A strong promoter, PMagpd, provides a tool for high gene expression in entomopathogenic fungus, Metarhizium acridum

Abstract

A glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter (PMagpd) was obtained from Metarhizium acridum and its active region analyzed by 5'-deletion strategy using β-glucuronidase (GUS) as a reporter. Sequence analysis revealed that typical regulatory elements of PMagpd were included in the 1.7 kb region upstream of the start codon of the Magpd gene. Deletion of the region from -1,691 bp to -1,463 bp, where the gpd box is harbored, did not significantly affect the PMagpd activity. Deletions of the regions upstream of -946 bp and upstream of -684 bp caused a major decrease of GUS activity. Compared with PgpdA (2.2 kb) in Aspergillus nidulans, PMagpd (1.4 kb) had a shorter sequence and significantly higher activity in M. acridum. This study provides an applicable promoter for over-expression of target genes in M. acridum.

Cloning, characterization and application of a glyceraldehyde-3-phosphate dehydrogenase promoter from Aspergillus terreus

“…The functions of the promoters were also investigated at the transcription level of sgfp by qRT-PCR [9]. The actin gene (ATEG_06973) was used as the endogenous control.…”

“…The efficient and compact native gpd promoter PgpdAt3 from A. terreus was identified. Some short and functional gpd promoters have been reported, including the 946-bp promoter from Metarhizium acridum [9], the 442-bp promoter from Lentinus edodes [29] and the 190-bp promoter from Candida bombicola [12]. It would appear that the gpd promoters from different microorganisms show different regulatory characteristics.…”

“…Thus, the constitutive promoters, which are not dependent upon on the carbon or nitrogen source or the specific inducers, are preferred. For example, constitutive promoters, such as the pkiA promoter of the A. niger protein kinase A [8] and the glyceraldehyde-3-phosphate dehydrogenase (gpd) promoters, have been successfully used to express the respective heterologous genes in filamentous fungi [9][10][11][12]. Recently, many promoters from Aspergillus species have been cloned, characterized and applied in the genetic engineering of these species [13][14][15].…”

“…Native promoters are the preferred promoters for genetic engineering of fungi because they are more efficient in directing gene expression than heterologous ones in some species of fungi [9,13,17,18]. Although A. terreus is an important producer of itaconic acid and lovastatin, the genetic tools for A. terreus, especially efficient native promoters, are lacking.…”

Overexpression of pucC improves the heterologous protein expression level in a Rhodobacter sphaeroides expression system

“…The gus gene was amplified with primer pairs from previous research (Cao et al, 2012). For the purification of heterologous protein, the His-tag was also introduced.…”