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Research on the metabolism and Gene Engineering of S-adenosyl-L-methionine Synthetase

Author: GuoJian
Tutor: HeGuangYuan
School: Huazhong University of Science and Technology
Course: Biochemical Engineering
Keywords: GAP promoter AOX1 promoter Pichia pastoris Adenosylmethionine synthetase
CLC: TQ920.1
Type: Master's thesis
Year: 2005
Downloads: 139
Quote: 1
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Abstract


S-adenosylmethionine (S-adenosyl-L-methionine, referred to as SAM or AdoMet ) is an important intermediate metabolites in vivo , and to participate in a variety of biochemical reactions , and has a wide and varied therapeutic effect . The thus extracted through microbial fermentation SAM SAM 's main industrial production pathway . SAM production has been mainly in S.cerevisiae fermentation . The laboratory through rational transformation of Pichia pastoris metabolic pathway , a constitutively express the SAM synthetase 2 (SAM2) the reorganization P.pastoris strain . May explore using the GAP promoter (P GAP ) - substituted AOX1 promoter ( P the AOX1 < / sub >) in the the Bijie yeast (P.pastoris) constitutive expression of the exogenous protein the sexual , the PCR method P.pastoris chromosome amplified GAP promoter in its replace the inducible expression vector pPIC9K of on P AOX1 , constructed constitutive expression vector pGAP9K . The synthase II adenosylmethionine (SAM) the recombinant in multicloning site of pGAP9K , obtain the recombinant plasmid Containing SAM gene pGAP9K -SAM . To conversion P.pastorisGS115 P.pastoris GS115 (pGAP9K-SAM) , to obtain high copy transformants constitutively expressed, while inducible transformants P.pastoris GS115 (pPIC9K-SAM) as controls . Constitutive transformants culture 4d after SAM2 expression of enzyme activity has reached its peak ; the peaked inducible transformants induced 6d , the expression of only the constitutive expression system 81% of the peak (4d) . The above results indicate that the constitutive GAP promoter construct expression system having a short fermentation time , the higher the expression level without methanol induction , the operating system is relatively simple , etc. the P GAP , can replace P < sub > AOX1 the SAM and exogenous protein expression in P.pastoris .

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