LaeA controls citric acid production through regulation of a citrate exporter encodingcexAinAspergillus luchuensismut.kawachii

Abstract

21The putative methyltransferase LaeA is a global regulator of metabolic and development 22 processes in filamentous fungi. We characterized the homologous laeA genes of the 23 white koji fungus Aspergillus luchuensis mut. kawachii (A. kawachii) to determine their 24 role in the hyperproduction of citric acid. The ΔlaeA strain exhibited a significant 25 reduction in citric acid productivity. Cap-analysis gene expression (CAGE) revealed 26 that laeA is required for the gene expression of a putative citrate exporter encoding cexA, 27 which is critical for citric acid production. The deficit in the productivity of citric acid 28 by the ΔlaeA strain was rescued by the overexpression of cexA to a level comparable 29 with that of the cexA-overexpressing ΔcexA strain. In addition, chromatin 30 immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) analysis indicated 31 that LaeA regulates the expression of cexA via methylation levels of the histones H3K4 32 and H3K9. These results indicate that LaeA is involved in citric acid production through 33 epigenetic regulation of cexA in A. kawachii. 34 35 IMPORTANCE 36 A. kawachii has been traditionally used for production of the distilled spirit shochu in 37 Japan. Citric acid produced by A. kawachii plays an important role in preventing 38 microbial contamination during the shochu fermentation process. This study 39 characterized homologous laeA genes; using CAGE, complementation test, and 40 ChIP-qPCR, it was found that laeA is required for citric acid production though 41 regulation of cexA in A. kawachii. The epigenetic regulation of citrate production 42 elucidated in this study will be useful for controlling the fermentation processes of 43 shochu. 44 45 KEYWORDS: Aspergillus luchuensis mut. kawachii; citric acid; putative 46 methyltransferase; laeA; citrate exporter; cexA 47 48 3Shochu is a traditional Japanese distilled spirit (1). The black koji fungus Aspergillus 49 luchuensis and its albino mutant white koji fungus Aspergillus luchuensis mut. kawachii 50 (A. kawachii) are primarily used for the production of shochu. A. luchuensis and A. 51 kawachii produce enzymes that decompose the starch contained in ingredients such as 52 rice, barley, buckwheat, and sweet potato (2). In addition, they also produce a large 53 amount of citric acid during the fermentation process to prevent the microbial 54 contamination. 55A. luchuensis is phylogenetically related to A. niger, which has been used for 56 industrial citric acid fermentation (3-5). Studies investigating the production of citric 57 acid have been performed in A. niger with respect to various aspects (6-8). In previous 58 studies, non-acidifying mutant strains of A. niger were analyzed and the laeA gene was 59 found to play a significant role in the production of citric acid and secondary 60 metabolites (9). In addition, laeA disruption also caused a significant decrease in the 61 citric acid production by Aspergillus carbonarius, a species closely related to A. niger 62 (10). LaeA was initially...