Identification Of Endophytical Fungus CH1307 From Cephalotaxus Mannii And Fermentation Condition For Homoharringtonine

The endophytic fungi from medicinal plants have been the focus in finding various biologically active substances. Cephalotaxus mannii Hook.f. was paid much attention because its roots, stems, leaves and fruits all have harringtonine compounds with anti-cancer activity. However Cephalotaxus mannii is extremely rare due to over-exploitation and growth condition deterioration in the past decades. So it is very important to find a new way to produce harringtonine.In order to find an alternative for extraction from Cephalotaxus mannii, an endophytic fungus, strain CH1307, isolated from Cephalotaxus mannii was identified and the fermentation condition was studied.At initial stage of cultivation, the colony in PDA medium was round with diameter 5-10mm, white in surface and yellow with regular edge on the back. The surface colony was villiform, with aerial hyphae about 1-2mm high. Later the colony color grew dark and brown, with the diameter 30-50mm. In PDB medium, Strain CH1307 growth amount was large. At initial stage of cultivation, white and even mycelia sphere was formed, and 5-7 days later pigments existed. Strain CH1307 could not produce any spore although many spore induction experiments were made in various different conditions. Under the microscope, the strain had slight and plexiform hyphae, with the mycelium transparent, the surface smooth. And it had a septum and round particles in hyphae, which was unique. Biochemical identification by Biolog system indicated that the strain had a SIM of 0.172 with Scopulariopsis brumptii Salvanet-Duval. The 830bp of the 18S rDNA partial sequences was obtained by molecular biology methods and the homology comparison indicated that the 18S rDNA sequence of the strain had a homology of only 97% with Pleosporales sp., Paraconiothyrium sp., and Coniothyrium sp.etc. ITS sequence comparison showed that CH1307 had only 84% with an uncultivatable soil fungus. Strain CH1307 might be a new species or genera in Pleosporales.Fermentation process study showed that homoharringtonine could be detected in the 5th day. Broth feeding in the 4th and 7th day was good for growth and also for harringtonine production. Precursor addition in the stationary phase, such as tyrosine and phenylalanine, helped to homoharringtonine production. After the single factor test such as different carbon source, carbon source concentration, inoculum, broth loading volume, a response surface methodology of rotation speed, beef extract, peptone, ammonium nitrate, tyrosine and phenylalanine was made to optimize the fermentation conditions. Regression equation was established.The optimal fermentation conditions were obtained as follows:25g/L of sucrose, 1g/L of beef extract,1.134g/L of peptone, and 1.5g/L of ammonium nitrate were added to PDB. Fermentation was at 200r/min,25℃for 15days. During the fermentation 5% of the feeding I was added on the 4th d, and 5% of the feedingⅡand 3mg/L of tyrosine and 2.79mg/L of phenylalanine was added on the 7th d. The homoharringtonine yield of the strain CH1307 increased from 122.1933μg/L to 214.0037μg/L.