Fatty acid elongation in methylotrophic yeast Hansenula polymorpha CBS 1976 and fatty acid auxotrophic mutants

Abstract

Fatty acid auxotrophic mutants (V1 to V10) of Hansenula polymorpha were isolated from the wild-type strain (leu1-1) of H. polymorpha CBS 1976 by mutagenization with ethyl methanesulfonate (EMS). All the mutants were screened as colonies which were unable to proliferate on YEPD after replication from initial YEPD containing a mixture of 1mM each of C14:0 C16:0 and C18:0 where the mutagenized cells were plated. In order to classify the mutants, their growth on media supplemented with various fatty acids of 1mM and/or 2mM C14:0 C16:0 and C18:0 were examined. Three groups of mutants possessing different phenotype were identified. The first group including six mutants (V1 to V6) did not grow on solid media supplemented with 1mM of C16:0, however, 2mM of C16:0 and 1-2mM of C18:0 could support the growth of this group. Ability of the first group which were unable to grow in 1mM C16:0 suggesting that this group of the mutants might have partially defect at the step of elongation of C16 to C18. The second group included only one mutant (V7) whose no growth when grown on media supplemented with 1mM of C14:0 and when cultivated on media supplemented with a mixture of C14:0 C16:0 and C18:0 noted that C14:0 was also increased when compared with those of the parental strain in the same media. Taken together, from these results suggesting that this mutant probably have the defect at the elongation of C14:0 to C16:0. Mutants in the third group (V8 V9 and V10) did not grow on medium cultivated with 1mM of C16:0 and 1mM of C18:0 supplementation but demonstrated their ability to proliferate on media with addition of C14:0 suggesting that this mutant might have the defect at the elongation before C14. From crosses of all the three groups of the mutants with an auxotrophic strain (ura3-1), all diploid hybrids were selected on minimal medium containing a mixture of C14:0, C16:0 and C18:0 fatty acids without uracil and MIN with uracil. The segregants of all the three groups were chosen as the recipient strain for complementation test. The diploid yeast cells were transformed with a plasmid YCpELO1.MOD harboring the coding sequences of S-ELO1 and containing the URA3 gene as a selective marker, in addition to the YCpGALELO2(U) harboring the coding sequences of S-ELO2 which containing the URA3 gene and galactose-inducible GAL promoter as a selective marker. The transformation of the yeast cells were performed using the lithium acetate method and then collected on fatty acid supplemented MIN medium. Transformation efficiency was determined by plating aliquots of transformants on the medium. Among them, only the ELO2 transformants could grow on the medium containing galactose without fatty acid supplementation. Fifteen of ~150 transformant cells designed as VT1 (URA3, FAE1) carring ELO2 were randomly chosen to further analysed the growth characteristic of VT1 transformant on solid and liquid medium containing various fatty acids related to wild-type strain. The results revealed the similar growth phenotype as found in solid and liquid medium by the growth of VT1 could reach closely to that of wild-type strain under any conditions. Moreover, comparison of chromatograms of fatty acid methyl esters (FEMEs) from the wild-type strain and VT1 mutant grown in YEPD media and YEPD supplemented with various fatty acids at 37 ํC for 24 h found that their fatty acids were similar in content and profile. Taken together, these results demonstrated that the ELO2 gene of S. cerevisiae could function in H. polymorpha V1, indicating a clear picture of the lesion in fatty acid biosynthesis in V1 mutant that the partial defect at least in the elongation step of C16 to C18. The information obtain from these studies could be exploited to create defective fatty acid elongase strains to be learnt about the mechanism of action of this yeast