Expression Vectors and Gene Fusions for the Directed Modification of the Carotenoid Biosynthesis Pathway in Mucor circinelloides.

Several fungal species, particularly some included in the Mucoromycotina, have been used to develop fermentation processes for the production of ß-carotene. Oxygenated derivatives of ß-carotene (xanthophylls) are desirable value-added products, and the preference by the market of carotenoids from biological sources has increased the research in different carotenoid-producing organisms. We currently use Mucor circinelloides f. lusitanicus as a model organism to develop strains with an increased content of new and more valuable carotenoids. The main carotenoid accumulated by M. circinelloides is ß-carotene, although it has some hydroxylase activity and produces low amounts of zeaxanthin. On the other hand, in astaxanthin-producing organisms two enzymatic activities are required for the production of astaxanthin from ß-carotene: a hydroxylase and a ketolase. In this chapter, we delineate part of our efforts to construct genetically modified strains that could advance in the improvement of carotenoid accumulation by this fungus and the diversification of its carotenoid content. Accordingly, we describe detailed and empirically tested protocols for the construction of functional expression vectors and gene fusions.

A Ras GTPase associated protein is involved in the phototropic and circadian photobiology responses in fungi.

Light is an environmental signal perceived by most eukaryotic organisms and that can have major impacts on their growth and development. The MadC protein in the fungus Phycomyces blakesleeanus (Mucoromycotina) has been postulated to form part of the photosensory input for phototropism of the fruiting body sporangiophores, but the madC gene has remained unidentified since the 1960s when madC mutants were first isolated. In this study the madC gene was identified by positional cloning. All madC mutant strains contain loss-of-function point mutations within a gene predicted to encode a GTPase activating protein (GAP) for Ras. The madC gene complements the Saccharomyces cerevisiae Ras-GAP ira1 mutant and the encoded MadC protein interacts with P. blakesleeanus Ras homologs in yeast two-hybrid assays, indicating that MadC is a regulator of Ras signaling. Deletion of the homolog in the filamentous ascomycete Neurospora crassa affects the circadian clock output, yielding a pattern of asexual conidiation similar to a ras-1 mutant that is used in circadian studies in N. crassa. Thus, MadC is unlikely to be a photosensor, yet is a fundamental link in the photoresponses from blue light perceived by the conserved White Collar complex with Ras signaling in two distantly-related filamentous fungal species.

Expansion of Signal Transduction Pathways in Fungi by Extensive Genome Duplication.

Plants and fungi use light and other signals to regulate development, growth, and metabolism. The fruiting bodies of the fungus Phycomyces blakesleeanus are single cells that react to environmental cues, including light, but the mechanisms are largely unknown [1]. The related fungus Mucor circinelloides is an opportunistic human pathogen that changes its mode of growth upon receipt of signals from the environment to facilitate pathogenesis [2]. Understanding how these organisms respond to environmental cues should provide insights into the mechanisms of sensory perception and signal transduction by a single eukaryotic cell, and their role in pathogenesis. We sequenced the genomes of P. blakesleeanus and M. circinelloides and show that they have been shaped by an extensive genome duplication or, most likely, a whole-genome duplication (WGD), which is rarely observed in fungi [3-6]. We show that the genome duplication has expanded gene families, including those involved in signal transduction, and that duplicated genes have specialized, as evidenced by differences in their regulation by light. The transcriptional response to light varies with the developmental stage and is still observed in a photoreceptor mutant of P. blakesleeanus. A phototropic mutant of P. blakesleeanus with a heterozygous mutation in the photoreceptor gene madA demonstrates that photosensor dosage is important for the magnitude of signal transduction. We conclude that the genome duplication provided the means to improve signal transduction for enhanced perception of environmental signals. Our results will help to understand the role of genome dynamics in the evolution of sensory perception in eukaryotes.

Bcmimp1, a Botrytis cinerea Gene Transiently Expressed in planta, Encodes a Mitochondrial Protein.

Botrytis cinerea is a widespread necrotrophic fungus which infects more than 200 plant species. In an attempt to characterize the physiological status of the fungus in planta and to identify genetic factors contributing to its ability to infect the host cells, a differential gene expression analysis during the interaction B. cinerea-tomato was carried out. Gene Bcmimp1 codes for a mRNA detected by differential display in the course of this analysis. During the interaction with the host, it shows a transient expression pattern with maximal expression levels during the colonization and maceration of the infected tissues. Bioinformatic analysis suggested that BCMIMP1 is an integral membrane protein located in the mitochondrial inner membrane. Co-localization experiments with a BCMIMP1-GFP fusion protein confirmed that the protein is targeted to the mitochondria. ΔBcmimp1 mutants do not show obvious phenotypic differences during saprophytic growth and their infection ability was unaltered as compared to the wild-type. Interestingly, the mutants produced increased levels of reactive oxygen species, likely as a consequence of disturbed mitochondrial function. Although Bcmimp1 expression is enhanced in planta it cannot be considered a pathogenicity factor.

Fungal cryptochrome with DNA repair activity reveals an early stage in cryptochrome evolution.

DASH (Drosophila, Arabidopsis, Synechocystis, Human)-type cryptochromes (cry-DASH) belong to a family of flavoproteins acting as repair enzymes for UV-B-induced DNA lesions (photolyases) or as UV-A/blue light photoreceptors (cryptochromes). They are present in plants, bacteria, various vertebrates, and fungi and were originally considered as sensory photoreceptors because of their incapability to repair cyclobutane pyrimidine dimer (CPD) lesions in duplex DNA. However, cry-DASH can repair CPDs in single-stranded DNA, but their role in DNA repair in vivo remains to be clarified. The genome of the fungus Phycomyces blakesleeanus contains a single gene for a protein of the cryptochrome/photolyase family (CPF) encoding a cry-DASH, cryA, despite its ability to photoreactivate. Here, we show that cryA expression is induced by blue light in a Mad complex-dependent manner. Moreover, we demonstrate that CryA is capable of binding flavin (FAD) and methenyltetrahydrofolate (MTHF), fully complements the Escherichia coli photolyase mutant and repairs in vitro CPD lesions in single-stranded and double-stranded DNA with the same efficiency. These results support a role for Phycomyces cry-DASH as a photolyase and suggest a similar role for cry-DASH in mucoromycotina fungi.

Canthaxanthin production with modified Mucor circinelloides strains.

Canthaxanthin is a natural diketo derivative of ß-carotene primarily used by the food and feed industries. Mucor circinelloides is a ß-carotene-accumulating zygomycete fungus and one of the model organisms to study the carotenoid biosynthesis in fungi. In this study, the ß-carotene ketolase gene (crtW) of the marine bacterium Paracoccus sp. N81106 fused with fungal promoter and terminator regions was integrated into the M. circinelloides genome to construct stable canthaxanthin-producing strains. Different transformation methods including polyethylene glycol-mediated transformation with linear DNA fragments, restriction enzyme-mediated integration and Agrobacterium tumefaciens-mediated transformation were tested to integrate the crtW gene into the Mucor genome. Mitotic stability, site of integration and copy number of the transferred genes were analysed in the transformants, and several stable strains containing the crtW gene in high copy number were isolated. Carotenoid composition of selected transformants and effect of culturing conditions, such as temperature, carbon sources and application of certain additives in the culturing media, on their carotenoid content were analysed. Canthaxanthin-producing transformants were able to survive at higher growth temperature than the untransformed strain, maybe due to the effect of canthaxanthin on the membrane fluidity and integrity. With the application of glucose, trehalose, dihydroxyacetone and L-aspartic acid as sole carbon sources in minimal medium, the crtW-expressing M. circinelloides strain, MS12+pCA8lf/1, produced more than 200 µg/g (dry mass) of canthaxanthin.

Gene fusions for the directed modification of the carotenoid biosynthesis pathway in Mucor circinelloides.

Several fungal species, particularly some included in the Mucorales, have been used to develop fermentation processes for the production of ß-carotene. Oxygenated derivatives of ß-carotene are more valuable products, and the preference by the market of carotenoids from biological sources has increased the research in different carotenoid-producing organisms. We currently use Mucor circinelloides as a model organism to develop strains able to produce new, more valuable, and with an increased content of carotenoids. In this chapter we describe part of our efforts to construct active gene fusions which could advance in the diversification of carotenoid production by this fungus. The main carotenoid accumulated by M. circinelloides is ß-carotene, although it has some hydroxylase activity and produces low amounts of zeaxanthin. Two enzymatic activities are required for the production of astaxanthin from ß-carotene: a hydroxylase and a ketolase. We used the ctrW gene of Paracoccus sp. N81106, encoding a bacterial ß-carotene ketolase, to construct gene fusions with two fungal genes essential for the modification of the pathway in M. circinelloides. First we fused it to the carRP gene of M. circinelloides, which is responsible for the phytoene synthase and lycopene cyclase activities in this fungus. The expected activity of this fusion gene would be the accumulation by M. circinelloides of canthaxanthin and probably some astaxanthin. A second construction was the fusion of the crtW gene of Paracoccus sp. to the crtS gene of Xanthophyllomyces dendrorhous, responsible for the synthesis of astaxanthin from ß-carotene in this fungus, but which was shown to have only hydroxylase activity in M. circinelloides. The expected result in M. circinelloides transformants was the accumulation of astaxanthin. Here we describe a detailed and empirically tested protocol for the construction of these gene fusions.

A gene for carotene cleavage required for pheromone biosynthesis and carotene regulation in the fungus Phycomyces blakesleeanus.

Mating and sexual development in fungi are controlled by molecular mechanisms that are specific for each fungal group. Mating in Phycomyces blakesleeanus and other Mucorales requires pheromones derived from ß-carotene. Phycomyces mutants in gene carS accumulate large amounts of ß-carotene but do not enter the sexual process. We show that carS encodes a ß-carotene-cleaving oxygenase that catalyzes the first step in the biosynthesis of a variety of apocarotenoids, including those that act as pheromones. Therefore carS mutants cannot stimulate their sexual partners, although they respond to them. CarS catalyzes the biosynthesis of a ß-ring-containing apocarotenoid that inhibits the activity of the carotenogenic enzyme complex in vegetative cells and provides a feedback regulation for the ß-carotene pathway. The carS gene product is a keystone in carotenogenesis and in sexual reproduction.

Functional analysis of the Phycomyces carRA gene encoding the enzymes phytoene synthase and lycopene cyclase.

Phycomyces carRA gene encodes a protein with two domains. Domain R is characterized by red carR mutants that accumulate lycopene. Domain A is characterized by white carA mutants that do not accumulate significant amounts of carotenoids. The carRA-encoded protein was identified as the lycopene cyclase and phytoene synthase enzyme by sequence homology with other proteins. However, no direct data showing the function of this protein have been reported so far. Different Mucor circinelloides mutants altered at the phytoene synthase, the lycopene cyclase or both activities were transformed with the Phycomyces carRA gene. Fully transcribed carRA mRNA molecules were detected by Northern assays in the transformants and the correct processing of the carRA messenger was verified by RT-PCR. These results showed that Phycomyces carRA gene was correctly expressed in Mucor. Carotenoids analysis in these transformants showed the presence of ß-carotene, absent in the untransformed strains, providing functional evidence that the Phycomyces carRA gene complements the M. circinelloides mutations. Co-transformation of the carRA cDNA in E. coli with different combinations of the carotenoid structural genes from Erwinia uredovora was also performed. Newly formed carotenoids were accumulated showing that the Phycomyces CarRA protein does contain lycopene cyclase and phytoene synthase activities. The heterologous expression of the carRA gene and the functional complementation of the mentioned activities are not very efficient in E. coli. However, the simultaneous presence of both carRA and carB gene products from Phycomyces increases the efficiency of these enzymes, presumably due to an interaction mechanism.

Expression of three isoprenoid biosynthesis genes and their effects on the carotenoid production of the zygomycete Mucor circinelloides.

The zygomycete Mucor circinelloides accumulates ß-carotene as the main carotenoid compound. In this study, the applicability of some early genes of the general isoprenoid pathway to improve the carotenoid production in this fungus was examined. The isopentenyl pyrophosphate isomerase gene (ipi) was cloned and used together with the genes encoding farnesyl pyrophosphate synthase (isoA) and geranylgeranyl pyrophosphate synthase (carG) in overexpression studies. Transformation experiments showed that the first bottleneck in the pathway, from the aspect of carotenoid production, is the step controlled by the carG gene, but overexpression of the ipi and isoA genes also contributes to the availability of the precursors. Transformations with these isoprenoid genes in combination with a bacterial ß-carotene ketolase gene yielded Mucor strains producing canthaxanthin and echinenone.

Protein-DNA interactions in the promoter region of the Phycomyces carB and carRA genes correlate with the kinetics of their mRNA accumulation in response to light.

Carotene biosynthesis in Phycomyces is photoinducible and carried out by phytoene dehydrogenase (encoded by carB) and a bifunctional enzyme possessing lycopene cyclase and phytoene synthase activities (carRA). A light pulse followed by periods of darkness produced similar biphasic responses in the expression of the carB and carRA genes, indicating their coordinated regulation. Specific binding complexes were formed between the carB-carRA intergenic region and protein extracts from wild type mycelia grown in the dark or 8min after irradiation. These two conditions correspond to the points at which the expression of both genes is minimal, suggesting that these binding complexes are involved in the down-regulation of photocarotenogenesis in Phycomyces. Protein extracts from carotene mutants failed to form the dark retardation complex, suggesting a role of these genes in the regulation of photocarotenogenesis. In contrast, protein extracts from phototropic mutants formed dark retardation complexes identical to that of the wild type.

The flavohemoglobin BCFHG1 is the main NO detoxification system and confers protection against nitrosative conditions but is not a virulence factor in the fungal necrotroph Botrytis cinerea.

Flavohemoglobins constitute a group of proteins involved in the metabolism of nitric oxide (NO). Botrytis cinerea was shown to have a single flavohemoglobin coding gene, Bcfhg1. Its expression was developmentally regulated, with maximum expression levels during germination of conidia, and was enhanced very quickly upon exposure to NO of germinating conidia, but not of mycelium growing and branching actively. Expression in planta paralleled the expression pattern during saprophytic growth with maximal expression occurring during the very early stages of the infection process. Bcfhg1 complemented the Saccharomyces cerevisiae yhb1 mutation, indicating that the encoded enzyme has NO dioxygenase activity. Biochemical and functional characterization of DeltaBcfhg1 mutants in comparison with the wild type strain demonstrated that, although BCFHG1 showed a high affinity for its substrate, appeared to represent the main inducible NO detoxification system and conferred protection against nitrosative stress in B. cinerea, the ability of the DeltaBcfhg1 mutant strains to infect different hosts was not affected.

Phycomyces MADB interacts with MADA to form the primary photoreceptor complex for fungal phototropism.

The fungus Phycomyces blakesleeanus reacts to environmental signals, including light, gravity, touch, and the presence of nearby objects, by changing the speed and direction of growth of its fruiting body (sporangiophore). Phototropism, growth toward light, shares many features in fungi and plants but the molecular mechanisms remain to be fully elucidated. Phycomyces mutants with altered phototropism were isolated approximately 40 years ago and found to have mutations in the mad genes. All of the responses to light in Phycomyces require the products of the madA and madB genes. We showed that madA encodes a protein similar to the Neurospora blue-light photoreceptor, zinc-finger protein WC-1. We show here that madB encodes a protein similar to the Neurospora zinc-finger protein WC-2. MADA and MADB interact to form a complex in yeast 2-hybrid assays and when coexpressed in E. coli, providing evidence that phototropism and other responses to light are mediated by a photoresponsive transcription factor complex. The Phycomyces genome contains 3 genes similar to wc-1, and 4 genes similar to wc-2, many of which are regulated by light in a madA or madB dependent manner. We did not detect any interactions between additional WC proteins in yeast 2-hybrid assays, which suggest that MADA and MADB form the major photoreceptor complex in Phycomyces. However, the presence of multiple wc genes in Phycomyces may enable perception across a broad range of light intensities, and may provide specialized photoreceptors for distinct photoresponses.

The gene coding for a new transcription factor (ftf1) of Fusarium oxysporum is only expressed during infection of common bean.

We report the isolation and analysis of the gene encoding ftf1 (Fusarium transcription factor 1), a previously undescribed putative transcription factor from highly virulent strains of Fusarium oxysporum f.sp. phaseoli that is transcribed specifically during early stages of infection of its host common bean (Phaseolus vulgaris L.). The predicted 1080 amino acid ftf1 protein contains a Zn(II)2-Cys6 binuclear cluster DNA-binding motif. ftf1 expression during axenic growth in culture was not detected by either Northern or RT-PCR. On the contrary, in planta transcription of ftf1 is increased about 24h after plant inoculation, as detected by real-time RT-PCR. This result suggests that ftf1 has a role in the establishment of the fungus within the plant and/or the progress of the disease. Multiple copies of ftf1 are present in highly virulent strains of F. oxysporum f.sp. phaseoli.

The Phycomyces madA gene encodes a blue-light photoreceptor for phototropism and other light responses.

Phycomyces blakesleeanus is a filamentous zygomycete fungus that produces striking elongated single cells that extend up to 10 cm into the air, with each such sporangiophore supporting a sphere containing the spores for dispersal. This organism has served as a model for the detection of environmental signals as diverse as light, chemicals, touch, wind, gravity, and adjacent objects. In particular, sporangiophore growth is regulated by light, and it exhibits phototropism by bending toward near-UV and blue wavelengths and away from far-UV wavelengths in a manner that is physiologically similar to plant phototropic responses. The Phycomyces madA mutants were first isolated more than 40 years ago, and they exhibit reduced sensitivity to light. Here, we identify two (duplicated) homologs in the White Collar 1 family of blue-light photoreceptors in Phycomyces. We describe that the madA mutant strains contain point mutations in one of these genes and that these mutations cosegregate with a defect in phototropism after genetic crosses. Thus, the phototropic responses of fungi through madA and plants through phototropin rely on diverse proteins; however, these proteins share a conserved flavin-binding domain for photon detection.

Heterologous expression of astaxanthin biosynthesis genes in Mucor circinelloides.

Most Mucor species accumulate beta-carotene as the main carotenoid. The crtW and crtZ astaxanthin biosynthesis genes from Agrobacterium aurantiacum were placed under the control of Mucor circinelloides expression signals. Expression vectors containing the bacterial genes were constructed, and PEG-mediated transformations were performed on a selected M. circinelloides strain. Transformants that exhibited altered carotene production were isolated and analyzed. Southern analysis showed that all plasmids behave as autoreplicative elements. Northern analysis detected the actual heterologous transcription products, whereas thin layer chromatography and high-performance liquid chromatography studies revealed the presence of new carotenoid compounds and intermediates among the transformants.

A novel fungal prenyl diphosphate synthase in the dimorphic zygomycete Mucor circinelloides.

Two Mucor circinelloides structural genes involved in isoprenoid biosynthesis were isolated and characterised. The isoA gene encodes a typical eukaryotic farnesyl diphosphate synthase (EC 2.5.1.10), whereas the isoB gene deduced amino acid sequence shows similarity to fungal medium-chain prenyl diphosphate synthases. By functional complementation in Escherichia coli, the isoB gene product was shown to be a solanesyl diphosphate synthase (EC 2.5.1.11), which is the first fungal enzyme reported having this specificity. In addition, a M. circinelloides one-marker-per-chromosome map was completed by contour-clamped homogeneous electric field localisation of isoA, isoB and three other isoprenoid biosynthesis genes to individual chromosomes.

Expression of the carG gene, encoding geranylgeranyl pyrophosphate synthase, is up-regulated by blue light in Mucor circinelloides.

A new structural gene, carG, involved in the biosynthesis of carotenoids in the fungus Mucor circinelloides was isolated by heterologous hybridisation, using a probe derived from the Gibberella fujikuroi ggs1 gene. Functional analyses in Escherichia coli showed that the encoded protein has geranylgeranyl pyrophosphate (GGPP) synthase activity. A comparison of the deduced protein with other GGPP synthases suggested that the carG gene might have evolved from other larger genes present in some fungi. The analysis of carG mRNA accumulation after blue light irradiation showed that the expression of this gene is up-regulated by blue light, as happens with the other structural genes involved in carotenogenesis in M. circinelloides. Analysis of the promoter region revealed the presence of several APE-like sequences, which participate in the blue-light regulation of the expression of different fungal genes. These sequences are also present in the above-mentioned Mucor genes and strongly support the idea that this gene plays an important role in the regulation of carotenoid synthesis, despite belonging to a more general metabolic pathway.

A gene coding for ornithine decarboxylase (odcA) is differentially expressed during the Mucor circinelloides yeast-to-hypha transition.

The differential display technique was used to identify genes from Mucor circinelloides involved in the yeast-to-hypha transition. Using a limited set of primer combinations, cDNA fragments corresponding to mRNAs differentially expressed during the dimorphic transition were isolated. Northern analyses showed that the accumulation of the transcript detected by hybridisation with one of the cDNA fragments increased during the transition and was undetectable at the mycelial stage. Sequence analysis and database searches of this fragment revealed high similarity to ornithine decarboxylase (ODC) encoding genes. The odcA gene of M. circinelloides was isolated from genomic and cDNA libraries and characterised. Electrophoretic karyotyping and hybridisations showed that the odcA gene is single-copy and linked to the leuA and rDNA genes. The single transcript detected (2.1 kb), was considerably longer than the deduced ORF. Through non-radioactive primer extension analysis four transcription initiation sites were mapped to positions -61, -167, -239 and -436 from the start codon. The ODC mRNA levels increased during the yeast-to-hypha transition, reaching a maximum at 120 min, which was accompanied by a rise in ODC enzymatic activity. The expression pattern of the odcA gene showed that in M. circinelloides the ODC levels are transcriptionally regulated, in contrast with other dimorphic fungi in which a post-transcriptional regulation has been proposed.

Interallelic complementation provides genetic evidence for the multimeric organization of the Phycomyces blakesleeanus phytoene dehydrogenase.

The Phycomyces blakesleeanus wild-type is yellow, because it accumulates beta-carotene as the main carotenoid. A new carotenoid mutant of this fungus (A486) was isolated, after treatment with ethyl methane sulfonate (EMS), showing a whitish coloration. It accumulates large amounts of phytoene, small quantities of phytofluene, zeta-carotene and neurosporene, in decreasing amounts, and traces of beta-carotene. This phenotype indicates that it carries a leaky mutation affecting the enzyme phytoene dehydrogenase (EC 1.3.-.-), which is specified by the gene carB. Biochemical analysis of heterokaryons showed that mutant A486 complements two previously characterized carB mutants, C5 (carB10) and S442 (carB401). Sequence analysis of the carB gene genomic copy from these three strains revealed that they are all altered in the gene carB, giving information about the nature of the mutation in each carB mutant allele. The interallelic complementation provides evidence for the multimeric organization of the P. blakesleeanus phytoene dehydrogenase.