Genetic study of fatty acid and lipid biosynthesis in Hansenula polymorpha

Two groups of Hansenula polymorpha mutants were screened by their fatty acid requirement, one requires saturated fatty acids for growth (sfa- mutamts) and the other requires unsaturated fatty acids (mfa- and pfa- mutants). Two of the sfa- mutants, S7 and S16, showed significantly difference in the fatty acid composition. S7 clearly defected in the production of C18:2∆9,12 and C18:3∆9,12,15, while S16 significantly accumulated medium-chain saturated fatty acids, C12:0 and C14:0. By tetrad analysis, the results showed that S7 had double mutation which composed of fatty acid synthesis mutation (Hpsfa7) and ∆12-desaturase mutation (Hpdes12). Two meiotic segregants, H69-2C and H69-2D harboring Hpdes12 gene, showed Sfa+ phenotype and grew on YPD without fatty acid supplementation indicating that the absence of C18 polyunsaturated fatty acids did not affect cell growth of the mutant. H69-2A and H69-2B segregants (Hpsfa7) showed Sfa- phenotype and required saturated fatty acids for growth. However, only the segregant H69-2B display similar properties to parental mutant (S7) on the growth on media supplemented with various fatty acids, while H69-2A was different. The comparison of the cloned ∆12-desaturase gene (HpDES12) of H. polymorpha SH4330 and S7 confirmed that the defect of C18:2∆9,12 และ C18:3∆9,12,15 synthesis was resulted from the mutation at ∆12-desaturase gene. Besides, the cloning gene also provided the characteristic of ∆12-desaturase of the yeast H. polymorpha. An open reading frame of the full length cDNA of ∆12-desaturase gene of H. polymorpha showed 1215 bp encoding for 404 amino acid residues (GenBank Accession No. GU226432). The deduce amino acids had the highest similarity to the ∆12-desaturase of Pichia pastoris that corresponds to the result of phylogenetic analysis. The cloned gene and mutants obtained in this study can be used as tools for studying association between fatty acid metabolism and cell physiology of eukaryotes.