Enrique A. Iturriaga

Blue-light regulation of phytoene dehydrogenase (carB) gene expression in Mucor circinelloides.

Abstract. The carB gene, encoding the phytoene dehydrogenase of Mucor circinelloides, was isolated by heterologous hybridisation with a probe derived from the corresponding gene of Phycomyces blakesleeanus. The cDNA and genomic copies complemented phytoene dehydrogenase defects in Escherichia coli and in carB mutants of M. circinelloides, respectively. Fluence-response curves for transcript accumulation were constructed after different blue-light pulses. The level of carB mRNA accumulation reached values up to 150-fold higher than basal levels in darkness. Several elements in the promoter of this gene resemble a consensus sequence identified in Neurospora crassa (APE) which is essential for blue-light regulation. Comparison of the available phytoene dehydrogenase sequences from plants, fungi, algae and bacteria suggests that the two known types of phytoene dehydrogenase are more closely related to each other than previously thought.

Heterologous transformation of Mucor circinelloides with the Phycomyces blakesleeanus leu1 gene.

SummaryThe leu1 gene of Phycomyces blakesleeanus was isolated within a HindIII-HindIII genomic DNA fragment by heterologous hybridization screening of a cosmid library, making use of the Mucor circinelloides leuA gene as a probe. The complete nucleotide sequence of this fragment reveals a single 2070 bp ORF with no introns, which presents at least 68% homology with that of the leuA gene. The P. blakesleeanus leu1 gene has also been expressed in the M. circinelloides mutant R7B (leu-), which was used to isolate the leuA gene by complementation. The homology with other known sequences shows that the leu1 gene encodes the P. blakesleeanus α-IPM (isopropylmalate) isomerase.

Development of a strain-specific SCAR marker for the detection of Trichoderma atroviride 11, a biological control agent against soilborne fungal plant pathogens

Abstract The genus Trichoderma includes biocontrol agents (BCAs) effective against soilborne plant pathogenic fungi. Several potentially useful strains for biological control are difficult to distinguish from other strains of Trichoderma found in the field. So, there is a need to find ways to monitor these strains when applied to natural pathosystems. We have used random amplified polymorphic DNA (RAPD) markers to estimate genetic variation among sixteen strains of the species T. asperellum, T. atroviride, T. harzianum, T. inhamatum and T. longibrachiatum previously selected as BCAs, and to obtain fingerprinting patterns. Analysis of these polymorphisms revealed four distinct groups, in agreement with previous studies. Some of the RAPD products generated were used to design specific primers. Diagnostic PCR performed using these primers specifically identify the strain T. atroviride 11, showing that DNA markers may be successfully used for identification purposes. This SCAR (sequence-characterised amplified region) marker can clearly distinguish strain 11 from other closely related Trichoderma strains.

ISOLATION AND MOLECULAR ANALYSIS OF THE OROTIDINE-5'-PHOSPHATE DECARBOXYLASE GENE (PYRG) OF PHYCOMYCES BLAKESLEEANUS

SummaryThe pyrG gene of Phycomyces was isolated from a Phycomyces genomic library, constructed in the cosmid pHS255, by hybridization with a 170 bp fragment of the pyrG gene of Aspergillus niger. This fragment includes a consensus sequence found in almost all species in which the orotidine-5′-phosphate decarboxylase (OMPdecase) gene has been sequenced. The complete nucleotide sequence of the cloned pyrG gene from Phycomyces was determined and the transcription start sites mapped. In the predicted amino acid sequence there are regions of strong homology to the equivalent genes of Saccharomyces cerevisiae, A. niger, Schizophyllum commune and Homo sapiens. Analysis of the sequence revealed the presence of two introns. The precise length and location of these introns was determined by sequencing the pyrG cDNA and comparing it with the genomic clone. Non-coding flanking regions showed obvious homology to the consensus TATA and CAAT boxes, and the polyadenylation signal “AATAAA”. The pyrG gene is the second Phycomyces gene that has been cloned and analysed. This is the first time that introns have been reported in Phycomyces.

Heterologous expression of astaxanthin biosynthesis genes in Mucor circinelloides

Most Mucor species accumulate β-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.

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

Abstract 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.

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

The zygomycete Mucor circinelloides accumulates β-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 β-carotene ketolase gene yielded Mucor strains producing canthaxanthin and echinenone.

Structure and function of the genes involved in the biosynthesis of carotenoids in the mucorales

Carotenoids are widely distributed natural pigments which are in an increasing demand by the market, due to their applications in the human food, animal feed, cosmetics, and pharmaceutical industries. Although more than 600 carotenoids have been identified in nature, only a few are industrially important (β-carotene, astaxanthin, lutein or lycopene). To date chemical processes manufacture most of the carotenoid production, but the interest for carotenoids of biological origin is growing since there is an increased public concern over the safety of artificial food colorants. Although much interest and effort has been devoted to the use of biological sources for industrially important carotenoids, only the production of biological β-carotene and astaxanthin has been reported. Among fungi, several Mucorales strains, particularlyBlakeslea trispora, have been used to develop fermentation process for the production of β-carotene on almost competitive cost-price levels. Similarly, the basidiomycetous yeastXanthophyllomyces dendrorhous (the perfect state ofPhaffia rhodozyma), has been proposed as a promising source of astaxanthin. This paper focuses on recent findings on the fungal pathways for carotenoid production, especially the structure and function of the genes involved in the biosynthesis of carotenoids in the Mucorales. An outlook of the possibilities of an increased industrial production of carotenoids, based on metabolic engineering of fungi for carotenoid content and composition, is also discussed.

Interallelic complementation at the pyrF locus and the homodimeric nature of orotate phosphoribosyltransferase (OPRTase) in Mucor circinelloides

Abstract Using 5-fluoroorotic acid (5-FOA) as a positive selection system we isolated mutants of Mucor circinelloides altered in the pyrimidine biosynthetic pathway. These mutants were found to be deficient either in orotidine-5′-monophosphate decarboxylase (OMPdecase), or in orotate phosphoribosyltransferase (OPRTase) activity. Complementation tests among mutants lacking OPRTase activity classified them into three groups, thus suggesting the possibility of interallelic complementation. To investigate this hypothesis a cDNA clone corresponding to the OPRTase-encoding gene of M. circinelloides was isolated by direct complementation of E. coli. The genomic copy transformed to prototrophy one member of each of the three classes of OPRTase-deficient mutants. We therefore concluded that they were all altered at the same locus, the pyrF locus. The corresponding alleles were cloned and sequenced. Comparisons of the amino acid sequence of M. circinelloides OPRTase with those of E. coli and S. typhimurium revealed a high degree of similarity in secondary and tertiary structure. As the two bacterial enzymes exist as dimers, a homodimeric quaternary structure of the M. circinelloides mature protein can be assumed. This would also explain the interallelic complementation between some pyrF mutants. The mutations found could affect either the active site or the structure of the dimer interface of the OPRTase.

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.