PubMed2006
Arom gene, encoding a single polypeptide that catalyses steps two to six of the aromatic amino acid (phenylalanine, tyrosine and tryptophan) biosynthetic pathway, has been amplified from Scleortinia sclerotiorum genomic DNA by PCR and sequenced. In order to identify the fragment encoding AROM protein experimentally and search a method of obtaining the enzyme in a large amount, the open reading frame of arom gene of S. sclerotiorum was amplified by Pyrobest DNA Polymerase and inserted between Kpn I and Not I sites of the vector pYES2 to construct the expression vector pYES2-arom. The construct was transformed into Saccharomyces cerevisiae H158 by the method of LiAc/ SSDNA/PEG. The rate of transformation was 2 x 10(2)/microg DNA, which was enough for the selection of the positive transformants. PCR using the extracted plasmids as the templates and restriction enzyme analysis of the plasmids extracted from E. coli cells transformed by the above plasmids were performed respectively to screen the positive S. cerevisiae transformants since the copy number of the plasmid in S. cerevisiae was low. Subsequently, the transformant activated by the SC-U medium containing 2% raffinose was inoculated into the SC-U medium containing 2% galactose and the SC-U medium containing 2% glucose respectively to induce and depress the expression of the foreign arom gene. The results of RT-PCR analysis showed: there was not any DNA band in the negative control without the anti-transcriptase, which indicated there was no DNA contamination in the extracted total RNA; there was an expected DNA band in the positive control using the expression vector pYES2-arom as the template, which indicated the used amplification condition was proper; there was not any DNA band in the negative control using total RNA from the depressed transformant as the template, which indicated the DNA bands amplified from total RNA of the induced transformant were not false; there were the expected DNA bands in the samples using total RNA of the transformant induced for 48h, 60h, 72h or 84h as the templates, which indicated the heterogeneous arom gene was transcribed in S. cerevisiae H158 cells. The result of Northern hybridization was consistent with that of RT-PCR, and showed that arom gene of S. sclerotiorum had been transcribed in S. cerevisiae H158 cells when the cells were induced for 48h in the SC-U medium containing 2% galactose at 30 degrees C at 180r/min. 5-enolpyruvylshikimate-3-phosphate synthase activity of the transformant, which was one of AROM protein activities, was measured by estimating the rate of Pi release to check the expressed AROM protein was active or not. The results of enzyme assay in the different culture period indicated that the transformant had 5-enolpyruvylshikimate-3-phosphate synthase activity and the activity reached the peak when the transformant was induced for 72h in SC-U medium at 30 degrees C at 180r/min. The molecular weight of AROM protein is high, it exists in cytoplasm as a dimmer and its expression is controlled by the amounts of amino acids. Therefore, it is very difficult to purify the enzyme. A great lot protein can be obtained by heterogeneous expression. S. cerevisiae expression system has the merits of safe status, authentic posttranslational modification, fast cultivation etc. and usually is the first choice eukaryotic expression system. S. cerevisiae expression system of AROM protein from S. sclerotiorum was successfully constructed for the first time, which provided the basis for the research on the catalysis mechanism of the enzyme and an economical means of simultaneously synthesizing five aromatic amino acid biosynthetic pathway enzymes.