Cloning, characterization and application of a glyceraldehyde-3-phosphate dehydrogenase promoter from Aspergillusterreus
It is important to develop native and highly efficient promoters for effective genetic engineering of filamentous fungi. Although Aspergillusterreus 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.
KeywordsAspergillusterreus Glyceraldehyde-3-phosphate dehydrogenase promoter Native promoter Promoter function Vitreoscilla haemoglobin
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