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Gene expression analysis of putative genes of industrial interest in knockouts mutants of Saccharomyces cerevisiae (PE-2 strain)

Grant number: 12/09447-5
Support type:Scholarships abroad - Research Internship - Master's degree
Effective date (Start): July 12, 2012
Effective date (End): October 11, 2012
Field of knowledge:Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
Principal researcher:Gonçalo Amarante Guimarães Pereira
Grantee:Nádia Maria Vieira Sampaio
Supervisor abroad: Juan Lucas Argueso Gomes de Almeida
Home Institution: Instituto de Biologia (IB). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
Research place: Colorado State University, United States  
Associated to the scholarship:10/14131-1 - Phenotypic and physiological characterization of Saccharomyces cerevisiae (PE-2 strain) mutants for putative genes of industrial interest, BP.MS

Abstract

The improvement of S. cerevisiae strains for higher bioethanol productivity and increased tolerance to industrial stress conditions is a key factor for making the production of renewable biofuels economically viable. In Brazil, the JAY270/PE-2 heterozygous diploid strain is used by many bioethanol distilleries due to its high productivity and vigorous. We previously reported the genome sequencing of JAY291, a JAY270/PE-2 derived haploid spore, which, among other findings, revealed a total of 21 predicted genes that are not found in the genome of the reference laboratory strain S288c. These two strains are significantly different in terms of their performance in fermentation and adaptation to the industrial environment. To investigate whether any of the 21 JAY291-specific genes contributes to its desirable properties we constructed a systematic collection of knockout mutants for these genes and are conducting phenotypic tests to establish their function. We deleted all 21 genes, comprising four cluster knockouts and three genes deleted individually. The mutant strains were then co-cultivated with the parental strain in 15-cycle competition assays performed in of sugar cane extract. Every 24 hours (one cycle), 0.5% of the culture was transferred to fresh media for growth and fermentation. The initial ratio of the JAY270/PE-2 parent to mutant cells at cycle zero was adjusted to approximately 1 (50% each), and changes to population composition were monitored periodically. After 15 cycles in co-culture, we noted decreases in all mutants relative to the parent, with final the representation ranging from 45% to 2%. In addition, fermentative assays showed a 4% yield decrease by a knockout strain for an eight-gene cluster. In order to acquire more data about the role of these genes, overexpression assays in JAY270 and insertion into S288c of genes with the most significant phenotypes are being performed. We also intend to analyze transcriptional responses of knockout strains during the fermentative process using microarrays. With all these data, we hope to identify in these regions fermentation-related characters that could become promising targets for genetic manipulation in industrial strains. (AU)

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