核盘菌arom基因的克隆及其在酿酒酵母中的表达

Cloning and expression of arom gene of Sclerotinia sclerotiorium

核盘菌(Sclerotinia sclerotiorum)arom基因已经被克隆、测序。该基因编码的AROM蛋白是芳香族氨基酸合成途径中催化第二步到第六步的五功能多肽。为了对扩增的核盘菌arom基因进行功能验证和探索大量获得AROM蛋白的成熟方法,克隆了arom基因的编码框序列,并将其与载体pYES2连接,构建了酵母表达载体pYES2-arom。用LiAc/SSDNA/PEG方法将该表达载体转入酿酒酵母H158(Saccharomyces cerevisiaeH158)中。酶活测定、RT-PCR和Northern杂交结果显示,核盘菌arom基因在酿酒酵母胞内获得了表达,转化子具有AROM蛋白结构域之一5-烯醇丙酮酰莽草酸-3-磷酸合酶的催化活性。不同培养时间取样样品的酶活检测结果表明,当转化子在30℃的条件下,在SC-U酵母尿嘧啶营养缺陷型选择培养基中以180r/min被振荡培养时,其外源AROM蛋白的5-烯醇丙酮酰莽草酸-3-磷酸合酶酶活在培养72h达到最高。

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 DNA by PCR and sequenced. In order to identify the fragment encoding AROM from Scleortinia sclerotiorum genomic 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 PyrobestTM DNA Polymerase and inserted between KpnⅠand NotⅠ 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 × 10^2/μg 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 tranformant as the template, which indicated the DNA bands amplified from total RNA of the induced tranformant were not false; there were the expected DNA bands in the samples using total RNA of the tranformant induced for 48h, 60h, 72h or 84h as the templates, which indicated the heterogeneous arom gene was transcripted in S. cerevisiae H158 ceils. The result of Northern hybridization was consistent with that of RT-PCR, and showed that arom gene of S. sclerotiorum had been transcripted in S. cerevisiae H158 ceils when the cells were induced for 48h in the SC-U medium containing 2% galactose at 30℃ 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 rcsults 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℃ 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.