Genes encoding enzymes related to 2-acetyl-1-pyrroline formation in Saccharomyces cerevisiae

• Main Authors: Hao-Zhen Zeng, 曾皓真
• Other Authors: Tzou-Chi Huang
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/23017765795320682920

碩士 === 國立屏東科技大學 === 生物科技研究所 === 95 === A popcorn-like flavor compound, 2-acetyl-1-pyrroline, has been reported as a potent flavor component of an aromatic rice, taro, soybean and pandan leaves. Results of tracer experiments indicated that the pyrroline source of 2-acetyl-1-pyrroline was proline, whereas the source of acetyl group was unknown. Methylglyoxal (MG), derived from dihydroxyacetone phosphate (DHAP) was proposed to be responsible for the acetyl group of 2-AP. Glycerol is common compatible osmolyte in water-stress organism. Glycerol kinase is key enzyme of the intermediate during metabolism. The concentration of MG level increased with increasing Glycerol concentration. We amplified the gene of glycerol kinase (GK), Glycerol-3-phosphate dehydrogenase (G3PDH) and Ornithine aminotransferase(OAT) from Saccharomyces cerevisiae by PCR. After confirming the sequences, we constructed the recombinant expression plasmid pYES2B-SCGK, pYES2B-SCG3PDH and pYES2A-SCOAT , and then transformed the recombinant genes into INVSC1 to express recombinant proteins. Model system composed of P5C synthesized in vitro by ScOAT and MG was set up to study the formation of 2-AP. In addition, the mRNA expression of the glycerol metabolism related genes: Glycerol kinase (GK)、Glycerol-3-phosphate dehydrogenase (G3PDH)、Glycerol dehydrogenase (GDHG)、Dihydroxyacetone kinase (DHAK) were analyzed with gene-specific primers by RT-PCR (Reverse Transcription-Polymerase Chain Reaction) in yeast treated various concentrations of NaCl and glycerol. Among the four genes tested, G3PDH overexpression of RNA was observed in NaCl treated yeast alls. The expression of G3PDH showed a dose dependent manner up to 5％ NaCl.