Evaluation of Acyl Coenzyme A Oxidase (Aox) Isozyme Function in the n-Alkane-Assimilating Yeast Yarrowia lipolytica

We have identified five acyl coenzyme A (CoA) oxidase isozymes (Aox1 through Aox5) in the n-alkane-assimilating yeast Yarrowia lipolytica, encoded by the POX1 through POX5 genes. The physiological function of these oxidases has been investigated by gene disruption. Single, double, triple, and quadruple disruptants were constructed. Global Aox activity was determined as a function of time after induction and of substrate chain length. Single null mutations did not affect growth but affected the chain length preference of acyl-CoA oxidase activity, as evidenced by a chain length specificity for Aox2 and Aox3. Aox2 was shown to be a long-chain acyl-CoA oxidase and Aox3 was found to be active against short-chain fatty acids, whereas Aox5 was active against molecules of all chain lengths. Mutations in Aox4 and Aox5 resulted in an increase in total Aox activity. The growth of mutant strains was analyzed. In the presence of POX1 only, strains did not grow on fatty acids, whereas POX4 alone elicited partial growth, and the growth of the double POX2-POX3-deleted mutant was normal excepted on plates containing oleic acid as the carbon source. The amounts of Aox protein detected by Western blotting paralleled the Aox activity levels, demonstrating the regulation of Aox in cells according to the POX genotype.     

Involvement of acyl coenzyme A oxidase isozymes in biotransformation of methyl ricinoleate into gamma-decalactone by Yarrowia lipolytica

We reported previously on the function of acyl coenzyme A (acyl-CoA) oxidase isozymes in the yeast Yarrowia lipolytica by investigating strains disrupted in one or several acyl-CoA oxidase-encoding genes (POX1 through POX5) (H. Wang et al., J. Bacteriol. 181:5140-5148, 1999). Here, these mutants were studied for lactone production. Monodisrupted strains produced similar levels of lactone as the wild-type strain (50 mg/liter) except for Deltapox3, which produced 220 mg of gamma-decalactone per liter after 24 h. The Deltapox2 Deltapox3 double-disrupted strain, although slightly affected in growth, produced about 150 mg of lactone per liter, indicating that Aox2p was not essential for the biotransformation. The Deltapox2 Deltapox3 Deltapox5 triple-disrupted strain produced and consumed lactone very slowly. On the contrary, the Deltapox2 Deltapox3 Deltapox4 Deltapox5 multidisrupted strain did not grow or biotransform methyl ricinoleate into gamma-decalactone, demonstrating that Aox4p is essential for the biotransformation.     

Involvement of Acyl Coenzyme A Oxidase Isozymes in Biotransformation of Methyl Ricinoleate into γ-Decalactone by Yarrowia lipolytica

We reported previously on the function of acyl coenzyme A (acyl-CoA) oxidase isozymes in the yeast Yarrowia lipolytica by investigating strains disrupted in one or several acyl-CoA oxidase-encoding genes (POX1 through POX5) (H. Wang et al., J. Bacteriol. 181:5140–5148, 1999). Here, these mutants were studied for lactone production. Monodisrupted strains produced similar levels of lactone as the wild-type strain (50 mg/liter) except for Δpox3, which produced 220 mg of γ-decalactone per liter after 24 h. The Δpox2 Δpox3 double-disrupted strain, although slightly affected in growth, produced about 150 mg of lactone per liter, indicating that Aox2p was not essential for the biotransformation. The Δpox2 Δpox3 Δpox5 triple-disrupted strain produced and consumed lactone very slowly. On the contrary, the Δpox2 Δpox3 Δpox4 Δpox5 multidisrupted strain did not grow or biotransform methyl ricinoleate into γ-decalactone, demonstrating that Aox4p is essential for the biotransformation.     

Expression of POX2 gene and disruption of POX3 genes in the industrial Yarrowia lipolytica on the γ-decalactone production

The yeast Yarrowia lipolytica growing on methyl ricinoleate can produce γ-decalactone, the worthy aroma compound, which can exhibit fruity and creamy sensorial notes, and recognized internationally as a safe food additive. Unfortunately, the yield is poor because of lactone degradation by enzyme Aox3 (POX3 gene encoded), which was responsible for continuation of oxidation after C(10) level and lactone reconsumption. In this paper, we chose the industrial Y. lipolytica (CGMCC accession number 2.1405), which is the diploid strain as the starting strain and constructed the recombinant strain Tp-12 by targeting the POX3 locus of the wild type, one copy of POX3 was deleted by CRF1+POX2 insertion. The other recombinant strain Tpp-11, which was a null mutant possessing multiple copies of POX2 and disrupted POX3 genes on two chromosomes, was constructed by inserting XPR2+hpt into the other copy of POX3 of Tp-12. The growth ability of the recombinants was changed after genetic modification in the fermentation medium. The production of γ-decalactone was increased, resulting from blocking β-oxidation at the C(10) Aox level and POX2 overexpression. The recombinant strain Tpp-11 was stable. Because there was no reconsumption of γ-decalactone, the mutant strain could be grown in continuous fermentation of methyl ricinoleate to produce γ-decalactone.     

Determination of Candida tropicalis acyl coenzyme A oxidase isozyme function by sequential gene disruption.

A recently developed transformation system has been used to facilitate the sequential disruption of the Candida tropicalis chromosomal POX4 and POX5 genes, encoding distinct isozymes of the acyl coenzyme A (acyl-CoA) oxidase which catalyzes the first reaction in the beta-oxidation pathway. The URA3-based transformation system was repeatedly regenerated by restoring the uracil requirement to transformed strains, either through selection for spontaneous mutations or by directed deletion within the URA 3 coding sequence, to permit sequential gene disruptions within a single strain of C. tropicalis. These gene disruptions revealed the diploid nature of this alkane- and fatty acid-utilizing yeast by showing that it contains two copies of each gene. A comparison of mutants in which both POX4 or both POX5 genes were disrupted revealed that the two isozymes were differentially regulated and displayed unique substrate profiles and kinetic properties. POX4 was constitutively expressed during growth on glucose and was strongly induced by either dodecane or methyl laurate and to a greater extent than POX5, which was induced primarily by dodecane. The POX4-encoded isozyme demonstrated a broad substrate spectrum in comparison with the narrow-spectrum, long-chain oxidase encoded by POX5. The absence of detectable acyl-CoA oxidase activity in the strain in which all POX4 and POX5 genes had been disrupted confirmed that all functional acyl-CoA oxidase genes had been inactivated. This strain cannot utilize alkanes or fatty acids for growth, indicating that the beta-oxidation pathway has been functionally blocked.     

Peroxisomal β-oxidation activities and γ-decalactone production by the yeast Yarrowia lipolytica

γ-Decalactone is a peachy aroma compound resulting from the peroxisomal β-oxidation of ricinoleic acid by yeasts. The expression levels of acyl-CoA oxidase (gene deletion) and 3-ketoacyl-CoA thiolase activities (gene amplification on replicative plasmids) were modified in the yeast Yarrowia lipolytica. The effects of these modifications on β-oxidation were measured. Overexpression of thiolase activity did not have any effect on the overall β-oxidation activity. The disruption of one of the acyl-CoA oxidase genes resulted in an enhanced activity. The enhancement led to an increase of overall β-oxidation activity but reduced the γ-decalactone production rates. This seemed to indicate a non-rate-limiting role for β-oxidation in the biotransformation of ricinoleic acid to γ-decalactone by the yeast Yarrowia lipolytica. All strains produced and then consumed γ-decalactone. We checked the ability of the different strains to consume γ-decalactone in a medium containing the lactone as sole carbon source. The consumption of the strain overexpressing acyl-CoA oxidase activity was higher than that of the wild-type strain. We␣concluded that peroxisomal β-oxidation is certainly involved in γ-decalactone catabolism by the yeast Y.␣lipolytica. The observed production rates probably depend on an equilibrium between production and consumption of the lactone. Received: 13 June 1997 / Received revision: 2 October 1997 / Accepted: 14 October 1997     

The acyl-CoA oxidases from the yeast Yarrowia lipolytica: characterization of Aox2p

One of the acyl-CoA oxidases from the yeast Yarrowia lipolytica, acyl-CoA oxidase 2 (Aox2p), has been expressed in Escherichia coli as an active, N-terminally tagged (His)(6) fusion protein. The specific activity of the purified enzyme, containing FAD, was 19.7 micromolmin(-1)mg(-1) using myristoyl-CoA as substrate. Using substrates with different chain lengths and different substituents, its kinetic properties were further analyzed. Straight-chain acyl-CoAs, with a chain length of 10-14C, are well oxidized, reflecting the properties of Aox2p as deduced from in vivo studies. Acyl-CoAs containing more than 14C were also desaturated, if their concentration was below 25 microM or if proteins capable of binding these CoA-esters, such as albumin or beta-casein, were added to the assay. These long-chain acyl-CoAs, although poor substrates, acted as competitors for the short- and medium-chain substrates. Compared to palmitoyl-CoA, activity toward hexadecadioyl-CoA, containing a omega-carboxy group, was similar. Taken together, these data suggest that micelles of long-chain acyl-CoAs are able to bind and inhibit Aox2p. The enzyme was also active toward acyl-CoA-esters containing a 2-methyl group, but only the 2S isomer was recognized.     

Lipid accumulation, lipid body formation, and acyl coenzyme A oxidases of the yeast Yarrowia lipolytica

Yarrowia lipolytica contains five acyl-coenzyme A oxidases (Aox), encoded by the POX1 to POX5 genes, that catalyze the limiting step of peroxisomal beta-oxidation. In this study, we analyzed morphological changes of Y. lipolytica growing in an oleic acid medium and the effect of POX deletions on lipid accumulation. Protrusions involved in the uptake of lipid droplets (LDs) from the medium were seen in electron micrographs of the surfaces of wild-type cells grown on oleic acid. The number of protrusions and surface-bound LDs increased during growth, but the sizes of the LDs decreased. The sizes of intracellular lipid bodies (LBs) and their composition depended on the POX genotype. Only a few, small, intracellular LBs were observed in the mutant expressing only Aox4p (Deltapox2 Deltapox3 Deltapox5), but strains expressing either Aox3p or both Aox3p and Aox4p had the same number of LBs as did the wild type. In contrast, strains expressing either Aox2p or both Aox2p and Aox4p formed fewer, but larger, LBs than did the wild type. The size of the LBs increased proportionately with the amount of triacylglycerols in the LBs of the mutants. In summary, Aox2p expression regulates the size of cellular triacylglycerol pools and the size and number of LBs in which these fatty acids accumulate.     

Role of beta-oxidation enzymes in gamma-decalactone production by the yeast Yarrowia lipolytica.

Some microorganisms can transform methyl ricinoleate into gamma-decalactone, a valuable aroma compound, but yields of the bioconversion are low due to (i) incomplete conversion of ricinoleate (C(18)) to the C(10) precursor of gamma-decalactone, (ii) accumulation of other lactones (3-hydroxy-gamma-decalactone and 2- and 3-decen-4-olide), and (iii) gamma-decalactone reconsumption. We evaluated acyl coenzyme A (acyl-CoA) oxidase activity (encoded by the POX1 through POX5 genes) in Yarrowia lipolytica in lactone accumulation and gamma-decalactone reconsumption in POX mutants. Mutants with no acyl-CoA oxidase activity could not reconsume gamma-decalactone, and mutants with a disruption of pox3, which encodes the short-chain acyl-CoA oxidase, reconsumed it more slowly. 3-Hydroxy-gamma-decalactone accumulation during transformation of methyl ricinoleate suggests that, in wild-type strains, beta-oxidation is controlled by 3-hydroxyacyl-CoA dehydrogenase. In mutants with low acyl-CoA oxidase activity, however, the acyl-CoA oxidase controls the beta-oxidation flux. We also identified mutant strains that produced 26 times more gamma-decalactone than the wild-type parents.     

Role of β-Oxidation Enzymes in γ-Decalactone Production by the Yeast Yarrowia lipolytica

Some microorganisms can transform methyl ricinoleate into γ-decalactone, a valuable aroma compound, but yields of the bioconversion are low due to (i) incomplete conversion of ricinoleate (C₁₈) to the C₁₀ precursor of γ-decalactone, (ii) accumulation of other lactones (3-hydroxy-γ-decalactone and 2- and 3-decen-4-olide), and (iii) γ-decalactone reconsumption. We evaluated acyl coenzyme A (acyl-CoA) oxidase activity (encoded by the POX1 through POX5 genes) in Yarrowia lipolytica in lactone accumulation and γ-decalactone reconsumption in POX mutants. Mutants with no acyl-CoA oxidase activity could not reconsume γ-decalactone, and mutants with a disruption of pox3, which encodes the short-chain acyl-CoA oxidase, reconsumed it more slowly. 3-Hydroxy-γ-decalactone accumulation during transformation of methyl ricinoleate suggests that, in wild-type strains, β-oxidation is controlled by 3-hydroxyacyl-CoA dehydrogenase. In mutants with low acyl-CoA oxidase activity, however, the acyl-CoA oxidase controls the β-oxidation flux. We also identified mutant strains that produced 26 times more γ-decalactone than the wild-type parents.     

Alcohol oxidase is a novel pathogenicity factor for Cladosporium fulvum, but aldehyde dehydrogenase is dispensable

Cladosporiumfulvum is a mitosporic ascomycete pathogen of tomato. A study of fungal genes expressed during carbon starvation in vitro identified several genes that were up regulated during growth in planta. These included genes predicted to encode acetaldehyde dehydrogenase (Aldh1) and alcohol oxidase (Aox1). An Aldh1 deletion mutant was constructed. This mutant lacked all detectable ALDH activity, had lost the ability to grow with ethanol as a carbon source, but was unaffected in pathogenicity. Aox1 expression was induced by carbon starvation and during the later stages of infection. The alcohol oxidase enzyme activity has broadly similar properties (Km values, substrate specificity, pH, and heat stability) to yeast enzymes. Antibodies raised to Hansenula polymorpha alcohol oxidase (AOX) detected antigens in Western blots of starved C. fulvum mycelium and infected plant material. Antigen reacting with the antibodies was localized to organelles resembling peroxisomes in starved mycelium and infected plants. Disruption mutants of Aox1 lacked detectable AOX activity and had markedly reduced pathogenicity as assayed by two different measures of fungal growth. These results identify alcohol oxidase as a novel pathogenicity factor and are discussed in relation to peroxisomal metabolism of fungal pathogens during growth in planta.     

Peroxisomal acyl-coenzyme A oxidase multigene family of the yeast Candida tropicalis; nucleotide sequence of a third gene and its protein product

We have determined the complete nucleotide sequence of gene POX2, which encodes one of the major peroxisomal polypeptides (PXPs) of Candida tropicalis. POX2 is linked to gene POX4, which codes for a subunit (PXP-4) of long-chain acyl-CoA oxidase. Southern blot analysis revealed that POX2 had a significant homology to POX4, and also to gene POX5 which encodes a subunit (PXP-5) of the isozyme of acyl-CoA oxidase. PXP-2, the protein product of POX2, was co-purified with PXP-4 from the isolated peroxisomes. PXP-2 itself was a flavoprotein and likely to form an equimolar complex with PXP-4, although its enzymatic activity was uncertain. POX2 corresponds to a single open reading frame of 724 amino acids and has no introns. The N-terminal sequence and the calculated Mr of the deduced polypeptide were consistent with those of isolated PXP-2. The primary structure was highly homologous to those of PXP-4 and PXP-5 in respect of the amino acid sequence and the hydropathy profile. We conclude that POX2 is a third gene of the peroxisomal acyl-COA oxidase multigene family.     

Repression of fatty-acyl-CoA oxidase-encoding gene expression is not necessarily a determinant of high-level production of dicarboxylic acids in industrial dicarboxylic-acid-producing Candida tropicalis

The synthesis of dicarboxylic acids (DCAs) in Candida tropicalis is thought to be induced by a decrease in fatty acyl-CoA-oxidase activity. However, in the present study we demonstrate that repression of the POX4 gene, encoding fatty acyl-CoA oxidase, does not directly lead to high-level production of DCAs. No fatty acyl-CoA-oxidase activity was detected if the POX4 gene of C. tropicalis strain 1098 (wild-type strain) was disrupted. Furthermore, introduction of the POX4 gene from C. tropicalis strain M1210A3, which is a mutant derived from strain 1098 and is used as an industrial DCA-producing strain, still exhibited low-level fatty acyl-CoA-oxidase activity. Nevertheless, production of DCA was not observed in either case. Furthermore, the increase in acyl-CoA-oxidase activity by expression of the POX4 gene in strain M1210A3 did not reduce high-level production of DCA. These results suggest that alterations in acyl-CoA-oxidase activity are not necessarily related to production of DCA in industrial DCA-producing C. tropicalis M1210A3.     

Peroxisome division in the yeast Yarrowia lipolytica is regulated by a signal from inside the peroxisome

We describe an unusual mechanism for organelle division. In the yeast Yarrowia lipolytica, only mature peroxisomes contain the complete set of matrix proteins. These mature peroxisomes assemble from several immature peroxisomal vesicles in a multistep pathway. The stepwise import of distinct subsets of matrix proteins into different immature intermediates along the pathway causes the redistribution of a peroxisomal protein, acyl-CoA oxidase (Aox), from the matrix to the membrane. A significant redistribution of Aox occurs only in mature peroxisomes. Inside mature peroxisomes, the membrane-bound pool of Aox interacts with Pex16p, a membrane-associated protein that negatively regulates the division of early intermediates in the pathway. This interaction inhibits the negative action of Pex16p, thereby allowing mature peroxisomes to divide.     

Ecoenzymatic stoichiometry of recalcitrant organic matter decomposition: the growth rate hypothesis in reverse

The flow of carbon and nutrients from plant production into detrital food webs is mediated by microbial enzymes released into the environment (ecoenzymes). Ecoenzymatic activities are linked to both microbial metabolism and environmental resource availability. In this paper, we extend the theoretical and empirical framework for ecoenzymatic stoichiometry from nutrient availability to carbon composition by relating ratios of ??-1,4-glucosidase (BG), acid (alkaline) phosphatase (AP), ??-N-acetylglucosaminidase (NAG), leucine aminopeptidase (LAP) and phenol oxidase (POX) activities in soils to measures of organic matter recalcitrance, using data from 28 ecosystems. BG and POX activities are uncorrelated even though both are required for lignocellulose degradation. However, the ratio of BG:POX activity is negatively correlated with the relative abundance of recalcitrant carbon. Unlike BG, POX activity is positively correlated with (NAG + LAP) and AP activities. We propose that the effect of organic matter recalcitrance on microbial C:N and C:P threshold element ratios (TER) can be represented by normalizing BG, AP and (NAG + LAP) activities to POX activity. The scaling relationships among these ratios indicate that the increasing recalcitrance of decomposing organic matter effectively reverses the growth rate hypothesis of stoichiometric theory by decreasing carbon and nutrient availability and slowing growth, which increases TER_N:P. This effect is consistent with the narrow difference between the mean elemental C:N ratios of soil organic matter and microbial biomass and with the inhibitory effect of N enrichment on rates of decomposition and microbial metabolism for recalcitrant organic matter. From these findings, we propose a conceptual framework for bottom-up decomposition models that integrate the stoichiometry of ecoenzymatic activities into general theories of ecology.     

Acyl-CoA oxidase is imported as a heteropentameric, cofactor-containing complex into peroxisomes of Yarrowia lipolytica

Five isoforms of acyl-CoA oxidase (Aox), designated Aox1p to Aox5p, constitute a 443-kD heteropentameric complex containing one polypeptide chain of each isoform within the peroxisomal matrix of the yeast Yarrowia lipolytica. Assembly of the Aox complex occurs in the cytosol and precedes its import into peroxisomes. Peroxisomal targeting of the Aox complex is abolished in a mutant lacking the peroxin Pex5p, a component of the matrix protein targeting machinery. Import of the Aox complex into peroxisomes does not involve the cytosolic chaperone Pex20p, which mediates the oligomerization and import of peroxisomal thiolase. Aox2p and Aox3p play a pivotal role in the formation of the Aox complex in the cytosol and can substitute for one another in promoting assembly of the complex. In vitro, these subunits retard disassembly of the Aox complex and increase the efficiency of its reassembly. Neither Aox2p nor Aox3p is required for acquisition of the cofactor FAD by other components of the complex. We provide evidence that the Aox2p- and Aox3p-assisted assembly of the Aox complex in the cytosol is mandatory for its import into peroxisomes and that no component of the complex can penetrate the peroxisomal matrix as a monomer.     

Metabolism of ricinoleic acid into gamma-decalactone: beta-oxidation and long chain acyl intermediates of ricinoleic acid in the genus Sporidiobolus sp

In order to study differences in gamma-decalactone production in yeast, four species of Sporidiobolus were cultivated with 5% of methyl ricinoleate as the lactone substrate. In vivo studies showed different time courses of intermediates of ricinoleic acid breakdown between the four species. In vitro studies of the beta-oxidation system were conducted with crude cell extracts of Sporidiobolus spp. and with ricinoleyl-CoA (RCoA) as substrate. The beta-oxidation was detected by measuring acyl-CoA oxidase, 3-hydroxyacyl-CoA dehydrogenase activities, and acetyl-CoA production. The time courses of the CoA esters resulting from RCoA breakdown by crude extract of Sporidiobolus spp. permit the proposal of different metabolic models in the yeast. These models explained the differences observed during in vivo studies.     

Lipid Accumulation, Lipid Body Formation, and Acyl Coenzyme A Oxidases of the Yeast Yarrowia lipolytica

Yarrowia lipolytica contains five acyl-coenzyme A oxidases (Aox), encoded by the POX1 to POX5 genes, that catalyze the limiting step of peroxisomal β-oxidation. In this study, we analyzed morphological changes of Y. lipolytica growing in an oleic acid medium and the effect of POX deletions on lipid accumulation. Protrusions involved in the uptake of lipid droplets (LDs) from the medium were seen in electron micrographs of the surfaces of wild-type cells grown on oleic acid. The number of protrusions and surface-bound LDs increased during growth, but the sizes of the LDs decreased. The sizes of intracellular lipid bodies (LBs) and their composition depended on the POX genotype. Only a few, small, intracellular LBs were observed in the mutant expressing only Aox4p (Δpox2 Δpox3 Δpox5), but strains expressing either Aox3p or both Aox3p and Aox4p had the same number of LBs as did the wild type. In contrast, strains expressing either Aox2p or both Aox2p and Aox4p formed fewer, but larger, LBs than did the wild type. The size of the LBs increased proportionately with the amount of triacylglycerols in the LBs of the mutants. In summary, Aox2p expression regulates the size of cellular triacylglycerol pools and the size and number of LBs in which these fatty acids accumulate.