Role of malate transporter in lipid accumulation of oleaginous fungus Mucor circinelloides

Zhao, Lina; Cánovas-Márquez, José Tomás; Tang, Xin; Chen, Haiqin; Chen, Yong Q.; Chen, Wei; Garre, Victoriano; Song, Yuanda; Ratledge, Colin

doi:10.1007/s00253-015-7079-y

Cited by 40 publications

(47 citation statements)

References 27 publications

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“…Previously, the malate transporter ( mt ) gene was overexpressed or knocked out in M. circinelloides to construct recombinant strains, which are as follows: MU522 ( mt -knockout, CCTCC M 2015552), MU241 (wild-type, as the control) and Mc-MT ( mt -overexpression, CCTCC M 2015551). The transformants of mt -overexpressing gene were named Mc-MT-1 and Mc-MT-2, respectively [16]. These engineered strains were respectively inoculated in high nitrogen (HN) and low nitrogen (LN) modified K & R medium with glucose as the sole carbon source.…”

Section: Resultsmentioning

confidence: 99%

“…Based on the genomic data search and sequences analysis of M. circinelloides CBS277.49, a gene (ID180302) encoding malate transporter was found in NCBI and named mt . Both null mutant (Mu522) and overexpressing strains (Mc-MT-1 and Mc-MT-2) of this gene were previously constructed in our laboratory [16].…”

Section: Methodsmentioning

confidence: 99%

“…Another important transporter which has a significant effect on citrate transfer in mitochondria is malate transporter. The correlation between the malate transporter and lipid accumulation in M. circinelloides was preliminarily studied by knockout and overexpression of the malate transporter ( mt ) gene [16]. Our results showed that the fatty acid content of the recombinant strain with overexpression of mt was increased by 70%, while that of the strain with knockout mt was decreased by 27% [17].…”

Section: Introductionmentioning

confidence: 99%

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13C metabolic flux analysis on roles of malate transporter in lipid accumulation of Mucor circinelloides

Wang

Zhang

et al. 2019

Microb Cell Fact

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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13C metabolic flux analysis on roles of malate transporter in lipid accumulation of Mucor circinelloides

Wang

Zhang

et al. 2019

Microb Cell Fact

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“…According to the genomic data of M. alpina , this study found only one gene encoding for D15D (Genebank accession number KF433065), which is 1212 bp long. For overexpression of the target gene, the coding region of D15D was cloned into the M. circinelloides expression vector pMAT1552, which contained the strong zrt1 promoter and flanking sequence of carRP locus to allow integration of the whole over-expressing construction by homologous recombination [26]. Integration in carRP locus produced white colonies, which were easily distinguishable from yellow transformants that did not integrate the D15D gene.…”

Section: Resultsmentioning

confidence: 99%

Genetic Modification of Mucor circinelloides to Construct Stearidonic Acid Producing Cell Factory

Khan

Yang

Hussain

et al. 2019

IJMS

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Stearidonic acid (SDA; 18:4, n-3) is the delta 15-desaturase product of gamma linolenic acid (GLA; 18:3, n-6) and delta 6-desaturase product of alpha linolenic acid (ALA; 18:3, n-3). Construction of engineered oleaginous microbes have been attracting significant interest in producing SDA because of its nutritional value and pharmaceutical applications. Mucor circinelloides is a GLA producing filamentous fungus, which can be a useful tool to produce SDA. This study has, therefore, overexpressed the delta-15 desaturase (D15D) gene from Mortierella alpina in this fungus to construct a SDA-producing cell factory. To produce SDA in M. circinelloides, the homologous overexpression of D15D gene was analyzed. When the gene was overexpressed in M. circinelloides CBS 277.49, up to 5.0% SDA was accumulated in this strain. According to current knowledge, this is the first study describing the construction of a SDA-producing cell factory by overexpression of D15D gene in oleaginous fungus M. circinelloides. A new scope for further research has been established by this work to improve SDA production in this fungus, specifically in its high lipid-producing strain, WJ11.

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“…It is hypothesis that, the A.niger didn't shows good intensity of key isoform (E), the isoform that related to lipid biosynthesis pathway in lipid accumulation with its relations on gene expression [27,28]…”

Section: Activity Of Key Enzymes Duringmentioning

confidence: 99%

Biochemical Characterization for Lipid Synthesis in Aspergillus niger

Journal¹

2016

Baghdad Sci.J

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A niger, a fungus which doesn't have high ability to production lipid, this fungus has been select to investigate the non oleaginicity. In this search, there are explorations about: i) growth profile ii) enzymes profile iii) isoforms. Growth profile shows that this fungus doesn't have ability to accumulate lipid more than 6% while bio mass are around 10g/l in spite of the presence of glucose in the media till the end of cultivation time and excision of nitrogen within 24 hrs. In enzyme study, we investigate all lipogenic enzymes Malic enzyme (ME), Fatty acid synthase (FAS), ATP: Citrate lays (ACL), NAD+ isocitrate dehydrogenase (NAD+ICDH), Glucose-6-phosphate (G6PD), and 6-phosphogluconate dehydrogenase (6PGD), all these enzymes show, activities till the end of cultivation time including ACL which is regarded the key enzyme to differentiate between the two species oleaginous and non oleaginous. So, there is no main reason to non oleaginicity for this fungus. A further experiment has been done using Polyacrylamide gel electrophoresis to identify ME isoforms. The result of Polyacrylamide gel electrophoresis shows multi isoforms (A, B, C, D & E), with low intensity of isoform E, the isoforms that may involve in lipid synthesis. We have now studied the biochemistry of A.niger grown under conditions designed to promote lipid accumulation and can now advance a coherent hypothesis to explain why A niger could not accumulate lipid more than 6%. So the absence of isoforme E is the main reason for non oleaginicity in A niger.

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Role of malate transporter in lipid accumulation of oleaginous fungus Mucor circinelloides

Cited by 40 publications

References 27 publications

13C metabolic flux analysis on roles of malate transporter in lipid accumulation of Mucor circinelloides

13C metabolic flux analysis on roles of malate transporter in lipid accumulation of Mucor circinelloides

Genetic Modification of Mucor circinelloides to Construct Stearidonic Acid Producing Cell Factory

Biochemical Characterization for Lipid Synthesis in Aspergillus niger

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