We cloned and characterized a gene encoding isocitrate lyase from the methylotrophic yeast Pichia pastoris. This gene was isolated from a P. pastoris genomic library using a homologous PCR hybridization probe, amplified with two sets of degenerate primers designed from conserved regions in yeast isocitrate lyases. The cloned gene was sequenced and consists of an open reading frame of 1563 bp encoding a protein of 551 amino acids. The molecular mass of the protein is calculated to be 60.6 kDa with high sequence similarity to isocitrate lyase from other organisms. There is a 64% identity between amino acid sequences of P. pastoris Icl and Saccharomyces cerevisiae Icl. Northern blot analyses showed that, as in S. cerevisiae, the steady-state ICL1 mRNA levels depend on the carbon source used for cell growth. Expression in P. pastoris of the dextranase gene (dexA) from Penicillium minioluteum under control of the ICL1 promoter proved that P ICL1 is a good alternative for the expression of heterologous proteins in this methylotrophic yeast. The sequence presented here has been deposited in the EMBL data library under Accession No. AJ272040.

A method to obtain K. marxianus mutants has been developed. Different auxotrophic mutants were isolated by nystatin and snail-enzyme enrichment procedures using an incubation time of 2 h before adding the antibiotic or the enzyme respectively. All his mutants analyzed by complementation tests turned out to belong to the same complementation group. Some of them were transformed and complemented by the S. cerevisiae HIS3 gene. These non-reverting his3 mutants contain no heterologous sequence, which is essential to make them acceptable for application in the food industry.