Study Of Saccharopine Reductase Gene Influence For Swainsonine Biosynthesis In Embellisia Fungi From Oxytropis Glabra

The swainsonine is produced in Embellisia fungus from Oxytropis glabra, and it is a toxic alkaloid to livestock accumulated to some extent in cells. Being a secondary metabolite, its synthesis pathway is really complicated. Presently, the pathway in Embellisia fungal endophyte in Oxytropis is still unknown.In this study, the wild type strain of Embellisia fungus in Oxytropis glabra was chosen as materrial as well as the mutant, and expression of saccharopine reductase gene was detected by reverse transcription PCR in this fungus. The swainsonine content in the mycelium was determined by enzyme analysis method, and the content of saccharopine and lysine in the mycelium was determined by LC-MS. The content changes of swainsonine and related compounds in the wild type strain as well as in the mutant were detected after supplement of L-?-aminoadipic acid, L-lysine, and L-pipecolic acid in media. The cDNA sequence of saccharopine reductase in the Embellisia fungus in Oxytropis was analyzed with bioinformatics software, and the sequence of amino acids of the enzyme was deduced. The constituent, structure and other charactors were investigate, and the hydrophilis, transmembrane region, signal peptide and the second and tertiary structures for this enzyme were predicted. The sequences of saccharopine reductase from eleven species of fungi were compared, and a phylogenetic tree was constructed. The main results were shown as follows:1. The expression of saccharopine reductase gene was detected by reverse transcription PCR in wild type strain, but no expression in the mutant. However, the expression of hygromycin phosphotransferase gene (hph) was detected in the mutant.2. The content of swainsonine in endopytes was determined by ?-mannosidase enzyme analysis method. The swainsonine content in the wild type strain was more than that in the mutant grown on PDA media. The swainsonine production was improved in both wild type strain and the mutant after supplement of L-lysine, L-a-aminoadipic acid and L-pipecolic acid. The increase of swainsonine production changed along with different contents of compounds.3. The content of swainsonine biosynthsis precursor-saccharopine and lysine was determined by LC-MS, and the content of saccharopine in the mutant was lower that of it in the wild type. However, the content of lysine lessly changed in the endophytes. The content of saccharopine in the wild type strain decreased with increase of addition content of a-aminoadipic acid. The content of lysine less changed in the wild type strain. The content of lysine and saccharopine was the most when the addition content of a-aminoadipic acid was reached to 1 ×10=4 mol/L, they were reached to 0.171?g/g and 0.563?g/g.4. The saccharopine reductase contained 472aa duduced from the cDNA sequence of the fungus including 61 acidity amino acids,57 alkalinity amino acids,111 polarity amino acids and 167 unpolarity amino acids. The deduced saccharopine reductase was a soluble protein without signal peptide and transmembrane domain, and its secondary structure was mainly based with random coils. In part regions, the secondary structure of the enzyme was disordering. Saccharopine reductase, and this enzymes amino acid sequence has compared with the other 10 species of saccharopine reductase in fungal, and build a phylogenetic tree. The sequences of saccharopine reductase from eleven species of fungi were compared, and the phylogenetic tree was constructed.