Construction Of Strains Producing Lipid With Cellulose By Protoplast Fusion

With the population growing and the acceleration of industrialization, the contradiction isgraveness between increase of lipid demand and shortage of natural source. The energy crisisand the resulting environmental pollution has become important problem that continued toplague human health, survival and development. Microbial lipid becomes a new type of lipidresources which has broad application prospects. Cellulose is aplenty reclaiming biopolymerson the earth. But80%of cellulose was not utilized or utilized un-reasonable. Selecting strainswhich can product lipid by cellulose, not only would release shortage of animals and plantslipid, but also would change waste into treasure and make for environment protection. Thathas important practice significance and utilization worth.Macrophomina phaseolina MOD-1, an endophytic oleaginous fungus isolated from thehealthy mulberry root, which has powerful lipid production ability and poor cellulosedegradation. The Trichoderma viride TP4and T44, Trichoderma longibrachiatum TL arethree strains with powerful ability of cellulose degradation which not produce lipid. In thisstudy, protoplasts preparation conditions of MOD-1, T44, TP4and TL were optimized. AndMOD-1is fused with three trichoderma separately to make fusion strains obtain the goodtraits of the parents, achieves the purpose of lipid production efficiently using cellulose. Themain contents were showed as follows.1. Optimization of enzymes and conditions for parents protoplasts preparation.With single factor, orthogonal test and central composite design, the enzymes andconditions of MOD-1protoplasts preparation were optimized. The best conditions of MOD-1protoplasts preparation were obtained as follow: Using0.66mol/L KCl as an osmoticstabilizer,44.58h cultured mycelium enzymolysised for4h at a concentration of20mg/mLlysozyrne+20mg/mL cellulase+5mg/mL snail cellulase enzyme mixture of enzyme solution,hydrolysis temperature of30C， regeneration medium for cultivation． Protoplastsconcentration reached7.02x10~6piece/mL，regeneration rate reached4.90%.With single factor, orthogonal test, the enzymes and conditions of T44, TP4and TLprotoplasts preparation were optimized. The best conditions of T44protoplasts preparationwere obtained as follow: Using0.7mol/L KCl as an osmotic stabilizer,36h culturedmycelium enzymolysised for3h at a concentration of15mg/mL lysozyrne+10mg/mL cellulase+25mg/mL snail cellulase enzyme mixture of enzyme solution, hydrolysistemperature of30C，regeneration medium for cultivation．Protoplast concentration reached6.35x10~6piece/mL，regeneration rate reached4.51%. TP4protoplasts preparation conditionswere：Using0.7mol/L KCl as an osmotic stabilizer,48h cultured mycelium enzymolysisedfor3h at a concentration of15mg/mL lysozyrne+15mg/mL cellulase+20mg/mL snailcellulase enzyme mixture of enzyme solution, hydrolysis temperature of30C，regenerationmedium for cultivation．Protoplasts concentration reached4.43x10~6piece/mL，regenerationrate rechaed3.26%. TL protoplasts preparation conditions were：Using0.7mol/L KCl as anosmotic stabilizer,36h cultured mycelium enzymolysised for4h at a concentration of20mg/mL lysozyrne+15mg/mL cellulase+15mg/mL snail cellulase enzyme mixture of enzymesolution, hydrolysis temperature of30C，regeneration medium for cultivation．Protoplastconcentration reached4.36x10~6piece/mL，regeneration rate reached4.12%.2. Optimization of the inactivation conditions of parents protoplasts.MOD-1, T44, TP4and TL protoplasts were inactivated Separately by UV-inactivated(irradiated at30W lamp distance10cm) and heat-inactivated (heated at55C constanttemperature water bath pot). The best inactivated conditions of parents protoplasts wereobtained as follow: MOD-1irradiated for10min or heated for15min, T44irradiated for25min or heated for25min, TP4irradiated for25min or heated for20min, TL irradiated30min or heated for20min, the parents protoplasts inactivation rate all reached100%.3. Fusion fusants screening.MOD-1inactivated protoplast wrer fused with T44or TP4or TL inactivated protoplastby other inactivated way separately. The fusants were prescreened according to colony andspore morphological character, screened again with strains,lipid yield in cellulose sodium assole carbon source for fermentation. MP-1which parents were MOD-1and TP4was screened,whose lipid production and lipid content achieved0.0824g/L、14.4594%partly. And thegenetic stability of MP-1were tested to be0.0824g/L and14.4594%after culture5generation. The RAPD result showed, fusion strain MP-1was the fusant of MOD-1with TP4,and closed to MOD-1in DNA lebel.