2010 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. 3.Progress Report Collaborating with Qingdao Institute of Bioenergy and Bioprocessing Technology (QIBEBT), Chinese Academy of Science, we have investigated genome sequence variations of ARS yeast strains Saccharomyces cerevisiae NRRL Y-50049 and its parental strain Y-12632. Both genomes were sequenced using a Genome Analyzer IIx (Illumina) using a paired end approach at QIBEBT. In addition, strain Y-50049 was sequenced using 454 Titanium sequencers (Roche). Single read and paired end technology were both used to improve the quality of the genome assembly. We have accomplished a total of 469,034 454 single reads and 15,948,887 solexa paired-end reads for the tolerant strain Y-50049, and 15,220,078 solexa paired-end reads for strain Y-12632. After hybrid assembly of the 454 and Solexa reads of the genome, we have collected at least 832 contigs for Y-50049 genome. For mutation analysis, we have identified over 16,000 site mutations of single nucleotide polymorphisms (SNPs) or insertions and deletions (InDels) for the Y-50049 genome. Data confirmation and analysis are underway. The original scheduled 1-year visiting scientist from QIBEBT to the National Center for Agricultural Utilization Research was canceled due to difficulties of staffing scientist arrangements and financial support from QIBEBT. The Authorized Departmental Officer's Designated Representative monitored the activities of this agreement via frequent e-mail contacts and teleconferences.