Cloning, characterization and application of a glyceraldehyde-3-phosphate dehydrogenase promoter from Aspergillus terreus
Cloning, characterization and application of a glyceraldehyde-3-phosphate dehydrogenase promoter from Aspergillus terreus
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DOI：
10.1007/s10295-013-1385-0
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发表时间：
2014-03-01
影响因子：
3.4
通讯作者：
Li, Jian-Jun
中科院分区：
文献类型：
--
作者：
Huang, Xuenian;Lu, Xuefeng;Li, Jian-Jun
开发天然高效的启动子对丝状真菌的高效基因工程具有重要意义。虽然土曲霉是生产衣康酸和洛伐他汀的重要工业真菌，但该微生物的可用遗传工具箱仍然相当有限。我们从A. terreus CICC 40205，随后使用sgfp（合成的绿色荧光蛋白）作为报道基因证实了其启动子功能。通过系统缺失进一步分析启动子PgpdAt的序列，以获得有效且紧凑的功能性启动子。两种截短形式的PgpdAt（1，081和630 bp）也能够驱动A. terreus这三个不同长度的PgpdAt启动子的活性进一步通过荧光、western blot和转录证实。其中最短的一个（630 bp）已成功应用于A. terreus表达的血红蛋白的功能通过CO（一氧化碳）差光谱和增强的氧摄取率、葡萄糖消耗和维生素A酸滴度来证明。我们的研究成功地开发和验证了一个高效和紧凑的天然启动子，用于A。terreus
It is important to develop native and highly efficient promoters for effective genetic engineering of filamentous fungi. Although Aspergillus terreus is an important industrial fungus for the production of itaconic acid and lovastatin, the available genetic toolbox for this microorganism is still rather limited. We have cloned the 5' upstream region of the glyceraldehyde-3-phosphate dehydrogenase gene (gpd; 2,150 bp from the start codon) from A. terreus CICC 40205 and subsequently confirmed its promoter function using sgfp (synthetic green fluorescent protein) as the reporter. The sequence of the promoter PgpdAt was further analysed by systematic deletion to obtain an effective and compact functional promoter. Two truncated versions of PgpdAt (1,081 and 630 bp) were also able to drive sgfp expression in A. terreus. The activities of these three PgpdAt promoters of varying different lengths were further confirmed by fluorescence, western blot and transcription. The shortest one (630 bp) was successfully applied as a driver of vgb expression in the genetic engineering of A. terreus. The function of expressed haemoglobin was demonstrated by the CO (carbon monoxide)-difference spectrum and enhanced oxygen uptake rate, glucose consumption and itaconic acid titer. Our study was successful in developing and validating an efficient and compact native promoter for genetic engineering of A. terreus.