Fungal Secondary Metabolism

Definition: Fungal secondary metabolites (SMs) comprise a vast collection of compounds expendable for these organisms under laboratory conditions. They exhibit enormous chemical diversity, and usu- ally belong to four major families: terpenoids, polyketides, non-ribosomal peptides, or a combination of the last two. Their functions are very diverse and are normally associated with a greater fitness of the producing fungi in their environment, which often compete with other microorganisms or interact with host plants. Many SMs have beneficial applications, e.g., as antibiotics or medical drugs, but others, known as mycotoxins, are harmful to health

The effects of disruption of phosphoglucose isomerase gene on carbon utilisation and cellulase production in Trichoderma reesei Rut-C30

BACKGROUND: Cellulase and hemicellulase genes in the fungus Trichoderma reesei are repressed by glucose and induced by lactose. Regulation of the cellulase genes is mediated by the repressor CRE1 and the activator XYR1. T. reesei strain Rut-C30 is a hypercellulolytic mutant, obtained from the natural strain QM6a, that has a truncated version of the catabolite repressor gene, cre1. It has been previously shown that bacterial mutants lacking phosphoglucose isomerase (PGI) produce more nucleotide precursors and amino acids. PGI catalyzes the second step of glycolysis, the formation of fructose-6-P from glucose-6-P. RESULTS: We deleted the gene pgi1, encoding PGI, in the T. reesei strain Rut-C30 and we introduced the cre1 gene in a Δpgi1 mutant. Both Δpgi1 and cre1(+)Δpgi1 mutants showed a pellet-like and growth as well as morphological alterations compared with Rut-C30. None of the mutants grew in media with fructose, galactose, xylose, glycerol or lactose but they grew in media with glucose, with fructose and glucose, with galactose and fructose or with lactose and fructose. No growth was observed in media with xylose and glucose. On glucose, Δpgi1 and cre1(+)Δpgi1 mutants showed higher cellulase activity than Rut-C30 and QM6a, respectively. But in media with lactose, none of the mutants improved the production of the reference strains. The increase in the activity did not correlate with the expression of mRNA of the xylanase regulator gene, xyr1. Δpgi1 mutants were also affected in the extracellular β-galactosidase activity. Levels of mRNA of the glucose 6-phosphate dehydrogenase did not increase in Δpgi1 during growth on glucose. CONCLUSIONS: The ability to grow in media with glucose as the sole carbon source indicated that Trichoderma Δpgi1 mutants were able to use the pentose phosphate pathway. But, they did not increase the expression of gpdh. Morphological characteristics were the result of the pgi1 deletion. Deletion of pgi1 in Rut-C30 increased cellulase production, but only under repressing conditions. This increase resulted partly from the deletion itself and partly from a genetic interaction with the cre1-1 mutation. The lower cellulase activity of these mutants in media with lactose could be attributed to a reduced ability to hydrolyse this sugar but not to an effect on the expression of xyr1

Identification and regulation of fusA, The polyketide synthase gene responsible for fusarin production in Fusarium fujikuroi

Fusarins are a class of mycotoxins of the polyketide family produced by different Fusarium species, including the gibberellin- producing fungus Fusarium fujikuroi. Based on sequence comparisons between polyketide synthase (PKS) enzymes for fusarin production in other Fusarium strains, we have identified the F. fujikuroi orthologue, called fusA. The participation of fusA in fusarin biosynthesis was demonstrated by targeted mutagenesis. Fusarin production is transiently stimulated by nitrogen avail- ability in this fungus, a regulation paralleled by the fusA mRNA levels in the cell. Illumination of the cultures results in a reduc- tion of the fusarin content, an effect partially explained by a high sensitivity of these compounds to light. Mutants of the fusA gene exhibit no external phenotypic alterations, including morphology and conidiation, except for a lack of the characteristic yellow and/or orange pigmentation of fusarins. Moreover, the fusA mutants are less efficient than the wild type at degrading cel- lophane on agar cultures, a trait associated with pathogenesis functions in Fusarium oxysporum. The fusA mutants, however, are not affected in their capacities to grow on plant tissues

A novel lncRNA as a positive regulator of carotenoid biosynthesis in Fusarium

The fungi Fusarium oxysporum and Fusarium fujikuroi produce carotenoids, lipophilic terpenoid pigments of biotechnological interest, with xanthophyll neurosporaxanthin as the main end product. Their carotenoid biosynthesis is activated by light and negatively regulated by the RING-finger protein CarS. Global transcriptomic analysis identified in both species a putative 1-kb lncRNA that we call carP, referred to as Fo-carP and Ff-carP in each species, upstream to the gene carS and transcribed from the same DNA strand. Fo-carP and Ff-carP are poorly transcribed, but their RNA levels increase in carS mutants. The deletion of Fo-carP or Ff-carP in the respective species results in albino phenotypes, with strong reductions in mRNA levels of structural genes for carotenoid biosynthesis and higher mRNA content of the carS gene, which could explain the low accumulation of carotenoids. Upon alignment, Fo-carP and Ff-carP show 75-80% identity, with short insertions or deletions resulting in a lack of coincident ORFs. Moreover, none of the ORFs found in their sequences have indications of possible coding functions. We conclude that Fo-carP and Ff-carP are regulatory lncRNAs necessary for the active expression of the carotenoid genes in Fusarium through an unknown molecular mechanism, probably related to the control of carS function or expressio

Carotenoid Cleavage Oxygenases from Microbes and Photosynthetic Organisms: Features and Functions

Apocarotenoids are carotenoid-derived compounds widespread in all major taxonomic groups, where they play important roles in different physiological processes. In addition, apocarotenoids include compounds with high economic value in food and cosmetics industries. Apocarotenoid biosynthesis starts with the action of carotenoid cleavage dioxygenases (CCDs), a family of non-heme iron enzymes that catalyze the oxidative cleavage of carbon–carbon double bonds in carotenoid backbones through a similar molecular mechanism, generating aldehyde or ketone groups in the cleaving ends. From the identification of the first CCD enzyme in plants, an increasing number of CCDs have been identified in many other species, including microorganisms, proving to be a ubiquitously distributed and evolutionarily conserved enzymatic family. This review focuses on CCDs from plants, algae, fungi, and bacteria, describing recent progress in their functions and regulatory mechanisms in relation to the different roles played by the apocarotenoids in these organisms.Ministerio de Ciencia y Tecnología AGL2015-70218, BIO2013-44239-R, AGL2012-34573, BIO2012-39716 y BIO2009-11131Junta de Andalucía P07-CVI-02813 y CTS-6638Generalitat Valenciana PROMETEOII/2014/02

Relation between CarS expression and activation of carotenogenesis by stress in Fusarium fujikuroi

Fusarium fujikuroi, a model organism for secondary metabolism in fungi, produces carotenoids, terpenoid pigments with antioxidant activity. Previous results indicate that carotenoid synthesis in F. fujikuroi is stimulated by light or by different stress conditions and downregulated by a RING finger protein encoded by carS gene. Here, we have analyzed the effects of three stressors, nitrogen scarcity, heat shock, and oxidative stress. We compared them with the effect of light in the wild type, a carS mutant that overproduces carotenoids, and its complemented strain. The assayed stressors increase the synthesis of carotenoids in the three strains, but mRNA levels of structural genes of carotenogenesis, carRA and carB, are only enhanced in the presence of a functional carS gene. In the wild-type strain, the four conditions affect in different manners the mRNA levels of carS: greater in the presence of light, without significant changes in nitrogen starvation, and with patent decreases after heat shock or oxidative stress, suggesting different activation mechanisms. The spores of the carS mutant are more resistant to H2O2 than those of the wild type; however, the mutant shows a greater H2O2 sensitivity at the growth level, which may be due to the participation of CarS in the regulation of genes with catalase domains, formerly described. A possible mechanism of regulation by heat stress has been found in the alternative splicing of the intron of the carS gene, located close to its 3′ end, giving rise to the formation of a shorter protein. This action could explain the inducing effect of the heat shock, but not of the other inducing conditions, which may involve other mechanisms of action on the CarS regulator, either transcriptionally or post-transcriptionally.Ministerio de Economía y Competitividad BIO 2015–69613-RMinisterio de Ciencia e Innovación RTI 2018-101902-B-I00Junta de Andalucía P10-CTS-6638, P20-0124

Impact of the White Collar Photoreceptor WcoA on the Fusarium fujikuroi Transcriptome

The proteins of the White Collar 1 family (WC) constitute a major class of flavin photoreceptors, widely distributed in fungi, that work in cooperation with a WC 2 protein forming a regulatory complex. The WC complex was investigated in great detail in Neurospora crassa, a model fungus in photobiology studies, where it controls all its major photoresponses. The fungus Fusarium fujikuroi, a model system in the production of secondary metabolites, contains a single WC-1 gene called wcoA. The best-known light response in this fungus is the photoinduction of the synthesis of carotenoids, terpenoid pigments with antioxidant properties. Loss of WcoA in F. fujikuroi results in a drastic reduction in the mRNA levels of the carotenoid genes, and a diversity of morphological and metabolic changes, including alterations in the synthesis of several secondary metabolites, suggesting a complex regulatory role. To investigate the function of WcoA, the transcriptome of F. fujikuroi was analyzed in the dark and after 15-, 60- or 240-min illumination in a wild strain and in a formerly investigated wcoA insertional mutant. Using a threshold of four-fold change in transcript levels, 298 genes were activated and 160 were repressed in the wild strain under at least one of the light exposures. Different response patterns were observed among them, with genes exhibiting either fast, intermediate, and slow photoinduction, or intermediate or slow repression. All the fast and intermediate photoresponses, and most of the slow ones, were lost in the wcoA mutant. However, the wcoA mutation altered the expression of a much larger number of genes irrespective of illumination, reaching at least 16% of the annotated genes in this fungus. Such genes include many related to secondary metabolism, as well as others related to photobiology and other cellular functions, including the production of hydrophobins. As judged by the massive transcriptomic changes exhibited by the wcoA mutant in the dark, the results point to WcoA as a master regulatory protein in F. fujikuroi, in addition to a central function as the photoreceptor responsible for most of the transcriptional responses to light in this fungus.España Ministerio de Economía y Competitividad, grant BIO2015-69613-RMinisterio de Ciencia e Innovación , MCI, Agencia Estatal de Investigación, AEI, and Fondo Europeo de Desarrollo Regional, FEDER, grant RTI2018-101902-B-I00)España Ministerio de Economía y Competitividad, grant BIO2015-71703-RED

Comparative transcriptomic analysis unveils interactions between the regulatory CarS protein and light response in Fusarium

Background The orange pigmentation of the agar cultures of many Fusarium species is due to the production of carotenoids, terpenoid pigments whose synthesis is stimulated by light. The genes of the carotenoid pathway and their regulation have been investigated in detail in Fusarium fujikuroi. In this and other Fusarium species, such as F. oxysporum, deep-pigmented mutants affected in the gene carS, which encodes a protein of the RING-finger family, overproduce carotenoids irrespective of light. The induction of carotenogenesis by light and its deregulation in carS mutants are achieved on the transcription of the structural genes of the pathway. We have carried out global RNA-seq transcriptomics analyses to investigate the relationship between the regulatory role of CarS and the control by light in these fungi. Results The absence of a functional carS gene or the illumination exert wide effects on the transcriptome of F. fujikuroi, with predominance of genes activated over repressed and a greater functional diversity in the case of genes induced by light. The number of the latter decreases drastically in a carS mutant (1.1% vs. 4.8% in the wild-type), indicating that the deregulation produced by the carS mutation affects the light response of many genes. Moreover, approximately 27% of the genes activated at least 2-fold by light or by the carS mutation are coincident, raising to 40% for an 8-fold activation threshold. As expected, the genes with the highest changes under both regulatory conditions include those involved in carotenoid metabolism. In addition, light and CarS strongly influence the expression of some genes associated with stress responses, including three genes with catalase domains, consistent with roles in the control of oxidative stress. The effects of the CarS mutation or light in the transcriptome of F. oxysporum were partially coincident with those of F. fujikuroi, indicating the conservation of the objectives of their regulatory mechanisms. Conclusions The CarS RING finger protein down-regulates many genes whose expression is up-regulated by light in wild strains of the two investigated Fusarium species, indicating a regulatory interplay between the mechanism of action of the CarS protein and the control by light.España, Ministerio de Economía y Competitividad, project BIO2015–69613-REspaña, Junta de Andalucía project CTS-6638 CTS-66

Use of chitin and chitosan to produce new chitooligosaccharides by chitinase Chit42: enzymatic activity and structural basis of protein specificity

Background Chitinases are ubiquitous enzymes that have gained a recent biotechnological attention due to their ability to transform biological waste from chitin into valued chito-oligomers with wide agricultural, industrial or medical applications. The biological activity of these molecules is related to their size and acetylation degree. Chitinase Chit42 from Trichoderma harzianum hydrolyses chitin oligomers with a minimal of three N-acetyl-d-glucosamine (GlcNAc) units. Gene chit42 was previously characterized, and according to its sequence, the encoded protein included in the structural Glycoside Hydrolase family GH18. Results Chit42 was expressed in Pichia pastoris using fed-batch fermentation to about 3 g/L. Protein heterologously expressed showed similar biochemical properties to those expressed by the natural producer (42 kDa, optima pH 5.5–6.5 and 30–40 °C). In addition to hydrolyse colloidal chitin, this enzyme released reducing sugars from commercial chitosan of different sizes and acetylation degrees. Chit42 hydrolysed colloidal chitin at least 10-times more efficiently (defined by the kcat/Km ratio) than any of the assayed chitosan. Production of partially acetylated chitooligosaccharides was confirmed in reaction mixtures using HPAEC-PAD chromatography and mass spectrometry. Masses corresponding to (d-glucosamine)1–8-GlcNAc were identified from the hydrolysis of different substrates. Crystals from Chit42 were grown and the 3D structure determined at 1.8 Å resolution, showing the expected folding described for other GH18 chitinases, and a characteristic groove shaped substrate-binding site, able to accommodate at least six sugar units. Detailed structural analysis allows depicting the features of the Chit42 specificity, and explains the chemical nature of the partially acetylated molecules obtained from analysed substrates. Conclusions Chitinase Chit42 was expressed in a heterologous system to levels never before achieved. The enzyme produced small partially acetylated chitooligosaccharides, which have enormous biotechnological potential in medicine and food. Chit42 3D structure was characterized and analysed. Production and understanding of how the enzymes generating bioactive chito-oligomers work is essential for their biotechnological application, and paves the way for future work to take advantage of chitinolytic activities. Electronic supplementary material The online version of this article (10.1186/s12934-018-0895-x) contains supplementary material, which is available to authorized users.España, MINECO BIO2013‑48779‑ C4‑1/‑2/‑4, BIO2016‑76601‑ C3‑1/‑2/‑3