A new regulatory mechanism controlling carotenogenesis in the fungus Mucor circinelloides as a target to generate β-carotene over-producing strains by genetic engineering
- Yingtong Zhang†1,
- Eusebio Navarro†2,
- José T. Cánovas-Márquez2,
- Lorena Almagro4,
- Haiqin Chen1,
- Yong Q. Chen1,
- Hao Zhang1,
- Santiago Torres-Martínez2,
- Wei Chen1, 3Email author and
- Victoriano Garre2Email author
© The Author(s) 2016
Received: 7 January 2016
Accepted: 23 May 2016
Published: 7 June 2016
Carotenoids 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.
This 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.
The 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.
β-carotene is a fat-soluble pigment that has several biological functions and great commercial value due to its diverse uses in food, pharmaceutical products, cosmetics and textiles [1–3]. Thus, the global carotenoid market value was $1.5 billion in 2014 and is expected to reach nearly $1.8 billion in 2019, with a compound annual growth rate of 3.9 % . Microbial β-carotene has attracted much attention because of its rapid generation and lack of influence from geographic location and climate. The microbial β-carotene currently produced by the industry comes from the alga Dunaliella salina  or the fungus Blakeslea trispora . B. trispora accumulates large amounts of carotenoids, as do other fungi of the subphylum mucormycotina, but the absence of molecular tools with which to manipulate its genome makes it difficult to improve carotenoid production. In contrast, the genome of Mucor circinelloides, which belongs to the same subphylum, has been sequenced (http://www.genome.jgi.doe.gov/Mucci2/Mucci2.home.html), and procedures for efficient genetic transformation, gene replacement and gene silencing by RNAi  allow improvement of carotene production by genetic manipulation.
Fungal carotenoids derive from 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) in the mevalonate pathway  and is regulated by complex genetic mechanisms. Light induction of carotenoid biosynthesis is considered to be conserved in filamentous fungi such as Neurospora crassa, Phycomyces blakesleeanus and M. circinelloides [9–11]. Transcription of structural genes for carotenoid biosynthesis increased enormously in response to light, in accordance with the improved carotene content. In the case of N. crassa, a model was established in which induction of carotenogenesis by light is mediated by the white-collar protein complex that is formed with the products of wc-1 and wc-2 genes [12, 13]. Genes homologous to wc-1 and wc-2 have been identified in several fungi, and one of the three homologs to wc-1 of M. circinelloides, mcwc-1c, was also shown to be involved in the induction of carotenogenesis by light . Deletion of mcwc-1c led to only a threefold increase in β-carotene levels, compared to the 21-fold increase in the wild-type strain after light exposure, which indicates that light-induced carotenogenesis in M. circinelloides requires mcwc-1c .
Another regulatory gene identified in M. circinelloides is crgA, which codes for a RING finger protein that was regarded as a negative regulator of carotenoid biosynthesis, because its disruption resulted in β-carotene overproduction in the dark as a result of a sharp increase of the carB and carRP mRNA levels . The presence of RING-finger zinc-binding domains, which define a family of ubiquitin ligases that mediate ubiquitylation of target proteins, suggests that CrgA acts as an E3 ubiquitin ligase. In fact, CrgA blocks the function of Mcwc-1b, a protein encoded by a second wc-1 homolog, by mono-ubiquitylation and di-ubiquitylation without degradation , which has also been observed in the regulatory process of the budding yeast transcription factor Met4 .
The crgA-mediated repression of carotenogenesis and light-induced carotenogenesis are independent regulatory pathways because the crgA mcwc-1c double mutant presented similar levels of β-carotene as the crgA single mutant in the dark but was not able to enhance the production to the same level as the crgA single mutant after illumination . The occurrence of multiple copies of wc-1 genes in mucoromycotina fungi such as M. circinelloides , P. blakesleeanus , Rhizopus oryzae  and Pilobolus crystallinus , and the existence of four transduction pathways in charge of different light responses in P. blakesleeanus, suggests that these basal fungi may have developed a more complex light regulatory system than dikaryotic fungi (ascomycetes and basiodiomycetes), which contains a single copy of the wc-1 gene [19, 20]. Moreover, the abundance of genes that regulate carotenogenesis, such as carC, carD, carS, carF and carI in P. blakesleeanus [21–24], suggests that carotene biosynthesis in M. circinelloides may be also regulated by a variety of elements.
In this report, we analysed 34 mutants in which carotenoid biosynthesis was affected to examine the regulatory mechanisms that control carotenoid biosynthesis in M. circinelloides and to generate an overproducing strain of interest for β-carotene production. Eight of these mutants lacked mutations on known structural or regulatory genes, suggesting that they contained mutations in unknown genes involved in carotenoid biosynthesis. Two of these mutants, MU206 and MU218, clearly carried mutations in regulatory genes because they accumulated greater amounts of β-carotene than the wild-type strain. Deletion of crgA in both strains produced an increase in β-carotene levels that was much greater in the crgA-null derivative of MU218, which suggests that the gene affected in MU218 is involved in a regulatory pathway other than that of crgA, whereas the gene mutated in MU206 may participate in the same regulatory pathway as crgA. Moreover, some of the overproducing strains may be of interest for industrial production of β-carotene.
Isolation of mutants affected in the regulation of carotenoid biosynthesis
The mycelium of M. circinelloides has a white-yellowish appearance in the dark because it accumulates a small amount of β-carotene. In the light, the biosynthesis of this pigment is stimulated and the mycelium becomes deep yellow. To identify new genes implicated in the regulation of carotenogenesis, mutagenized spores of the wild-type strain R7B were allowed to follow a complete vegetative cycle to permit expression of recessive mutations, since initial mutants are heterokaryons due to the multinucleate nature of R7B. The recycled spores were used to screen for colour mutants both in the dark and after exposure to light.
Mutants in carotenoid biosynthesis analysed in this work
Color in the dark
Color in the light
Mutation in carotenogenic genes
MU212, 213, 214
5′ splice site intron 1 mcwc1c
5′ splice site intron 4 mcwc1c
T308 M CarB
20 bp deletion carB
MU265, MU267, MU280
MU268, MU269, MU271, MU272
MU274, MU275, MU277, MU284
Deletion in region carB-carRP
Deletion in region carB-carRP
Analysis of carotenogenic genes in mutants
The phenotypes shown by the mutants could be due to mutations in the structural carotenogenic genes (carB and carRP) and in known (mcwc-1c)  or unknown regulatory genes, including the genes that code for putative homologs to N. crassa wc-2  and P. blakesleeanus madB . Mutations in the repressor crgA were not considered because none of the mutants showed a bright yellow colour, which is a hallmark of mutations in that gene . Thus, the carB-carRP genomic region, including coding and promoter sequences, and coding and promoter sequences (~200 bp) of mcwc-1c and the four putative mcwc-2 genes (ID78608, ID85699, ID153812 and ID157130) of the 34 colour mutants were amplified, and the polymerase chain reaction (PCR) fragments were directly sequenced without previous cloning to avoid detection of mutations produced by the PCR. The obtained sequences were compared to the corresponding wild-type alleles of the reference genome (http://www.genome.jgi.doe.gov/Mucci2/Mucci2.info.html) to identify mutations. The results of this analysis show that 25 strains presented different mutations in mcwc1c, carB and carRP, including deletions of carB-carRP, characterised by Southern blot hybridisation (data no shown), in two strains (Table 1). Several mutants carried the same mutation suggesting that they derived from an original mutant that was amplified in the spore-recycling step. Surprisingly, no mutations were found in the four mcwc-2 genes. In contrast, nine mutants showed no mutations in the analysed genes and should thus carry mutations in uncharacterised genes that are probably involved in the regulation of carotenoid biosynthesis.
Carotene content in carotenogenic mutants with uncharacterised mutations
Amount of β-carotene accumulated by mutants affected in carotenoid biosynthesis
β-carotene (μg/g dry mass)
Wild type (R7B)
8.6 ± 2.6ab
170.8 ± 11.9d
81.6 ± 9.4d
254.7 ± 24.1e
4.8 ± 0.4ab
119.7 ± 16.9c
6.0 ± 0.6ab
75.7 ± 8.1b
63.1 ± 6.3c
736.1 ± 20.3f
0.0 ± 0.0a
0.0 ± 0.0a
8.2 ± 0.2ab
165.8 ± 8.5d
5.5 ± 0.8ab
71.3 ± 11.5b
12.0 ± 2.0b
54.8 ± 5.5b
8.9 ± 1.6ab
47.2 ± 9.0b
In contrast, the white mutant MU264 showed undetectable levels of β-carotene in the dark and light (Table 2; Additional file 1: Figure S1). Special attention was dedicated to this mutant and carotenoids accumulated by this mutant were analysed by HPLC (Additional file 2: Figure S2). No carotenoids were detected in light, including phytoene, indicating that MU264 carries a mutation that blocks completely the carotenoid pathway. In addition to the mutants with low β-carotene levels, two strains, MU206 and MU218, accumulated high levels of β-carotene both in the dark and in light, suggesting that they are affected in genes involved in the regulation of carotenoid biosynthesis. The levels of β-carotene in those strains in the dark were roughly ten times higher than the levels in the wild-type strain R7B, whereas increases in light of 1.5 and 4.5 times were seen in MU206 and MU218, respectively (Table 2).
Characterisation of carotenogenic mutant genomes
The devised strategy for identification of mutations that provoked alterations in β-carotene levels was sequencing of the entire genome followed by comparison to the M. circinelloides reference genome. Although eight strains showed altered β-carotene levels, six strains (MU206, MU216, MU217, MU218, MU264 and MU286) with different pattern of β-carotene accumulation were selected for whole-genome sequencing and two (MU283 and MU285) were preserved for future analyses. In addition to the six strains with altered carotenogenesis, the genome of parental strain R7B was sequenced for use as a reference because this strain was obtained by UV light treatment and could harbour additional mutations to that in the 3-isopropylmalate dehydratase gene (leuA) gene that makes it auxotrophic for leucine .
The genome sequences of the seven genomes were obtained using SOLiD technology, and the sequences were mapped against the M. circinelloides reference genome (http://www.genome.jgi.doe.gov/Mucci2/Mucci2.info.html) to detect base substitutions and small deletions and insertion (indels). More than 99.7 % of the reference genome was successfully mapped for each sample, and the mean coverage depth over that mapped region ranged from 39.8 to 53.6× (Additional file 3: Table S1). As a proof of the quality of the genome sequences, the known single-base substitution in the leuA gene that causes a change from glutamic acid to lysine at position 220  was detected in all genomes (Additional files 4, 5: Tables S2, S3). As expected, the R7B genome contained additional mutations, which included three base substitutions that resulted in amino acid changes in a putative E3 ubiquitin-protein ligase (P111S), in a DUF1688 domain–containing protein (D331Y) and in a putative helicase-2 (S308G) (Additional files 4, 5: Tables S2, S3). In addition, 11 base substitutions were found in intergenic regions, one in the intron 1 of basic helix-loop-helix transcription factor-like protein (ID113859) and two others that did not produce amino acid changes (ID76084 and ID164535). Each of these base substitutions was present in the six mutants, except the mutation in the DUF1688 domain–containing protein and two base substitutions in intergenic regions that only appear in MU264 and MU286. The strains without those substitutions were obtained from spores treated with NTG in 1993 and kept frozen until 2009, whereas the strains with the substitutions were isolated in 2009. The genomic DNA from R7B was isolated from the strain used in 2009 and, therefore, it is possible that those changes occurred spontaneously in the R7B strain in our laboratory between 1993 and 2009. In any case, the presence of the mutation in the gene coding for the DUF1688 domain–containing protein in R7B discarded the notion that this mutation is responsible for the phenotype of the mutants.
Mutations present in the carotenogenic mutants
Scaffold 2: 2919466
Scaffold 4: 538767
Scaffold 11: 599712
Scaffold 6: 2888792
E3 ubiquitin ligase
Scaffold 2: 3372803
Scaffold 1: 2734563
Scaffold 1: 3851481
Scaffold 1: 5070681
Scaffold 1: 5222094
Scaffold 1: 5816574
Scaffold 2: 2533223
Scaffold 3: 2140181
Scaffold 3: 2748240
Scaffold 4: 2327078
Scaffold 4: 2465882
Scaffold 5: 541005
Scaffold 5: 2147324
Scaffold 6: 389132
Scaffold 6: 2797394
Scaffold 7: 408056
Scaffold 8: 1129513
Scaffold 11: 585629
Cdc4 and related F-box and WD-40 proteins
Similar to dentin sialophosphoprotein
Hypothetical protein (WD40 domain)
Hypothetical protein (PX domain)
Type IA hybrid histidine kinase
DEAD/DEAH box DNA helicase (Mer3)
Putative DNA-binding protein
Serine/threonine protein kinase
Chromodomain helicase DNA binding protein
DNA excision repair protein (Rad5)
Autophagic serine protease Alp2
Exocyst comlex component Sec10
Scaffold 1: 1190571
Scaffold 1: 3673453
Scaffold 1: 4109183
Scaffold 2: 460034
Scaffold 2: 46601146
Scaffold 3: 1851291
Scaffold 3: 4242409, 4243539
Scaffold 4: 580831
Scaffold 4: 647221
Scaffold 4: 3597815
Scaffold 5: 2893384
Scaffold 6: 218062
Scaffold 6: 835657
Scaffold 6: 2366334
Scaffold 7: 1528520
Scaffold 7: 2134543
Scaffold 8: 1828638
Scaffold 9: 869061
Scaffold 9: 965023
Scaffold 10: 586865
Mitocondrial carrier protein
Nuclear distribution protein NUDC
Related to plant expansions
E3 Ring finger ubiquitin ligase
E3 Ubiquitin ligase (HECT)
Basic-leucine zipper (bZIP) transcription factor
DnaJ domain protein
WD repeat-containing protein
Golgi SNAP receptor complex member
Thioesterase/thiol ester dehydrase-isomerase
Sec5 subunit of exocyst complex
Kinesin like protein
Iron/zinc ion transporter
Translation elongation factor EF1B
Zinc finger, PHD-type
Scaffold 1: 1206002
Scaffold 4: deletion 1887965–1893565
Scaffold 6: 2888792
Scaffold 7: 2443177
Septin family protein
Adenylyl cyclase class 3–4/guanylyl cyclase
F-box protein containing LRR
Scaffold 5: 2245854
Scaffold 1: deletion 635987–635988
Scaffold 3: deletion 4425722–4425732
Scaffold 5: 2245854
Scaffold 6: 2888792
Lipase class 3
Vacuolar sorting protein VPS1
Autophagy-related protein 9
Lipase class 3
To validate the whole-genome sequencing, affected genes from mutants with the fewest affected genes (MU206, MU264 and MU286) were amplified by PCR and sequenced. In all cases, the sequences of the amplified fragments contained the mutations identified in whole-genome sequencing (data not shown), validating the whole-genome sequencing. Moreover, those mutants were transformed with self-replicative plasmids that contained the wild-type alleles corresponding to the mutated genes, and functional complementation was not observed in any case (data not shown), which suggests either the genes in the plasmids were not efficiently expressed or that the phenotype could be provoked for mutations in putative intergenic regions. The lack of complementation is particularly relevant in the case of mutant MU206 because it showed just one base substitution in one gene and was used as a parental strain in the experiments described below.
Generation of crgA null mutants in MU206 and MU218 backgrounds
Analysis of carotenogenesis in crgA-null derivatives of MU206 and MU218
β-carotene content in crgA-null derivatives of MU206 and MU218
β-carotene(ug/g dry mass)
25.9 ± 2.6a
667.3 ± 49.1a
1481.8 ± 131.0c
2003.1 ± 146.0c
2621.0 ± 204.0f
3559.0 ± 262.5f
3001.9 ± 232.1g
4009.2 ± 206.8g
93.0 ± 16.6a
1007.8 ± 40.0ab
97.2 ± 9.9a
1230.9 ± 54.1a
1817.6 ± 139.6d
2328.6 ± 152.9cd
441.1 ± 16.1b
1094.6 ± 47.4ab
2089.8 ± 117.6e
2879.0 ± 127.6e
1931.2 ± 97.9de
2591.6 ± 154.3de
661.3 ± 32.0b
1425.1 ± 51.4b
The β-carotene content both in the dark and in light in all crgA mutants derived from MU206 (MU609, MU611 and MU612) and from MU218 (MU605 and MU606) was higher than that seen in MU221 and the prototrophic MU206 strain (MU613). In contrast, the transformants that carried an ectopic integration of the replacement fragment (MU610 for MU206 and MU607 for MU218) showed β-carotene levels similar to those of the corresponding prototrophic strains (Table 4). Although the simultaneous presence of mutations in MU206 or MU218 and crgA deletion produced an increase in the β-carotene content compared to MU221, the effect was much greater with an MU218 background (Table 4).
New regulatory elements in the control of M. circinelloides carotenoid biosynthesis
Several partially characterised regulatory networks control carotenogenesis in fungi that belong to the mucoromycotina subphylum. In P. blakesleeanus, the biosynthesis of carotenoid biosynthesis is regulated by a complex regulatory network triggered with several environmental factors (blue light, sexual interaction, retinol and aromatic chemicals) that act in independent ways . In M. circinelloides, two regulatory pathways have been identified that control different aspects of carotenoid biosynthesis . Thus, the induction of carotenoid biosynthesis by light is mediated by the gene mcwc-1c , whereas the basal levels of carotenoids are under the control of a different regulatory pathway in which crgA and mcwc-1b are the main actors . However, there should be an additional unknown regulatory mechanism, as suggested by the ability of light to stimulate carotene biosynthesis in mcwc-1c–null strains, although the effect is very weak .
The results of this work support the existence of additional regulatory mechanisms, because eight mutants were isolated that showed altered β-carotene levels even though they lacked mutations in the genes of the two previously known regulatory pathways. Five of the mutants (MU216, MU217, MU283, MU285 and MU286) showed reduced β-carotene levels only in light, which suggests that they are affected by a regulatory pathway involved in the induction by light of carotenoid biosynthesis. However, the absence of common mutations in the sequenced genomes of three of these mutants (MU216, MU217 and MU286) points to the existence of more than one regulatory pathway, although the notion that they carry mutations in different elements of the same pathway, even the mcwc-1c regulatory pathway , cannot be dismissed. The other three mutants (MU206, MU218 and MU264) showed alterations of β-carotene both in the dark and in light, although MU206 and MU218 retained their ability to respond to light, which indicates that they carry mutations in genes involved in mechanisms that control the basal carotenoid levels but not its induction by light. Interestingly, MU264 showed a complete blockage of carotenoid synthesis suggesting it defines a pivotal regulatory pathway that controls carotene biosynthesis. Moreover, the absence of shared mutations amongst these mutants suggests that they define either distinct regulatory pathways or different elements of the same pathway. In fact, the genes affected in these mutants, particularly mutants with high β-carotene levels (MU206 and MU218), could code for proteins that participate in the crgA regulatory pathway. The crgA gene represses carotenoid biosynthesis by proteolysis-independent mono-ubiquitilation and di-ubiquitilation of Mcwc-1b, which activates transcription of the structural carotenogenic genes carB and carRP when it is non-ubiquitilated . As a result, deletion of crgA led to significant improvement in carotene accumulation even though carotenogenesis was still induced by light . The β-carotene content in the mutants MU206 and MU218 somehow resembled the phenotype of the crgA-null mutant MU221, which suggests that they could be affected in genes that participate in the same regulatory pathway as the crgA gene. In fact, MU206 and MU218 also showed higher mRNA levels of the structural carotenogenic genes than the wild-type strain (Fig. 3), which suggests that the mutations in these mutants increase the transcription of structural carotenogenic genes in a fashion similar to that of loss-of-function mutations of crgA .
To gain insight into the underlying regulatory mechanism altered in mutants MU206 and MU218 and its relationship with the crgA gene, this gene was disrupted in both MU206 and MU218. Disruption of the crgA gene in those mutants resulted in enhancement of both the mRNA levels of carotenogenic genes (carB and carRP) and the β-carotene content in comparison to the recipient strains. Moreover, both MU206-derived and MU218-derived mutants were able to increase both mRNA levels of carotenogenic genes and β-carotene content in response to light, which indicates that the regulatory mechanism that controls induction by light was operating in both types of mutants. However, the expression enhancement of carotenogenic genes in the MU218-derived mutants was much greater than that in the MU206-derived mutants both in the dark and in light, and it was linked to significantly greater accumulation of β-carotene. In fact, the mRNA levels of carotenogenic genes in the crgA-null mutants derived from MU218 greatly exceeded those shown by MU221 both in the dark and in light (Table 4). Even more interesting was that they doubled the amount of β-carotene present in MU221 both in the dark and in light. These results suggest that some of the mutations in MU218 affect one or some of the genes that control the expression of carotenoid genes independently of crgA, thus revealing the existence of an additional mechanism involved in the regulation of carotenoid biosynthesis in M. circinelloides. This mechanism should repress the expression of carotenoid structural genes to a lesser extent than crgA because mRNA levels of carotenogenic genes and carotenoid content are much lower in MU218 than in MU221, particularly in the dark, where there is no interference by light stimulation (Table 4). Therefore, these two regulatory mechanisms may work in parallel, and the mutants for both mechanisms showed consequently higher expression of carotenogenic structural genes and β-carotene content than mutants for just one mechanism. Unfortunately, the presence in MU218 of mutations in several genes renders analysis of the identified regulatory mechanism impossible at this time. Further work is necessary to identify the gene responsible for the phenotype of MU218 and to understand the manner in which it regulates carotenoid biosynthesis.
In contrast, although crgA mutants derived from MU206 also showed higher levels of both mRNAs of carotenogenics genes and β-carotene than MU221 both in the dark and in light, the increases were much more discrete than those seen in the crgA mutants derived from MU218 (Fig. 3; Table 4). Although the existence of a fourth regulatory pathway in the control of carotenogenesis, which is affected in MU206, cannot be discarded, it is more probable that the gene responsible for the MU206 phenotype participates in the crgA regulatory pathway. This idea is supported not only by the discrete increase in expression of carotenogenic genes and carotene content observed in the crgA mutants derived from MU206 over MU221, but also by the similarity in the patterns of β-carotene accumulation shown by MU206 and MU221 (Table 4). Therefore, it is tempting to speculate that MU206 carries a mutation in a gene that acts upstream of crgA or participates with crgA in the repression of carotenogenic structural genes. Further experiments are required to identify the gene responsible for the phenotype of MU206 and understand the relationship with crgA in the regulation of carotenogenesis.
A putative candidate for industrial production of β-carotene
As one of the essential pigments, β-carotene is the precursor of vitamin A, retinal and retinoic acid, which are of critical importance in vision, nutrition and cellular growth [31–33]. β-carotene biosynthesis has been described extensively in the zygomycetes B. trispora, P. blakesleeanus and M. circinelloides, which are promising organisms for industrial production. Compared to the other two fungi, M. circinelloides could be an interesting alternative as a biotechnological β-carotene source because of the efficient tools available for genetic manipulation. This work represents a proof of concept of this idea because the disruption of the crgA gene in mutants (MU206 and MU218) that are affected in the regulation of carotene biosynthesis generated several β-carotene–overaccumulating strains. Amongst these strains, the largest producer of β-carotene, MU606, accumulated 3001.9 ± 103.7 and 4009.23 ± 180.4 μg/g β-carotene of dry mass in the dark and in light, respectively. Therefore, it yielded approximately five and two times more β-carotene than the wild-type strain and MU221. Despite these increases in β-carotene levels, the amount of β-carotene in this strain is far from the main current industrial source of β-carotene , B. trispora, which is capable of producing 30 mg/g  by mixing two strains of opposite mating types in an optimised enrichment medium. Industrial β-carotene production using M. circinelloides could be simpler than the complicated fermentation process used with B. trispora. In addition, the yeast-like morphology of this dimorphic fungus facilitates biotechnological production . Therefore, the β-carotene–overaccumulating strain obtained in this study is a strong putative candidate for industrial production of β-carotene because additional genetic manipulation or optimisation of growth conditions could generate improved strains comparable to industrial strains of B. trispora.
In conclusion, we adopted a classical forward strategy combined with modern reverse genetic techniques to deepen the regulation of carotenoid biosynthesis and the generation of strong candidate strains of industrial interest for β-carotene production. The results suggest the existence of a new regulatory mechanism that represses carotenoid biosynthesis independently and in parallel to the previously known repressor crgA. Like crgA, it acts at the level of mRNA accumulation keeping the transcript levels of carotenogenic genes low. Although the key gene involved in this new regulatory mechanism is pending on determination, a set of candidates has been identified by whole-genome sequencing. In addition, a double mutant for crgA and the new identified regulatory element was capable of accumulating up to 4 mg/g of β-carotene, being a candidate to be target of further improvements for biotechnological β-carotene production.
Strains, plasmids and transformation conditions
The strains generated in this work were derived from R7B, which is a leucine auxotroph of M. circinelloides f. lusitanicus CBS277.49  that keeps a wild-type phenotype for carotenoid biosynthesis . Spores of this strain were mutagenized with UV light (50 mJ/cm2) or NTG in conditions that produced a 1–5 % survival rate as previously described . Due to the multinucleate state of R7B spores, spores were allowed to complete a vegetative cycle in complete medium to unveil the expression of recessive mutations. To reduce the probability to isolate the same mutant several times, spores to be mutagenized were split in different tubes and kept separately after recycling.
The strains MU241 and MU221 were also derived from R7B; MU221 is a crgA-null mutant, and MU241 is a prototrophic wild-type strain after replacement of the previous leuA mutant allele with the wild-type allele . Plasmid pLEU4 , which is an auto-replicative plasmid that harbours a wild-type leuA allele, was used to complement the leuA auxotrophy of the recipient strains. Plasmid pVEN172  that contains a 6.5-kb PstI fragment of the wild-type allele of leuA gene flanked by DNA regions adjacent to the crgA gene was used to disrupt the crgA gene of MU206 and MU218. Cultures were grown in minimal medium (YNB)  or complete medium (YPG) . l-leucine (20 μg/ml) was supplied when required. The transformation procedure has been described previously .
Nucleic acid manipulation and analysis
Genomic DNA of M. circinelloides was prepared as described previously . Plasmid was isolated by the rapid boiling method  and purified following the manufacturer’s instructions (Thermo Scientific, USA). The presence of mutations in carotenogenic genes in the mutants was analysed by PCR amplification of carB, carRP, mcwc-1c and mcwc-2 genes (ID78608, ID85699, ID153812 and ID157130) using the primers listed in Additional file 7: Table S5 and PfuUltra II Fusion HS DNA Polymerase (Agilent). The PCR fragments were directly sequenced without cloning to avoid interference with mutations produced during PCR, and the obtained sequence was compared to the reference genome of M. circinelloides CBS277.49 (http://www.genome.jgi.doe.gov/Mucci2/Mucci2.info.html).
Linear DNA fragments for transformation were released by treating the plasmid pVEN172 with PstI, and then purified from agarose gel with GeneJET Gel Extraction Kit (Thermo Scientific, USA). To determine whether the fragment was integrated into the genome correctly, PCR amplification in an “Eppendorf Mastercycler personal” was performed with genomic DNA as a template and Herculase II fusion DNA polymerase (Agilent Technologies, USA) following the manufacturer’s instructions. Primers cigC-F1 and crgA-R1 were designed for amplification of the recombinant fragment (Additional file 7: Table S5). The samples were incubated at 95 °C for 3 min for pre-denaturation and subjected to 30 cycles of denaturation (95 °C for 30 s), annealing (55 °C for 30 s) and extension (72 °C for 3 min 30 s) with an additional 5-min extension step.
The transcription level of the structural genes carB and carRP was analysed with the Droplet Digital PCR technique (ddPCR). Total RNA was isolated using Trizol reagent following the instructions of the manufacturer (Invitrogen). Reverse transcription was performed with PrimeScript RT Reagent Kit with gDNA Eraser (Takara), and the ddPCR experiments were carried out with QX200™ ddPCR™ EvaGreen Supermix (BioRad). The PCR reaction mix was partitioned into water-in-oil emulsion droplets with a QX100 Droplet Generator. The droplets were then transferred to a 96-well PCR plate and put into a thermal cycler (Bio Rad C1000). Thermal cycling conditions were [95 °C × 5 min; 40 cycles of (95 °C × 0.5 min, 60 °C × 1 min); 4 °C × 5 min; 98 °C × 5 min (ramp rate set to 2 °C/s)]. The PCR plate was then moved to the QX100 Droplet reader for automatic reading of samples. The copy numbers of carB and carRP genes from various samples were normalised to the actin reference gene (ID157034; scaffold_07: 2052804–2054242).
For whole-genome sequencing of the DNA of carotenogenic mutants, genomic DNA was isolated by cesium chloride gradient , sequenced using SOLiD technology and analysed at the Centre for Genomic Research in the University of Liverpool. A sample of mutations was validated by direct sequencing of PCR products from mutated genes in strains MU206, MU264 and MU286 using specific primers (Additional file 7: Table S5). The sequence reads have been deposited in the European Nucleotide Archive and the accession number is PRJEB13169.
Analysis of carotenes
Carotenes were extracted from mycelia grown on YNB solid medium or YNB supplemented with leucine for leucine auxotrophic strains, for 84 h in the dark and 60 h in the dark followed by 24 h in light at 26 °C. The mycelia were washed and dried between paper towels and triturated with a pestle in the presence of liquid nitrogen. After lyophilisation, 2 mg of the freeze-dried mycelium powder was weighed, suspended in 2 ml of methanol and extracted several times with 2 ml of petroleum until the mycelium was colourless . For spectrophotometric quantification of β-carotene, the absorption coefficients given by Davies  were applied.
To HPLC analysis of the carotenoids, the organic phase extracted as described above were mixed and evaporated at 30 °C in vacuum. All samples were suspended in 500 µL ethyl acetate and filtered (0.22 μm) prior to HPLC with diode-array detection HPLC–DAD analysis. Carotenoids were identified and quantified using a column ZORBAX Eclipse XDB-C18 end capped 5 μm, 4.6 × 150 mm reverse phase column (Agilent Technologies, USA). The eluents used were (A) acetonitrile: water (9:1, v/v) and (B) ethyl acetate. The column separation was allowed via a series of gradient such as follows: 0–40 % solvent B (0–20 min), 40–60 % solvent B (20–25 min), 60–100 % solvent B (25–25, 1 min), 100 % solvent B (25, 1–35 min) and 100–0 % solvent B (35–50 min) at a flow rate of 1 ml/min. The injection volume was 15 μl each one. Detection of individual carotenoids was made at 284 and 453 nm, which are the wavelength of maximum absorption of phytoene and β-carotene. These compounds were identified by co-chromatography with standards and by elucidation of their spectral characteristics using a photo-diode array detector. The concentrations of carotenoids were estimated on basis of an adequated standard curve using their respective standard compounds .
All experiments were carried out independently at least three times, and the mean values ± SD were presented. Statistical analysis was performed with one-way analysis of variance with SPSS 20.
YZ participated in knocking out crgA gene, isolation mutants, carotene content analysis of mutants and the manuscript writing. EN participated in the isolation of carotenogenic mutants, performed their phenotypic characterization, analyzed their genomic sequences, validated the mutations and supervised the complementation experiments. JTCM performed the complementation experiments. LA participated in the HPLC analysis of carotenoids. HC participated in analysis of carotene content. YQC and HZ helped conceive and coordinate the study. STM participated in the isolation of carotenogenic mutants. WC supervised the experiments. VG designed and supervised the experiments and participated in writing of the manuscript. All authors read and approved the final manuscript.
We thank J. A. Madrid for technical assistance. This work was funded by the Fundación Séneca (Comunidad Autónoma de la Región de Murcia, Spain), Project no. 08802/PI/08, the Program for New Century Excellent Talents (NCET-13-0831), the Program for Changjiang Scholars and Innovative Research Team in University (IRT1249).
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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