Overexpression of the Mitochondrial Malic Enzyme Genes (malC and malD) Improved the Lipid Accumulation in Mucor circinelloides WJ11.
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ABSTRACT:
SUBMITTER: Fazili ABA
PROVIDER: S-EPMC9260706 | biostudies-literature |
REPOSITORIES: biostudies-literature
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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.
| S-EPMC7916304 | biostudies-literature
Project description:Mucor circinelloides is an oleaginous, dimorphic zygomycete fungus species that produces appreciable levels of ethanol when grown under aerobic conditions in the presence of high glucose, indicating the fungus is a Crabtree-positive microorganism. Engineering efforts to redirect carbon flux from ethanol to lipid biosynthesis may shed light on the critical role of ethanol biosynthesis during aerobic fermentation in M. circinelloides. Therefore, in this study, the alcohol dehydrogenase gene (ADH1) of M. circinelloides WJ11 was deleted, and its effects on growth, lipid production, and fatty acid content were analyzed. Our results showed that knocking out of adh1∆ reduced the ethanol concentration by 85-90% in fermented broth, indicating that this gene is the major source of ethanol production. Parallel to these findings, the lipid and fatty acid content of the mutant was decreased, while less change in the growth of WJ11 was observed. Furthermore, a fermentation study showed the lipid and fatty acid content was restored in the mutant strain when the fermentation media was supplemented with 0.5% external ethanol, indicating the importance of alcohol dehydrogenase and its product on growth and lipid biosynthesis in M. circinelloides. To our knowledge, this is the first study to show a link between alcohol dehydrogenase and lipid production in M. circinelloides.
| S-EPMC9503276 | biostudies-literature
Project description:The filamentous fungus Mucor circinelloides VI 04473 (indicated by MC1 in these data) was exposed to treatments known to trigger the accumulation of lipids in fungal cells, in order to investigate the genomic basis of this trait. Treatments consisted of different levels of calcium and inorganic phosphorus substrate in the growth media used for cultivation: For calcium, the two levels were calcium present in a concentration of 0.1 g/L (Ca1), and absence of calcium (Ca0). For phosphorus, there were three different levels: The reference concentration P1 (phosphate salts in a concentration of 7 g/L KH2PO4 and 2 g/L Na2HPO4), four times the reference concentration (P4), and half the reference concentration (P0.5). The combination of the different levels of calcium and phosphorus resulted in six different growth media, indicated in these data by numbers _1 - _6: 1 = Ca1P1, 2 = Ca1P4, 3 = Ca1P0.5, 4 = Ca0P1, 5 = Ca0P4, 6 = Ca0P0.5. There were 3-4 replicates per unique growth media, indicated by R1-R4 . The biomass was washed and harvested after 5 days of cultivation. RNA was extracted from the biomass and sequenced, followed by analysis of the transcriptome (genes differentially expressed between treatments).
2023-09-15 | E-MTAB-13309 | biostudies-arrayexpress
Project description:The genome-scale metabolic model of a lipid-overproducing strain of Mucor circinelloides WJ11 was developed. The model (iNI1159) contained 1,159 genes, 648 EC numbers, 1,537 metabolites, and 1,355 metabolic reactions, which were localized in different compartments of the cell. Using flux balance analysis (FBA), the iNI1159 model was validated by predicting the specific growth rate. The metabolic traits investigated by phenotypic phase plane analysis (PhPP) showed a relationship between the nutrient uptake rate, cell growth, and the triacylglycerol production rate, demonstrating the strength of the model. A putative set of metabolic reactions affecting the lipid-accumulation process was identified when the metabolic flux distributions under nitrogen-limited conditions were altered by performing fast flux variability analysis (fastFVA) and relative flux change. Comparative analysis of the metabolic models of the lipid-overproducing strain WJ11 (iNI1159) and the reference strain CBS277.49 (iWV1213) using both fastFVA and coordinate hit-and-run with rounding (CHRR) showed that the flux distributions between these two models were significantly different. Notably, a higher flux distribution through lipid metabolisms such as lanosterol, zymosterol, glycerolipid and fatty acids biosynthesis in iNI1159 was observed, leading to an increased lipid production when compared to iWV1213. In contrast, iWV1213 exhibited a higher flux distribution across carbohydrate and amino acid metabolisms and thus generated a high flux for biomass production. This study demonstrated that iNI1159 is an effective predictive tool for the pathway engineering of oleaginous strains for the production of diversified oleochemicals with industrial relevance.
| S-EPMC6613434 | biostudies-literature
Project description:The fungus, Mucor lusitanicus, is of great interest for microbial lipids, because of its ability to accumulate intracellular lipid using various carbon sources. The biosynthesis of fatty acid requires the reducing power NADPH, and acetyl-CoA, which is produced by the cleavage of citrate in cytosol. In this study, we employed different strategies to increase lipid accumulation in the low lipid-producing fungi via metabolic engineering technology. Hence, we constructed the engineered strain of M. lusitanicus CBS 277.49 by using malate transporter (mt) and 2-oxoglutarate: malate antiporter (sodit) from M. circinelloides WJ11. In comparison with the control strain, the lipid content of the overexpressed strains of mt and sodit genes were increased by 24.6 and 33.8%, respectively. These results showed that mt and sodit can affect the distribution of malate in mitochondria and cytosol, provide the substrates for the synthesis of citrate in the mitochondria, and accelerate the transfer of citrate from mitochondria to cytosol, which could play a significant regulatory role in fatty acid synthesis leading to lipids over accumulation.
| S-EPMC8640210 | biostudies-literature
Project description:The mitochondrial citrate transporter (MCT) plays an important role in citrate efflux from the mitochondria in eukaryotes, and hence provides a direct correlation between carbohydrate metabolism and lipid synthesis. Our previous studies on transporters confirmed the presence of two MCTs (TCT and CT) in oleaginous Mucor circinelloides WJ11 associated with high lipid accumulation. However, the molecular mechanism of citrate efflux from the mitochondria by MCT in M. circinelloides is still unclear. To study the citrate transport mechanism of CT, the citrate transporter gene was expressed in Escherichia coli, and its product was purified. The citrate transport activity of the protein was studied in CT reconstituted liposomes. Our results showed high efficiency of CT for [14C] citrate/citrate exchange with K m 0.01 mM at 25°C. Besides citrate, other molecules such as oxaloacetate, malate, fumarate, succinate aconitate, oxoadipate, isocitrate, and glutamate also promote citrate transport. In addition, the ct overexpression and knockout plasmids were constructed and transferred into M. circinelloides WJ11, and the mitochondria were isolated, and the transport activity was studied. Our findings showed that in the presence of 10 mM malate, the mitochondria of ct-overexpressing transformant showed 51% increase in the efflux rate of [14C] citrate, whereas the mitochondria of the ct-knockout transformant showed 18% decrease in citrate efflux compared to the mitochondria of wild-type WJ11. This study provided the first mechanistic evidence of citrate efflux from the mitochondria by citrate transporter in oleaginous filamentous fungus M. circinelloides, which is associated with high lipid accumulation.
| S-EPMC8160456 | biostudies-literature
Project description:Mucor circinelloides strain:WJ11 Genome sequencing and assembly
| PRJNA290220 | ENA
Project description:The citrate transporter protein (CTP) plays an important role in citrate efflux from the mitochondrial matrix to cytosol that has great importance in oleaginous fungi. The cytoplasmic citrate produced after citrate efflux serves as the primary carbon source for the triacylglycerol and cholesterol biosynthetic pathways. Because of the CTP's importance, our laboratory has extensively studied its structure/function relationships in Mucor circinelloides to comprehend its molecular mechanism. In the present study, the tricarboxylate citrate transporter (Tct) of M. circinelloides WJ11 has been cloned, overexpressed, purified, kinetically, and structurally characterized. The Tct protein of WJ11 was expressed in Escherichia coli, isolated, and functionally reconstituted in a liposomal system for kinetic studies. Our results showed that Tct has a high affinity for citrate with Km 0.018 mM. Furthermore, the tct overexpression and knockout plasmids were created and transformed into M. circinelloides WJ11. The mitochondria of the tct-overexpressing transformant of M. circinelloides WJ11 showed a 49% increase in citrate efflux, whereas the mitochondria of the tct-knockout transformant showed a 39% decrease in citrate efflux compared to the mitochondria of wild-type WJ11. To elucidate the structure-function relationship of this biologically important transporter a 3D model of the mitochondrial Tct protein was constructed using homology modeling. The overall structure of the protein is V-shaped and its 3D structure is dimeric. The transport stability of the structure was also assessed by molecular dynamics simulation studies. The activity domain was identified to form hydrogen bond and stacking interaction with citrate and malate upon docking. Tricarboxylate citrate transporter has shown high binding energy of -4.87 kcal/mol to citric acid, while -3.80 kcal/mol to malic acid. This is the first report of unraveling the structural characteristics of WJ11 mitochondrial Tct protein and understanding the approach of the transporting toward its substrate. In conclusion, the present findings support our efforts to combine functional and structural data to better understand the Tct of M. circinelloides at the molecular level and its role in lipid accumulation.
| S-EPMC8696028 | biostudies-literature
Project description:The genome of a high lipid-producing fungus Mucor circinelloides WJ11 (36% w/w lipid, cell dry weight, CDW) was sequenced and compared with that of the low lipid-producing strain, CBS 277.49 (15% w/w lipid, CDW), which had been sequenced by Joint Genome Institute. The WJ11 genome assembly size was 35.4 Mb with a G+C content of 39.7%. The general features of WJ11 and CBS 277.49 indicated that they have close similarity at the level of gene order and gene identity. Whole genome alignments with MAUVE revealed the presence of numerous blocks of homologous regions and MUMmer analysis showed that the genomes of these two strains were mostly co-linear. The central carbon and lipid metabolism pathways of these two strains were reconstructed and the numbers of genes encoding the enzymes related to lipid accumulation were compared. Many unique genes coding for proteins involved in cell growth, carbohydrate metabolism and lipid metabolism were identified for each strain. In conclusion, our study on the genome sequence of WJ11 and the comparative genomic analysis between WJ11 and CBS 277.49 elucidated the general features of the genome and the potential mechanism of high lipid accumulation in strain WJ11 at the genomic level. The different numbers of genes and unique genes involved in lipid accumulation may play a role in the high oleaginicity of strain WJ11.
| S-EPMC4564205 | biostudies-literature
Project description:BACKGROUND:Mitochondrial and cytoplasmic malate transporter proteins are responsible for transmembrane transport of malate, thereby linking malate metabolism in various subcellular regions of the cell. These transporters play an important role in fatty acid biosynthesis of oleaginous microorganisms. Our previous studies have found that lipid content of the recombinant Mucor circinelloides (M. circinelloides) strain with mitochondrial malate transporter (mt) gene overexpression was increased by 70%, while that of strain with mt gene knockout was decreased by 27%. However, the mechanism of malate transporter promoting the transport of mitochondrial malate and citrate related to lipid accumulation is not clear. Therefore, 13C-labeled glucose metabolic flux analysis was carried out to identify the metabolic network topology and estimate intracellular fluxes of genetically engineered M. circinelloides strains for the purpose of better understanding the roles of malate transporters in citrate transport systems and lipid accumulation. RESULTS:The metabolic flux distribution analysis suggested that tricarboxylic acid (TCA) cycle flux ratio of mt-overexpression strains was decreased compared to that of the control strain, but in contrast, glyoxylic acid (GOX) cycle flux ratio was increased. Accordingly, the mt-knockout strain showed an opposite phenomenon with a higher TCA cycle flux ratio and a lower GOX cycle flux ratio than the control strain. GOX cycle might be more effective than TCA cycle in producing malate and oxaloacetate replenishment. Moreover, a relatively higher flux ratio of the pentose phosphate (PP) pathway was obtained in mt-overexpression strains, but no significant difference in the malic enzyme flux between recombinant strains and the control strain. Our results confirmed that PP pathway might play an important role for supplying NADPH and malic enzyme is not a limiting factor for fatty acid synthesis in oleaginous fungus M. circinelloides strains. CONCLUSION:Intracellular metabolic flux information suggested that mt-overexpression strains had higher flux in PP pathway and GOX cycle, lower flux in TCA cycle, and no difference in malic enzyme cycle. Together, the role of malate transporter was assumed to further participate in transporting cycle of acetyl-CoA and drive PP pathway to supply NADPH required for lipid accumulation in recombinant M. circinelloides strains.
| S-EPMC6737672 | biostudies-other