Investigation of Malic Acid Production in Aspergillus oryzae under Nitrogen Starvation Conditions

Knuf, Christoph; Nookaew, Intawat; Brown, Stephen H.M.; McCulloch, Michael; Berry, Alan; Nielsen, Jens

doi:10.1128/aem.01445-13

Cited by 72 publications

(53 citation statements)

References 29 publications

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“…Recently, a study in Aspergillus oryzae revealed that nitrogen starvation promotes intracellular malic acid biosynthesis (34). With its large expansion of low-pH secretory hydrolytic enzymes (35), A. oryzae is becoming a promising species for the industrial production of malic acid.…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…Additionally, the muC strain was grown in the low oxygen tension condition, which made TCA cycle very weak. Thus, the malic acid production should use the pathway (1), which involved PEP carboxykinase converting PEP to oxaloacetate and malate dehydrogenase converting oxaloacetate to malate and this pathway was found in another aerobe: Aspergillus oryzae , which was a malate producer . The native anaplerotic reaction such as the reaction converting pyruvate to oxaloacetate by pyruvate carboxylase was found in T. fusca .…”

Section: Resultsmentioning

confidence: 99%

“…Thus, the malic acid production should use the pathway (1), which involved PEP carboxykinase converting PEP to oxaloacetate and malate dehydrogenase converting oxaloacetate to malate and this pathway was found in another aerobe: Aspergillus oryzae, which was a malate producer. 16 The native anaplerotic reaction such as the reaction converting pyruvate to oxaloacetate by pyruvate carboxylase was found in T. fusca. There is another route converting the malate to pyruvate or pyruvate to malate, catalyzed by malate dehydrogenase (oxaloacetate-decarboxylating).…”

Section: Pathways To Malic Acidmentioning

confidence: 99%

“…To thoroughly compare the metabolite files between different strains, malate, succinate, butyrate, lactate and propionate were measured by HPLC. The effect of yeast extract on malic acid production by T. fusca muC‐16 was tested because the nitrogen sources affected the malate secretion . The batch fermentation on ∼100 g/L cellulose and high concentration of corn stover were studied to achieve the high titer of malic acid.…”

Section: Introductionmentioning

confidence: 99%

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Metabolic engineering of a laboratory-evolvedThermobifida fuscamuC strain for malic acid production on cellulose and minimal treated lignocellulosic biomass

Deng

Mao

Zhang

2016

Biotechnol Progress

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Malic acid is mainly used as an acidulant and taste enhancer in the beverage and food industry. Previously, a mutant strain Thermobifida fusca muC, obtained by adaptive evolution was found to accumulate malic acid on cellulose with low yield. In this study, the malic acid synthesis pathway in T. fusca muC was confirmed to be from phosphoenolpyruvate to oxaloacetate, followed by reduction of oxaloacetate to malate. To increase the yield of malic acid by the muC strain significantly, the carbon flux from pyruvate was redirected to oxaloacetate by expressing an exogenous pyruvate carboxylase (PCx) gene from Corynebacterium glutamicum ATCC 13032 in the chromosome of T. fusca muC-16. The yield of malic acid in the engineered strain muC-16 was increased by 47.9% compared to the parent strain muC. The muC-16 strain was then grown on ∼100 g/L cellulose and the highest titer of malic acid was 62.76 g/L by batch fermentation. T. fusca muC-16 strain converted milled corn stover to malic acid with the highest titer of 21.47 g/L with minimal treatment.

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“…However, this microbe produces aflatoxin, which makes it unsuitable for use in food-grade chemical production. Other species of Aspergillus, such as Aspergillus niger ATCC10577 (West, 2011), Aspergillus oryzae NRRL3488 (Knuf et al, 2013), and Aspergillus oryzae DSM 1863 (Knuf et al, 2014), are found to produce 17, 30.2, and 42 g/L L-malate, respectively. In addition, the isolated microorganisms such as Schizophyllum commune and Zygosaccharomyces rouxii are found to accumulate 18 and 75 g/L L-malate, respectively, but the yield and productivity are low, limiting their usefulness for industrial production (Kawagoe et al, 1997;Taing and Taing, 2007).…”

Section: Introductionmentioning

confidence: 99%

Metabolic engineering of Escherichia coli W3110 to produce L‐malate

Dong

Chen

Qian

et al. 2016

Biotech & Bioengineering

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A four-carbon dicarboxylic acid L-malate has recently attracted attention due to its potential applications in the fields of medicine and agriculture. In this study, Escherichia coli W3110 was engineered and optimized for L-malate production via one-step L-malate synthesis pathway. First, deletion of the genes encoding lactate dehydrogenase (ldhA), pyruvate oxidase (poxB), pyruvate formate lyase (pflB), phosphotransacetylase (pta), and acetate kinase A (ackA) in pta-ackA pathway led to accumulate 20.9 g/L pyruvate. Then, overexpression of NADP -dependent malic enzyme C490S mutant in this multi-deletion mutant resulted in the direct conversion of pyruvate into L-malate (3.62 g/L). Next, deletion of the genes responsible for succinate biosynthesis further enhanced L-malate production up to 7.78 g/L. Finally, L-malate production was elevated to 21.65 g/L with the L-malate yield to 0.36 g/g in a 5 L bioreactor by overexpressing the pos5 gene encoding NADH kinase in the engineered E. coli F0931 strain. This study demonstrates the potential utility of one-step pathway for efficient L-malate production. Biotechnol. Bioeng. 2017;114: 656-664. © 2016 Wiley Periodicals, Inc.

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Investigation of Malic Acid Production in Aspergillus oryzae under Nitrogen Starvation Conditions

Cited by 72 publications

References 29 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

Metabolic engineering of a laboratory-evolvedThermobifida fuscamuC strain for malic acid production on cellulose and minimal treated lignocellulosic biomass

Metabolic engineering of Escherichia coli W3110 to produce L‐malate

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