# Production of Fungal Pigments: Molecular Processes and Their Applications
## 2 Pigmented Fungi and Biosynthesis of Pigments
### Azaphilones
The ensuing step is catalyzed by a ketoreductase (coded by _mppA_), allowing the formation of the corresponding alcohol, namely FK17-P2a, the first stable intermediate in the biosynthetic pathway. FK17-P2a is subsequently oxidized by FAD-dependent monooxygenase (coded by _mppF_) to generate azanigerone E, giving rise to the pyranoquinone skeleton, the so-called polyketide chromophore for azaphilone biosynthesis. In parallel to the above-stated polyketide synthase pathway, MpFas2, the canonical fatty acid synthetase, is also involved in the biosynthesis of MonAzps. MpFas2 has been described to generate short-chain 3-oxo-fatty acyl thioesters, the side chain moiety for _Monascus_ azaphilones. The _M. purpureus_ biosynthetic gene cluster harbors an O-acyl transferase (coded by _mppB_), which is proposed as the catalyst for the transfer of the MpFas2 products (the side-chain fatty acyl moiety) to the C-4 hydroxyl group. Further steps result in the production of the classical yellow pigments monascin and anakfalavin, as well as orange pigments rubropunctatin and monascorubrin (Figure 4). Together, the main pathway of _Monascus_ azaphilone biosynthesis contributes dominantly to the above-described four kinds of classical azaphilone pigments. However, the pyran rings of the classical orange pigments rubropunctatin and monascorubrin are vulnerable to spontaneous O-to-N substitution by readily reacting with endogenous amines to yield the \(\gamma\)-vinyllogous pyridines of the red pigments termed rubropunctamine and monascorubramine [80]. Comparative transcriptome studies with _M. purpureus_ YY-1 have found that carbon starvation stress, from the use of relatively low-quality carbon sources, might result in high pigment production [78]. The above-mentioned phenomenon was attributed to the suppression of the central carbon metabolism and enhancement of the acetyl-CoA pool, which would provide important substrate to produce azaphilone pigments. Two different carbon sources, soluble starch and glycerol, have been investigated in _M. purpureus_ FAFU618 for their effects on production of MonAzPs [81]. The production of intracellular and extracellular pigments apparently differed between the soluble starch group (SSG) and glycerol group (GCG). Moreover, the analyses of intracellular pigments determined by UPLC-QTOF-MS/MS showed significant increases of monascin and anakfalavin in the GCG and the elevation of rubropunctatin and monascorubrin in the SSG. The expression levels of MonAzP biosynthetic genes revealed by RT-qPCR demonstrated that genes _mppA_, _mppC_,  
Figure 4: Schematic diagram of various azaphilone pigment biosynthesis via polyketide synthase (PKS) pathway and fatty acid synthase (FAS) pathway in _Monascus purpureus_. _mpppR1_, and _mppR2_ were down-regulated, while _MpPKS5_, _MpFasA2_, _MpFasB2_, _mppB_, _mppD_, and _mppE_ were up-regulated. Meanwhile, the study found 27 differentially expressed proteins of mycelia between SSG and GCG, among which 18 proteins were upregulated, and these proteins were associated with the glycolytic pathway, translation, energy generation, and proteolysis. The remaining nine proteins were downregulated in GCG, and they include ribosomal proteins, heat shock proteins (HSPs), and others. Taken together, their investigation strongly suggests that the control of MonAzPs production is not only closely linked to translational levels of key proteins (enzymes) in the polyketide biosynthetic routes but also tightly associated with the levels of primary metabolism-generated molecules serving as substrates for polyketide synthesis [81].