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

Abstract

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.

Identification of an itaconic acid degrading pathway in itaconic acid producing Aspergillus terreus

“…The spores were cultured on potato dextrose agar (PDA) at 30°C for 5 days. The 2.5 × 10 7 spores were inoculated into 55 ml itaconic acid production medium (IPM, pH 3.25) (Huang et al 2014a) in a 500-ml shake flask, and the mycelium precultivation was carried out on a rotary shaker at 220 rpm and 37°C. After cultivation for 20 h, 5 ml of the seed cultures were directly transferred into 50 ml of IPM (the normal condition) or IPM with extra 40 g/L of itaconic acid (the I40 condition, pH 3.25) in 500-ml shake flasks and cultivated on a rotary shaker at 220 rpm and 37°C.…”

Direct production of itaconic acid from liquefied corn starch by genetically engineered Aspergillus terreus

“…So far, the only investigated native promoter from A. terreus is the gpd promoter, which has been successfully applied in driving vgb (the Vitreoscilla hemoglobin gene) overexpression in A. terreus [16]. PgpdA from A. nidulans was by far the most frequently used promoter for genetic modification of A. terreus [17,18].…”

Establishing an efficient gene-targeting system in an itaconic-acid producing Aspergillus terreus strain

“…The spores were cultured on AMMB agar [Aspergillus minimal medium (AMM, http://www.fgsc.net/ methods/anidmed.html) with 5 g wheat bran l -1 ] at 32°C for 7 days. Further cultivation was carried out in 500 ml non-baffled shake-flasks containing 55 ml itaconic acid production medium (IPM) on a rotary shaker at 200 rpm and 37°C (Huang et al 2014c). …”

“…The genetargeting element of ATEG_04633 was constructed as follows: The selectable marker hph was amplified from pG3H (Huang et al 2014c) using primer hph-F/ hph-R. The flanking regions of ATEG_04633 were amplified from the genome of A. terreus CICC40205 using primer pairs U4633-F/U4633-R and D4633-F/ D4633-R respectively, and then fused with hph by fusion PCR.…”

“…To examine the uracil auxotrophy, 5-FOA and PT resistant of mutants, the WT, At-Dku80, At-DpyrG, AtloxP-pyrG An , At-loxP-DpyrG An strains were incubated on AMM plates for 4 days, AMM plates supplemented 0.1 mg PT l -1 and 10 mM uracil for 7 days, and AMM plates supplemented 10 mM 5-FOA and 10 mM uracil for 7 days. The itaconic acid productivities and growth rates of the WT, AtDku80, At-loxP-pyrG An mutants were investigated by cultivation in shake flasks as described previously (Huang et al 2014c).…”