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ABSTRACT Inducible promoters are indispensable elements when considering the possibility to modulate gene expression on demand. Desirable traits of conditional expression systems include their capacity for tight downregulation, high overexpression, and in some instances for fine-tuning, to achieve a desired product’s stoichiometry. Although the number of inducible systems is slowly increasing, suitable promoters comprising these features are rare. To date, the concomitant use of multiple regulatable promoter platforms for controlled multigene expression has been poorly explored. This work provides pioneer work in the human pathogenic fungus Aspergillus fumigatus, wherein we investigated different inducible systems, elucidated three candidate promoters, and proved for the first time that up to three systems can be used simultaneously without interfering with each other. Proof of concept was obtained by conditionally expressing three antifungal drug targets within the ergosterol biosynthetic pathway under the control of the xylose-inducible PxylP system, the tetracycline-dependent Tet-On system, and the thiamine-repressible PthiA system. IMPORTANCE In recent years, inducible promoters have gained increasing interest for industrial or laboratory use and have become key instruments for protein expression, synthetic biology, and metabolic engineering. Constitutive, high-expressing promoters can be used to achieve high expression yields; however, the continuous overexpression of specific proteins can lead to an unpredictable metabolic burden. To prevent undesirable effects on the expression host’s metabolism, the utilization of tunable systems that allow expression of a gene product on demand is indispensable. Here, we elucidated several excellent tunable promoter systems and verified that each can be independently induced in a single strain to ultimately develop a unique conditional multigene expression system. This highly efficient, modular toolbox has the potential to significantly advance applications in fundamental as well as applied research in which regulatable expression of several genes is a key requirement.

ABSTRACT Aspergillus fumigatus is one of the deadliest fungal species, causing hundreds of thousands of deaths each year. Because azoles provide the preferred first-line option for treatment of aspergillosis, the increase in rates of resistance and the poor therapeutic outcomes for patients infected with a resistant isolate constitute a serious global health threat. Azole resistance is frequently associated with specific tandem repeat duplications of a promoter element upstream of cyp51A, the gene that encodes the target for this drug class in A. fumigatus. This promoter element is recognized by the activating transcription factors SrbA and AtrR. This region also provides a docking platform for the CCAAT-binding complex (CBC) and HapX, which cooperate in the regulation of genes involved in iron-consuming pathways, including cyp51A. Here, we studied the regulatory contributions of SrbA, AtrR, CBC, and HapX binding sites to cyp51A expression and azole resistance under different iron availability employing promoter mutational analysis and protein-DNA interaction analysis. This strategy revealed iron status-dependent and -independent roles of these regulatory elements. We show that promoter occupation by both AtrR and SrbA is required for iron-independent steady-state transcriptional activation of cyp51A and its induction during short-term iron exposure relies on HapX binding. We further reveal the HapX binding site as a repressor element, disruption of which increases cyp51A expression and azole resistance regardless of iron availability. IMPORTANCE First-line treatment of aspergillosis typically involves the use of azole antifungals. Worryingly, their future clinical use is challenged by an alarming increase in resistance. Therapeutic outcomes for such patients are poor due to delays in switching to alternative treatments and reduced efficacy of salvage therapeutics. Our lack of understanding of the molecular mechanisms that underpin resistance hampers our ability to develop novel therapeutic interventions. In this work, we dissect the regulatory motifs associated with azole resistance in the promoter of the gene that encodes the azole drug target Cyp51A. These motifs include binding platforms for SrbA and AtrR, as well as the CCAAT-binding complex and HapX. Employing mutational analyses, we uncovered crucial cyp51A-activating and -repressing functions of the binding sites. Remarkably, disrupting binding of the iron regulator HapX increased cyp51A expression and azole resistance in an iron-independent manner.