BackgroundAs a strong fermentator, Saccharomyces cerevisiae has the potential to be an excellent host for ethanol production by consolidated bioprocessing. For this purpose, it is necessary to transform cellulose genes into the yeast genome because it contains no cellulose genes. However, heterologous protein expression in S. cerevisiae often suffers from hyper-glycosylation and/or poor secretion. Thus, there is a need to genetically engineer the yeast to reduce its glycosylation strength and to increase its secretion ability.ResultsSaccharomyces cerevisiae gene-knockout strains were screened for improved extracellular activity of a recombinant exocellulase (PCX) from the cellulose digesting fungus Phanerochaete chrysosporium. Knockout mutants of 47 glycosylation-related genes and 10 protein-trafficking-related genes were transformed with a PCX expression construct and screened for extracellular cellulase activity. Twelve of the screened mutants were found to have a more than 2-fold increase in extracellular PCX activity in comparison with the wild type. The extracellular PCX activities in the glycosylation-related mnn10 and pmt5 null mutants were, respectively, 6 and 4 times higher than that of the wild type; and the extracellular PCX activities in 9 protein-trafficking-related mutants, especially in the chc1, clc1 and vps21 null mutants, were at least 1.5 times higher than the parental strains. Site-directed mutagenesis studies further revealed that the degree of N-glycosylation also plays an important role in heterologous cellulase activity in S. cerevisiae.ConclusionsSystematic screening of knockout mutants of glycosylation- and protein trafficking-associated genes in S. cerevisiae revealed that: (1) blocking Golgi-to-endosome transport may force S. cerevisiae to export cellulases; and (2) both over- and under-glycosylation may alter the enzyme activity of cellulases. This systematic gene-knockout screening approach may serve as a convenient means for increasing the extracellular activities of recombinant proteins expressed in S. cerevisiae.

To begin characterizing DNA repair capability among Xiphophorus species, we adapted oligonucleotide-based DNA repair assays to extracts of fish tissues. Here, we report the initial results of relative base excision repair (BER) capability among 3 inbred Xiphophorus fish lines representing 2 species (X. maculatus and X. couchianus), and interspecies hybrid F(1) animals produced by crossing them. Overall, data from uracil- N-glycosylase (UNG)-initiated BER assay (UNG-BER) indicate that brain tissue extracts generally possess higher BER activity than do gill and liver extracts. UNG-BER activities in gill and liver extracts were similar. The BER activity in the tissues of F(1) interspecies hybrids followed the activity pattern of the X. couchianus parent in gill and liver extracts, was similar to the X. maculatus parent in brain extracts, but exhibited a reduced repair capacity in gill tissue extracts compared with either parent. We discuss the use of oligonucleotide-based DNA repair assays to elucidate the role that inheritance of DNA repair potential may play in susceptibility to disease and tumorigenesis in the intact organism.

ABSTRACT. Primers for eight microsatellites were developed; they successfully amplified DNA from 20 domesticated Formosan Sambar deer (Cervus unicolor swinhoei). All loci were polymorphic, with 10-19 alleles per locus. The average observed heterozygosity across loci and samples was 0.310, ranging from 0 to 0.750 at each locus. All loci but one, CU18, deviated from Hardy-Weinberg equilibrium due to excessive homozygosity in these domesticated broodstocks, reflecting inbreeding. These microsatellite loci will be useful, not only for assessment of population structure and genetic variability, but also for conservation of wild deer populations in Taiwan.

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