniques such as Delitto Perfetto to achieve improvement in the industrial fermentation traits of the wild strain. (AU)