多拷贝脂肪酶基因在黑曲霉中表达研究

Expression of multicopy lipase gene in Aspergillus niger

文章在使用含6个gla A box强启动子Pgla A6R和高效分泌信号肽Sgla A调控黑曲霉脂肪酶基因Anlip A表达基础上,用pyr G双向筛选标记,应用多拷贝黑曲霉脂肪酶基因策略,构建含1个拷贝Anlip A基因表达载体p SZHA6R-Anlip A和含2个拷贝Anlip A基因表达载体p SZHA6R-2Anlip A,通过农杆菌介导法转化黑曲霉CICC2462(Δpyr GΔas AA::pyr G),获得as AA基因位点整合纯合同源重组转化子CICC2462(p SZHA6R-Anlip A)和CICC2462(p SZHA6R-2Anlip A)。重组菌株经摇瓶发酵后,作荧光定量PCR、SDS-PAGE和脂肪酶活力检测。结果表明,重组菌株可分泌表达约37.0 ku目的蛋白条带,CICC2462(p SZHA6R-2Anlip A)中脂肪酶表达在m RNA水平和酶活水平皆高于CICC2462(p SZHA6R-Anlip A)。发酵第5天CICC2462(p SZHA6R-2Anlip A)中Anlip A基因转录水平为CICC2462(p SZHA6R-Anlip A)1.26倍。二者酶活力在发酵第8天达峰值,此时CICC2462(p SZHA6R-2Anlip A)是CICC2462(p SZHA6R-Anlip A)1.32倍,酶活力达1 480 U·m L-1。对重组菌株CICC2462(p SZHA6R-2Anlip A)第8天发酵液作酶学性质分析,结果表明,最适温度为35℃,最适p H为6.0。获得1株高效分泌表达脂肪酶食品级黑曲霉工程菌。

The strong promoter PglaA6R containing six copies of the glaA box and efficient secretion of signal peptide SglaA were used to regulate the expression of AnlipA.Constructed expression vectors pSZHA6R-AnlipA containing one copy of AnlipA and pSZHA6R-2AnlipA containing two copies of AnlipA using pyrG bidirectional selection marker and applying the multicopy Aspergillus niger lipase gene strategy.Two expression vectors were transferred into Aspergillus niger CICC2462(ΔpyrGΔasAA::pyrG)by Agrobacterium-mediated method,then the homologous recombination homozygotes CICC2462(pSZHA6RAnlipA)and CICC2462(pSZHA6R-2AnlipA)at the asAA integration site were obtained.After fermentation in shake flask,detections of fluorescence quantitative PCR,SDS-PAGE and lipase activity indicated the pure homologous recombination strains had about 37.0 ku protein band.The mRNA and enzyme activity levels of lipase in CICC2462(pSZHA6R-2AnlipA)were both higher than those in CICC2462(pSZHA6R-AnlipA).The transcription level of CICC2462(pSZHA6R-2AnlipA)was 1.26 times greater than CICC2462(pSZHA6RAnlipA)on the 5th day.The enzyme activity of the two homologous recombination strains peaked on the 8th day of fermentation.At this time,CICC2462(pSZHA6R-2AnlipA)was 1.32 times greater than CICC2462(pSZHA6R-AnlipA)and the enzyme activity reached 1 480 U·mL-1.The enzymatic properties of the 8th day fermentation broth of the recombinant strain CICC2462(pSZHA6R-2AnlipA)was analyzed.The results showed that the optimum temperature was 35°C and the optimum pH was 6.0.In this way,food grade Aspergillus niger engineering strain that secreted lipase efficiently was obtained.