Inducible Selectable Marker Genes to Improve Aspergillus fumigatus Genetic Manipulation

Abstract

The hygromycin B phosphotransferase gene from Escherichia coli and the pyrithiamine resistance gene from Aspergillus oryzae are two dominant selectable marker genes widely used to genetically manipulate several fungal species. Despite the recent development of CRISPR/Cas9 and marker-free systems, in vitro molecular tools to study Aspergillus fumigatus, which is a saprophytic fungus causing life-threatening diseases in immunocompromised hosts, still rely extensively on the use of dominant selectable markers. The limited number of drug selectable markers is already a critical aspect, but the possibility that their introduction into a microorganism could induce enhanced virulence or undesired effects on metabolic behavior constitutes another problem. In this context, here, we demonstrate that the use of ptrA in A. fumigatus leads to the secretion of a compound that allows the recovery of thiamine auxotrophy. In this study, we developed a simple modification of the two commonly used dominant markers in which the development of resistance can be controlled by the xylose-inducible promoter PxylP from Penicillium chrysogenum. This strategy provides an easy solution to avoid undesired side effects, since the marker expression can be readily silenced when not required.

Modular Inducible Multigene Expression System for Filamentous Fungi

“…PthiA , on the contrary, has not been widely used in general and the regulation of this promoter is still unclear. Enticed by the PthiA promoter’s strength and inducibility, which can be deduced from other studies ( 30 , 38 ), we decided to include it in our work to further evaluate its suitability as molecular tool. Adequate comparison of the regulation profile of these promoters required the exclusion of any possible locus-related effects; hence, we selected the previously described endogenous marker fcyB ( 37 ) for the integration of the constructs.…”

“…Initially, we analyzed each promoter separately, to better establish the optimal conditions for their induction or repression. PxylP has been widely used in A. fumigatus ( 38 , 41 – 44 ), and the general conditions to obtain a high level of expression were set to 1% glucose and 1% xylose in minimal medium, while glucose alone would be suitable to efficiently downregulate PxylP -driven gene expression. We conducted a titration analysis using 1% glucose and xylose in a concentration range from 0% to 3%, and we confirmed, as previously described ( 38 ), that a concentration as low as 0.09% xylose was sufficient for induction ( Fig.…”

“…A variety of promoters regulated by xylose have been well established in several species, including diverse species of Aspergillus and Penicillium , Acremonium chrysogenum , and Trichoderma reesei ( 27 , 53 – 58 ). In A. fumigatus , the xylanase promoter PxylP from P. chrysogenum has been well characterized and broadly used ( 27 , 38 , 42 ). The tetracycline-regulatable expression systems, after being identified in bacteria, have been modified and optimized for use in eukaryotic organisms, again having major applications among Aspergillus species ( 19 , 20 , 28 , 59 , 60 ).…”

“…It is synthesized in bacteria, fungi, and plants and has to be supplied with the diet in mammals ( 66 ). In a previous study, we investigated a point-mutated version of PthiA that render the promoter activity independent of thiamine regulation ( 38 ). Expression patterns suggested that, when active, PthiA should mediate high expression levels.…”

Characterization and engineering of the xylose-inducible xylP promoter for use in mold fungal species

“…The xylP promoter (p xylP ) controlling expression of a xylanase from Penicillium chrysogenum allows high induction by xylan or its degradation product xylose with low basal activity in the absence of an inducer ( Haas et al, 1993 ; Zadra et al, 2000 ). p xylP was demonstrated to permit conditional gene expression of diverse genes in various mold species including P. chrysogenum ( Bugeja et al, 2010 , 2013 ; Huber et al, 2019 ; Janus et al, 2009 ; Kopke et al, 2013 ; Pongsunk et al, 2005 ; Sigl et al 2010 , 2011 ), Penicillium marneffei ( Bugeja et al, 2010 , 2013 ; Pongsunk et al, 2005 ), Aspergillus nidulans ( Monahan et al, 2006 ; Wong et al, 2007 ; Wong et al 2008 ; Wong et al 2009 ; Tribus et al, 2010 ; Ma et al, 2018 ; Pidroni et al, 2018 ; Wang et al, 2021 ; Li et al, 2021 ; Unkles et al, 2014 ), Aspergillus fumigatus ( Yasmin et al, 2012 ; Gsaller et al, 2012 ; Fazius et al, 2012 ; Altwasser et al, 2015 ; Baldin et al 2015 , 2021 ; Vaknin et al, 2016 ; Misslinger et al, 2019 ; Bauer et al, 2019 ; López-Berges et al, 2021 ; Handelman et al, 2021 ; Fabri et al, 2021 ) and Sordaria macrospora ( Kopke et al, 2010 ). A. fumigatus is a ubiquitous saprobic fungus but at the same time the most common mold pathogen of humans.…”