Yeast Candida Maltosa Research Articles

Crude Aqueous Root Extracts of Solanum spp. Show Nematotoxic Activity Against Meloidogyne incognita

Brazilian crops are strongly affected by the root-knot nematode Meloidogyne incognita, which compromises food production and causes serious economic losses around the world. Synthetic nematicides are still some of the main control strategies for this pathogen, despite the high risk to human health and the environment. Therefore, sustainable biotechnology represents a viable alternative for nematode control. Plants of the Solanaceae family are present in several biomes and are a source of secondary metabolites of various chemical classes. In this context, the nematotoxic effect of crude aqueous root extracts (CAREs) of three Solanum species was investigated: Solanum americanum, S. subinerme and S. lycocarpum. CARE showed high nematicidal activity (94.6±9.2% mortality, p ≤ 0.001) against second-stage juveniles (J2) of M. incognita, even after heating to 50 °C. These CAREs also inhibited hatching of juveniles (98.4±0.7% of mortality, p ≤ 0.001) in addition to presenting a mean lethal dose of 336 μg ml-1. No toxic activity was observed against non-target organisms, such as fungi (Trichoderma harzianum and Trichoderma asperellum), yeast (Candida maltosa), nitrogen-fixing bacteria (Bradyrhizobium diazoefficiaens, B. japonicum), two strains of Azospirillum brasilense, AbV 5 and AbV 6 and Bacillus [2538 Ba]. Bioassay performed in a greenhouse demonstrated that CAREs of these species were effective in reducing the number of egg masses and galls (> 85%, p ≤ 0.0001), and did not show a phytotoxic effect on soybean seedlings. Our findings indicate that CAREs from plants of the Solanaceae family, especially S. lycocarpum, demonstrated nematotoxic activity on J2 and inhibited the hatching of Meloidogyne incognita eggs, representing an ecologically appropriate tool to control nematode infection in crops worldwide.

Changes in cell wall structure and protein set in Candida maltosa grown on hexadecane.

The yeast Candida maltosa is a model organism for studying adaptive changes in the structure and function of the cell wall when consuming water-insoluble nutrient sources. The cells of C. maltosa that utilize hydrocarbons contain supramolecular structures, so-called "canals" in the cell wall. Differences in protein profiles of culture liquids and cell wall extracts of C. maltosa grown on glucose and hexadecane were analyzed. Three proteins specific of cells grown on hexadecane were revealed using mass spectrometry: glycosyl hydrolase EPD2 in the culture liquid; a protein belonging to the cytochrome C family in the 0.5 mol/L NaCl extract; and PPIA_CANAL protein known as chaperone, in the 0.1% SDS extract. The possible role of these proteins in cell wall structures responsible for adaptation to hexadecane utilization is discussed.

Fungal biotransformation of short-chain n-alkylcycloalkanes.

The cycloalkanes, comprising up to 45% of the hydrocarbon fraction, occur in crude oil or refined oil products (e.g., gasoline) mainly as alkylated cyclohexane derivatives and have been increasingly found in environmental samples of soil and water. Furthermore, short-chain alkylated cycloalkanes are components of the so-called volatile organic compounds (VOCs). This study highlights the biotransformation of methyl- and ethylcyclohexane by the alkane-assimilating yeast Candida maltosa and the phenol- and benzoate-utilizing yeast Trichosporon mucoides under laboratory conditions. In the course of this biotransformation, we detected 25 different metabolites, which were analyzed by HPLC and GC-MS. The biotransformation process of methylcyclohexane in both yeasts involve (A) ring hydroxylation at different positions (C2, C3, and C4) and subsequent oxidation to ketones as well as (B) oxidation of the alkyl side chain to hydroxylated and acid products. The yeast T. mucoides additionally performs ring hydroxylation at the C1-position and (C) oxidative decarboxylation and (D) aromatization of cyclohexanecarboxylic acid. Both yeasts also oxidized the saturated ring system and the side chain of ethylcyclohexane. However, the cyclohexylacetic acid, which was formed, seemed not to be substrate for aromatization. This is the first report of several new transformation reactions of alkylated cycloalkanes for eukaryotic microorganisms.

Modelling the effects of lactic acid, sodium benzoate and temperature on the growth of Candida maltosa.

The strain of Candida maltosaYP1 was originally isolated from air filters that ensured clean air overpressure in yoghurt fermentation tanks. Its growth in contaminated yoghurts manifested outwardly through surface growth, assimilation lactic acid and slight production of carbon dioxide. This was the opportunity to model the effects of lactic acid and sodium benzoate on growth and predict its behaviour in foods. The approach used in this study provides knowledge about microbiological quality development during storage of the fermented milk products as well as some preserved foods for storage and consumption.

Modifications of the cell wall of yeasts grown on hexadecane and under starvation conditions.

Electron-microscopic examinations have demonstrated local modifications in the cell wall of the yeast Candida maltosa grown on hexadecane. In our earlier studies, these modified sites, observed in other yeasts grown on oil hydrocarbons, were conventionally called 'canals'. The biochemical and cytochemical studies of C. maltosa have revealed a correlation between the formation of 'canals' and decrease in the amount of cell wall polysaccharides, glucan and mannan. The ultrathin sections and surface replicas have shown that the 'canals' are destroyed by pronase, thus indicating that a significant proportion of their content is represented by proteins. This finding was compatible with our earlier data on the localization of oxidative enzymes in 'canals' and possible participation of the 'canals' in the primary oxidation of hydrocarbons. A completely unexpected and intriguing phenomenon has been the appearance of 'canals' in the yeast C. maltosa under starvation conditions. Unlike the yeasts grown on hexadecane, mannan almost disappears in starving cells, while the quantity of glucan first decreases and then is restored to its initial level. The role of 'canals' in starving cells is as yet unclear; it is assumed that they acquire exoenzymes involved in the utilization of products of cell lysis in the starving population. In the future, 'canals' of starving cells will be studied in connection with their possible participation in apoptosis.

Biotransformation of cyclohexane and related alicyclic hydrocarbons by Candida maltosa and Trichosporon species

The hydrocarbons of crude oil and oil products such as gasoline or diesel oil comprise an aliphatic, an aromatic, and an alicyclic fraction. The alicyclic fraction represents a group of substances which, along with polycyclic hydrocarbons and tar-like components, are among the most biodegradation resistant components of oil. The major part of the alicyclic oil fraction is made up of cyclohexane and its derivatives. Little is currently known about the microbial, especially fungal, degradation of alicyclic hydrocarbons. In this study, the ability of yeasts of the genera Candida and Trichosporon to use cyclohexane and some related alicyclic hydrocarbons as substrates was investigated. The anamorphic ascomycetous yeast Candida maltosa and different strains of the anamorphic basidiomycetous yeast genus Trichosporon are not able to utilize cyclohexane as carbon source when present at 0.005%–1.0% and at concentrations of over about 0.5% cyclohexane is toxic for them. However at concentrations of 0.25%–0.4% the yeasts can oxidize cyclohexane and transform it via cyclohexanol to cyclohexanone. Cyclohexanone seems to be a dead-end-product for these yeasts because no further conversion of cyclohexanone to ε-caprolactone could be demonstrated. Analysis of the cycloalkane oxidation after cell cultivation with different carbon sources revealed that non-specific enzymes were involved and no additional enzyme induction seems to occur in the presence of cycloalkanes.

Metabolism of alkenes and ketones by Candida maltosa and related yeasts.

Knowledge is scarce about the degradation of ketones in yeasts. For bacteria a subterminal degradation of alkanes to ketones and their further metabolization has been described which always involved Baeyer-Villiger monooxygenases (BVMOs). In addition, the question has to be clarified whether alkenes are converted to ketones, in particular for the oil degrading yeast Candida maltosa little is known. In this study we show the degradation of the aliphatic ketone dodecane-2-one by Candida maltosa and the related yeasts Candida tropicalis, Candida catenulata and Candida albicans as well as Trichosporon asahii and Yarrowia lipolytica. One pathway is initiated by the formation of decyl acetate, resulting from a Baeyer-Villiger-oxidation of this ketone. Beyond this, an initial reduction to dodecane-2-ol by a keto reductase was clearly shown. In addition, two different ways to metabolize dodec-1-ene were proposed. One involved the formation of dodecane-2-one and the other one a conversion leading to carboxylic and dicarboxylic acids. Furthermore the induction of ketone degrading enzymes by dodecane-2-one and dodec-1-ene was shown. Interestingly, with dodecane no subterminal degradation products were detected and it did not induce any enzymes to convert dodecane-2-one.

Identification and Characterization of a Gene Encoding an ABC Transporter Expressed in the Dicarboxylic Acid-Producing YeastCandida maltosa

A gene, CmCDR1, encoding an ABC transporter of the dicarboxylic acid (DCA)-producing yeast Candida maltosa was cloned. Transcription of CmCDR1 was upregulated in a DCA-hyper-producing mutant of C. maltosa in a later phase of culture on n-dodecane, but not in its parental strain. CmCDR1 expression was significantly induced by the longer-chain DCA in this mutant.

Degradation of phenylalkanes and characterization of aromatic intermediates acting as growth inhibiting substances in hydrocarbon utilizing yeast Candida maltosa

Phenylalkanes constitute up to 28% of refined gasoline fuel oil. Because of the widespread use of petroleum, oil and gasoline and the environmental risks associated with accidental spills, the ability to degrade these compounds is a major goal of environmental research. We here report our investigation in the use of yeast to degrade phenylalkanes. Among the studied strains of the genera Candida, Hansenula, Pichia, Saccharomyces and Arxula the alkane-assimilating ones grew on phenylalkanes with alkyl side chains longer than C7, utilizing them as sole carbon and energy source. Detailed studies on phenylalkane metabolism were carried out with Candida maltosa SBUG 700. During incubation of this yeast with phenyldecane, phenyloctane and phenylhexane 4-phenylbutanoic acid, 4-phenyl-3-butenoic acid and phenylacetic acid were produced whereas benzoic acid was the only detectable degradation product of phenylnonane and phenylheptane. Phenylacetic acid and benzoic acid were accumulated in high amounts due to the inability of C. maltosa to oxidize these acids further. Oxidation of phenylalkanes was inhibited in the presence of 1-aminobenzotriazole acting as inhibitor of cytochrome P450. Furthermore, toxic effects of benzoic acid and phenylacetic acid amplified by fallen pH values of growth medium strongly inhibited the metabolism of phenylalkanes as well as glucose assimilation and this led to incomplete phenylalkane degradation.

Role of Speciation in Organotin Toxicity to the Yeast Candida maltosa

Assessment of organometal pollution requires an understanding of the various processes that influence the bioavailability and toxicity of the contaminant. Organotins may exist as both cationic species and neutral hydroxides in aqueous solution, with the formation of chloride species in the presence of Cl-. Although these species have different chemical properties, there is very little information on the influence of speciation on organotin and microbial cell interactions. Tributyltin (TBT) and triphenyltin (TPT) interactions with the yeast Candida maltosa were investigated between pH 3.5 and 7.5 and in up to 0.5 M NaCl at pH 5.5. Toxicity increased with both pH and NaCl concentration and the mechanisms of interaction depended on the species present in solution. TBT and TPT interacted by different mechanisms, as evidenced by action on membrane fluidity. Furthermore, there was a strong correlation between toxicity and overall octanol-water distribution ratio (D(OW)) of organotin compounds. Triorganotin cations are less toxic than triorganotin hydroxides, which are in turn less toxic than triorganotin chlorides. These findings underline the importance of speciation effects on organotin interactions in the environment.

A Gcn4p Homolog Is Essential for the Induction of a Ribosomal Protein L41 Variant Responsible for Cycloheximide Resistance in the Yeast Candida maltosa

Cycloheximide (CYH) resistance in the yeast Candida maltosa is based on the inducible expression of genes encoding a variant of ribosomal protein L41-Q, with glutamine at position 56 instead of the proline found in normal L41. The promoter of L41-Q2a, one of the L41-Q gene alleles encoding L41-Q, has an element similar to the Gcn4p-responsive element of Saccharomyces cerevisiae. In a previous study, this element was shown to be essential for the induction of L41-Q by CYH. In the present study, a C. maltosa GCN4 homolog, C-GCN4, was cloned. It had a long 5'-leader region with three upstream open reading frames. Enhanced expression of the C-GCN4 reporter fusion gene upon the addition of 3-aminotriazole or by mutations in start codons of all three upstream open reading frames indicates that C-GCN4 expression is under translation repression as was seen with GCN4. The C-GCN4-depleted mutant was unable to grow in a nutrient medium containing CYH and did not express L41-Q genes. Recombinant C-Gcn4p bound to the consensus DNA element for Gcn4p, 5'-(G/A)TGACTCAT-3', located upstream of L41-Q2a. Thus, C-Gcn4p, which likely functions in the general control of amino acid biosynthesis, is essential for the expression of L41-Q genes.

Rapid monitoring of the biodegradation of phenol-like compounds by the yeast Candida maltosa using BOD measurements

Abstract Since phenol-like compounds can have serious environmental effects, microbiological and biochemical features of their aerobic degradation are of great interest, and monitoring oxygen uptake can give valuable data about the processes involved. It is shown here that the AQUALYTIC® Sensomat System can provide reliable, continuous measurement of biological oxygen demand (BOD), and is suitable for small-scale studies of the degradation of phenol-like compounds by the soil-borne yeast Candida maltosa. It was found that C. maltosa can use phenol and catechol as sole sources of carbon and energy at concentrations up to 1.7 g l −1 and 1.5 g l −1 , respectively, and it is unaffected by resorcinol, even at 2 g l −1 . It can also co-metabolise p-cresol, but cannot utilise benzoate or salicylate. These results may have practical implications for the use of C. maltosa in soil bioremediation, e.g. in bioventing. Enzyme tests were also performed to help interpret the data. Phenol hydroxylase activity reached maximum levels at the beginning of the exponential phase during cultivation on phenol. After the phenol had been completely utilised, the enzyme was slowly degraded.

Inorganic tin and organotin interactions with Candida maltosa.

As a consequence of the widespread industrial and agricultural applications of organotins, contamination of various ecosystems has occurred in recent decades. Understanding how these compounds interact with microorganisms is important in assessing the risks of organotin pollution. The organotins, tributyltin (TBT), trimethyltin (TMT) and inorganic tin, Sn(IV), were investigated for their physical interactions with non-metabolising cells and protoplasts of the yeast Candida maltosa, an organism that is often associated with contaminated environments. Uptake, toxicity and membrane-acting effects of these compounds, at concentrations approximating those found in polluted environments, were assessed. Sn(IV) and TBT uptake occurred by different mechanisms. Uptake of Sn(IV) was 2-fold greater in intact cells than protoplasts, underlining the importance of cell wall binding, whereas TBT uptake levels by both cell types were similar. TBT uptake resulted in cell death and extensive K+ leakage, while Sn(IV) uptake had no effect. TMT did not interact with cells. Of the three compounds, TBT alone altered membrane fluidity, as measured by the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene incorporated into cells. Anisotropy of 1-(4-trimethylaminophenyl-6-phenyl-1,3,5-hexatriene) was not affected, implying that TBT is not confined to the surface of the cytoplasmic membrane, but acts within membrane lipids. These results indicate that the cell wall is the dominant site of Sn(IV) interactions with yeast, while lipophilic interactions play an important role in uptake and toxicity of TBT.

Microbiology and physiology of Cachaça (Aguardente) fermentations.

Cachaça (aguardente) is a rum-style spirit made from sugar cane juice by artisanal methods in Brazil. A study was made of the production, biochemistry and microbiology of the process in fifteen distilleries in Sul de Minas. Identification of 443 yeasts showed Saccharomyces cerevisiae to be the predominant yeast but Rhodotorula glutinis and Candida maltosa were predominant in three cases. Bacterial infection is a potential problem, particularly in older wooden vats, when the ratio of yeasts:bacteria can be 10:1 or less. A study of daily batch fermentations in one distillery over one season in which 739 yeasts were identified revealed that S. cerevisiae was the predominant yeast. Six other yeast species showed a daily succession: Kluyveromyces marxianus, Pichia heimii and Hanseniaspora uvarum were present only at the beginning, Pichia subpelliculosa and Debaryomyces hansenii were detected from mid to the end of fermentation, and Pichia methanolica appeared briefly after the cessation of fermentation. Despite a steady influx of yeasts from nature, the species population in the fermenter was stable for at least four months suggesting strong physiological and ecological pressure for its maintenance. Cell densities during the fermentation were: yeasts - 4 x 108/ml; lactic acid bacteria -4 x 10(5)/ml; and bacilli - 5 x 10(4)/ml. Some acetic acid bacteria and enterobacteriaceae appeared at the end. Sucrose was immediately hydrolysed to fructose and glucose. The main fermentation was complete after 12 hours but not all fructose was utilised when harvesting after 24 hours.

Cyanobacteria—a potential source of new biologically active substances

Hydrophilic and lipophilic extracts of twelve cyanobacterial strains, isolated from fresh and brackish water, and two waterblooms, collected during the summer from the Baltic Sea, were investigated for their antibiotic activities against seven microorganisms. No inhibitory effects were found against the three Gram-negative bacteria Escherichia coli, Proteus mirabilis and Serratia marcescens and the yeast Candida maltosa. Of all cyanobacterial samples, extracts from seven species inhibited the growth of at least one of the Gram-positive bacteria Micrococcus flavus, Staphylococcus aureus and Bacillus subtilis. M. flavus proved to be the most sensitive bacterium in the agar diffusion test system. In particular, the hexane and dichlormethane extracts showed antimicrobial effects. But only one water extract, prepared from material of a natural waterbloom, was found to be active.

Studies on cycloheximide-sensitive and cycloheximide-resistant ribosomes in the yeast Candida maltosa.

Cycloheximide sensitivity or resistance in yeast is under the control of genes encoding different forms of ribosomal protein L41. In our previous studies, we have shown by isolating L41-Q1a, L41-P1a and their respective allelic genes, L41-Q1b and L41-P1b, from the partial diploid genome of C. maltosa, that this species, which is inducibly resistant to CYH, has both types of the L41 genes and that the expression of at least one of the L41-Q genes is induced by CYH, whereas L41-P genes are constitutively expressed. Here, we have identified another L41 (L41-Q2a), its allelic gene (L41-Q2b) and a third gene (L41-Q3) from the genome of C. maltosa. By gene disruption experiments, we now show that L41-Q1a and L41-Q1b are not responsible for the resistance to CYH and that the DeltaL41-Ps strain, which has only functional L41-Q genes, shows constitutive resistance to CYH, but grows more slowly than the DeltaL41-Qs strain, which has only functional L41-P genes, in the absence of CYH. Our results also show that in vitro, ribosomes containing L41-Q-type are less active in translation than those containing L41-P-type, although only the former ribosomes are active in the presence of CYH. These data suggest that ribosomes containing L41-Q-type are less active under normal growth conditions, but that this activity is not affected in the presence of CYH. We discuss the possible multi-step evolutionary event(s) by which C. maltosa has acquired the property of inducible resistance to CYH.

Oxygenation Cascade in Conversion of n-Alkanes to α,ω-Dioic Acids Catalyzed by Cytochrome P450 52A3

Purified recombinant cytochrome P450 52A3 and the corresponding NADPH-cytochrome P450 reductase from the alkane-assimilating yeast Candida maltosa were reconstituted into an active alkane monooxygenase system. Besides the primary product, 1-hexadecanol, the conversion of hexadecane yielded up to five additional metabolites, which were identified by gas chromatography-electron impact mass spectrometry as hexadecanal, hexadecanoic acid, 1, 16-hexadecanediol, 16-hydroxyhexadecanoic acid, and 1, 16-hexadecanedioic acid. As shown by substrate binding studies, the final product 1,16-hexadecanedioic acid acts as a competitive inhibitor of n-alkane binding and may be important for the metabolic regulation of the P450 activity. Kinetic studies of the individual sequential reactions revealed high Vmax values for the conversion of hexadecane, 1-hexadecanol, and hexadecanal (27, 23, and 69 min-1, respectively), whereas the oxidation of hexadecanoic acid, 1, 16-hexadecanediol, and 16-hydroxyhexadecanoic acid occurred at significantly lower rates (9, 9, and 5 min-1, respectively). 1-Hexadecanol was found to be the main branch point between mono- and diterminal oxidation. Taken together with data on the incorporation of 18O2-derived oxygen into the hexadecane oxidation products, the present study demonstrates that a single P450 form is able to efficiently catalyze a cascade of sequential mono- and diterminal monooxygenation reactions from n-alkanes to alpha, omega-dioic acids with high regioselectivity.

Mutual conversion of fatty-acid substrate specificity by a single amino-acid exchange at position 527 in P-450Cm2 and P-450Alk3A.

The two eukaryotic fatty-acid hydroxylases P-450Cm2 and P-450Alk3A, which represent CYP52A4 variants naturally occurring in the yeast Candida maltosa, were characterized with respect to their substrate specificity. Whereas P-450Cm2 was found to catalyse lauric acid omega-hydroxylation with greater efficiency, P-450Alk3A had higher palmitic acid turnover numbers compared to P-450Cm2, resulting in ratios of lauric acid to palmitic acid turnover rates of nearly 11 and 3 for P-450Cm2 and P-450Alk3A, respectively. As shown by means of chimeric enzymes and site-directed mutagenesis, the key residue determining these differences in substrate specificity was found to be a single amino acid at position 527. Interestingly, the mutual exchange of valine (P-450Cm2) and leucine (P-450Alk3A) led to a direct transposition of specificity, suggesting that amino acids at this site may determine the efficiency of fatty-acid hydroxylation relatively independently of other active-site residues. This was further supported by the finding that P-450Cm2 and P-450Alk3A with methionine at position 527 displayed almost identical hydroxylation activities. Moreover, methionine to leucine substitutions at the corresponding alignment position in P-450Cm1 (CYP52A3), P-450Alk2A (CYP52A5) and P-450Alk5A (CYP52A9) altered the fatty-acid specificity of these enzymes. In comparison to the structure of the bacterial P-450BM3 (CYP102), we propose that the amino acid at position 527 may serve to close the substrate-binding pocket near to the haem in the fatty-acid-omega-hydroxylating P-450 of the CYP52 family.

Isozyme Function of n-Alkane-inducible Cytochromes P450 in Candida maltosa Revealed by Sequential Gene Disruption

An n-alkane-assimilating yeast Candida maltosa contains multiple n-alkane-inducible forms of cytochromes P450 (P450alk), which can be assumed to catalyze terminal hydroxylation of n-alkanes in the assimilation pathway. Eight structurally related P450alk genes have been identified. In the present study, the function of four major isoforms of P450alk (encoded by ALK1, ALK2, ALK3, and ALK5 genes) was investigated by sequential gene disruption. Auxotrophic markers used for the selection of disrupted strains were regenerated repeatedly through either mitotic recombination between heterozygous alleles of the diploid genome or directed deletion of the marker gene, to allow sequential gene disruptions within a single strain. The strain depleted of all four isoforms could not utilize n-alkanes for growth, providing direct evidence that P450alk is essential for n-alkane assimilation. Growth properties of a series of intermediate disrupted strains, plasmid-based complementation, and enzyme assays after heterologous expression of single isoforms revealed (i) that each of the four individual isoforms is alone sufficient to allow growth on long chain n-alkane; (ii) that the ALK1-encoding isoform is the most versatile and efficient P450alk form, considering both its enzymatic activity and its ability to confer growth on n-alkanes of different chain length; and (iii) that the ALK5-encoding isoform exhibits a rather narrow substrate specificity and thus cannot support the utilization of short chain n-alkanes.

Characterization of then-Alkane and Fatty Acid Hydroxylating Cytochrome P450 Forms 52A3 and 52A4

Two enzymes, P450 52A3 (P450Cm1) and 52A4 (P450Cm2), the genes of which belong to theCYP52multigene family occuring in the alkane-assimilating yeastCandida maltosa,have been characterized biochemically and compared in terms of their substrate specificities. For this purpose, both the P450 proteins and the correspondingC. maltosaNADPH-cytochrome P450 reductase were separately produced by expressing their cDNAs inSaccharomyces cerevisiae,purified, and reconstituted to active monooxygenase systems. Starting from microsomal fractions with a specific content of 0.75 nmol P450Cm1, 0.34 nmol P450Cm2, and 10.5 units reductase per milligram of protein, respectively, each individual recombinant protein was purified to homogeneity. P450 substrate difference spectra indicated strong type I spectral changes and high-affinity binding ofn-hexadecane (Ks= 26 μM) andn-octadecane (Ks= 27 μM) to P450Cm1, whereas preferential binding to P450Cm2 was observed using lauric acid (Ks= 127 μM) and myristic acid (Ks= 134 μM) as substrates. These substrate selectivities were further substantiated by kinetic parameters, determined forn-alkane and fatty acid hydroxylation in a reconstituted system, which was composed of the purified components and phospholipid, as well as in microsomes obtained after coexpressing each of the P450 proteins with the reductase. The highest catalytic activities within the reconstituted system were measured forn-hexadecane hydroxylation to 1-hexadecanol by P450Cm1 (Vmax= 27 μM× min−1,Km= 54 μM) and oxidation of lauric acid to 16-hydroxylauric acid by P450Cm2 (Vmax= 30 μM× min−1,Km= 61 μM). We conclude that P450Cm1 and P450Cm2 exhibit overlapping but distinct substrate specificities due to different chain-length dependencies and preferences for eithern-alkanes or fatty acids.