Molecular Cloning of XYL3 ( d -Xylulokinase) from Pichia stipitis and Characterization of Its Physiological Function

Abstract

XYL3, which encodes a D-xylulokinase (EC 2.7.1.17), was isolated from Pichia stipitis CBS 6054 genomic DNA by using primers designed against conserved motifs. Disruption of XYL3 eliminated D-xylulokinase activity, but D-ribulokinase activity was still present. Southern analysis of P. stipitis genomic DNA with XYL3 as a probe confirmed the disruption and did not reveal additional related genes. Disruption of XYL3 stopped ethanol production from xylose, but the resulting mutant still assimilated xylose slowly and formed xylitol and arabinitol. These results indicate that XYL3 is critical for ethanol production from xylose but that P. stipitis has another pathway for xylose assimilation. Expression of XYL3 using its P. stipitis promoter increased Saccharomyces cerevisiae D-xylulose consumption threefold and enabled the transformants to produce ethanol from a mixture of xylose and xylulose, whereas the parental strain only accumulated xylitol. In vitro, D-xylulokinase activity in recombinant S. cerevisiae was sixfold higher with a multicopy than with a single-copy XYL3 plasmid, but ethanol production decreased with increased copy number. These results confirmed the function of XYL3 in S. cerevisiae.

Activating and Elucidating Metabolism of Complex Sugars in Yarrowia lipolytica

“…The alignment results were also edited using the Jalview 2.8 tool (49) for enhanced visual presentation. To find the metabolic enzymes responsible for the xylose and cellobiose degradation, we used the XYL1, XYL2, and XYL3 proteins of P. stipitis (50,51) as well as BGLs of A. niger (BGL1; Q30BH9) (52), A. fumigatus (BTGE; B0Y9Q9) (53), and Candida wickerhamii (BGLA; Q12602) (54) as the templates. We further applied the PSORTII tool (55) to predict the cellular localization of the BLASTPsearched putative BGL genes of Y. lipolytica.…”

Bioethanol a Microbial Biofuel Metabolite; New Insights of Yeasts Metabolic Engineering

“…It was reported that XYL1 overexpressing S. cerevisiae mutant, produced xylitol in sequential or continuous batches [189]. Later, Jan and his collages (2003) reported that proportional increasing XYL2 expression to XYL1, potentially decreased xylitol levels [172]. They thought that the xylitol production by engineered S. cerevisiae strains resulted from an excess of NADPH relatively to NADH for the early assimilation step of xylose [80].…”

“…Later, XK from P. stipites (PsXK) has been overexpressed in an engineered S. cerevisiae background for high expression levels of XYL1 and XYL2 [171,172]. The specificity of P. stipites XK enzyme for D-xylulose was much higher, with less D-ribulose activity than the XK from S. cerevisiae [168].…”

“…In native xylose-fermenting yeast P. stipitis, the primary ADH is heavily expressed with decreasing the availability of O 2 [116,188]. Moreover, P. stipitis produces xylitol and arabitol, when the xylulokinase is deleted [172].…”

“…In C. shehatae, a proton symport seems to mediate the L-arabinose uptake [201]. Besides, D-arabinitol dehydrogenase, which has a role in another pathway, links D-xylulose to D-ribulose metabolism [172,202].…”