Fungal Gene Expression on Demand: an Inducible, Tunable, and Metabolism-Independent Expression System for Aspergillus niger

Abstract

Filamentous fungi are the cause of serious human and plant diseases but are also exploited in biotechnology as production platforms. Comparative genomics has documented their genetic diversity, and functional genomics and systems biology approaches are under way to understand the functions and interaction of fungal genes and proteins. In these approaches, gene functions are usually inferred from deletion or overexpression mutants. However, studies at these extreme points give only limited information. Moreover, many overexpression studies use metabolism-dependent promoters, often causing pleiotropic effects and thus limitations in their significance. We therefore established and systematically evaluated a tunable expression system for Aspergillus niger that is independent of carbon and nitrogen metabolism and silent under noninduced conditions. The system consists of two expression modules jointly targeted to a defined genomic locus. One module ensures constitutive expression of the tetracyclinedependent transactivator rtTA2 S -M2, and one module harbors the rtTA2 S -M2-dependent promoter that controls expression of the gene of interest (the Tet-on system). We show here that the system is tight, responds within minutes after inducer addition, and allows fine-tuning based on the inducer concentration or gene copy number up to expression levels higher than the expression levels of the gpdA promoter. We also validate the Tet-on system for the generation of conditional overexpression mutants and demonstrate its power when combined with a gene deletion approach. Finally, we show that the system is especially suitable when the functions of essential genes must be examined.

Exploration of Sulfur Assimilation of Aspergillus fumigatus Reveals Biosynthesis of Sulfur-Containing Amino Acids as a Virulence Determinant

“…To further confirm that the metH gene is indispensable for A. fumigatus, we constructed a conditional expression strain, AfS180, in which the expression of the coding sequence is driven by a doxycycline-dependent Tet-ON module (see Fig. S7 in the supplemental material) (74,75), a system that has successfully been used in A. fumigatus to prove gene essentiality (76). In the presence of doxycycline, the metH gene is properly expressed to support growth of AfS180 (Fig.…”

Role of the Guanine Nucleotide Exchange Factor Rom2 in Cell Wall Integrity Maintenance of Aspergillus fumigatus

“…pCH003 was constructed by cloning rho1 (PCR amplified with primers Rho1-6g06900fufnew and Rho1-6g06900-rev with chromosomal template DNA) in pJW121. pJW121 was constructed by cloning the tetracycline-dependent transactivator, the crgA terminator, and the tetO7::Pmin (a blunt-ended fragment derived from pVG2.2 after digestion with EcoRI and PmeI [22]) into the PmeI site of pSK379. pJW103 and pSK379 were described previously (7,23).…”

Molecular genetic analysis of vesicular transport in Aspergillus niger reveals partial conservation of the molecular mechanism of exocytosis in fungi

“…7f). Note that it is unlikely that Tet-On mediated expression is insufficient to induce overexpression of the endogenous ssoA, as it was previously shown that the Tet-On system enables expression levels similar to gpdA expression (Meyer et al, 2011a), which in fact would be 80-fold higher than ssoA expression (data not shown).…”

“…Mutant alleles of ssoA (encoding SsoA L81F , SsoA L81G , SsoA R212K and SsoA R212P ) were generated by PCR using primers carrying the respective mutations and XhoI-EcoRI ends. The For the creation of a conditional ssoA overexpression construct using the Tet-On system, the ssoA ORF or ssoA ORF with a truncated transmembrane domain (ssoADTM) was cloned into pVG2.2 (Meyer et al, 2011a) and the resulting plasmid was transformed for targeted integration at the pyrG locus using the pyrG* marker. After Southern blot analysis, strains were selected (MK22.3 and MK24.20) that contained the wild-type ssoA gene or ssoADTM gene at the pyrG locus under control of the tetracycline promoter.…”

“…Next, another approach to create a conditional mutant was tried by using the Tet-On system that we recently established for A. niger (Meyer et al, 2011a). The SsoA protein contains a N-terminal syntaxin domain and a Cterminal transmembrane domain (280-302 aa; Fig.…”