COMPARATIVE GENOME SEQUENCE ANALYSIS AND GENETIC ENGINEERING OF TOLERANT ETHANOLOGENIC YEAST SACCHAROMYCES CEREVISIAE NRRL Y-50049
Bioenergy Research Unit
2012 Annual Report
1a.Objectives (from AD-416):
Identify genomic basis and relationships between enhanced inhibitor tolerance and efficient heterogeneous sugars utilization derived from lignocelluolosic biomass; engineer tolerant Saccharomyces cerevisiae NRRL Y-50049 for improved and balanced utilization of pentoses and hexoses from lignocellulosic hydrolysates.
1b.Approach (from AD-416):
Comparative genome sequence analysis will be carried out to underline genetic fundamentals of the tolerance and in situ detoxification of the biomass conversion inhibitors.
Comparative genome expression analysis will be carried out to establish gene regulatory networks involving tolerance and heterogeneous sugar utilizations including pentoses and hexoses.
Genetic engineering will be carried out to incorporate heterogeneous genes into the tolerant Y-50049 for improved pentose utilization.
The research focused mainly on comparative genome sequence analysis. Ethanologenic yeast strain NRRL Y-12632 and its stress-tolerant derivative NRRL Y-50049 were sequenced and compared in this study. A total of 5,380 and 5,384 polypeptide-encoding genes were predicted in Y-12632 and Y-50049 genomes, respectively, using the gene model of strain S288C as the reference. The gene annotation was performed by searching genome databases. Evolutionary analysis showed that both Y-12632 and Y-50049 are highly diverged with respect to any other strains and will be useful in characterizing the sequence diversity present in S. cerevisiae. Mutational discovery analysis was also performed. We also integrated the genomic variations with transcriptomics changes of the genome to analyze the key genes, pathways, and adaptive mechanisms of the yeast to the inhibitor, hydroxy-methyl furfural (HMF). The MAPK signaling pathway was characterized as the essential signaling pathway of the HMF stimulus and several pathways or metabolic activities were identified as playing important roles in this process, including enhancement in DNA repair function, functional variation of membrane lipid and proteins, and alterations in pyridine nucleotide precursor metabolism, as well as other metabolic modifications. ARS scientist, in conjunction with the 8th USDA-Ministry of Science and Technology (MOST) Joint Working Group Meeting in China, 2011, was able to visit the QIBEBT team and review the research progress. Currently, ARS scientists are working on biological confirmation of the informatics predictions. Scientists from both sides continue to collaborate closely on developing a manuscript to be published in a peer-reviewed journal in 2013.