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Category: Applied and Industrial Microbiology
Engineering the Pichia stipitis Genome for Fermentation of Hemicellulose Hydrolysates, Page 1 of 2
< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555815547/9781555819057_Chap03-1.gif /docserver/preview/fulltext/10.1128/9781555815547/9781555819057_Chap03-2.gifAbstract:
Pichia stipitis is a source of genes for engineering xylose metabolism in Saccharomyces cerevisiaea task undertaken in numerous laboratories around the world. Increasing the capacity of P. stipitis for rapid xylose fermentation can greatly improve its usefulness in commercial xylose fermentations. Genetic transformation with URA3 is more efficient than transformation with the modified Sh ble marker. Cultivation conditions can strongly affect expression of fermentative enzymes in P. stipitis. Unlike S. cerevisiae, which regulates fermentation by sensing the presence of glucose, P. stipitis induces fermentative activity in response to oxygen limitation. The coupling of Xyl1 and Xyl2 activities therefore tends to result in the consumption of NADPH and accumulation of NADH. Preliminary data based on expressed sequence tags indicate that transcripts for fatty acid synthase (FAS2) stearoyl-coenzyme A desaturase (OLE1) are induced under oxygen-limiting conditions. Xylanase production by P. stipitis has been recognized, and the organism has also been transformed with heterologous xylanases to increase xylanase activity. A genetic system has been developed that includes the auxotrophic markers URA3 and LEU3 along with modified forms of the phleomycin D1 resistance marker, Sh ble, and the Cre recombinase. Many genes in P. stipitis are found in functionally related clusters. Further metabolic engineering and strain selection are needed to increase the overall fermentation rate and ethanol tolerance of P. stipitis for the commercial bioconversion of hemicellulose hydrolysates.
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Mutants developed from P. stipitis CBS 6054.
Relative expression of transcripts for the glutamate decarboxylase bypass. Abbreviations: IDH1 and IDH2, isocitrate dehydrogenase 1 and 2; GDH2, NAD-specific glutamate dehydrogenase; GDH3, NADP-specific glutamate dehydrogenase;GAD2, glutamate decarboxylase 2; UGA1.1 and UGA1.2, 4-aminobutyrate aminotransferase; UGA2 and UGA22, succinate semialdehyde dehydrogenase; KGD2, 2-ketoglutarate dehydrogenase; Isoct, isocitrate; AKG, 2-keto-glutarate; LGlu, L-glutamate; 4-AB, 4-aminobutyrate; Suc-SA, succinate semialdehyde; Succ, succinate; Fum, fumarate; GA, glucose aerobic; XA, xylose aerobic; GOL, glucose oxygen limited; XOL, xylose oxygen limited.
Induction of transcripts for lipid synthesis under oxygen-limiting conditions. Abbreviations: GA, glucose aerobic; XA, xylose aerobic; GOL, glucose oxygen limited; XOL, xylose oxygen limited.
Gene clusters of β-glucosidases and endoglucanases with sugar transporters in the P. stipitis genome. Approximate chromosome coordinates are shown. All constructs were derived from the original sequence deposit at the Joint Genome Institute website (http://genome.jgi-psf.org/Picst3/Picst3.home.html). The complete genome is also found in GenBank.
Phylogenetic relationships among hexose transporters and beta-glucosidases found in clusters in P. stipitis. Gene names correspond to designations given on the Joint Genome Institute website (see legend to Fig. 4 for URL) and in GenBank. The method used was Neighbor Joining with Best Tree; distances were uncorrected, and gaps were distributed proportionally.
Gene clusters of α-glucosidases with putative maltose permeases in the P. stipitis genome. Approximate chromo-some coordinates are shown. All constructs were derived from the original sequence deposit at the Joint Genome Institute website (see legend to Fig. 4 for URL). The complete genome is also found in GenBank.
Phylogenetic relationships of putative sugar transporters from P. stipitis. Except as noted, all genes are from P. stipitis. Gene names correspond to designations given on the Joint Genome Institute website and in GenBank. Sequences for CiGXF1 and CiGXS1 are from C. intermedia; sequences for Xylhp and Xylhp homolog are from D. hansenii. The method used was Neighbor Joining with Best Tree; distances were uncorrected, and gaps were distributed proportionally.
Phylogenetic relationships of α-glucosidases from P. stipitis. Gene names correspond to designations given on the Joint Genome Institute website and in GenBank. The method used was Neighbor Joining with Best Tree; distances were uncorrected, and gaps were distributed proportionally.
Galactose and lactose gene clusters in P. stipitis chromosome 3.
Fermentation characteristics of three strains of P. stipitis grown in shake flasks