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.