# Caroenoid Biosynthesis in _Fusarium_
## 6 Regulation by Light-Independent Factors  
Nitrogen availability controls the production of different _Fusarium_ secondary metabolites. Well-known examples are the productions of gibberellins, bikaverin, and fusarubin, induced by the absence of nitrogen through a complex regulatory network [85]. However, other metabolites, as fusarins and fusaric acid, exhibit an opposite regulatory pattern, with a higher production under excess of nitrogen [86]. Experiments with immobilized mycelia of _F. fuijkuroi_ incubated under low nitrogen conditions, to which excess nitrogen was subsequently added, revealed a higher synthesis of carotenoids under nitrogen starvation [87]. The negative effect of nitrogen was confirmed with the exchange of media between immobilized mycelia samples with different nitrogen contents, resulting in enhanced or reduced synthesis depending on the pass to low or high nitrogen conditions, respectively, indicating that the repressing effect is reversible. In a different approach, the effect of nitrogen was studied in shake cultures of wild type and _carS_ mutants. The production of carotenoids was higher in low nitrogen medium (low N/C ratio) than in high nitrogen medium (high N/C) in either of the tested strains [84]. A different set of experiments, in which cultures grown under an excess of nitrogen were transferred to a nitrogen-free solution, showed an increase of transcript levels of the structural genes _carRA_ and _carB_ after the transfer, which was accompanied by an enhanced accumulation of carotenoids. As found for photoinduction (see Section 3), the transcriptional increase was transitory, with maximal levels found in this case after more than 10 h following nitrogen deprivation. The stimulatory effect of nitrogen starvation on _F. fuijkuroi_ carotenogenesis, either at mRNA or carotenoid levels, is additive with the one produced by light [84], indicating different activating mechanisms. The regulation of carotenogenesis by nitrogen has been also described in _N. crassa_, where the mRNA levels for the _carRA_ and _carB_ orthologs _al-1_ and _al-2_ increase considerably under nitrogen limitation compared to standard nitrogen conditions, either in the wild type or in blind _wc_ mutants [88]. The regulation by nitrogen might involve control of expression at the level of chromatin structure, as indicated by the differences in histone methylation found for the genes of the _car_ cluster in a mutant of the methyltransferase KMT6 in _Fusarium graminearum_[89]. In _F. fuijkuroi_,the expression of several nitrogen-regulated clusters for secondary metabolite production correlate with H3K9 acetylation [90]. This suggests a similar mechanism in the regulation of the structural _car_ genes, which is currently under investigation. Other regulatory circuits may also influence the control of carotenoid biosynthesis. A major regulatory pathway is the one involving cAMP, produced by adenylate cyclase under the stimulation of a G\(\alpha\) unit from a heterotrimeric G protein. cAMP acts on other proteins, including protein kinases that phosphorylate target proteins to module their activity. The mutation of the adenylate cyclase gene _acyA_ in _F. Fujikuroi_ results in higher levels of carotenoids in the dark but in a reduced photoinduction [91]. The mutation affects other phenotypes, as the growth pattern or the production of other secondary metabolites. Another study in the same species, that extended the analysis to the effect of mutations of two adenylyl cyclase-stimulating G\(\alpha\) subunits and two cAMP-dependent PKs, confirmed different alterations in secondary metabolism and development [92], but in this case the effect on carotenogenesis was not investigated. Carotenogenesis may also have a regulatory connection with sexual development, as suggested by the lower carotenoid photoinduction of the mutants of the MAT1-2-1 mating-type gene of _F. verticillioides_[69]. In _N. crassa_, besides the regulation by light, the synthesis of carotenoids is coupled to condiiation in a light-independent manner [70].