Strain selection and metabolic engineering of Pichia stipitis for increased ethanol production from xylose

In Pichia stipitis strain TJ26, transformation with a XYL1-containing plasmid increased the xylose reductase activity within the organism as compared to a control transformant when both strains were cultivated in xylose-containing medium under high aeration conditions. This same TJ26 transformant showed increased xylose reductase activity as compared to a control transformant activity when grown on glucose-containing medium. Overexpression of XYL1 did not improve ethanol production by TJ26. Rather, ethanol production was lowered.; Overexpression of XYL1 was studied in another P. stipitis strain, PSU1. A transformant harboring a XYL1 -containing plasmid was compared to a control transformant in medium containing peptone (and other chemicals) and in medium lacking peptone. In the medium including peptone, the engineered strain showed increases in ethanol production when compared to the control strain.; P. stipitis strains with extra copies of both XYL1 and XYL2 were constructed. The extra copies of these genes were born on an autonomous plasmid or on a plasmid which integrated into the chromosomal deoxyribonucleic acid at the XYL2 locus. A medium including peptone and other chemicals allowed for increases in the ethanol production by the engineered strain harboring the autonomous plasmid when compared to the control transformant, while results with medium lacking peptone were inconsistent (ethanol production by the engineered strain was sometimes lowered when compared to the control transformant). The integrative transformant produced statistically the same amount or lower amounts of ethanol as the host strain in medium containing yeast extract, peptone and xylose or when this medium was used in inoculum preparation. However, the integrative transformant produced more ethanol than wild type P. stipitis in medium containing yeast nitrogen base, urea, peptone, and xylose when cells from frozen stocks were used.; XYL2 complemented a mutation in a P. stipitis strain with slowed growth on both L-arabinose and D-xylose. Transformation of this strain with a XYL2-containing plasmid allowed for increased growth on both pentoses, and it also increased xylitol dehydrogenase activity. Wild type P. stipitis grown on L-arabinose had both L-arabinose reductase and D-xylose reductase activities.