Plate ethanol-screening assay for selection of the Pichia stipitis and Hansenula polymorpha yeast mutants with altered capability for xylose alcoholic fermentation

Abstract

A new method for the selection of Pichia stipitis and Hansenula polymorpha yeast mutants with altered capability to ferment xylose to ethanol was developed. The method is based on the ability of P. stipitis and H. polymorpha colonies to grow and produce ethanol on agar plates with xylose as the sole carbon and energy source. Secreted ethanol, in contrast to xylose, supports growth of cells of the indicator xylose-negative strains (the wild-type strain of Saccharomyces cerevisiae or Deltaxyl1 mutant of H. polymorpha) mixed with agar medium. The size of the tester culture-growth zone around xylose-grown colonies appeared to be dependent on the amount of secreted ethanol. Mutants with altered (decreased or elevated) ethanol production in xylose medium have been isolated using this method. The mutants exhibited pleiotropic alterations in enzymatic activities of the intermediary xylose metabolism.

Development of a Genome-Wide Random Mutagenesis System Using Proofreading-Deficient DNA Polymerase δ in the Methylotrophic Yeast Hansenula polymorpha

“…This result suggests that to obtain a mutant H. polymorpha stain with enhanced ability to ferment xylose, an additional round of mutagenesis should be performed in combination with a proper screening strategy. For example, it may be possible to select such a mutant on a xylose medium based on the ability to support the growth of an indicator strain that can grow on ethanol, as previously reported [11]. It will also be possible to construct a reporter strain using the recently identified ethanol-inducible promoter of the HpADH3 gene of H. polymorpha [39].…”

Remaining Challenges in the Metabolic Engineering of Yeasts for Biofuels

“…Significant organisms include Neurospora crassa, 164 Hansenula polymorpha, 165,166 Pachysolen tannophilus, 167 Candida arabinofermentans, 168 and Pichia guillermondii. 168 A recent report describes the progress of and interest in the yeast Spathaspora passalidarum as a unique host for fuels production.…”

Selection of Xilose-Fermenting Yeast Strains

“…The plate test for ethanol production was carried out according Grabek-Lejko et al (2006). In this technique, pentose-fermenting yeasts are placed to grow in a solid culture medium containing Dxylose as the sole carbon source and the presence of ethanol is detected by growth of a reference yeast strain that does not grow on xylose but grows ethanol.…”