The native xylose fermenting yeast Pichia stipitis has the capacity to ferment hemicellulosic sugars.  For this organism to produce ethanol on an industrial scale, improvements must be made in the fermentative rate and overall ethanol production.  To facilitate these changes in metabolic function, a strategy was developed to manipulate wild-type, non-auxotrophic strains.  Two drug resistance genes known to function in S. cerevisiae (Nat1 and hph) were synthetically constructed to facilitate expression in both P. stipitis and S. cerevisiae.  P. stipitis uses the yeast alternative codon, so the use of synthetic construction was necessary to avoid the use of the CUG codon which codes for leucine in most organisms, but codes for serine in P. stipitis.  The Nat1 and hph genes were both synthesized using DNA2.0.  Following synthesis, promoters and terminators were added to both genes, and functionality of the synthetic genes was evaluated in both P. stipitis and S. cerevisiae.  Both of the functional drug markers were added to P. stipitis expression vectors, into which genes of interest can be cloned.  Single genes have been transformed into non-auxotrophic strains of P. stipitis, followed by excision of the drug marker using the flanking LoxP sites and cre recombinase.  Removal of the drug marker allowed for the addition of additional genes using the same drug marker.  This strategy has allowed us to make significant gains in both fermentative capacity and ethanol production.