?xEPA
United States
Environmental Protection
Agency
Hazardous Waste Engineering
Research Laboratory
Cincinnati OH 45268
Research and Development
EPA/600/M-88/001 Jan. 1988
Air Force No. ESL-TR-86-52
ENVIRONMENTAL
RESEARCH BRIEF
Gene Engineering of Yeasts for the Degradation of Hazardous Waste
John C. Loper*
Abstract
Environmentally recalcitrant compounds including
2,3,7,8-tetrachlorodibenzo-p-dioxin and hexachloroben-
zene are metabolized in mammalian liver via reactions
characteristic of cytochrome P-450 monooxygenase
systems. This research examined the structure and
function of cytochrome P-450 genes in yeast as a model
for gene engineering such eukaryotic P-450 enzymes for
biodegradation of hazardous waste by yeasts. Sacchar-
pmyces cerevisiae and Candida tropicalis are two yeasts
known to produce major P-450 enzymes. These enzymes
were purified and antibodies produced in rabbits were then
used in the isolation or characterization of clones
containing a P-450 gene from each organism. DNA
sequence was determined for the gene isolated from S.
cerevisiae and for several hundred bases of chromosomal
DMA on each side of the gene, and deletion experiments
in the promoter region were conducted. The deduced
protein sequence from this-gene was compared to those
of the other known subfamilies of eukaryotic P-450 genes;
the several features-identified included a new homology
region for these protein sequences. The C. tropicalis gene
and its promoter region has been partially sequenced.
Introduction
This is a report of the research conducted under a
Cooperative Agreement between the U.S. Environmental
Protection Agency (EPA) and the University of Cincinnati.
The work examined the yeasts Saccharomyces cerevisiae
and Candida tropicalis, eukaryotic microorganisms known
to produce major cytochrome P-450 enzymes. Cytochrome
P-450 monooxygenases are the only class of enzymes
known to catalyze the specific degradation of certain highly
'Department of Microbiology and Molecular Genetics, University of
Cincinnati College of Medicine, Cincinnati, OH 45221.
- recalcitrantcblorinated_ar,omatic -hydrocarbons.. A long-^
range goal of this research direction is to obtain yeasts
which combine the uptake and degradation of these target
compounds with the capacity to survive or to survive and
grow in toxic environments. This" result would provide a
system for controlled, low-cost biodegradation of such
environmentally stable toxic wastes. The work under this
Cooperative Agreement sought the characterization of
yeast cytochrome P-450 genes, as a model for the gene
engineering of eukaryotic cytochromes P-450 in yeast.
The Role of Gene Engineered Eukaryotic
Cytochrome P-450 Monooxygenases
Among the targets for engineered biodegradation are
compounds which by definition are recalcitrant, that is,
not observed to be degraded by natural processes. In
general, the most toxic and most stable recalcitrant organic
compounds are those with a high degree of chlorination.
Of particular concern are polychlorinated aromatic
hydrocarbons having no adjacent unsubstituted carbons,
for example, hexachlorobenzene, highly chlorinated
biphenyls, and 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD). Such compounds are not attacked by bacterial
dioxygenases, which are enzymes whose aromatic
substrates contain adjacent carbons free of constituents
other than hydrogen. However, there is evidence for the
hydroxylation/dechlorination of such recalcitrant com-
pounds in some bacteria and particularly in eukaryotic
organisms via cytochrome P-450 monooxygenases. These
enzymes can modify a substrate one carbon at a time,
and because of their number and their occasional
overlapping specificity, they provide for a broad range of
oxidative and reductive reactions.
Such P-450 catalyzed reactions play essential roles in
mammalian metabolism in (a) the synthesis of the major
cell constituents cholesterol and fatty acids and of
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hormones including glucocorticoids, sex hormones and
prostaglandins, and (b) the metabolism of therapeutic
drugs and other xenobiotic compounds. Knowledge of P-
450s has been gained primarily from mammalian systems,
and the data for the P-450 catalyzed metabolism of TCDD
and similarly recalcitrant compounds are from in vivo and
In vitro studies of mammalian liver. Knowledge of yeast
P-450 systems is less advanced. Nevertheless, it has been
demonstrated that P-450 monooxygenase pathways are
constituted similarly among higher and lower eukaryotes
and that mammalian P-450 gene products can function
in yeasts. Thus, it is likely that P-450 genes for degradation
of recalcitrant compounds either can be identified in yeasts
or can be introduced for expression in yeasts.
Engineering the gene expression of these appropriate P-
450 monooxygenases in yeasts would make possible the
testing of these microorganisms for biodegradation of
recalcitrant hazardous compounds (8). In some'cases the
establishment of the expressed P-450 gene may be the
only additional trait required to equip the cell for the desired
degradation; in others it is likely that additional properties
will be important, for compound uptake and for survival,
etc. The primary effort of this study was to characterize
cytochrome P-450 genes in S. cerevisiae, the yeast
organism most amenable for such molecular genetic
studies. Genes engineered in this organism subsequently
can be transferred to other strains. Since different species
of yeast are likely to tolerate better the ecological
conditions presented by various noxious environments,
strains isolated from those environments could be used
as recipients of functional P-450 gene sets to establish
the preferred detoxicating variants. Candida tropicalis is
a likely candidate as one of these ecologically suitable
organisms. This yeast already contains an elaborate
machinery for the assimilation of hydrophobic long chain
hydrocarbons enabling its growth on crude oil. Secondary
to the studies in S. cerevisiae, P-450 genes in this
organism were also examined.
Characterization of Yeast P-450 Genes: Isolation
and Sequence Determination
When this study started, considerable data were available
on a few P-450 enzymes of yeast but no yeast P-450 genes
were available. In S. cerevisiae the best characterized P-
450 enzyme was known to demethylate lanosterol and
the same or a quite similar form was known to hydroxylate
benzo{a)pyrene. The gene for this demethylase was chosen
for isolation as a model system and as a gene of potential
use in general biodegradation.
Another well studied yuast P-450 enzyme is alkane u-
hydroxylase. This enzyme catalyzes the first step in the
metabolism of long chain n-alkanes as a carbon and energy
source. It is found in a variety of yeasts other than S.
cerevisiae; Candida tropicalis is a major example. The C.
tropicalis gene for this enzyme was chosen for isolation
as the second model. This choice was based upon the
likelihood that this P-450 may be of direct use in
biodegradation, and because utilization of alkanes involves
processes for cell uptake of hydrophobic substrates which
are likely to be of use in the uptake of hydrophobic
hazardous wastes.
As a useful step toward P-450 gene isolation and
identification, microsomal preparations were first isolated
from these two microorganisms and the S. cerevisiae
lanosterol demethylase, and the C. tropicalis w-
hydroxylase were purified. Antibodies in rabbits were
prepared and physical and immunological similarities of
the two enzymes were examined (1,2). Then using a
recently described method based upon gene dosage effects
for isolation of genes in yeast, we isolated a lanosterol
demethylase gene from S. cerevisiae (3).
Using a cloned subfragment of DNA containing the entire
gene, overlapping deletions were produced which then
were used as templates for DNA sequencing of one DNA
strand of the gene by the M13 dideoxy method. The
com"plementary-srra7Ta~was^^
method, using oligonucleotide primers synthesized in the
Department. As a result, the DNA sequence of this gene
has been determined together with the sequence of several
hundred nucleotide base pairs upstream and downstream
of the protein coding region (4).
When genes are sequenced, a useful procedure is to use
the universal genetic code to identify the predicted amino
acid sequence of the protein product. Since the isolation
and sequence characterization of mammalian P-450 genes
is a major area of research among numerous laboratories,
several P-450 genes have now been examined in this
procedure and their protein sequence determined. When
the protein sequence of the yeast P-450 was compared
to those of higher eukaryotes, many similar features were
identified (4). Chief among these are (a) the presence of
a strongly hydrophobic region near the amino terminal
end, which could serve as a typical anchor peptide for
attachment of the protein to intracellular membranes, and
(b) a 21 amino acid segment near the C terminal region
which conforms t,o a homologous region detected for all
other P-450 genes. This homologous region apparently
is required for the binding of heme to the apoprotein.
These features of the deduced protein sequence identify
itnsj?. cerevisiae gene as a cytpchrome P-450 gene. Based
on these and upon overall DNA homology properties, this
yeast P-450 protein comprises an additional subfamily
among the superfamily of eukaryotic P-450s. It is clear
also that this S. cerevisiae P-450 gene has more in
common with other subfamilies of eukaryotic P-450 genes
than it does with the structure of the known bacterial P-
450 gene (4,5). Thus, the sequence characterization of
a yeast P-450 gene has provided unique information on
evolutionary relationships among the P-450 superfamily.
For isolation of the w-hydroxylase P-450 gene from C.
tropicalis, direct use was made of the anti-enzyme antibody
for gene detection in a gene expression library. This
procedure led to the isolation of the complete u>-
hydroxylase gene (6). Total sequence characterization of
this gene is in progress, using a double strand plasmid
sequencing procedure that employs templates from sets
of deletions extending across either end of the isolated
gene.
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A New Region of Sequence Similarity Among All
Eukaryotic P-450s
As noted, the S. cerevisiae P-450 comprises a new P-
450 subfamily. With the advantage of this new information
a detailed comparison of homology among all seven of
the subfamilies in this sytem was conducted. The analysis
identified a new region of sequence similarity among all
the eukaryotic P-450s (5,7). This advance is important to
research on the structure and function relationships of
all P-450s since it indicates protein sites where site-
specific mutagenesis would be particularly informative.
Regulation of P-450 Expression in Yeast
Understanding and gene engineering of regulatory signals
are important for the controlled expression of a P-450 gene
* for degradation reactionsrCbnsKierable progress has been'
made on several aspects of this question. ForS. cerevisiae,
the DNA sequence analysis has included over 800
nucleotide bases proximal to the structural gene coding
region. Deletion analysis of this region has shown that
the major upstream portion can be removed without
preventing gene expression (9). Also, additional genes can
alter the level of lanosterol demethylase maintained in
this organism. Recombinant strains have been constructed
that provide for stable high cytoplasmic levels of this
enzyme (3).
The signals for induction of w-hydroxylase in C. tropicalis
are of particular interest for gene engineering the
biodegradation of toxic hydrophobic compounds. This work
indicates that transcription of this enzyme is induced one
hundred-fold or more as the cells adapt to assimilation
of mineral oil alkanes. It appears that over 24 additional
genes are involved in this assimilation process. Expression
of these genes provides for changes in surfactant products,
lipophilic changes in cell wall structure and production
of intracellular organelles involved in catabolism of the
alkanes. The result is a yeast extensively adapted for
utilization of these hydrophobic compounds, and it is likely
that they then can take up more hazardous hydrophobic
compounds as well. A strategy has been outlined for
engineering P-450 enzymes for mpnooxygenation of
specific, hazardous hydrocarbons into such yeasts, with
the inserted P-450 genes under the inductive control of
the w-hydroxylase P-450 gene promoter. This promoter
has been isolated as part of the C. tropicalis DNA segment
containing the w-hydroxylase gene (6). Sequencing of this
promoter was in progress in this laboratory as this
Cooperative Agreement ended.
Publications
The following publications describe research supported by
this Cooperative Agreement.
1. Loper, J. C., Chen, C. and Dey, C. R. Gene engineering
in yeast for biodegradation: immunological cross-
reactivity among cytochrome P-450 system proteins
of S. cerevisiae and C. tropicalis. Hazardous Waste
Hazard. Mat. 2,131 -141, 1985.
2. Loper, J., Chen, C. and Dey, C. Gene engineering
of yeasts for the biodegradation of hazardous wastes,
in Proceedings of the Eleventh Annual Research
Symposium on Incineration and Treatment of
Hazardous Waste, EPA/600/9-85/028, September
1985, pp. 112-119.
3. Kalb, V. F., Loper, J. C., Dey, C. R., Woods, C. W.
and Sutler, T. R. Isolation of a cytochrome P-450
structural gene from S. cerevisiae. Gene, 45(3):237-
245, 1986.
4. Kalb, V. F., Woods, C. W., Turi, T. G., Dey, C. R.,
Sutler, T. R. and Loper, J. C. Primary struclure of
the P450 lanosterol demelhylase gene from Sac-
charomyces cerevisiae. DNA, 6(6):529-537 (1 987).
5. Kalb, V. F^and Loper, J._£._A_s^grnenled region of
"sequence' similarity found "in eight different eukary-
otic P450 families {Manuscript submitted for review.)
6. Sanglard, D., Chen, C. and Loper, J. C. Isolation of
the alkane inducible cytochrome P-450 (P450alk)
gene from the yeast Candida tropicalis. Biochem.
Biophys. Res. Comm. 144,251-257(1987).
7. Chen, C., Turi, T. G., Sanglard, D. and Loper, J. C.
Isolation of the Candida tropicalis gene for P450
lanosterol demethylase and its expression in
Saccharomyces cerevisiae. Biochem. Biophys. Res.
Comm. 146, 1311-1317(1987).
8. Chen, C., Dey, C. R., Kalb, V. F., Sanglard, D., Sutler,
T. R., and Loper, J. C. Engineering P450 genes in
yeast in Land Disposal Remedial Action, Incineration
and Trealmenl of Hazardous Wasle. Proceedings of
ihe thirteenlh annual research symposium. U.S.
EPA, EPA/600/9-87/01 5, July 1 987, pp 403-410.
9. Turi, T., Dey, C. R. and Loper, J. C. A forthcoming
paper on genelic regulalion of ihe P-450i4DM gene
in Saccharomyces cerevisiae.
Abstracts
The following published abslracls have recorded research
supported by Ihis Cooperalive Agreement.
1. Loper, J. C., Kalb, V. F. and Chen, C. Cylochrome
P-450 genes in Candida tropicalis, 6lh International
Symposium Microsomes and Drug Oxidations,
Brighlon, England, 5-10 August 1984.
2. Chen, C., Dey, C. R. and Loper, J. C. Mulliple
cylochrome P-450 proleins from S. cerevisiae and
C. tropicalis: immunological comparisons, American
Sociely for Microbiology Genelics and Molecular
Biology of Industrial Microorganisms Conference,
Bloominglon, IN, Sepl. 30-Ocl. 3, 1984.
3. Suller, T. R., Kalb, V. F., Woods, C., Dey, C. R. and
Loper, J. C. Yeasl cylochrome P-450 genes. 1 986.
Annual Meeling Society of Toxicology, New Orleans,
LA, March, 1986.
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4. Kalb, V. K., Woods, C. W., Dey, C. R., Sutler, T. R.
and Loper, J. C. Isolation and sequence of a
cytochrome P-450 structural gene from Saccharo-
myces cerevisiae 55th Annual Meeting Genetics
Society of America, Urbana, IL, June, 1986.
5. Sanglard, D., Chen. C. and Loper, J. C. Isolation of
the genes for the cytochrome P-450 w-hydroxylase
system of the alkane assimilating yeast Candida
tropicalis. 55th Annual Meeting Genetics Society of
America, Urbana, IL, June, 1986.
United Slates
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
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