Construction Of The Strain Of Fujian Monascus Into High Yield Pigment And Low Yield Citrinin

Monascus is an important microbial resources, it can produce pigment, monacolin K, y-aminobutyric acid, ergosterol and so on. However, people found citrinin which with renal toxicity, carcinogenicity and teratogenicity, this is a tremendous impact on China’s red yeast production, exports. Therefore, to obtain high yield pigment and low yield even does not produce citrinin is an urgent task.In this study, I research optimum medium carbon, nitrogen, inoculation, fermentation temperature, fermentation time, liquid volume and speed in Fujian Monascus M-CL. The result was that rice flour as carbon source, peptone as nitrogen source, inoculum was 10%, liquid volume was 50ml/250ml, speed was 180r/min, fermentation temperature was 30℃, fermentation time was 6 days.Fujian Monascus M-CL as the starting strain, by overlap extension PCR technology integration PtrpC promoter gene, MpigE gene and TrpC terminator gene, then insert fusion fragment into PSKH plasmid which has hygromycin resistance to build overexpression vector HPMT. After sequencing and double digestion verification shows that overexpression vector was successfully constructed.Select protoplast PEG-mediated transformation method for transformation, optimized protoplast preparation conditions, when the combination of lyase was:0.4% lysozyme,0.6% Snailase,0.8% cellulase, osmotic stabilizer was 0.6mol/L MgSO4, hydrolysis temperature was 30℃, hydrolysis time was 3h the number of its protoplast was maximum.After successfully transformed the original strain of Monascus M-CL, use hygromycin which concentration is 100μg/ml to screen mutant strains. Fermented original strain and mutant strains in liquid medium, measured pigment content by UV spectrophotometry, determined citrinin content by high performance liquid chromatography. The results showed that overexpression strain than the original strain citrinin production decreased 41.8%, and the pigment production increased by 11%.In order to further reduce the content of citrinin successfully constructed gene knockout components by overlap extension PCR method. Both ends of the knockout fragment is homologous arms and in the middle of it was the hygromycin resistance gene. By sequence analysis, indicates that we have successfully established a Monascus knockout assembly,then may shift to the overexpression strain.It laied the foundation for a more superior species.