AUTHOR=Wang Gang , Li Yujie , Yang Bolei , Li Erfeng , Wu Wenqing , Si Peidong , Xing Fuguo 

TITLE=AwAreA Regulates Morphological Development, Ochratoxin A Production, and Fungal Pathogenicity of Food Spoilage Fungus Aspergillus westerdijkiae Revealed by an Efficient Gene Targeting System

JOURNAL=Frontiers in Microbiology

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2022.857726

DOI=10.3389/fmicb.2022.857726

ISSN=1664-302X

ABSTRACT=<p><italic>Aspergillus westerdijkiae</italic>, the producer of ochratoxin A (OTA), which is of worldwide concern, is an import fungal species in agriculture, food, and industry. Here, we got the uridine auxotrophic mutant of <italic>A. westerdijkiae</italic> by deleting <italic>AwpyrG</italic>. The Δ<italic>AwpyrG</italic> could be used for bio-transformation with exogenous <italic>AfpyrG</italic> expression cassette as a selection marker. In order to enhance the efficiency of gene targeting, <italic>Awku70</italic> and <italic>Awlig4</italic> were homologously deleted from Δ<italic>AwpyrG</italic>. The efficiencies of homologous replacement for Δ<italic>Awku70</italic> and Δ<italic>Awlig4</italic> were 95.7 and 87.0% in the deletion of <italic>AwAreA</italic>, respectively, demonstrating a drastic increase from 4.3% of the wild type (WT) strain. Furthermore, the function of <italic>AwAreA</italic> was identified with <italic>AwAreA</italic> deletion mutant and the control strain Δ<italic>Awku70</italic>. <italic>AwAreA</italic> regulated the growth and conidiation of <italic>A. westerdijkiae</italic> in response to nitrogen sources. The concentration of OTA for Δ<italic>Awku70</italic> was in the range of 19.4 to 186.9 ng/cm<sup>2</sup> on all kinds of nitrogen sources. The OTA production influenced by the deletion of <italic>AwAreA</italic> was different based on nitrogen sources. Pathogenicity assays on pears, grapes, salted meat, and cheese showed that <italic>AwAreA</italic> acted as a negative regulator in the infection of food substrates. Therefore, the genetic methods and engineered strains enable us to substantially expand the use of <italic>A. westerdijkiae</italic>, one of more than twenty OTA-producing fungi, in the study of mycotoxin biosynthesis and regulation, and consequently to aim at providing new ways for controlling this pathogen.</p>