A new regulatory mechanism controlling carotenogenesis in the fungus Mucor circinelloides as a target to generate β-carotene over-producing strains by genetic engineering

Abstract

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.

Comparative Analysis of β-Carotene Production by Mucor circinelloides Strains CBS 277.49 and WJ11 under Light and Dark Conditions

“…Similar to our findings, Zhang et al, obtained 667.3 µg/g of β-carotene accumulation in wild-type strain of CBS277.49 (MU241) upon continuous illumination, while investigating the regulatory pathway of β-carotene production. They also found a manifold increase in β-carotene production upon continuous illumination, as compared to the culture grown in dark similar to our study [7]. In another study, wild-type of M. circinelloides 277.49 produced 859 µg/g of β-carotene, using minimal agar and liquid media under light condition [32].…”

“…In the present study, two strains of M. circinelloides CBS 277.49 and WJ11 were selected for comparative study of β-carotenoid production under light and dark conditions. These strains were reported as an excellent source of lycopene, β-carotene, lipids, and other bioactive compounds [7,28]. It was previously demonstrated that lipid accumulation is correlated with an increase in carotenoid accumulation [13] so we speculated that this high lipid producing strain [29] might produce high amount of β-carotene.…”

“…The light response in M. circinelloides and P. blakesleeanus for carotenoid biosynthesis is well-known [18,23]. Transcriptional analysis of structural genes of β-carotene biosynthesis (carB and carRP) showed increased mRNA levels in response to light, in accordance with the improved carotene content [7]. In N. crassa, the most prominent carotenogenic synthesis by light was observed [24,25].…”

“…For instance, shaking cultivation of B. trispora has been accompanied with a decline in carotenoid accumulation, so surface cultivation must be employed; and the optimal growth temperature for these two fungus should never be above 25 • C [6]. Furthermore it has been suggested recently that industrial production of carotenoid using M. circinelloides could be much simpler than the complex fermentation process used for β-carotene production by B. trispora [7]. M. circinelloides is more favorable among other zygomycetes for genetic studies due to the availability of molecular tools, as well as its genomic sequence (http://www.genome.jgi.doe.gove/Mucci2.home.html).…”

Comparative genomics applied to Mucor species with different lifestyles

“…Carotenoid synthesis has been described in M. circinelloides and in particular in overproducing isolates [3,58,59]. Although the different species within the Mucoromycotina subphylum are expected to produce and accumulate large amount of carotenoids, differences might exist among species [60]. Terpenes play also an important role in flavour production which is an important trait for cheese-ripening fungi that could have been under human-directed selection in species used for cheese making [61].…”