Establishment of
            <i>Agrobacterium tumefaciens</i>
            -Mediated Transformation of an Oleaginous Fungus,
            <i>Mortierella alpina</i>
            1S-4, and Its Application for Eicosapentaenoic Acid Producer Breeding

Ando, Akinori; Sumida, Yosuke; Negoro, Hiroaki; Suroto, Dian Anggraini; Ogawa, Jun; Sakuradani, Eiji; Shimizu, Sakayu

doi:10.1128/aem.00648-09

Cited by 95 publications

(87 citation statements)

References 26 publications

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“…Agrobacterium tumefaciens C58C1 was cultured in YEP medium, which consists of 10 g/liter tryptone, 10 g/liter yeast extract, and 5 g/liter NaCl. The compositions of synthetic complete (SC) medium, minimal medium (MM), and induction medium (IM) were described previously (8,14,15).…”

Section: Methodsmentioning

confidence: 99%

Metabolic Engineering of Mortierella alpina for Enhanced Arachidonic Acid Production through the NADPH-Supplying Strategy

Hao

Chen

et al. 2016

Appl Environ Microbiol

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NADPH is known to be a key cofactor required for fatty acid synthesis and desaturation. Various enzymatic reactions can generate NADPH. To determine the effect of NADPH sources on lipogenesis, glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (PGD), isocitrate dehydrogenase (IDH), and malic enzyme (ME) were overexpressed in Mortierella alpina. Our results showed that G6PD2 had the most significant effect on fatty acid synthesis, with a 1.7-fold increase in total fatty acid, whereas ME2 was more effective in desaturation, with a 1.5-fold increase in arachidonic acid (AA) content over control. Co-overexpression of G6PD2 and ME2 improved both fatty acid synthesis and desaturation. Within 96 h of fermentation using the fed-batch method, the co-overexpressing strain accumulated AA at a productivity of 1.9 ؎ 0.2 g/(liter · day), which was 7.2-fold higher than that in the M. alpina control that was cultured in a flask. IMPORTANCEThis study proved that the pentose phosphate pathway is the major NADPH contributor during fatty acid synthesis in M. alpina. The NADPH sources may be differently responsible for fatty acid synthesis or desaturation. Co-overexpression of G6PD2 and ME2 significantly increases AA production. NADPH is a critical factor for many biological processes in oleaginous microbes (1, 2). However, the function of the various sources of NADPH in fatty acid synthesis is still not fully understood. NADPH can be generated by several enzymatic reactions. Malic enzyme (ME; EC 1.1.1.40) was considered the sole NADPH provider for fatty acid synthesis in oleaginous microbes (3). Some evidence, however, indicated that ME is only one of several NADPH sources (4, 5). We and others have recently demonstrated that the pentose phosphate pathway (PPP), specifically the glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) and the 6-phosphogluconate dehydrogenase (PGD; EC 1.1.1.44) genes, plays a major role during microbial fatty acid synthesis (1, 2, 6). Isocitrate dehydrogenase (IDH; EC 1.1.1.42) is another potential NADPH source and was proven to play a key role in fatty acid metabolism in adipocytes (7).NADPH also plays an important role during fatty acid desaturation, which could be significantly improved by overexpressing a mitochondrial ME2 in Mortierella alpina (8) and Mucor circinelloides (9). These results implied that NADPH generated by cytosolic and membrane-bound enzymes may affect fatty acid synthesis and desaturation, respectively. Therefore, the role of the various sources of NADPH needs to be systematically evaluated and explored as an effective approach to increase the production of total fatty acids (TFA) and especially of unsaturated fatty acids.Mortierella alpina is an oleaginous fungus used for the commercial production of arachidonic acid (AA); this organism can accumulate fatty acids of up to 50% of dry weight (10,11). An expression system using the uracil auxotroph strain CCFM 501 was recently established and reliably applied for gene manipulation (1,5,8,12, 13). In ...

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Section: Methodsmentioning

confidence: 99%

Metabolic Engineering of Mortierella alpina for Enhanced Arachidonic Acid Production through the NADPH-Supplying Strategy

Hao

Chen

et al. 2016

Appl Environ Microbiol

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“…GY medium was used for extracting M. alpina genomic DNA; when this medium was used for positive selection of uracil auxotrophs, 5-FOA (1 mg/ml) and uracil (0.05 mg/ml) were added. The compositions of synthetic complete (SC) medium, minimal medium (MM), and induction medium (IM) were described before (12,13 6.0. The incubation protocols were as previously described (15).…”

Section: Methodsmentioning

confidence: 99%

Role of Malic Enzyme during Fatty Acid Synthesis in the Oleaginous Fungus Mortierella alpina

Hao

Chen

Wang

et al. 2014

Appl Environ Microbiol

142

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The generation of NADPH by malic enzyme (ME) was postulated to be a rate-limiting step during fatty acid synthesis in oleaginous fungi, based primarily on the results from research focusing on ME in Mucor circinelloides. This hypothesis is challenged by a recent study showing that leucine metabolism, rather than ME, is critical for fatty acid synthesis in M. circinelloides. To clarify this, the gene encoding ME isoform E from Mortierella alpina was homologously expressed. ME overexpression increased the fatty acid content by 30% compared to that for a control. Our results suggest that ME may not be the sole rate-limiting enzyme, but does play a role, during fatty acid synthesis in oleaginous fungi. Oleaginous fungi, such as the commercial production species Mortierella alpina and Mucor circinelloides, can accumulate fatty acids to more than 20% of their cell dry weight (1). However, the mechanism of fatty acid biosynthesis in these organisms is still not fully understood. The carbon flux pathway and provision of NADPH are two major events during fatty acid synthesis. NADPH is particularly important as the sole source of reducing power during fatty acid synthesis in oleaginous fungi (2-5).The NADPH-generating enzyme malic enzyme (ME) (NADP ϩ dependent; EC 1.1.1.40), catalyzing the decarboxylation of malate to pyruvate (malate ϩ NADP ϩ ϭ pyruvate ϩ CO 2 ϩ NADPH), was speculated to play a pivotal role during fatty acid synthesis in oleaginous fungi (3, 5). When ME activity was inhibited, the cell fatty acid content of M. circinelloides decreased from 24% to 2% (6). Moreover, in the M. circinelloides strain R7B, overexpression of the ME genes from M. alpina and M. circinelloides produced a 2.5-fold increase in fatty acid content (7). These results led to the conclusion that a ratelimiting step in fatty acid biosynthesis is the generation of NADPH by ME (7). However, a recent study revealed that leucine auxotrophy caused a 2.5-fold decrease in cell fatty acid content and that leuA gene expression restored its level in M. circinelloides strain R7B. ME overexpression, however, did not alter the fatty acid content despite a significant increase in ME activity (8). It was thus proposed that the leucine metabolic pathway, by participating in acetyl coenzyme A (acetyl-CoA) generation, may be critical during fatty acid synthesis in M. circinelloides (8,9).M. circinelloides R7B, generated by random mutagenesis (10), may contain other, unknown mutations in addition to that in the leuA gene, which can complicate the interpretation of the abovementioned results. Therefore, a recipient strain with a known genetic mutation(s) and stable fatty acid synthesis characteristics will be advantageous for future research. To this end, a uracil auxotroph of M. alpina ATCC 32222 was generated via homologous recombination. The M. alpina ATCC 32222 cytosolic ME (isoform E)-encoding gene (11), named malE1, was cloned and homologously overexpressed in order to evaluate the role of ME in fatty acid synthesis. MATERIALS AND METHODSStrains and gr...

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“…However, these species might serve as platform organisms to overproduce hydrocarbons if the pathways to the hydrocarbons are identified and engineered into the oleaginous species. This assumes that these oleaginous strains can be genetically manipulated; this is 8.2 certainly the case for many of the yeasts and at least one of the filamentous fungi named above (Ando et al 2009). Using these oleaginous fungi as platforms would be potentially beneficial for two reasons.…”

Section: Accomplishments and Resultsmentioning

confidence: 99%

PNNL Fungal Biotechnology Core DOE-OBP Project

Baker¹,

Bruno²,

Butcher³

et al. 2009

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SummaryIn 2009, we continued to address barriers to fungal fermentation in the primary areas of morphology control, genomics, proteomics, fungal hyperproductivity, biomass-to-products via fungal based consolidated bioprocesses, and filamentous fungal ethanol. "Alternative renewable fuels from fungi" was added as a new subtask. Plans were also made to launch a new advanced strain development subtask in FY2010. Project subtask highlights included: Morphology: Key accomplishments included analysis of genes involved in control of fungal bioprocess organism morphology and a morphology scoring system for quantitative measurement. This task was closed out and replaced beginning in FY10 with Advanced Strain Development.

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Establishment of Agrobacterium tumefaciens -Mediated Transformation of an Oleaginous Fungus, Mortierella alpina 1S-4, and Its Application for Eicosapentaenoic Acid Producer Breeding

Cited by 95 publications

References 26 publications

Metabolic Engineering of Mortierella alpina for Enhanced Arachidonic Acid Production through the NADPH-Supplying Strategy

Metabolic Engineering of Mortierella alpina for Enhanced Arachidonic Acid Production through the NADPH-Supplying Strategy

Role of Malic Enzyme during Fatty Acid Synthesis in the Oleaginous Fungus Mortierella alpina

PNNL Fungal Biotechnology Core DOE-OBP Project

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