*4.3. Yield Improvement*

Yield is another key bottle neck in the way to produce fungal pigments to supply industrial demand. Yield improvement can start early in wet bench step, selecting promising species from under-studied niches. Marine environment has gained prominence in this area in recent decades. In terms of chemical structures, marine metabolites are frequently halogenated in comparison to metabolites biosynthesized by non-marine microorganisms. Halogenated fungal metabolites reach 59.2% of metabolites isolated from marine fungi and, among these metabolites, several halogenated pigments of the azaphyllone class have been reported, as penicilazaphilones D (**113**) and E (**114**) isolated from *Penicillium sclerotiorum* (Figure 13) [38,93]. It is noteworthy that fungal species isolated from marine environment can also be isolated from terrestrial sources, such as *P. sclerotiorum*, that, despite being isolated from soil, was also reported of being capable of producing halogenated derivatives (**115** and **116**) (Figure 13) [94,95].

**Figure 13.** Halogenated azaphilones produced by marine and terrestrial fungi. **113–114**: penicilazaphilones D and E; **115**: sclerotiorin; **116**: N-ethylbenzene-sclerotioramine [95,96].

Enhancement of metabolites yield can be achieved applying stressing conditions during fungal development, aiming at activating unconventional metabolic routes related to the production of substances linked to defense (biotic stress) or adaptation (abiotic stress). This technique is particularly interesting for the production of fungal pigments, since these metabolites are associated with defense against various types of abiotic stress [96]. Abiotic stress is usually caused by altering nutrients (carbon, nitrogen, minerals) and conditions (temperature, length, oxygen supply) in the culture medium, improving pigments production, although independently of directing to a single pigment color. Increase in biomass development is not a must to enhance pigments production, as optimized conditions for development of fungal biomass not necessarily guarantee maximum production of metabolite [82]. In general, in the search for better yields, both, biomass and metabolite yield should increase [97].

The relationship between fungal development and pigments secretion was reported for *T. albobiverticillius* (IBT31667). When cultured on Czapek Yeast Agar (CYA), a malt-free extract, this species produced atrorosins, pigments already reported as metabolites of *T. atroroseus* IBT 11181 [32]. Production of atrorosins by *T. atroroseus* was accomplished on a complex culture medium containing metals solution supplemented with single amino acids as the sole nitrogen source in the range of pH 4–5. In sequence, Tolborg et al. (2019) [97] demonstrated that individual amino acids as the sole nitrogen source led to high biomass production but not necessarily to high amounts of red pigment in *T. atroroseus*. Tolborg's group also reported that some amino acids can avoid the cocktail pigments phenomenon directing *T. atroroseus* to produce single atrorosins. Corroborating their work, only atrorosin S (**92**) was detected in the fermentation broth when serine was used as the sole nitrogen source. Addition of glutamic acid as a second nitrogen source induced the production of atrorosin E (**94**). Interestingly, only some aminoacids induced atrorosins biosynthesis, since individual supplementation of proline, lysine, asparagine and tryptophan as the sole nitrogen source did not result in atrorosins production by *T. atroroseus* [32]. This strain produced two new azaphilone pigments, talaralbols A and B, along with five known

azaphilone metabolites, when subjected to growth under submerged fermentation in malt extract medium (ME) (28 ◦C, 120 rpm) during 14 days [58].

Pigments production by *T. atroroseus* strain GH2 was studied in two different culture media (pH 5.0, 30 ± 2 ◦C, 200 rpm, 8 days) [98]. The first one was composed by synthetic Czapek-dox modified medium containing high levels of xylose, with and without nutrients supplementation and the second medium was composed by hydrolyzed corncob, a lignocellulosic waste. *T. atroroseus* GH2 demonstrated a significantly different response to the carbon and nitrogen composition of the culture media, with improved growth and enhanced pigments production in the hydrolyzed corncob medium without any nutrient supplementation. Therefore, *T. atroroseus* was pointed by the authors as a promising pigment-producing microorganism for economically competitive large-scale fermentation at lower cost [98].

Carbon source in the fermentation is a very major parameter to direct fungal metabolism. Parul et al. [83] demonstrated that mannitol is the best carbon source for reproduction and growth of *T. purpureogenus* strain F, but the growth is accompanied by low yield of pigment production, while sucrose causes the opposite effect. The authors correlate this fact to species and strain-specific capacity to produce specific enzymes that will dictate the fungus priorities. Under no stressing conditions and abundant carbon availability, primary metabolism is prioritized and the metabolism will be directed to biomass productions instead of secondary metabolites production [83]. In addition, the rate of carbon source depletion is also important. In large-scale industrial production, the rapid growth of the fungus occurs together with rapid decrease in the carbon source concentration. To avoid decrease of metabolite production rate, the carbon source must be constantly added to the fed-batch fermentation to guarantee a constant concentration of this substrate and, consequently, uninterrupted production of pigments [99]. In the same way, culture medium agitation and aeration ensure better distribution of nutrients and better growth, but at the expense of faster depletion of carbon sources. Therefore, agitation and aeration are factors that must be strictly controlled in industrial production [83].

Another tool to improve the yield of fungi metabolites is to create stress conditions during fungal development, thar results in activation and/or suppression of gene clusters to allow fungal adaptation and survival. Co-cultivation two fungal species is an example of stressing condition that generates metabolic responses to allow survival in multispecies environment. Oppong-Danquah et al. [100] described a specific co-cultivation gene cluster, when studying the co-culture of pigment producer fungus *Plenodomus influorescens* with *Pyrenochaeta nobilis*, where five polyketides were produced, including the yellow azaphilones spiciferinone (**117**) and 8a-hydroxy-spiciferinone (**118**) (Figure 14). The cultivation of *Trichoderma guizhouense* NJAU 4742 in the presence of *Fusarium oxysporum* cells also resulted in increase in azaphilone production, which was demonstrated experimentally by the increased activity of the gene cluster responsible for pigment production. This fungal response was drove to neutralize the high concentration of H2O2, produced as a defense mechanism during co-cultivation, since azaphilones are capable of neutralizing free radicals, especially the superoxide anion [101]. The same effect is observed in other oxidative stress conditions related to H2O2, such as fungal cultivation in the presence of the fungicides amphotericin B, miconazole and ciclopirox. The production of azaphilones increases as a survival mechanism directed to the neutralization of fungicide effects rather than a decrease in antifungal concentration [101].

**Figure 14.** Azaphilones produced by *Plenodomus influorescens* in co-cultivation with *Pyrenochaeta nobilis*: **117**: spiciferinone; **118**: 8a-hydroxy-spiciferinone [102].

Cost minimization for industrial production of azaphilones can be reduced by using agro-industrial waste as material for fungal growth, which also helps to solve the problem of pollution associated with the disposal of residues in the environment [19,98]. Liu et al. [102] used rice straw hydrolysate for pigment production by *M. purpureus* M630 but reported that this substrate and does not have the ideal carbon content required by the fungus. Although supplementation may be necessary in some cases, the use of agroindustrial residues has been reported to be economically viable also adding sustainability to the process.

As aforementioned, another approach to achieve yield improvement and consequently increase the viability of industrial production of fungal metabolites is the use of mutant strains and genetic engineering [99]. The current knowledge of the metabolic pathways and secondary metabolism precursors allow to manipulate fungi as "real industrial cell factories" [103] and take advantage of the entire pigment gene cassette to improve pigment yield [104]. In this way, Liu et al. [99] managed to knock-out a cAMP phosphodiesterase gene in *M. purpureus* HJ11, which led to the accumulation of intracellular cAMP causing a stimulating effect in secondary metabolism that resulted in 2.3-fold increase in pigment production.
