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Construction of DGLA producing cell factory by genetic modification of Mucor circinelloides.

ABSTRACT: BACKGROUND:Dihomo-gamma linolenic acid (DGLA, 20:3, n-6) is the elongated product of Gamma linolenic acid (GLA, 18:3, n-6) catalyzed by the enzyme delta-6 elongase (D6E) or gamma linolenic acid elongase (GLELO). Construction of engineered oleaginous microbes have been attracting significant interest to produce DGLA because of its nutritional value and medicinal applications. Mucor circinelloides is a GLA producing filamentous fungus which can be a useful tool to produce DGLA. We have, therefore, overexpressed the D6E (GLELO) gene in this fungus to construct DGLA producing cell factory. RESULT:To produce DGLA in M. circinelloides, homologous overexpression of D6E (GLELO) gene was analyzed. When the gene was overexpressed in M. circinelloides CBS277.49, up to 5.72% DGLA was produced in this strain. CONCLUSION:To our knowledge, this is the first report describing the overexpression of D6E (GLELO) gene in M. circinelloides to construct DGLA producing cell factory. A new scope for further research has been established by this work for improved production of DGLA in this fungus, specifically in its high lipid-producing strain, WJ11.

SUBMITTER: Khan MAK

PROVIDER: S-EPMC6448318 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Construction of DGLA producing cell factory by genetic modification of Mucor circinelloides.

Khan Md Ahsanul Kabir MAK Yang Junhuan J Hussain Syed Ammar SA Zhang Huaiyuan H Liang Li L Garre Victoriano V Song Yuanda Y

Microbial cell factories 20190403 1

<h4>Background</h4>Dihomo-gamma linolenic acid (DGLA, 20:3, n-6) is the elongated product of Gamma linolenic acid (GLA, 18:3, n-6) catalyzed by the enzyme delta-6 elongase (D6E) or gamma linolenic acid elongase (GLELO). Construction of engineered oleaginous microbes have been attracting significant interest to produce DGLA because of its nutritional value and medicinal applications. Mucor circinelloides is a GLA producing filamentous fungus which can be a useful tool to produce DGLA. We have, th ...[more]

PMID: 30943965

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Project description:BackgroundMucor circinelloides WJ11 is a high-lipid producing strain and an excellent producer of γ-linolenic acid (GLA) which is crucial for human health. We have previously identified genes that encode for AMP-activated protein kinase (AMPK) complex in M. circinelloides which is an important regulator for lipid accumulation. Comparative transcriptional analysis between the high and low lipid-producing strains of M. circinelloides showed a direct correlation in the transcriptional level of AMPK genes with lipid metabolism. Thus, the role of Snf-β, which encodes for β subunit of AMPK complex, in lipid accumulation of the WJ11 strain was evaluated in the present study.ResultsThe results showed that lipid content of cell dry weight in Snf-β knockout strain was increased by 32 % (from 19 to 25 %). However, in Snf-β overexpressing strain, lipid content of cell dry weight was decreased about 25 % (from 19 to 14.2 %) compared to the control strain. Total fatty acid analysis revealed that the expression of the Snf-β gene did not significantly affect the fatty acid composition of the strains. However, GLA content in biomass was increased from 2.5 % in control strain to 3.3 % in Snf-β knockout strain due to increased lipid accumulation and decreased to 1.83 % in Snf-β overexpressing strain. AMPK is known to inactivate acetyl-CoA carboxylase (ACC) which catalyzes the rate-limiting step in lipid synthesis. Snf-β manipulation also altered the expression level of the ACC1 gene which may indicate that Snf-β control lipid metabolism by regulating ACC1 gene.ConclusionsOur results suggested that Snf-β gene plays an important role in regulating lipid accumulation in M. circinelloides WJ11. Moreover, it will be interesting to evaluate the potential of other key subunits of AMPK related to lipid metabolism. Better insight can show us the way to manipulate these subunits effectively for upscaling the lipid production. Up to our knowledge, it is the first study to investigate the role of Snf-β in lipid accumulation in M. circinelloides.

Project description: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.

Project description:Mucor circinelloides is a zygomycete fungus and an emerging opportunistic pathogen in immunocompromised patients, especially transplant recipients and in some cases otherwise healthy individuals. We have discovered a novel example of size dimorphism linked to virulence. M. circinelloides is a heterothallic fungus: (+) sex allele encodes SexP and (-) sex allele SexM, both of which are HMG domain protein sex determinants. M. circinelloides f. lusitanicus (Mcl) (-) mating type isolates produce larger asexual sporangiospores that are more virulent in the wax moth host compared to (+) isolates that produce smaller less virulent sporangiospores. The larger sporangiospores germinate inside and lyse macrophages, whereas the smaller sporangiospores do not. sexMΔ mutants are sterile and still produce larger virulent sporangiospores, suggesting that either the sex locus is not involved in virulence/spore size or the sexP allele plays an inhibitory role. Phylogenetic analysis supports that at least three extant subspecies populate the M. circinelloides complex in nature: Mcl, M. circinelloides f. griseocyanus, and M. circinelloides f. circinelloides (Mcc). Mcc was found to be more prevalent among clinical Mucor isolates, and more virulent than Mcl in a diabetic murine model in contrast to the wax moth host. The M. circinelloides sex locus encodes an HMG domain protein (SexP for plus and SexM for minus mating types) flanked by genes encoding triose phosphate transporter (TPT) and RNA helicase homologs. The borders of the sex locus between the three subspecies differ: the Mcg sex locus includes the promoters of both the TPT and the RNA helicase genes, whereas the Mcl and Mcc sex locus includes only the TPT gene promoter. Mating between subspecies was restricted compared to mating within subspecies. These findings demonstrate that spore size dimorphism is linked to virulence of M. circinelloides species and that plasticity of the sex locus and adaptations in pathogenicity have occurred during speciation of the M. circinelloides complex.

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Construction of DGLA producing cell factory by genetic modification of Mucor circinelloides.

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Construction of DGLA producing cell factory by genetic modification of Mucor circinelloides.

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