Role of the Phenylalanine-Hydroxylating System in Aromatic Substance Degradation and Lipid Metabolism in the Oleaginous Fungus Mortierella alpina

Wang, Hongchao; Chen, Haiqin; Hao, Ge‐Fei; Yang, Bo; Feng, Yun; Wang, Yu; Feng, Lu; Zhao, Jianxin; Song, Yuanda; Zhang, Hao; Chen, Yong Q.; Wang, Lei; Chen, Wei

doi:10.1128/aem.00238-13

Cited by 31 publications

(37 citation statements)

References 80 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Recently, acetyl-CoA generated from the endogenous leucine metabolic pathway was postulated to be another rate-limiting step during fatty acid synthesis in M. circinelloides (8), indicating that the amino acid metabolism may intersect with fatty acid synthesis as an acetyl-CoA resource. Likewise, in M. alpina, phenylalanine and tyrosine were suggested to be metabolized by the phenylalanine-hydroxylating system to contribute NADPH and acetyl-CoA for lipid metabolism (32). The supplement of acetyl-CoA might play a more important role during fatty acid synthesis in oleaginous fungi than previously speculated (7).…”

Section: Discussionmentioning

confidence: 47%

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

Hao

Chen

Wang

et al. 2014

Appl Environ Microbiol

Self Cite

142

View full text Add to dashboard Cite

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...

show abstract

Section: Discussionmentioning

confidence: 47%

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

Hao

Chen

Wang

et al. 2014

Appl Environ Microbiol

Self Cite

142

View full text Add to dashboard Cite

show abstract

“…S7). Although we cannot exclude that this may be due to the inability of adt3 to take up or metabolize Tyr in the form we supplied, it is tempting to speculate that Phe could be metabolized by a pathway that does not encompass the Tyr conversion step (Wang et al, 2013).…”

Section: Phe Role In Nutrient Availabilitymentioning

confidence: 99%

The dehydratase ADT3 affects ROS homeostasis and cotyledon development

et al. 2016

View full text Add to dashboard Cite

During the transition from seed to seedling, emerging embryos strategically balance available resources between building up defenses against environmental threats and initiating the developmental program that promotes the switch to autotrophy. We present evidence of a critical role for the phenylalanine (Phe) biosynthetic activity of AROGENATE DEHYDRATASE3 (ADT3) in coordinating reactive oxygen species (ROS) homeostasis and cotyledon development in etiolated Arabidopsis (Arabidopsis thaliana) seedlings. We show that ADT3 is expressed in the cotyledon and shoot apical meristem, mainly in the cytosol, and that the epidermis of adt3 cotyledons contains higher levels of ROS. Genome-wide proteomics of the adt3 mutant revealed a general down-regulation of plastidic proteins and ROS-scavenging enzymes, corroborating the hypothesis that the ADT3 supply of Phe is required to control ROS concentration and distribution to protect cellular components. In addition, loss of ADT3 disrupts cotyledon epidermal patterning by affecting the number and expansion of pavement cells and stomata cell fate specification; we also observed severe alterations in mesophyll cells, which lack oil bodies and normal plastids. Interestingly, up-regulation of the pathway leading to cuticle production is accompanied by an abnormal cuticle structure and/or deposition in the adt3 mutant. Such impairment results in an increase in cell permeability and provides a link to understand the cell defects in the adt3 cotyledon epidermis. We suggest an additional role of Phe in supplying nutrients to the young seedling.

show abstract

“…The function of BH 4 in Mort. alpina is suggested to contribute NADPH and acetyl-CoA for lipid accumulation by the utilization of phenylalanine and tyrosine (Wang et al, 2013). While the importance of folate metabolism has been long recognized and attributed to its function of producing one-carbon units for nucleic acid synthesis, another crucial function of this pathway is generating NADPH (Fan et al, 2014).…”

Section: Discussionmentioning

confidence: 99%

“…The BH 4 de novo synthesis and regeneration pathways in Mortierella alpina were first purified and characterized in fungi in our laboratory (Wang et al, 2011a;Wang et al, 2013). Though the function of DHFR in folate metabolism has been characterized in the yeast Candida albicans, Candida glabrata and Saccharomyces cerevisiae (Baccanari et al, 1989;Daly et al, 1994;Lagosky et al, 1987;Liu et al, 2008), the role of DHFR in fungal BH 4 salvage pathway has never been explored before.…”

Section: Introductionmentioning

confidence: 99%

“…In our previous study, a requirement for BH 4 in phenylalanine hydroxylation has been reported in Mort. alpina, which has been shown to be functionally significant in lipid metabolism by contributing NADPH (Wang et al, 2013). Recently, quantitative flux analysis of NADPH in immortalized baby mouse kidney epithelial cells revealed the crucial function of folate metabolism in generating NADPH, with a majority of this NADPH consumed by fatty acid synthesis (Fan et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Role of dihydrofolate reductase in tetrahydrobiopterin biosynthesis and lipid metabolism in the oleaginous fungus Mortierella alpina

et al. 2016

Self Cite

View full text Add to dashboard Cite

Mortierella alpina is a well-known polyunsaturated fatty acid-producing oleaginous fungus. Analysis of the Mort. alpina genome suggests that there is a putative dihydrofolate reductase (DHFR) gene playing a role in the salvage pathway of tetrahydrobiopterin (BH 4 ), which has never been explored in fungi before. DHFR is the sole source of tetrahydrofolate and plays a key role in maintaining BH 4 levels. Transcriptome data analysis revealed that DHFR was up-regulated by nitrogen exhaustion, when Mort. alpina starts to accumulate lipids. Significant changes were found in the fatty acid profile in Mort. alpina grown on medium containing DHFR inhibitors compared to Mort. alpina grown on medium without inhibitors. To explore the role of DHFR in folate/BH 4 metabolism and its relationship to lipid biosynthesis, we expressed heterologously the gene encoding DHFR from Mort. alpina in Escherichia coli and we purified the recombinant enzyme to homogeneity. The enzymatic activity was investigated by liquid chromatography and MS and VIS-UV spectroscopy. The kinetic parameters and the effects of temperature, pH, metal ions and inhibitors on the activity of DHFR were also investigated. The transcript level of cytosolic NADPH-producing gene involved in folate metabolism is down-regulated by DHFR inhibitors, which highlights the functional significance of DHFR in lipid biosynthesis. The relationship between DHFR and lipid metabolism is thus of major importance, and folate metabolism may be an alternative NADPH source in fatty acid synthesis. To our knowledge, this study is the first to report the comprehensive characterization of a BH 4 salvage pathway in a fungus.

show abstract

Role of the Phenylalanine-Hydroxylating System in Aromatic Substance Degradation and Lipid Metabolism in the Oleaginous Fungus Mortierella alpina

Cited by 31 publications

References 80 publications

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

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

The dehydratase ADT3 affects ROS homeostasis and cotyledon development

Role of dihydrofolate reductase in tetrahydrobiopterin biosynthesis and lipid metabolism in the oleaginous fungus Mortierella alpina

Contact Info

Product

Resources

About