Glycerol catabolism in Aspergillus nidulans

Hondmann, D.H.A.; Busink, R.; Witteveen, C.F.B.; Visser, Jaap

doi:10.1099/00221287-137-3-629

Cited by 33 publications

(54 citation statements)

References 28 publications

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“…Many microorganisms can use glycerol as a carbon source (9,10,11). The bacterial glycerol metabolic pathway is known as a dehydrogenation pathway and a phosphorylation pathway (9).…”

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confidence: 99%

l -Lactate Production from Biodiesel-Derived Crude Glycerol by Metabolically Engineered Enterococcus faecalis: Cytotoxic Evaluation of Biodiesel Waste and Development of a Glycerol-Inducible Gene Expression System

Doi

2015

Appl Environ Microbiol

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Biodiesel waste is a by-product of the biodiesel production process that contains a large amount of crude glycerol. To reuse the crude glycerol, a novel bioconversion process using Enterococcus faecalis was developed through physiological studies. The E. faecalis strain W11 could use biodiesel waste as a carbon source, although cell growth was significantly inhibited by the oil component in the biodiesel waste, which decreased the cellular NADH/NAD ؉ ratio and then induced oxidative stress to cells. When W11 was cultured with glycerol, the maximum culture density (optical density at 600 nm [OD 600 ]) under anaerobic conditions was decreased 8-fold by the oil component compared with that under aerobic conditions. Furthermore, W11 cultured with dihydroxyacetone (DHA) could show slight or no growth in the presence of the oil component with or without oxygen. These results indicated that the DHA kinase reaction in the glycerol metabolic pathway was sensitive to the oil component as an oxidant. The lactate dehydrogenase (Ldh) activity of W11 during anaerobic glycerol metabolism was 4.1-fold lower than that during aerobic glycerol metabolism, which was one of the causes of low L-lactate productivity. The E. faecalis pflB gene disruptant (⌬pfl mutant) expressing the ldhL1 LP gene produced 300 mM L-lactate from glycerol/crude glycerol with a yield of >99% within 48 h and reached a maximum productivity of 18 mM h ؊1 (1.6 g liter ؊1 h ؊1 ). Thus, our study demonstrates that metabolically engineered E. faecalis can convert crude glycerol to L-lactate at high conversion efficiency and provides critical information on the recycling process for biodiesel waste. Biodiesel is one of the alternatives to fossil fuel that reduces greenhouse effects and is being produced worldwide (1, 2). Biodiesel is produced from triacylglycerol, which is a main component of vegetable oils and animal fats, by either chemical or enzymatic reactions (1, 3, 4). When biodiesel is produced by a chemical process, triacylglycerol is exposed to methanol under highly alkaline conditions to cleave the ester bond between glycerol and fatty acids, and the resulting methylated fatty acids are used as biodiesel (3). Consequently, this chemical process generates a by-product containing a large amount of crude glycerol, together with methanol and salts, that is called biodiesel waste. Microbial bioconversion processes to convert glycerol to other valuable materials, such as alcohols and organic acids, are being actively developed to reuse the crude glycerol included in biodiesel waste (5). When biodiesel waste contains impurities, such as methanol and residual fatty acids, it is pretreated to remove these impurities (6, 7) because raw biodiesel waste may be cytotoxic to producers (7,8). However, cytotoxic substances have not been identified in biodiesel waste.Many microorganisms can use glycerol as a carbon source (9, 10, 11). The bacterial glycerol metabolic pathway is known as a dehydrogenation pathway and a phosphorylation pathway (9). In the dehydrogenation ...

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“…Many microorganisms can use glycerol as a carbon source (9,10,11). The bacterial glycerol metabolic pathway is known as a dehydrogenation pathway and a phosphorylation pathway (9).…”

mentioning

confidence: 99%

l -Lactate Production from Biodiesel-Derived Crude Glycerol by Metabolically Engineered Enterococcus faecalis: Cytotoxic Evaluation of Biodiesel Waste and Development of a Glycerol-Inducible Gene Expression System

Doi

2015

Appl Environ Microbiol

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“…Many microorganisms, including lactic acid bacteria, utilize glycerol in a phosphorylation pathway rather than in a dehydrogenation pathway (4,5,6,24,25,26). In contrast, W11 utilized glycerol efficiently in only the dehydrogenation pathway (Table 3).…”

Section: Discussionmentioning

confidence: 99%

Pyruvate Formate-Lyase Is Essential for Fumarate-Independent Anaerobic Glycerol Utilization in the Enterococcus faecalis Strain W11

Doi

Ikegami

2014

J Bacteriol

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Although anaerobic glycerol metabolism in Enterococcus faecalis requires exogenous fumarate for NADH oxidation, E. faecalis strain W11 can metabolize glycerol in the absence of oxygen without exogenous fumarate. In this study, metabolic end product analyses and reporter assays probing the expression of enzymes involved in pyruvate metabolism were performed to investigate this fumarate-independent anaerobic metabolism of glycerol in W11. Under aerobic conditions, the metabolic end products of W11 cultured with glycerol were similar to those of W11 cultured with glucose. However, when W11 was cultured anaerobically, most of the glucose was converted to L-lactate, but glycerol was converted to ethanol and formate. During anaerobic culture with glycerol, the expression of the L-lactate dehydrogenase and pyruvate dehydrogenase E1␣␤ genes in W11 was downregulated, whereas the expression of the pyruvate formate-lyase (Pfl) and aldehyde/alcohol dehydrogenase genes was upregulated. These changes in the expression levels caused the change in the composition of end products. A pflB gene disruptant (⌬pfl mutant) of W11 could barely utilize glycerol under anaerobic conditions, but the growth of the ⌬pfl mutant cultured with either glucose or dihydroxyacetone (DHA) under anaerobic conditions was the same as that of W11. Glucose metabolism and DHA generates one NADH molecule per pyruvate molecule, whereas glycerol metabolism in the dehydrogenation pathway generates two NADH molecules per pyruvate molecule. These findings demonstrate that NADH generated from anaerobic glycerol metabolism in the absence of fumarate is oxidized through the Pfl-ethanol fermentation pathway. Thus, Pfl is essential to avoid the accumulation of excess NADH during fumarate-independent anaerobic glycerol metabolism. E nterococci are the most common lactic acid bacteria in the human intestinal tract, and some strains are well-known opportunistic pathogens (1, 2). Physiological studies have shown that Enterococcus faecalis can use glycerol as a carbon source (3). Microorganisms utilize glycerol through a dehydrogenation pathway, a phosphorylation pathway, or both. The dehydrogenation pathway begins with the dehydrogenation of glycerol to produce dihydroxyacetone (DHA), which is catalyzed by glycerol dehydrogenase (GldA). The DHA produced is then phosphorylated by DHA kinase (4). In the phosphorylation pathway, glycerol is first phosphorylated to glycerol-3-phosphate (glycerol-3P) by a glycerol kinase and then oxidized to dihydroxyacetone phosphate by a glycerol-3P oxidase or a glycerol-3P dehydrogenase (4). In either pathway, the resulting dihydroxyacetone phosphate enters glycolysis. Lactic acid bacteria, such as several Lactobacillus species and Pediococcus pentosaceus, generally utilize glycerol through the phosphorylation pathway, rather than the dehydrogenation pathway (5, 6), although E. faecalis strains can utilize glycerol through either the dehydrogenation pathway or the phosphorylation pathway (7).In some E. faecalis strains, glycerol metabolis...

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“…The missing link in this pathway is an L-threo-3-deoxy-hexulosonate aldolase that would convert L-threo-3-deoxy-hexulosonate to pyruvate and L-glyceraldehyde. It was earlier suggested that D-galacturonate is metabolized through a non-phosphorylating pathway in the mold Aspergillus nidulans (9,10). However it was speculated that D-glyceraldehyde is an intermediate (11).…”

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The Missing Link in the Fungal D-Galacturonate Pathway

Hilditch

Berghäll

Kalkkinen

et al. 2007

Journal of Biological Chemistry

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The fungal path for the catabolism of D-galacturonate is only partially known. It is however distinctly different to the wellknown bacterial path. The known elements of the fungal path are D-galacturonate reductase converting D-galacturonate to L-galactonate and L-galactonate dehydratase converting L-galactonate to L-threo-3-deoxy-hexulosonate (2-keto-3-deoxy-L-galactonate). Here we describe the missing link in this pathway, an aldolase converting L-threo-3-deoxy-hexulosonate to pyruvate and L-glyceraldehyde. Fungal enzymes converting L-glyceraldehyde to glycerol have been described previously. The L-threo-3-deoxy-hexulosonate aldolase activity was induced in the mold Hypocrea jecorina (Trichoderma reesei) during growth on D-galacturonate. The enzyme was purified from this mold and a partial amino acid sequence obtained. This sequence was then used to identify the corresponding gene from the H. jecorina genome. The deletion of the gene resulted in a strain unable to grow on D-galacturonate and accumulating L-threo-3-deoxy-hexulosonate. The open reading frame was cloned from cDNA and functionally expressed in the yeast Saccharomyces cerevisiae. A histidine-tagged protein was expressed, purified, and characterized. The enzyme catalyzed reaction was reversible. With L-threo-3-deoxy-hexulosonate as substrate the K m was 3.5 mM and with pyruvate and L-glyceraldehyde the K m were 0.5 and 1.2 mM, respectively. D-Galacturonate is an important carbon source for microorganisms living on decaying plant material because D-galacturonate is the principal component of pectin. It is also of relevance in biotechnology when cheap raw materials such as pectin-rich materials like citrus peel or sugar beet pulp are to be exploited. However, knowledge about the microbial pathways for D-galacturonate catabolism is rather limited. A bacterial catabolic pathway has been described previously, whereas a fungal path is only partly known. The bacterial pathway consists of five enzymes converting D-galacturonate to pyruvate and D-glyceraldehyde-3-phosphate. The intermediate metabolites are D-ta-gaturonate, D-altronate, D-erythro-3-deoxy-hexulosonate, and D-erythro-3-deoxy-hexulosonate-6-phosphate. The enzymes in this path are uronate isomerase (EC 5.3.1.12) (1), NADHutilizing D-tagaturonate reductase (EC 1.1.1.5) (2), altronate dehydratase (EC 4.2.1.7) (3), 2-dehydro-3-deoxy-D-gluconate kinase (EC 2.7.1.45) (4), and 2-dehydro-3-deoxy-D-gluconate-6-phosphate aldolase (EC 4.1.2.14) (5). The fungal path is distinctly different. The first two steps of this pathway have been described. In the first step D-galacturonate is reduced to L-galactonate by an NADPH-coupled D-galacturonate reductase (6). In Hypocrea jecorina (Trichoderma reesei) this enzyme activity was induced when grown on D-galacturonate but the activity was absent during growth on other carbon sources. The D-galacturonate reductase gene gar1 is identified and the purified protein characterized (6). In the second step L-galactonate is converted to L-threo-3-deoxy-hexulosonate (2-keto-...

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Glycerol catabolism in Aspergillus nidulans

Cited by 33 publications

References 28 publications

l -Lactate Production from Biodiesel-Derived Crude Glycerol by Metabolically Engineered Enterococcus faecalis: Cytotoxic Evaluation of Biodiesel Waste and Development of a Glycerol-Inducible Gene Expression System

l -Lactate Production from Biodiesel-Derived Crude Glycerol by Metabolically Engineered Enterococcus faecalis: Cytotoxic Evaluation of Biodiesel Waste and Development of a Glycerol-Inducible Gene Expression System

Pyruvate Formate-Lyase Is Essential for Fumarate-Independent Anaerobic Glycerol Utilization in the Enterococcus faecalis Strain W11

The Missing Link in the Fungal D-Galacturonate Pathway

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