BackgroundCarotenoids are natural pigments with antioxidant properties that have important functions in human physiology and must be supplied through the diet. They also have important industrial applications as food colourants, animal feed additives and nutraceuticals. Some of them, such as β-carotene, are produced on an industrial scale with the use of microorganisms, including fungi. The mucoral Blakeslea trispora is used by the industry to produce β-carotene, although optimisation of production by molecular genetic engineering is unfeasible. However, the phylogenetically closely related Mucor circinelloides, which is also able to accumulate β-carotene, possesses a vast collection of genetic tools with which to manipulate its genome.ResultsThis work combines classical forward and modern reverse genetic techniques to deepen the regulation of carotenoid synthesis and generate candidate strains for biotechnological production of β-carotene. Mutagenesis followed by screening for mutants with altered colour in the dark and/or in light led to the isolation of 26 mutants that, together with eight previously isolated mutants, have been analysed in this work. Although most of the mutants harboured mutations in known structural and regulatory carotenogenic genes, eight of them lacked mutations in those genes. Whole-genome sequencing of six of these strains revealed the presence of many mutations throughout their genomes, which makes identification of the mutation that produced the phenotype difficult. However, deletion of the crgA gene, a well-known repressor of carotenoid biosynthesis in M. circinelloides, in two mutants (MU206 and MU218) with high levels of β-carotene resulted in a further increase in β-carotene content to differing extents with respect to the crgA single-null strain; in particular, one strain derived from MU218 was able to accumulate up to 4 mg/g of β-carotene. The additive effect of crgA deletion and the mutations present in MU218 suggests the existence of a previously unknown regulatory mechanism that represses carotenoid biosynthesis independently and in parallel to crgA.ConclusionsThe use of a mucoral model such as M. circinelloides can allow the identification of the regulatory mechanisms that control carotenoid biosynthesis, which can then be manipulated to generate tailored strains of biotechnological interest. Mutants in the repressor crgA and in the newly identified regulatory mechanism generated in this work accumulate high levels of β-carotene and are candidates for further improvements in biotechnological β-carotene production.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0493-8) contains supplementary material, which is available to authorized users.
Based on the newly-released genomic data of Mucor circinelloides CBS 277.49, we have annotated five genes encoding for malic enzyme: all code for proteins that contain conserved domains/motifs for malic acid binding, NAD(+) binding and NAD(P)(+) binding. Phylogenetic analysis for malic enzyme genes showed that genes ID 78524 and 11639 share ~80% amino acid identity and are grouped in cluster 1; genes ID 182779, 186772 and 116127 share ~66% amino acid identity are grouped in cluster 2. Genes ID 78524, 11639 and 166127 produce proteins that are localized in the mitochondrion, while the products from genes 182779 and 186772 are localized in the cytosol. Based on the comparative analysis published previously by Song et al. (Microbiology 147:1507-1515, 2001), we propose that malic enzyme genes ID 78524, 166127, 182779, 186772, 11639, respectively, represent protein isoforms I, II, III/IV, V, and VI.
A lycopene-overaccumulating strain of the fungus M. circinelloides was generated that could be an alternative for the industrial production of lycopene. Vital catalytic residues for lycopene cyclase activity and the potential mechanism of lycopene formation and accumulation were identified.
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