Identification of phosphatidylserine decarboxylases 1 and 2 from Pichia pastoris.
Genetic manipulation of lipid biosynthetic enzymes allows modification of cellular membranes. We made use of this strategy and constructed mutants in phospholipid metabolism of Pichia pastoris, which is widely used in biotechnology for expression of heterologous proteins. Here we describe identification of two P. pastoris phosphatidylserine decarboxylases (PSDs) encoded by genes homologous to PSD1 and PSD2 from Saccharomyces cerevisiae. Using P. pastoris psd1Delta and psd2Delta mutants we investigated the contribution of the respective gene products to phosphatidylethanolamine synthesis, membrane composition and cell growth. Deletion of PSD1 caused loss of PSD activity in mitochondria, a severe growth defect on minimal media and depletion of cellular and mitochondrial phosphatidylethanolamine levels. This defect could not be compensated by Psd2p, but by supplementation with ethanolamine, which is the substrate for the cytidine diphosphate (CDP)-ethanolamine pathway, the third route of phosphatidylethanolamine synthesis in yeast. Fatty acid analysis showed selectivity of both Psd1p and Psd2p in vivo for the synthesis of unsaturated phosphatidylethanolamine species. Phosphatidylethanolamine species containing palmitic acid (16:0), however, were preferentially assembled into mitochondria. In summary, this study provides first insight into membrane manipulation of P. pastoris, which may serve as a useful method to modify cell biological properties of this microorganism for biotechnological purposes.
Wriessnegger
Tamara
T
Institute of Biochemistry, Graz University of Technology, Graz, Austria.
Sunga
Anthony Jay
AJ
Cregg
James M
JM
Daum
Guenther
G
D000595:Amino Acid Sequence; D002262:Carboxy-Lyases / Q000235:genetics* / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry; D005227:Fatty Acids / Q000032:analysis; D017353:Gene Deletion; D053858:Metabolic Networks and Pathways / Q000235:genetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D010714:Phosphatidylethanolamines / Q000378:metabolism*; D010802:Phylogeny; D010843:Pichia / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D005227:Fatty Acids; D010714:Phosphatidylethanolamines; C483858:phosphatidylethanolamine; D002262:Carboxy-Lyases; C022353:phosphatidylserine decarboxylase