Breeding Of Saccharomyces Cerevisiae With Higher Yield S-Adenosyl-L-Methionine By Composite Mutagenesis And Protoplast Fusion

S-adenosyl-L-methionine (SAM) is a kind of intracellular physiological activator, it has many physiological fuctions such as transmethylation, transaminopropylation and transsulfenylation. Domestic and international markets need much more SAM because SAM can prevent and cure arthritis, despression and liver disease. However, SAM can not be produced industrially in our country due to strain problem and immature technology. This experiment aims to improve the yield of SAM from S.cerevisiae with composite mutagenesis and the technique of protoplast fusion. Major achievements list as follows:1. The freeze thawing, ethanol and perchloric method had different effects to extract SAM by cell discruption. The freeze thawing and ethanol method influenced the stability of SAM seriously and made much loss of SAM, so they were not better ways to extract SAM. The result of cell discruption by perchloric method was the best, it could freed most quantity of SAM and be selected to extract SAM. In the study of SAM analytical methods, HPLC to analyse SAM was accurate and high-efficiency, the retention time was about 7th minute, one SAM sample can be determined in 10 minutes.2. Thirteen starting strains were identified to study whether they can bear spore, their biomass and yield of SAM were determined, too. Then separated haploid from the strain named R1 with good growth condition and high yield of SAM,60 haploids were obtained. Applied composite mutagenesis of UV and DES to treat hR10 which had higher yield of SAM,4 strains named DR1-3, NM14, NM39, NM45 were screened in solid medium containing 10μg/mL of nystatin. In comparison with that of their own starting strains, the yield of SAM increased by 34.3%,10.9%,14.1%,18.7% respectively.3. The parental strains DR1-3, NM14,NM39. NM45 were signed with parent-inactivation, then their protoplasts realized fusion under the induction of PEG and 90 fusants were obtained in solid medium containing 20^g/mL of nystatin, after determining their biomass and SAM content, a fusant called F35 with improved yield of SAM were selected. The yield of SAM produced by F35 reached 22.5mg/L, which was increased by 103% in comparison with that of the starting strain R1. SAM content in dry cell was 1.97mg/gDCW, which was twice as much as that of R1. The subculture experiments indicated that the hereditary character of high producing was stable.4. Single factor experiments were done to optimize the medium and conditions of fermentation for F35 producing SAM. The results showed the optimal fermentation conditions were:maltose for carbon source, peptone for nitrogen source, pH 6, culture temperature for 30℃. Then the components of the medium and the fermentation time were optimized by orthogonal test design. The test determined the effect degree of factors to SAM fermentation: peptone> NH4C1> maltose> fermentation time> NH4H2PO4; It is concluded that a medium containing 10% of maltose,1% of peptone,0.4% of NH4H2PO4, 0.5% of NH4C1,0.01% of MgSO4,0.6% of KH2PO4, and 72h of fermentation time was suitable for SAM production by F35.