# Caroenoid Biosynthesis in _Fusarium_
## 5 Down-Regulation of Caroenoid Production: The _CarS_ Gene  
Standard mutagenesis protocols allow the identification of _Fusarium_ mutants affected in the synthesis of carotenoids, easy to detect because of their changes in pigmentation. Such mutants are more easily identified on minimal medium, such as the DG minimal agar [73], than on richer media, e.g., Potato Dextrose Agar (PDA), which frequently results in the accumulation of other unrelated pigments which may mask the color of carotenoids. Caroenoid mutants usually identified by visual inspection are either albino in the light, as those lacking an early enzymatic step of the carotenoid pathway, or deeply pigmented in the dark, affected in a down-regulating function. The deep-orange mutants, exhibiting a light-independent carotenoid production and generically called _carS_, have been described in _F. fujikuroi_[22] and _F. oxysporum_[68]. The _carS_ mutants have been investigated in detail in _F. fujikuroi_, where they accumulate an increasing amount of carotenoids with aging [55] and contain large amounts of mRNA for the structural genes, from _carRA_ to _carD_[38; 42; 48; 59] (Figure 3). This up-regulation results in an increase of carotenogenic enzymes, as deduced from the study of in vitro carotenogenesis in a cell-free system of a _carS_ mutant compared to the wild type [74]. Because of their high carotenoid content, the effect of light is less apparent in the _carS_ mutants, but expression studies have found a detectable photoinduction of the transcript levels for the structural genes. This has been found in _F. fujikuroi_[42] and in _F. oxysporum_[68], and it has been recently corroborated in _F. fujikuroi_ in RNA-seq analyses (Figure 3). The qualitative mixture of carotenoids accumulated by two _carS_ mutants of _F. fujikuroi_ obtained in different genetic backgrounds is similar to that of their corresponding wild types under light, with minor differences, mainly a lower torulene content in the _carS_ strains (Figure 4). Because of the large accumulation of carotenoids in the dark, these mutants are also useful to investigate possible damaging effects of light on the carotenoids. The comparison of the chromatograms from dark or light-grown cultures in two _carS_ mutants reveal again only minor changes, with a decrease in torulene and an increase in \(\beta\)-carotene in the samples from the illuminated cultures (Figure 4), which could be due to different sensitivities of these carotenes to light. Because of their intense pigmentation, the _carS_ mutants have been useful tools to detect mutants with qualitative alterations in the carotenoid mixtures, e.g., making possible the identification of torulene accumulating mutants [22] or mutants with alterations in substrate recognition by the CarB desaturas [30]. The _carS_ mutants have been also an optimal background to check the effect of possible carotenoid inhibitors on _Fusarium_ carotenogenesis, revealing the differential efficiency of some cyclase inhibitors that were fully active in other species [55]. The gene _carS_ was found to code for a protein of the RING finger (RF) family. The assignation was based in the identification of relevant mutations in all the _carS_ mutants analyzed so far, the deep-pigmented phenotype resulting from the targeted mutation of the gene in _F. oxysporum_[68] and the complementation of the _carS_ mutation in _F. fujikuroi_[76]. The predicted CarS protein has sequence similarity with the protein CrgA of _M. circinelloides_, whose mutation results in a similar carotenoid overproducing pattern in this species [77; 78]. The degree of similarity between the CarS and CrgA proteins is not very high, as expected for two taxonomically distant fungi, but it  
Figure 4: HPLC analysis of carotenoids produced by two different wild types of _F. fujikuroi_ and the _carS_ mutants SG39 [22] and SF116 [48]. The strains were grown for 7 days on DG minimal medium in the dark or under light (7 W m\({}^{-2}\) white light). The method for HPLC separation has been described [75].