Introduction
The filamentous fungus Monascus spp. was initially identified by the French scientist Van Tieghem in 1884 (Van Tieghem, 1884). The growth of Monascus spp. occurs within a temperature range of 26°C to 42°C, with the most favorable temperatures falling between 30°C and 35°C. Optimal pH for growth ranges from 3.5 to 5.0, showing a specific preference for lactophilic conditions(Park et al., 2017). Therefore,Monascus spp. has been extensively utilized in China since ancient times, with red yeast rice being the most prevalent form of application. Red yeast rice is typically derived from solid-state fermentation of the Monascus spp. on a rice substrate, resulting in a delightful flavor and distinctive color during the fermentation process (Zhang et al., 2023). Meanwhile, Monascu s is known to produce a range of beneficial secondary metabolites, such asMps s, Monacolin K, and γ-aminobutyric acid, during fermentation. However, it should be noted that certain strains ofMonascus may also generate the mycotoxin citrinin under specific fermentation conditions.
The nitrogen source is an indispensable element in the growth process of fungi, serving as a crucial constituent of nucleotides, amino acids, and other vital components. It exerts a profound influence on fungal growth and development, spores production capacity, as well as the synthesis of secondary metabolites(Tudzynski, 2014). Fungi possess the capacity to utilize a diverse array of nitrogen sources, albeit with varying degrees of proficiency. In environments where ammonium nitrogen and glutamine are available, fungi exhibit a preferential utilization for these nitrogen sources. Conversely, in the absence of such nitrogen sources, uptake of nitrate nitrogen, urea, uric acid, and purine-pyrimidine as alternative nitrogen sources is observed(Marzluf, 1997). In certain fungal species, the processes of response, uptake, and assimilation of nitrogen sources necessitate the involvement of multiple regulators in nitrogen metabolism. This enables the activation or inhibition of specific structural genes involved in nitrogen metabolism based on the form of available nitrogen sources to meet the organism’s demand for this essential element.(Stajić et al., 2006).
The expression of genes involved in nitrogen metabolism is subject to global regulatory mechanisms, while simultaneously being specifically regulated by individual substrates.(Qian et al., 2019). The GATA family of transcription factors serves as the primary regulators of nitrogen metabolism in fungi. These GATA transcription factors were initially discovered within the promoter region of the chicken bead protein.(Evans et al., 1988). The DNA-binding proteins, characterized by a conserved zinc-finger amino acid sequence of Cys-X2 -Cys-X17 -Cys-X2 -Cys, exhibit a strong affinity towards the (T/A) GATA (A/G) motif.(Lentjes et al., 2016). The GATA transcription factors in fungi have been extensively investigated and found to play crucial roles in fungal nitrogen metabolism, sexual and asexual development, as well as iron carrier biosynthesis(Liu et al., 2018). The yeast saccharomyces cerevisiae has identified four GATA transcription factors involved in nitrogen metabolism, namely Gatl1p and Gatl3p, which exert positive regulation on nitrogen metabolism, and Gzf3p and Dal80p, which exert negative regulation on nitrogen metabolism. (Magasanik and Kaiser, 2002) . The filamentous fungi possess only two GATA-type nitrogen regulators, namelyAreA and AreB .
The two GATA global transcriptional regulators of nitrogen metabolism in filamentous fungi, AreA and AreB , play distinct roles:AreA activates genes associated with the utilization of secondary nitrogen sources, while AreB inhibits the activation ofAreA .(Tudzynski, 2014). The AreA gene was previously investigated in Aspergillus nidulans (Hynes, 1975), thereby confirming its involvement in the regulation of nitrogen metabolism in filamentous fungi and its impact on secondary metabolites as well as pathogenicity.
The red Monsacus pigments exhibit a chemical similarity to the orange ones, differing only in the substitution of an eNHe group for the eOe group. This substitution is believed to occur through the amination reaction between NH3 units and orange pigments(Haws et al., 1959; Kumasaki et al., 1962).The water-soluble red pigment derivatives are primarily located extracellularly and are synthesized through the reaction between the orange pigment and amino acids(Jung et al., 2003; Lin and Demain, 1994). Therefore, the nitrogen source is considered a crucial regulator, and its impact on red pigment production has been extensively investigated by numerous researchers. It has been demonstrated that specific nitrogen sources significantly influence the composition of MPs . Ammonium chloride and ammonium sulfate facilitate the formation of orange or yellow pigments(Kang et al., 2014; Liang, 2009).The formation of red pigments is facilitated by nitrates and organic nitrogen sources, such as monosodium glutamate (MSG), amino acids, or yeast extracts (Jung et al., 2003; Lee et al., 2001).
In this study, the original C100 strain was used to screen out the knockout and overexpression strains by homologous recombination and co-culture with Agrobacterium tumefaciens. Subsequently, the three strains were cultured by solid state fermentation, and the products were analyzed. In this study, the expression of key genes related to growth and development and pigment synthesis was analyzed to elucidate the function of the MareA gene.