Characterization and disruption of Saccharopine reductase gene from swansonine producing fungus Undifilum oxytropis

Locoweed plants found in southwestern United States often harbor an Ascomycete-fungus Undifilum oxytropis, a slow growing endophyte that produces a toxic alkaloid, swainsonine. Consumption of U. oxytropis by grazing animals induces a neurological disorder called locoism. Swainsonine is a alpha-mannosidase inhibitor with potential anti-tumorigenic ability. Little is known about the biosynthetic pathway of swainsonine in endophytic fungi, but previous studies on taxonomically related fungi indicate that pipecolic acid and saccharopine are key swainsonine-forming intermediates, which may be regulated by the enzyme, saccharopine reductase. The aims of this study were to identify the saccharopine reductase gene from U. oxytropis, develop a transformation system for the fungus, and study saccharopine reductase function using gene disruption. To identify saccharopine reductase, degenerate primers were designed from Magnaporthe grisea and Penicillum chrysogenum, which identified a one kb segment of the gene. RACE-PCR and Inverse PCR techniques were used to decipher the 3' UTR and 5' termini of saccharopine reductase gene. Taken together, 1903 bp of the saccharopine reductase gene was deciphered, which included the 1494 bp open reading frame (ORF), the start codon (ATG), the stop codon (TAA) and the poly A+ tail. The saccharopine reductase sequence of U. oxytropis is 92% homologous to that of M. grisea. Using an Ascomycete-fungus specific vector, Ppd-EGFP a transformation system for U. oxytropis was established. Briefly, U. oxytropis protoplasts were transformed with Ppd-EGFP, which drives the expression of Enhanced Green Fluorescent Protein (EGFP). The quality of transformed protoplasts and transformation efficiency were monitored during the process. Ppd-EGFP confers resistance to hygromycin 13 and throughout the monitoring process resistance to antibiotic hygromycin B was maintained. Using the deciphered sequence and the established transformation system, disruption of saccharopine reductase was performed using homologous recombination. Disruption of saccharopine reductase in U. oxytropis led to decreased levels of saccharopine and lysine. Accumulation of 1-piperideine, 6-carboxylic acid (P6C), pipecolic acid and swainsonine occurred as a result of this disruption, as measured using LC-mass spectrometry. Thus, saccharopine reductase was shown to be involved in the degradation of swainsonine and its precursor pipecolic acid in U. oxytropis.