From the Laboratory of Cellular Genetics, Department
of Biotechnology, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan,
and the ¶ Max Delbrück Center for Molecular Medicine,
Robert-Rössle-Strasse 10, 13122 Berlin-Buch, Germany
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ABSTRACT |
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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.
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INTRODUCTION |
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Cytochromes P450 are heme-containing monooxygenases that are distributed widely among living organisms (1). Higher eukaryotes generally contain multiple forms of cytochrome P450 catalyzing diverse oxidative reactions in the metabolism of a large number of endogenous and xenobiotic compounds. Depending on the induction level and substrate specificity of individual cytochromes P450, cellular metabolic processes are often effected by a specific ensemble of isoforms, making it difficult to distinguish their biological function.
In microorganisms, such a cytochrome P450 multiplicity only rarely can be found. One of the microorganisms harboring a cytochrome P450 multigene family is an n-alkane-assimilating yeast Candida maltosa (2-6). Resembling the situation in Candida tropicalis (7), this yeast species contains n-alkane-inducible forms of P450 (P450alk), and eight structurally related P450alk genes belonging to the CYP52 family were identified. Coupled with NADPH-cytochrome P450 reductase (8), they are assumed to catalyze the terminal hydroxylation of n-alkanes, which represents the first and rate-limiting step in the n-alkane assimilation pathway. The functions of the C. maltosa cytochromes P450 were first apparent from in vitro reconstitution of n-alkane hydroxylase and NADPH-cytochrome P450 reductase purified from n-alkane-grown C. maltosa cells (9) and could be confirmed later by in vivo CO inhibition studies (10). In addition to these investigations, recent studies revealed the enzymatic characters of some P450alk forms by means of heterologous expression in Saccharomyces cerevisiae (11-13). However, investigations about the cellular significance of individual P450alk isoforms for the n-alkane assimilation pathway are still fragmental. In particular, it remains to be clarified whether indeed such a P450alk multiplicity is required for the n-alkane assimilation pathway of C. maltosa and to what extent each of the gene products of eight P450alk genes contributes to the respective n-alkane-assimilating phenotype.
Recent development of the genetic engineering systems including host-vector systems as well as the gene disruption method, which enabled us to disrupt stepwise both alleles of a certain gene in the diploid genome by using two selectable markers, have greatly facilitated molecular biological analyses of C. maltosa (14-19). As an initial step to address the cellular function of individual isoforms of P450alk, we previously disrupted the first isolated P450alk gene and found that the disrupted stain retained the ability to utilize n-alkanes as a sole carbon and energy source because of the n-alkane hydroxylation activity of the other P450alk forms. In the present paper we describe the sequential disruption of multiple P450alk genes and show the effect of these disruptions on the n-alkane-assimilating property of C. maltosa. The results in this paper provide evidence that the difference in substrate specificity among multiple isoforms of P450alk in vivo can be detected clearly by using the gene disruption technique even in a nonconventional yeast.
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EXPERIMENTAL PROCEDURES |
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Strains and Media--
The C. maltosa strains used in
this study are listed in Table I. Media
for C. maltosa were YPD (1% yeast extract, 2%
Bacto-pepton, and 2% glucose) and YNB (0.67% Yeast Nitrogen Base
(Difco) without amino acids and either 2% glucose or 1%
n-alkanes), which was supplied with appropriate nutrients.
5-Fluoro-orotic acid (5FOA)1
was added at a final concentration of 2 mg/ml. The
n-alkane-assimilating property of the C. maltosa
strains was tested both in the liquid and solid YNB media. The
bacterial strain Escherichia coli MV1190 ((srl-recA)306::Tn10(tetr)
(lac-pro) thi
, supE
(F', proAB,
lacIq,
lacZ
M15, traD36)) was used for
plasmid preparations and was grown in LB broth.
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Plasmid Construction and Yeast Transformation for Gene
Disruptions--
A 1.8-kb HindIII-EcoT22I
fragment of pCMU6 carrying the C. maltosa URA3 gene (18) was
subcloned into the HindIII-PstI sites of pUC19 to
construct pURAD. A 1.6-kb SspI-DraIII fragment of the C. maltosa ADE1 gene (16) was cloned into the
SalI site of pUC119 through the SalI linker to
construct pUADE. A 6.7-kb SalI-BamHI fragment
carrying entire ALK2-B and ALK3-B genes and their
5'- and 3'-noncoding regions (4) was subcloned into pUC19 to construct
pUA23. For gene disruption of the ALK2-ALK3
locus, a 1.6-kb EcoRV-BamHI fragment of pURAD and
a 1.5-kb DraI-BamHI fragment of pUADE replaced a
4.1-kb BglII-EcoRV fragment of pUA23 to construct
pUD23U and pUD23A, respectively. The
alk2-alk3::URA3 and the
alk2-alk3:: ADE1 cassettes
for gene disruption were excised from pDA23U and pDA23A, respectively,
with PstI-BamHI double digestions in both cases,
and transformed into C. maltosa. The transformation of
C. maltosa was performed by a modified lithium acetate
method (14). For regeneration of the ADE1 marker,
pDA23ade was constructed by ligation of a 1.3-kb
PstI-EcoRV fragment from pUD23A and a 1.3-kb
EcoRV-BamHI fragment from pUA23 into the
PstI-BamHI sites of pUC19, and the
alk2-alk3::
ade1 cassette
was generated by digestions with PstI-BamHI for
the C. maltosa transformation. In these three cassettes, the
complete ALK2 coding region, NH2-terminal
one-third of the coding region of ALK3, and the
ALK2-ALK3 internal region (1.0 kb) were
replaced.
Southern Blot Hybridization-- Total DNA of C. maltosa was isolated from a culture of 10 ml of YNB-glucose medium as described (18). Southern blot analysis was performed with an ECL (enhanced chemiluminescence) gene detection system (Amersham) in accordance with the instruction of the supplier. For detection of the appropriate gene replacements of the ALK2-ALK3 locus, total DNA from the transformants was digested with BamHI and ClaI and probed with a 0.6-kb HindIII fragment of ALK3-B (see Fig. 2). For the ALK5 and ALK1 replacements, total DNA from the transformants was digested with HindIII and PstI-BglII, respectively, and probed with a 2.1-kb HindIII fragment of ALK5-A and a 2.3-kb HindIII fragment of ALK1-A, respectively. The appropriate replacements were confirmed as follows. Both of the intact alleles of ALK5 gave a 2.2-kb doublet band. The genomic blot of the first step transformant gave a 3.3-kb band as predicted for the replacement with URA3 in addition to the 2.2-kb band of the undisrupted allele. That of the second step transformant gave new 1.0- and 2.0-kb bands as predicted for the replacement with ADE1 in addition to the 3.3-kb band for the URA3-replacement, and the 2.2-kb band for the intact ALK5 allele disappeared. In ALK1 disruptions the genomic blots of the transformants for the replacements with URA3, ADE1, and HIS5 gave 8.2-, 9.1-, and 9.8-kb bands, respectively. Disruption of both alleles of ALK1 was confirmed by detection of each of the two respective bands for the replacements and by the disappearance of the 7.0-kb band for the intact allele of ALK1. A 0.9-kb SalI fragment of pURAD (18), a 0.7-kb EcoRI-EcoRV fragment of ADE1 (16), and a 2.3-kb EcoRI-HindIII fragment of HIS5 (15) were also used as probes to confirm the replacements of URA3, ADE1, and HIS5, respectively.
P450 Induction Experiments-- To determine the expression level of spectrally active P450 in the wild-type strain CHAU1 as well as in the disruptant strains DA235-24, DA123-14, and DA1235-12, respective cultures were first grown to a density of about 1 × 107 cells/ml in a yeast minimal medium containing 1.34% Yeast Nitrogen Base, 2% glucose and, as required, histidine (50 mg/liter), adenine (100 mg/liter), and uracil (40 mg/liter). Then cells were washed in the fresh medium without glucose and cultivated under the same conditions except for 1% dodecane as a sole carbon source. P450 contents were determined after an induction time of 4 h by means of CO difference spectra (10).
Plasmid Construction for Plasmid-based Complementation-- A 1.8-kb PstI-XhoI fragment of the TRA region, which contains an autonomously replicating sequence and a centromeric DNA of C. maltosa (19), was isolated from the vector pTRA11 (14) and cloned into the PstI site of pUC18 through a PstI linker. From the resulting plasmid a 1.8-kb PstI fragment was inserted into the AatII site of pUC19 through blunt end ligation. Then a 1.2-kb DraI-XbaI fragment of URA3 from pURAD was inserted into the XhoI site of the TRA region through an XhoI linker to construct pUTU1. A 2.8-kb BamHI-SphI fragment of ALK1-A isolated from pUA1 was inserted into the BamHI-SphI sites of pUTU1 to construct pUTU-ALK1. A 2.6-kb BglII fragment of ALK2-A and a 3.3-kb EcoT22I-EcoRV fragment of ALK3-A were inserted into the BamHI site and the PstI-SmaI sites of pUTU1 to construct pUTU-ALK2 and pUTU-ALK3, respectively. A 3.5-kb PstI-EcoRI fragment of ALK5-A was inserted into the PstI-SmaI sites through blunt end ligation to construct pUTU-ALK5.
Exchanges of the promoter regions of ALK1 and ALK5 genes were done by polymerase chain reaction as follows. To obtain recombinant products having ALK1 promoter and ALK5 coding region, primers 5'-AACATCTGGTCATGATTGATGAAATACTTCCT-3' and 5'-TCATCAATCATGACCAGATGTTATAAATG-3' were used. For the product having ALK5 promoter and ALK1 coding region, primers 5'-TAAACTAAAAATATGGCTATAGAACAAATTATTG-3' and 5'-GTTCTATAGCCATATTTTTAGTTTAATCTTATTTAAA-3', respectively, were used. Underlines correspond to the translational initiation codons. As flanking primers, M13 forward and reverse sequencing primers were used. Plasmids pUTU-ALK1 and pUTU-ALK5 were used as templates for the first polymerase chain reaction step to obtain the respective promoter and coding region. Then the second polymerase chain reaction step generated exchanged recombinant products. The products were cloned into the KpnI-SphI sites of pUTU1 to construct pUTU-A1/A5 (ALK1 promoter and ALK5 coding region) and pUTU-A5/A1 (ALK5-promoter and ALK1-coding region), respectively.Heterologous Expression and Enzyme Assay-- Construction of the four coexpression vectors used for the simultaneous production of the individual C. maltosa P450alk forms and NADPH-cytochrome P450 reductase was described previously (13). Heterologous expression in S. cerevisiae was done as described previously (11). The enzyme assay was carried out using the microsomal fraction using [1-14C]dodecane (Sigma) and [1-14C]hexadecane (Amersham) as substrates as described previously (13).
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RESULTS |
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Disruption of Single Loci-- We have shown that four out of the eight P450alk genes of C. maltosa are significantly induced by n-alkanes and may thus encode P450 enzymes directly involved in n-alkane assimilation in the previous study (6). These four genes have been designated ALK1, ALK2, ALK3, and ALK5. Each of them occurs in two allelic variants in agreement with the diploid nature of C. maltosa genome. Previous gene disruption experiments revealed that a C. maltosa strain defective in ALK1 retained the ability to grow on long chain n-alkanes (17). Therefore, it has been the first objective of the present study to disrupt each of the other three n-alkane-inducible P450alk genes (for the experimental strategy applied, see Fig. 1) and to examine on this basis their contribution to the phenotype of n-alkane assimilation.
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Regeneration of Selectable Markers--
To investigate the
cellular function of individual P450alk isoforms, the respective
P450alk genes had to be disrupted simultaneously within a single
strain. However, because the initial round of disruption resulted in a
Ura+ and Ade+ phenotype and only the
HIS5 marker was available, methods to restore the selectable
markers to the disrupted strains should have been developed to permit
reutilization of the markers for further disruption of the remaining
P450alk genes. In one method, the introduced selectable marker
ADE1 was substituted for a deletion derivative of it by
means of cotransformation with a selectable plasmid to facilitate the
selection of the replacement. The ADE1-deleting cassette alk2-alk3::ade1 was
transformed into strain DA23-81 along with an
HIS5-containing autonomously replicating plasmid, pBTH10B
(15). Among the resulting His+ transformants, some
developed red colored colonies characteristic of the ade1
mutant. They were tested for adenine auxotrophy, and the appropriate
replacement with the alk2-alk3::
ade1 cassette was confirmed by Southern blot analysis of the genomic DNA of an
Ade
strain, DA23-816 (Fig. 2, lane 4). Then,
the plasmid pBTH10B was cured from the strain DA23-816 by growth on
nonselective medium.
Development of Multiple P450alk Gene Disruptions and Their Effects on n-Alkane Assimilation-- Mutants containing multiple P450alk gene disruption were generated according to the scheme shown in Fig. 1. The complete set of disruption in all the three P450alk loci, ALK1, ALK2-ALK3, and ALK5, was obtained by sequential disruption. The complete genotypes of the strains generated are listed in Table I. In each step, the appropriate genotypes were confirmed by Southern blot analysis of the isolated strains (see "Experimental Procedures"), as shown in the case of the ALK2-ALK3 disruption.
The growth properties of P450alk gene disruptants on n-alkanes of different chain lengths as sole carbon sources were examined (see Table II). Strain DA1235-12, in which all four of the genes were disrupted, could not utilize n-alkanes of any chain lengths for growth, although this strain was able to grow on n-alcohols and fatty acids as sole carbon sources. The growth property of this strain indicated that the sequential gene disruption resulted in a complete functional block of the n-alkane assimilation at the first step. Strains DA123-14, DA235-24, and DA15-23, in which two of the three loci were disrupted, showed good growth on C16, indicating that each of the products of the three loci was alone sufficient for the assimilation of long chain n-alkane. Interestingly, a specificity of the n-alkane-assimilating property was observed depending on the chain length of n-alkanes. The strain DA123-14 could not utilize C12 at all for growth, whereas the other strains grew significantly on this substrate, although some of them showed weak growth. And the strain DA123-14 alone showed weak growth on C14. This growth property of the strain DA123-14 indicated that the ALK5 product that remained functional in this strain could function fully only on the long chain n-alkane. Strains devoid of ALK1 showed merely poor growth on C12, whereas strains expressing ALK1 showed good growth on C12. These results indicated that the ALK1 product played an important role for assimilation of short chain n-alkanes. To prove the expression of spectrally active P450 in the gene disruptant strains, P450s were induced by C12 as described under "Experimental Procedures." Whereas the wild-type strain CHAU1 expressed a significant amount of P450 (0.11 nmol/108 cells), no significant expression of P450 was observed in the P450 disruptant strain DA1235-12. Under the same conditions, the presence of P450 could be detected clearly in strain DA235-24 (0.09 nmol/108 cells) and DA123-14 (0.02 nmol/108 cells). Interestingly, in the strain DA235-24 in which only the ALK1 product remained functional, almost 80% of the P450 amount of the wild-type strain could be found, indicating that this P450 form was most abundant P450 in C. maltosa. In contrast, the amount of P450 in the strain DA123-14 in which only ALK5 remained functional was less than 20% of the wild-type strain.Plasmid-based Complementation with Each P450alk Gene-- Function of individual P450alk genes toward n-alkane assimilation was analyzed further by plasmid-based complementation. Each of four entire genes having 5'- and 3'-flanking regions was cloned into pUTU1, which carried both an autonomously replicating sequence and a centromeric DNA sequence of C. maltosa (19), transformed into the strain DA1235-121, which was devoid of all four of the genes and in which the URA3-selectable marker could be utilized, and examined for n-alkane assimilating property. Table III summarizes the results of the complementation experiments. Complementation with ALK1 resulted in the complete recovery of n-alkane assimilating property, to utilize n-alkanes of different chain length as sole carbon sources for growth. Although the disruption experiments could not distinguish between the function of the products of ALK2 and ALK3, the results of the complementation indicated that both the ALK2 and the ALK3 products solely could function significantly in assimilation of n-alkanes of different chain length. However, both complemented strains grew only weakly on C12 as in the cases of the ALK1-disrupted strains. Unlike these three genes, ALK5 was able to complement the assimilating property of n-alkanes with only C16 but not with C12 and C14.
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Enzyme Activities of Individual P450alk Isoforms-- Enzymatic activities of individual P450alk isoforms were characterized by means of turnover rates toward n-alkanes. Because expression levels of P450 in the complemented strains were limited, heterologous overexpressions of individual P450alk forms in another yeast, S. cerevisiae, in which the related activity is absent, were applied. A highly active P450 monooxygenase reconstitution system consisting of P450alk and NADPH-cytochrome P450 reductase from C. maltosa has been established already in vivo in S. cerevisiae (11, 13). Microsomal fractions were prepared from the respective strains overexpressing P450alk and then assayed for P450alk-encoded hydroxylase activities toward C12 and C16 (see Table IV). The ALK1 product displayed the strongest hydroxylase activity, suggesting that it was the most important P450alk form for the primary hydroxylation of n-alkanes. Products of ALK2 and ALK3 showed n-alkane hydroxylation activities of approximately half of the ALK1 product, respectively. No preference of the activity depending on n-alkane chain lengths was observed for these three P450alk forms. The ALK5 product showed relatively weak but significant activity toward C16 (approximately one-forth of the ALK1 product), whereas it showed very little activity toward C12. The substrate specificity of the ALK5 product toward the long chain n-alkanes was congruent with the results of the sequential gene disruption.
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DISCUSSION |
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To our knowledge, this is the first report about gene disruption
of multiple forms of cytochromes P450 and about its direct effect on
phenotype. The sequential gene disruption of the P450alk genes resulted
in a C. maltosa strain (DA1235-12) that could not utilize
n-alkanes as sole carbon sources for growth because of the
lack of the functional P450alk. Because only the P450alk genes were
manipulated in the genome, this result provides direct evidence that
P450alk participates in the n-alkane assimilation pathway. In the proposed n-alkane assimilation pathway,
n-alkane is first hydroxylated at the terminal position to
produce n-alcohol and then oxidized successively to fatty
acids. The growth property of the disrupted strain that could not
assimilate n-alkanes but could grow on n-alcohols
and fatty acids as sole carbon sources clearly correlated with the
in vitro n-alkane-hydroxylating activity of the P450alk
enzymes shown here and in previous studies (5, 9, 12, 13), confirming
that P450alk catalyzes the first step of n-alkane
assimilation. This growth property also indicates that none of the four
isoforms analyzed is essential in the further downstream steps of the
assimilation pathway, although considerable fatty acid
-hydroxylation activity has been shown for some of them (5,
11-13).
The overlapping function of the four P450alk forms toward hydroxylation of at least long chain n-alkanes is supported by the results that disruption of all four of the genes was necessary to generate the n-alkane-nonassimilating strain and that each of the four genes could complement the assimilating ability of the disrupted strain. Furthermore, their n-alkane hydroxylation activities were confirmed directly by an in vitro assay using a heterologous overexpression system.
The ALK1 product is the most important P450alk isoform because when it was present in the cells, the growth was significant on n-alkanes of any chain length. Its functional importance was supported by the strongest in vitro enzyme activity among the P450alks examined as well as by the considerable amount in the cells as shown in the strain DA235-24 in which only the ALK1 product was the functional P450alk. The absence of the functional ALK1 product in the series of disruptants resulted in weak growth on the short chain n-alkanes, indicating that the ALK1 product is not essential but necessary for full activity for the assimilation of short chain n-alkanes. Because the previous analysis of the gene disruption of ALK1 had been done using only n-hexadecane (17), we could not find this importance. The ALK2 and the ALK3 products can also hydroxylate n-alkanes of any chain length, although their function on short chain n-alkanes is restricted. However, the ALK5 product demonstrated a narrow spectrum toward long chain n-alkanes. The in vivo complementation experiment using the promoter-exchanged constructs and the direct in vitro enzyme assay (see Tables III and IV) showed that the functional specificity was affiliated not to the expression level of the enzyme but to the enzyme activity itself.
Previous analysis on the induction of individual P450alk genes (6) revealed that ALK1 is induced strongly by short chain n-alkanes but relatively weakly by long chain n-alkanes. This result is congruent with the functional importance of the ALK1 product on short chain n-alkanes. However, ALK5 is induced by both long and short chain n-alkanes, whereas its product shows the substrate preference toward long chain n-alkane. Furthermore, although the four P450alk isoforms are not essential for the following steps of n-alkane assimilation pathway, three genes, ALK1, ALK2, and ALK5, are induced weakly but reproducibly by n-alcohols and fatty acids. The regulation of the expression of individual P450alk forms is not simply correlated with their functions.
The P450alk gene family in C. maltosa consists of at least eight members (6). The four isoforms characterized here are significantly induced when the carbon sources are n-alkanes, whereas the other four genes (ALK4, ALK6, ALK7, and ALK8) are induced only at low levels by n-alkanes as well as n-alcohols and fatty acids. The growth property of the n-alkane-nonassimilating strain indicates that these undisrupted four P450alk genes cannot function fully on the assimilation. However, further analysis such as in vitro assay using heterologous overexpression is necessary to determine whether they have enzymatic activity toward n-alkanes.
Methods to restore the auxotrophic requirements to cells previously transformed to respective prototrophy with the selectable marker genes have been developed here in C. maltosa to allow sequential gene disruption within a single strain. According to the strategy used previously (17), only both alleles of a single gene could be disrupted in a stepwise manner using two selectable markers. The cotransformation with a selectable autonomously replicating plasmid facilitated the selection of the regeneration of the ade1 auxotrophy. Moreover, the strain regenerating the ade1 marker could be distinguished easily because of red color development of the colony. Although the condition for the cotransformation was not optimized, a frequency of one ade1 strain among approximately 300 His+ transformants was obtained. Such a cotransformation strategy has also been applied in C. albicans where the gene replacement was selected by colony hybridization (20).
The 5FOA selection method also allowed us to regenerate the ura3 marker by means of the positive selection of a ura3 mutant. The strain having heterozygous disrupted alleles, one replaced with URA3 and the other by another marker or its deletion derivative, was rendered homozygous through mitotic recombination. Such a recombination was observed frequently in our previous study, in which ura3 mutants homozygous for disruption were obtained from the strain heterozygous for disruption by selecting the 5FOA resistance (18). Almost all ura3 mutants isolated by the 5FOA selection in this and in the previous studies were homozygous for disruption with a few exceptions, in which a local mutation in the URA3-carrying allele occurred. Such a strategy for the disruption of both alleles of a single gene has been applied previously to C. albicans, where UV irradiation has been used to facilitate recombination (21). The repeated use of URA3 for sequential disruptions has also been reported in C. tropicalis (22). In the case of C. tropicalis, since only URA3 has been available as a selectable marker in the host strain, the regeneration required the nystatin enrichment of ura3 strains. In principle, the combination of the two methods for regenerating the auxotrophic markers would allow us disruption of a nonlimiting number of genes in C. maltosa.
The disrupted strain unable to assimilate n-alkanes may
provide a useful host for further investigation of structure-function relationships of P450alk because mutant-type P450alks, the wild-type of
which is nonfunctional on a specific substrate, can be positively selected by its n-alkane assimilating phenotype if they are
functional. Because the structures of the four isozymes are
significantly similar (56-67% amino acid identities) to each other
(4, 6), P450alk is a suitable model for such a study. Industrially,
C. maltosa may also be useful for the production of some
hydrophobic metabolic intermediates produced by a selected P450alk form
such as dicarboxylic acids. Some P450alk isoforms should play important roles for the production because considerable fatty acid
-hydroxylation activity has been reported for them (5, 11-13). In
combination with the functional block of the
-oxidation system by
gene disruption as described in C. tropicalis (22, 23) and
C. maltosa (24) and gene disruption of the appropriate
P450alk forms as described here, it may be possible to construct an
efficient strain for production.
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ACKNOWLEDGEMENTS |
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We are grateful to Rose-Marie Rosler for technical assistance.
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FOOTNOTES |
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* This work was supported in part by a grant-in-aid for science research from the Ministry of Education, Science, and Culture of Japan and by Grant 0310257 from the Bundesministerium für Forschung und Technologie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-01, Japan.
Recipient of the Japan Society for the Promotion of Science
Fellowships.
** To whom correspondence should be addressed. Tel.: 81-3-3812-2111 (ext. 5169); Fax: 81-3-3812-9246; E-mail: amtakag{at}hongo.ecc.u-tokyo.ac.jp.
1 The abbreviations used are: 5FOA, 5-fluoro-orotic acid; kb, kilobase(s).
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REFERENCES |
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