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author:("magro, Lorena")
1.  A new regulatory mechanism controlling carotenogenesis in the fungus Mucor circinelloides as a target to generate β-carotene over-producing strains by genetic engineering 
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.
Electronic supplementary material
The online version of this article (doi:10.1186/s12934-016-0493-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4897934  PMID: 27266994
β-carotene; Carotenogenesis regulation; Whole-genome sequencing; Knockout mutants; crgA
2.  Dissecting the Transcriptional Response to Elicitors in Vitis vinifera Cells 
PLoS ONE  2014;9(10):e109777.
The high effectiveness of cyclic oligosaccharides like cyclodextrins in the production of trans-resveratrol in Vitis vinifera cell cultures is enhanced in the presence of methyl jasmonate. In order to dissect the basis of the interactions among the elicitation responses triggered by these two compounds, a transcriptional analysis of grapevine cell cultures treated with cyclodextrins and methyl jasmonate separately or in combination was carried out. The results showed that the activation of genes encoding enzymes from phenylpropanoid and stilbene biosynthesis induced by cyclodextrins alone was partially enhanced in the presence of methyl jasmonate, which correlated with their effects on trans-resveratrol production. In addition, protein translation and cell cycle regulation were more highly repressed in cells treated with cyclodextrins than in those treated with methyl jasmonate, and this response was enhanced in the combined treatment. Ethylene signalling was activated by all treatments, while jasmonate signalling and salicylic acid conjugation were activated only in the presence of methyl jasmonate and cyclodextrins, respectively. Moreover, the combined treatment resulted in a crosstalk between the signalling cascades activated by cyclodextrins and methyl jasmonate, which, in turn, provoked the activation of additional regulatory pathways involving the up-regulation of MYB15, NAC and WRKY transcription factors, protein kinases and calcium signal transducers. All these results suggest that both elicitors cause an activation of the secondary metabolism in detriment of basic cell processes like the primary metabolism or cell division. Crosstalk between cyclodextrins and methyl jasmonate-induced signalling provokes an intensification of these responses resulting in a greater trans-resveratrol production.
PMCID: PMC4196943  PMID: 25314001
3.  Methyl jasmonate induces extracellular pathogenesis-related proteins in cell cultures of Capsicum chinense 
Plant Signaling & Behavior  2011;6(3):440-442.
Suspension cultured cells of Capsicum chinense secrete proteins to the culture medium in both control conditions and under methyl jasmonate treatment. The exogenous application of methyl jasmonate induced the accumulation of putative pathogenesis-related proteins, class I chitinase, leucin-rich repeat protein, NtPRp27-like protein and pectinesterase which were also found in suspension cultured cells of C. annuum elicited with methyl jasmonate. However, a germin-like protein, which has never been described in methyl jasmonate-elicited C. chinense suspension cultured cells, was found. The different effects described as being the result of exogenous application of signalling molecules like methyl jasmonate on the expression of germin-like protein suggest that germin-like proteins may play a variety of roles in protecting plants against pathogen attacks and different stresses. Further studies will be necessary to characterize the differential expression of these pathogenesis-related proteins and to throw light on the complexity of their regulation.
PMCID: PMC3142433  PMID: 21346408
capsicum; cell cultures; methyl jasmonate; pathogenesis-related proteins
4.  Synergistic effect of methyljasmonate and cyclodextrin on stilbene biosynthesis pathway gene expression and resveratrol production in Monastrell grapevine cell cultures 
BMC Research Notes  2008;1:132.
Plant cell cultures have been shown as feasible systems for the production of secondary metabolites, being the elicitation with biotic or abiotic stimuli the most efficient strategy to increase the production of those metabolites. Vitaceae phytoalexins constitute a group of molecules belonging to the stilbene family which are derivatives of the trans-resveratrol structure and are produced by plants and cell cultures as a response to biotic and abiotic stresses. The potential benefits of resveratrol on human health have made it one of the most thoroughly studied phytochemical molecules. The aim of this study was to evaluate the elicitor effect of both cyclodextrin (CD) and methyljasmonate (MeJA) on grapevine cell cultures by carrying out a quantitative analysis of their role on resveratrol production and on the expression of stilbene biosynthetic genes in Vitis vinifera cv Monastrell albino cell suspension cultures.
MeJA and CD significantly but transiently induced the expression of stilbene biosynthetic genes when independently used to treat grapevine cells. This expression correlated with resveratrol production in CD-treated cells but not in MeJA-treated cells, which growth was drastically affected. In the combined treatment of CD and MeJA cell growth was similarly affected, however resveratrol production was almost one order of magnitude higher, in correlation with maximum expression values for stilbene biosynthetic genes.
The effect of MeJA on cell division combined with a true and strong elicitor like CD could be responsible for the observed synergistic effect of both compounds on resveratrol production and on the expression of genes in the stilbene pathway.
PMCID: PMC2628674  PMID: 19102745

Results 1-4 (4)