Identification of a Classical Mutant in the Industrial HostAspergillus nigerby Systems Genetics: LaeA Is Required for Citric Acid Production and Regulates the Formation of Some Secondary Metabolites

Abstract

The asexual filamentous fungus Aspergillus niger is an important industrial cell factory for citric acid production. In this study, we genetically characterized a UV-generated A. niger mutant that was originally isolated as a nonacidifying mutant, which is a desirable trait for industrial enzyme production. Physiological analysis showed that this mutant did not secrete large amounts of citric acid and oxalic acid, thus explaining the nonacidifying phenotype. As traditional complementation approaches to characterize the mutant genotype were unsuccessful, we used bulk segregant analysis in combination with high-throughput genome sequencing to identify the mutation responsible for the nonacidifying phenotype. Since A. niger has no sexual cycle, parasexual genetics was used to generate haploid segregants derived from diploids by loss of whole chromosomes. We found that the nonacidifying phenotype was caused by a point mutation in the laeA gene. LaeA encodes a putative methyltransferase-domain protein, which we show here to be required for citric acid production in an A. niger lab strain (N402) and in other citric acid production strains. The unexpected link between LaeA and citric acid production could provide new insights into the transcriptional control mechanisms related to citric acid production in A. niger. Interestingly, the secondary metabolite profile of a ΔlaeA strain differed from the wild-type strain, showing both decreased and increased metabolite levels, indicating that LaeA is also involved in regulating the production of secondary metabolites. Finally, we show that our systems genetics approach is a powerful tool to identify trait mutations.

The global regulator LaeA controls production of citric acid and endoglucanases inAspergillus carbonarius

“…Regulatory effects of laeA ortholog on secretion of cellulases have been shown in T. reesei [37] and the effects on mycotoxins have also been shown previously in A. carbonarius [9]. Findings of our study suggest that the regulatory effect extends even further to production of organic acids as it is also suggested by Niu et al [30] and Dai and Baker [10]. It was also shown that at the same time as LaeA regulates endoglucanase in A. carbonarius, it does not regulate two of the other important cell wall degrading enzymes, namely beta-glucosidases and xylanases.…”

“…In a recent patent in A. niger, the overexpression of laeA alone or together with the disruption of alg3 contributed to an increase in citric acid production indicating that it is not only secondary metabolites, reproduction and morphology that are regulated by LaeA [10]. A point mutation in the laeA gene in A. niger was very recently shown to create a non-acidifying mutant with reduced production of citric and oxalic acid supporting the hypothesis that it is not only secondary metabolites LaeA regulates [30]. It is not only in Aspergillus species that LaeA has a regulatory effect.…”

“…All three isolates produced naphtho-γ-pyrones and pyranonigrin A, but the relative amounts of a polar naphtho-γ-pyrone-related compound (BMS 192548) was higher in the laeA knockout than in the WT and laeA-overexpressed strains. The naphtho-γ-pyrones are related to the black pigment in Aspergillus section Nigri conidia [19,30], but BMS 192548 may also be secreted into the medium as a yellow pigment if overproduced as in the laeA knockout.…”

Revitalization of a Forward Genetic Screen Identifies Three New Regulators of Fungal Secondary Metabolism in the Genus Aspergillus

“…These uncharacterized MRB mutants were cryogenically stored, awaiting a faster and more economical strategy for identification. Whole-genome sequencing has been used to identify mutant alleles in multiple model organisms (27)(28)(29), including several fungi (30)(31)(32)(33)(34)(35). Here we describe our success in utilizing next-generation sequencing of mutants using Illumina and Ion Torrent platforms to quickly and effectively identify the genetic basis of 12 of the remaining MRB mutants.…”

Diverse data supports the transition of filamentous fungal model organisms into the post-genomics era

“…Because of its importance for both enzyme and organic acid production, multiple strains of A. niger have sequenced and analysed (Pel et al 2007). The reference sequence data from these strains has been critical for development of ‘fast forward’ genetic approaches for association of genes with mutant phenotypes (Niu et al 2016a, 2016b). All of this has built upon early sharing of research tools.…”