CAJ | 학술논문

构建含酿酒酵母组成型强启动子PGK、G418抗性基因及rDNA片段的整合型载体pScIKP,利用rDNA多个同源重组位点,将外源基因以多拷贝整合到酵母染色体上,无需诱导即可持续表达;利用载体位于表达盒两端同尾酶,可插入多个基因表达盒,实现多基因稳定共表达.为检验共表达情况,反转录从绿色木霉中获得纤维素酶基因eg3和cbh2, 克隆并转化获得重组酵母菌株S.cerevisiae-ec. 该重组酵母能降解羧甲基纤维素形成水解圈;用羧甲基纤维素还原糖法和滤纸酶活力法测定酶活力,其最适温度和最适pH值与所表达单酶相似,表明共表达未影响两种酶的生物学特性;且双酶具有协同作用,能更有效降解非结晶纤维素.pScIKP载体能成功用于多个外源基因共表达和产物协同作用的研究,为构建能直接降解纤维素的酿酒酵母菌株,实现纤维素可再生能源的生物利用奠定了基础.
구건함양주효모조성형강계동자PGK、G418항성기인급rDNA편단적정합형재체pScIKP,이용rDNA다개동원중조위점,장외원기인이다고패정합도효모염색체상,무수유도즉가지속표체;이용재체위우표체합량단동미매,가삽입다개기인표체합,실현다기인은정공표체.위검험공표체정황,반전록종록색목매중획득섬유소매기인eg3화cbh2, 극륭병전화획득중조효모균주S.cerevisiae-ec. 해중조효모능강해최갑기섬유소형성수해권;용최갑기섬유소환원당법화려지매활역법측정매활력,기최괄온도화최괄pH치여소표체단매상사,표명공표체미영향량충매적생물학특성;차쌍매구유협동작용,능경유효강해비결정섬유소.pScIKP재체능성공용우다개외원기인공표체화산물협동작용적연구,위구건능직접강해섬유소적양주효모균주,실현섬유소가재생능원적생물이용전정료기출.
To construct a Saccharomyces cerevisiae integrated expression vector,the 3-phosphoglycerate kinase (pgk) promoter and terminator,the G418 resistance gene and the S.cerevisiae rDNA sequence were cloned into a backbone vector pBluescript II SK(-) to generate a shuttle vector pScIKP.This vector can integrate foreign genes into the yeast genome with multiple copies by homologous recombination,and constitutively express these genes without induction.Inserting several expression cassettes into the vector is a simple process that facilitates multiple genes co-expression.To test the co-expression ability,two cellulase genes endoglucanase 3 and cellobiohydrolase 2 were obtained from Trichoderma viride AS3.3711 by RT-PCR and inserted into pSCIK in order to generate the co-expression vector pScIKP-ec.The S.cerevisiae-ec transformants could stably express eg3 and cbh2 with the highest CMCase activity of 4.60 U/mL and the filter paper assay activity of 0.70 U/mL.These co-expressed enzymes were able to degrade phosphoric acid-swollen cellulose more efficiently than eg3 or cbh2 alone.These results demonstrate that the S.cerevisiae integrated vector pScIKP can co-express multiple foreign genes and be applied to the studying of the synergism of co-expression products.This system can also be used to construct S.cerevisiae, which has the ability to express multiple cellulases and directly degrade cellulosic materials.