Cloning and expression in phospholipid containing cultures of the gene encoding the specific phosphatidylglycerol/phosphatidylinositol transfer protein from Aspergillus oryzae: evidence that the pg/pi-tp is tandemly arranged with the putative 3-ketoacyl-CoA thiolase gene

Abstract

The phosphatidylglycerol/phosphatidylinositol transfer protein (PG/PI-TP) is a new and original phospholipid transfer protein (PLTP) isolated from the Deuteromycete, Aspergillus oryzae. We have isolated a genomic clone of the A. oryzaepg/pi-tp using a probe derived from the corresponding cDNA and sequenced the complete gene. The DNA sequence analysis revealed that pg/pi-tp gene is composed of three exons encoding a 18,823 Da protein of 175 amino acids as previously described and of two introns as deduced by cDNA and genomic sequence alignment. The isolated pg/pi-tp gene do not show similarity with other PLTP genes or the deduced PG/PI-TP protein with proteins already known. Comparison of the encoded PG/PI-TP with other deduced proteins from recent genomic or cDNA sequence from databases revealed that the PG/PI-TP was close to two encoded proteins deduced from the cDNA database of Aspergillus nidulans (54% identity and 68% similarity) and the second from Neurospora crassa (53% identity and 76% similarity). Therefore, we suggested that both proteins might belong to the PLTP family. Southern blot analysis of A. oryzae genomic DNA show that the PG/PI-TP was encoded by a single gene. Expression of pg/pi-tp was performed in phospholipid containing cultures with increasing carbon source concentrations in order to study the regulation of the PLTPs in the filamentous fungus cell. This was done to know if a high density culture could yield a high amount of biomass with high phospholipid transfer activity. Results showed that phospholipids as compared to glucose in standard cultures stimulated mycelial growth and global phospholipid transfer activity, but not the pg/pi-tp transcript accumulation. However, high concentration of both carbon sources yielded an inhibition of the expression of the pg/pi-tp gene and of the global phospholipid transfer activity. In conclusion, both carbon sources are not suitable to increase the PLTP production in high density cultures for biotechnological applications. Finally, using the gene walking sequencing method it is demonstrated that the pg/pi-tp is tandemly arranged on opposite DNA strands in a tail-to-tail orientation with a putative gene encoding the 3-ketoacyl-CoA thiolase (EC 2.3.1.16). Unlike the pg/pi-tp gene, this thiolase gene show a putative ‘β-oxidation box’ and encodes a putative 44,150 Da protein of 321 amino acids composed of a putative N-terminal PTS2 (Peroxisomal Targeting Signal) consensus sequence for the peroxisome targeting. Comparison of the amino acid sequence of the A. oryzae thiolase to that of the Yarrowia lipolytica showed a 50% identity and a 69% similarity.