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Gene engineering of yeasts for the degradation of hazardous waste
323
of Hazardous Materials, 19 (1988) 323-326 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands Journal
CSEPA ENVIRONMENTAL RESEARCH Gene
Engineering
of Yeasts
for the Degradation
BRIEF of Hazardous
Waste
John C. Loper” Abstract Envrronmentally recalcitrant compounds including 2.3.7.6.tetrachlorodibenzo-p-dioxin and hexachlorobenzene are metabolized I” mammalian lwer via reactions character!stic of cytochrome P-450 monooxygenase systems. This research examined the structure and function of cytochrome P-450 genes I” yeast es a model for gene engineering such eukaryotlc P-450 enzymes for btodegradation of hazardous waste by yeasts Saccharomyces cerevisiae and Candida tropicalis are two yeasts known to produce major P-450 enzymes. These enzymes were purified and antIbodIes produced I” rabbits were then used I” the lsolatio” or characterizatbon of clones conta~nong a P-450 gene from each organrsm. DNA sequence cerevwae
was and
determtned for several
for the hundred
gene bases
isolated from S of chromosomal
DNA on each side of the gene, and deletion experiments rn the promoter region were conducted The deduced protan sequence from this gene was compared to those of the other known subfamilies of eukaryottc P-450 genes: the several features identlfled Included a new homology region for these proteln sequences. The C fropica/rs gene and 0s promoter regmn has been partially sequenced.
Introduction This IS a report of the research conducted under a Cooperatwe Agreement between the U S. Environmental Protection Agency (EPA) and the Unrverslty of Clnclnnati. The work examined the yeasts Saccharomyces cerevisfae and Candida trop~calis. eukaryotlc mlcroorganlsms known io produce ma,or cytochrome P-450 enzymes Cytochrome P-450 monooxygenases are the only class of enzymes known to catalyze the specific degradatton of certain highly
recalcitrant chlorinated aromatic hydrocarbons. A longrange goal of this research directlo” is to obtain yea?.ts whbch combine the uptake and degradatlo” of these target compounds with the cepeaty to surwve or to survive and grow I” toxic environments. This result would system for controlled, low-cost biodegradation
provide a of such
enwronmentally stable towc wastes. The work under this Cooperative Agreement sought the characterization of yeast cytochrome P-450 genes, es a model for the gene engrneerlng of eukaryotlc cytochromes P-450 I” yeast.
The Role of Gene Engineered Eukaryotic Cytochrome P-450 Monooxygenases Among
the
targets
for
engineered
baodegradatlon
are
compounds whach by defmltlon are recalcitrant, that IS. not observed to be degraded by natural processes In general, the most toxic and most stable recalcitrant organw compounds are those with a high degree of chlonnation Of particular concern are polychlorlnated aromatjc hydrocarbons having no adjacent unsubstltuted carbons. for example. hexachlorobenzene, highly chlorinated biphenyls, and 2.3.7.8.tetrachlorodrbenzo-p-dloxln (TCDDJ. Such compounds are not attacked by bacterial dioxygenases. which are enzymes whose aromatlc substrates contan adjacent carbons free of constituents other than hydrogen However, there is evidence for the hydroxylatlon/dechlorlnatlon of such recalcmant compounds an some bacterta and particularly I” eukaryotlc organisms we cytochrome P-450 monooxygenases These enzymes can modify a substrate one carbon at a ttme, end because of thetr overlapprng speclfaclty, cwdatwe and reductive
number and their occasional they prowde for a broad range of reactIons
Such P.450 catalyzed reactIons play essenttal roles in mammalian metabolrsm in (a) the synthesis of the ma,or cell constituents cholesterol and fatty acjds and of
This material was originally published by the U.S. Environmental Protection Agency as EPA/ 600/M-88/001, January 1988.
324
hormones lncludlng glucocortlcords, sex hormones and prostaglandlns, and (b) the metabolism of therapeutic drugs and other xenobloitc compounds Knowledge of P450s has been galned prtmarllyfrom mammalian systems, and the data for the P-450 catalyzed metabolism of TCDD and s~mllarly recalcitrant compounds are from in viva and ,n vlfro studies of mammalian liver Knowledge of yeast P-450 systems IS less advanced Nevertheless. I, has been demonstrated that P-450 monooxygenase pathways are constituted slmllarly among hlgher and lower eukaryotes and that mammaljan P-450 gene products can function in yeasts Thus, it 1s likely that P-450 genes for degradation of recalcttrant compounds enher can be identlfled I” yeasts or can be introduced for expression in yeasts Englneerlng the gene expression of these approprlete P450 monooxygenases in yeasts would make possible the testing of these m,croorganlsms for blodegradatmn of recalcitrant hazardous compounds (8) Ln some cases the establishment of the expressed P-450 gene may be the onlyaddmonal tra~trequ~redtoequ~pthecellforthedes~red degradation, ,n others It IS likely that addltlonal properties will be Important. for compound uptake and for survival, etc The primary effort of this study was to characterrze cytochrome P-450 genes I” S cerev,s,ae. the yeast organism most amenable for such molecular genetnc studres Genes englneered I” this orgitnlsm subsequently can be transferred to other strabns. Since dlfferent species of yeast are lrkely to tolerate better the ecological condltnons presented by various noxious environments, strains isolated from those enwonments could be used as recipients of functional P-450 gene sets to establish the preferred detoxlcatlng YarNants CandIda tropcahs IS a likely candldate as one of these ecologically suitable organisms This yeast already contains an elaborate machinery for the asslmllatlon of hydrophobrc long cham hydrocarbons enabling Its growth on crude 011 Secondary to the studies I” S. cerevis~ae, P-450 genes I” this orgamsm were also examined
Characterization of Yeast P-450 and Sequence Determination
Genes:
Isolation
When th,w study started. conslderable data were wallable on a few P-450 enzymes of yeast but no yeast P-450 genes were avaIlable In S cerevwae the best characterized P450 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 1solatnon as a model system and as a gene of potential use I” general bmdegradatlon Another well stud& yiast P-450 enzyme IS alkane whydroxylase This enzyme catalyzes the fwst step nn the metabolism of long cham n-alkanes as a carbon and energy source It is found in a variety of yeasts other than S. cerevrs~ae; Can&da rropica/!s IS a major example The C tropicalis gene for this enzyme was chosen for lsolatlon as the second model. This chmce was based upon the llkelthood that this P-450 may be of direct use tn blodegradatlon. and because utlllzatian of alkanes involves processes for cell uptake of hydrophobic substrates whwh
are likely hazardous
to be wastes
of
use
I”
the
uptake
of
hydrophobic
As a useful step toward P-450 gene lsolatlon and Identification. mxrosomal preparatmns were first Isolated from these two mlcroorganlsms and the S cerevrsiae lanosterol demerhylase, and fhe C tropIcaIls whydraxylase were punfled AntIbodIes ,n rabbjts were prepared and physical and rmmvnologlcal slmilar~tles of the two enzymes were examined 11.21 Then using a recently described method based upon gene dosage effects for lsolatlon of genes I” yeast. we Isolated a lanosterol demethylase gene from S cerevis~ae (3) Uswg a cloned subfragment of DNA contanng the entnre gene. overlapplng deletions were produced which then were used as templates for DNA sequencing of one DNA strand of the gene by the Ml3 dldeoxy method The complementary strand was also sequenced by the dldeoxy method, using ollgonucleotlde primers synthewed I” the Department As a result, the DNA sequence of this gene has beendetermined together wth thesequence of several hundred nucleotlde base pairs upstream and downstream of the protein coding rrgfon (4) When genes the unwersal acid sequence and sequence
are sequenced, a useful procedure IS to use genetIc code to Identify the predlcted am~no of the protean product Since the !solatlon characterlratlon of mammalian P-450 genes
1s a malor area several P-450 procedure and
of research among numerous laboratories. genes have now been examined in thrs their protein sequence determlned When
the protein sequence of the yeast P-450 was compared to those of hlgher eukaryotes, many similar features were Identtfled (41 Chtef among these are (a) the presence of a strongly hydrophobx regfon “ear the amdno terminal end, which could serve as a typical anchor peptlde for attachment of the protem to intracellular membranes. and (b) a 21 arn~no aced segment near the C terminal region which conforms to a homologous region detected for all other P-450 genes This homologous reg!on apparently IS required for the bIndIng of heme to the apoprotw” These features of the deduced prote,” sequence ldentlfy fhlsS. cerevwae gene as a cytochrome P-450 gene Based on these and upon overall DNA homology propertIes. this yeast P-450 protean comprises an addmonal subfamily among the superfamily of eukaryotlc P-450s It IS clear also that this S cerevisiae P-450 gene has more I” common with other subfamilies of eukaryow P-450 genes than It does wlih the structure of the known bacterral P450 gene (4.5) Thus, the sequence chatacterlzatlon of a yeast P-450 gene has provided unique lnformatlon on evolut8onary relatlonshlps among the P-450 superfamlly For
lsolatlon
of
the w-hydroxylase P-450 gene from C use was made of the am-enzyme antibody for gene detectlon I” a gene expresslo” lnbrary This procedure led to the nsolat1on of the complete w. hydroxylase gene (6). Total sequence characterwatmn of this gene IS in progress. using a doilble strand plasmld sequencing procedure that employs templates from sets of deletions exlendrng across eather end of the ~sola~wl
rrop~cehs. dwect
gene.
325
A
New
Region
Eukaryotic
of
Sequence
Similarity
Among
Loper, J Chen. C and Dey. ot yeasts for the blodegradatlon
All
P-4505
in Proceedings Symposium
As noted, the S. cerevisiae P-450 comprises a new P450 subfamily. With the advantage of this new lnformatmn a detalled comparison of homology among all seven of the subfamilies in this sytem was conducted. The analysis ldentlfied a new region of sequence slmilarrty among all the eukaryotic P-450s (5.71 This advance is important to research on the structure and function relatlonshlps of all P-450s since ,t lndlcates prote,” sites where sitespeclflc mutac~enes~s would be particularly lnformatwe
Regulation
of
P-450
Expression
Hazardous 1985,pp
The
245,
for
gnductlon
of w-hydroxylase
in C
Sanglard. D and Loper. Chew C, Turi, T G lsolatlon of the Candida trop~calts gene for lanosterol demethylase and Its expresslo” Saccharomyces cerevwae Blochem Blophys Comm. 146, 1311.1317(1987).
segment of this as this
Publications
1.
describe
research
supported
by
Lope,.J C..Chen. C. andDey, C.R. Geneengmeer~ng in yeast for bmdegradatlonimmunological crossreactwty among cytochrome P-450 system protems of S cerewsiae and C rropicak Hazardous Waste Hazard. Mat. 2.131-141, 7985
)
J C. P450 I” Res
Chew C Dey. C R., Kalb. V F, Sanglard, D Sutter. T R.. and Lope,, J C Englneerlng P450 genes I” yeast ,n Land Disposal Remedlal Actton. lnclneration and Treatment of Hazardous Waste Proceedings of the thirteenth annual research symposium US EPA, EPA/600/9-87/015. July 1987. pp 403.410
Expression products, production
has been isolated as part of the C. trop!ca/,s DNA contalnmg the w-hydroxylase gene(6). Sequencing promote, was I” progress I” this laboratory Cooperative Agreement ended
followng publlcatlons Cooperatwe Agreement
1986
Sanglard. 0.. Chen, C and Loper. J. C lsolatlon of the alkane lnduclble cytochrome P~450 (P450alk) g,?ne from the yeast CandIda rroprcal~ Blochem Blophys Res Comm 144, 261.257 (19871
rrop~ca/is
of Intracellular organelles Involved m catabohsm of the alkanes. The result IS a yeast extensrvely adapted for utllwatlon 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 monooxygenation of specifvz, hazardous hydrocarbons Into such Yeasts. with the inserted P-450 genes under the unductlve control of the w-hydroxylase P-450 gene promoter. This promote,
The this
September
Kalb. V F and Loper, J. C A segmented region of sequence slmllarlty found I” e!ght different eukaiyolrc P450 famtlies (Manuscript submItted for review
are of particular Interest for gene eng~neerlng the biodegradation of toxic hydrophobic compounds This work lndlcates that transcrlptlon of this enzyme is Induced one hundred-fold or more as the cells adapt to asslmllation of mIneral oil alkanes It appears that over 24 addttronal genes are involved in this asslmllation process. of these genes prcwdes for changes I” surfactant llpophllnc changes !n cell wall structure and
EPA/600/9-85/02B.
Kalb, V F, Woods. C W, Turi. T. G, Dey. C A Sutte,. T R and Looer. J C Prlmarv structure of the P450 lanoste,ol demethylax gene from Sack charomyces cerevr+,ae DNA. 616) 529.537 (1987)
in Yeast
(3).
signals
Waste, 112-119
Annual Research and Treatment of
Woods, C W Kalb. V F, Loper, J C, Dey. C R and Sutter. T R lsolatlon of a cytochrome P-450 structuial gene from S cerevrsrae Gene, 45(3J 237.
UnderstandIng and gene eng,neer,ng of regulatory slgrals are important for rhe controlled expresslo” of a P-450 gene for degradatjon reactions Considerable progress has been made on several aspects of this questIon For S. cerevis~ae, the DNA sequence analysis has Included over 800 nucleotide bases proximal to the structural gene codtng region. Deletion analysis of this region has shown that the major upstream porno” can be removed wthout preventing gene expression (9) Also, addItIonal genes can alter the level of lanosterol demethylase maIntanned in th!so,ganlsm. Recombinant st,alns have beenconstructed that provide for stable high cytoplasmlc levels of this enzyme
C. Gene enguneerlng of hazardous wastes,
of the Eleventh on lncineratmn
Turl,
T , Dey,
C
paper on genetlc I” Saccharomyces
R
and
Loper,
regulation cerev,s!ae
J
C. A forthcomlng
of the
P-450,ro~
gene
Abstracts The
followng
supported
publIshed by this
abstracts
Cooperative
have
recorded
research
Agreement
Loper, J C, Kalb, V. F and Chen. C Cvtochrome P-450 genes I” CandIda frop/calrs. 6th lnternatlonal Svmpos~um Mlcrosomes and Drug Owldarlons. B,ighton. England, 5-10 August. 1984 Chen. C. Dey, C R and Loper, J. C Multtple cytochrome P-450 prore~ns from S cerevrs,ae and C trop~airs lmmunologlcal comparnsons. American Socrety for Mlcroblologv Genetlcs and Molecular Biology of Industrial MIcroorganIsms Conference. Bioomlngton. IN. Sept 3O~Oct 3. 1984 Sutter. T R Kalb, V. F Woods, C Looer. J. C Yeast cvtochrome P-450 Annual Meeting Society of Tox+xlogy. LA. March. 1986
Dey. C R and oenes 1986 New Orleans.
326
4
Kalb. V K.. Woods. C. W.. Dey. C. R Sutter. T R. and Loper, J. C lsolaf~on and sequence of a cytochrome P-450 structural gene from Saccharomyces cerevisiae 55th Annuef Meeting Genetics Society of America. Urbana, IL, June. 1986.
5.
Sanglard, D Chen, C. and Loper. J. C. lsolatlon of the genes for the cytochrome P-450 w-hydroxylase system of the alkane asslmllating yeast Candida 55th Annual Meeting Genetics Soclety of America. Urbana. IL, June. 1966.
rroprcahs
of Hazardous Materials, 19 (1988) 323-326 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands Journal
CSEPA ENVIRONMENTAL RESEARCH Gene
Engineering
of Yeasts
for the Degradation
BRIEF of Hazardous
Waste
John C. Loper” Abstract Envrronmentally recalcitrant compounds including 2.3.7.6.tetrachlorodibenzo-p-dioxin and hexachlorobenzene are metabolized I” mammalian lwer via reactions character!stic of cytochrome P-450 monooxygenase systems. This research examined the structure and function of cytochrome P-450 genes I” yeast es a model for gene engineering such eukaryotlc P-450 enzymes for btodegradation of hazardous waste by yeasts Saccharomyces cerevisiae and Candida tropicalis are two yeasts known to produce major P-450 enzymes. These enzymes were purified and antIbodIes produced I” rabbits were then used I” the lsolatio” or characterizatbon of clones conta~nong a P-450 gene from each organrsm. DNA sequence cerevwae
was and
determtned for several
for the hundred
gene bases
isolated from S of chromosomal
DNA on each side of the gene, and deletion experiments rn the promoter region were conducted The deduced protan sequence from this gene was compared to those of the other known subfamilies of eukaryottc P-450 genes: the several features identlfled Included a new homology region for these proteln sequences. The C fropica/rs gene and 0s promoter regmn has been partially sequenced.
Introduction This IS a report of the research conducted under a Cooperatwe Agreement between the U S. Environmental Protection Agency (EPA) and the Unrverslty of Clnclnnati. The work examined the yeasts Saccharomyces cerevisfae and Candida trop~calis. eukaryotlc mlcroorganlsms known io produce ma,or cytochrome P-450 enzymes Cytochrome P-450 monooxygenases are the only class of enzymes known to catalyze the specific degradatton of certain highly
recalcitrant chlorinated aromatic hydrocarbons. A longrange goal of this research directlo” is to obtain yea?.ts whbch combine the uptake and degradatlo” of these target compounds with the cepeaty to surwve or to survive and grow I” toxic environments. This result would system for controlled, low-cost biodegradation
provide a of such
enwronmentally stable towc wastes. The work under this Cooperative Agreement sought the characterization of yeast cytochrome P-450 genes, es a model for the gene engrneerlng of eukaryotlc cytochromes P-450 I” yeast.
The Role of Gene Engineered Eukaryotic Cytochrome P-450 Monooxygenases Among
the
targets
for
engineered
baodegradatlon
are
compounds whach by defmltlon are recalcitrant, that IS. not observed to be degraded by natural processes In general, the most toxic and most stable recalcitrant organw compounds are those with a high degree of chlonnation Of particular concern are polychlorlnated aromatjc hydrocarbons having no adjacent unsubstltuted carbons. for example. hexachlorobenzene, highly chlorinated biphenyls, and 2.3.7.8.tetrachlorodrbenzo-p-dloxln (TCDDJ. Such compounds are not attacked by bacterial dioxygenases. which are enzymes whose aromatlc substrates contan adjacent carbons free of constituents other than hydrogen However, there is evidence for the hydroxylatlon/dechlorlnatlon of such recalcmant compounds an some bacterta and particularly I” eukaryotlc organisms we cytochrome P-450 monooxygenases These enzymes can modify a substrate one carbon at a ttme, end because of thetr overlapprng speclfaclty, cwdatwe and reductive
number and their occasional they prowde for a broad range of reactIons
Such P.450 catalyzed reactIons play essenttal roles in mammalian metabolrsm in (a) the synthesis of the ma,or cell constituents cholesterol and fatty acjds and of
This material was originally published by the U.S. Environmental Protection Agency as EPA/ 600/M-88/001, January 1988.
324
hormones lncludlng glucocortlcords, sex hormones and prostaglandlns, and (b) the metabolism of therapeutic drugs and other xenobloitc compounds Knowledge of P450s has been galned prtmarllyfrom mammalian systems, and the data for the P-450 catalyzed metabolism of TCDD and s~mllarly recalcitrant compounds are from in viva and ,n vlfro studies of mammalian liver Knowledge of yeast P-450 systems IS less advanced Nevertheless. I, has been demonstrated that P-450 monooxygenase pathways are constituted slmllarly among hlgher and lower eukaryotes and that mammaljan P-450 gene products can function in yeasts Thus, it 1s likely that P-450 genes for degradation of recalcttrant compounds enher can be identlfled I” yeasts or can be introduced for expression in yeasts Englneerlng the gene expression of these approprlete P450 monooxygenases in yeasts would make possible the testing of these m,croorganlsms for blodegradatmn of recalcitrant hazardous compounds (8) Ln some cases the establishment of the expressed P-450 gene may be the onlyaddmonal tra~trequ~redtoequ~pthecellforthedes~red degradation, ,n others It IS likely that addltlonal properties will be Important. for compound uptake and for survival, etc The primary effort of this study was to characterrze cytochrome P-450 genes I” S cerev,s,ae. the yeast organism most amenable for such molecular genetnc studres Genes englneered I” this orgitnlsm subsequently can be transferred to other strabns. Since dlfferent species of yeast are lrkely to tolerate better the ecological condltnons presented by various noxious environments, strains isolated from those enwonments could be used as recipients of functional P-450 gene sets to establish the preferred detoxlcatlng YarNants CandIda tropcahs IS a likely candldate as one of these ecologically suitable organisms This yeast already contains an elaborate machinery for the asslmllatlon of hydrophobrc long cham hydrocarbons enabling Its growth on crude 011 Secondary to the studies I” S. cerevis~ae, P-450 genes I” this orgamsm were also examined
Characterization of Yeast P-450 and Sequence Determination
Genes:
Isolation
When th,w study started. conslderable data were wallable on a few P-450 enzymes of yeast but no yeast P-450 genes were avaIlable In S cerevwae the best characterized P450 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 1solatnon as a model system and as a gene of potential use I” general bmdegradatlon Another well stud& yiast P-450 enzyme IS alkane whydroxylase This enzyme catalyzes the fwst step nn the metabolism of long cham n-alkanes as a carbon and energy source It is found in a variety of yeasts other than S. cerevrs~ae; Can&da rropica/!s IS a major example The C tropicalis gene for this enzyme was chosen for lsolatlon as the second model. This chmce was based upon the llkelthood that this P-450 may be of direct use tn blodegradatlon. and because utlllzatian of alkanes involves processes for cell uptake of hydrophobic substrates whwh
are likely hazardous
to be wastes
of
use
I”
the
uptake
of
hydrophobic
As a useful step toward P-450 gene lsolatlon and Identification. mxrosomal preparatmns were first Isolated from these two mlcroorganlsms and the S cerevrsiae lanosterol demerhylase, and fhe C tropIcaIls whydraxylase were punfled AntIbodIes ,n rabbjts were prepared and physical and rmmvnologlcal slmilar~tles of the two enzymes were examined 11.21 Then using a recently described method based upon gene dosage effects for lsolatlon of genes I” yeast. we Isolated a lanosterol demethylase gene from S cerevis~ae (3) Uswg a cloned subfragment of DNA contanng the entnre gene. overlapplng deletions were produced which then were used as templates for DNA sequencing of one DNA strand of the gene by the Ml3 dldeoxy method The complementary strand was also sequenced by the dldeoxy method, using ollgonucleotlde primers synthewed I” the Department As a result, the DNA sequence of this gene has beendetermined together wth thesequence of several hundred nucleotlde base pairs upstream and downstream of the protein coding rrgfon (4) When genes the unwersal acid sequence and sequence
are sequenced, a useful procedure IS to use genetIc code to Identify the predlcted am~no of the protean product Since the !solatlon characterlratlon of mammalian P-450 genes
1s a malor area several P-450 procedure and
of research among numerous laboratories. genes have now been examined in thrs their protein sequence determlned When
the protein sequence of the yeast P-450 was compared to those of hlgher eukaryotes, many similar features were Identtfled (41 Chtef among these are (a) the presence of a strongly hydrophobx regfon “ear the amdno terminal end, which could serve as a typical anchor peptlde for attachment of the protem to intracellular membranes. and (b) a 21 arn~no aced segment near the C terminal region which conforms to a homologous region detected for all other P-450 genes This homologous reg!on apparently IS required for the bIndIng of heme to the apoprotw” These features of the deduced prote,” sequence ldentlfy fhlsS. cerevwae gene as a cytochrome P-450 gene Based on these and upon overall DNA homology propertIes. this yeast P-450 protean comprises an addmonal subfamily among the superfamily of eukaryotlc P-450s It IS clear also that this S cerevisiae P-450 gene has more I” common with other subfamilies of eukaryow P-450 genes than It does wlih the structure of the known bacterral P450 gene (4.5) Thus, the sequence chatacterlzatlon of a yeast P-450 gene has provided unique lnformatlon on evolut8onary relatlonshlps among the P-450 superfamlly For
lsolatlon
of
the w-hydroxylase P-450 gene from C use was made of the am-enzyme antibody for gene detectlon I” a gene expresslo” lnbrary This procedure led to the nsolat1on of the complete w. hydroxylase gene (6). Total sequence characterwatmn of this gene IS in progress. using a doilble strand plasmld sequencing procedure that employs templates from sets of deletions exlendrng across eather end of the ~sola~wl
rrop~cehs. dwect
gene.
325
A
New
Region
Eukaryotic
of
Sequence
Similarity
Among
Loper, J Chen. C and Dey. ot yeasts for the blodegradatlon
All
P-4505
in Proceedings Symposium
As noted, the S. cerevisiae P-450 comprises a new P450 subfamily. With the advantage of this new lnformatmn a detalled comparison of homology among all seven of the subfamilies in this sytem was conducted. The analysis ldentlfied a new region of sequence slmilarrty among all the eukaryotic P-450s (5.71 This advance is important to research on the structure and function relatlonshlps of all P-450s since ,t lndlcates prote,” sites where sitespeclflc mutac~enes~s would be particularly lnformatwe
Regulation
of
P-450
Expression
Hazardous 1985,pp
The
245,
for
gnductlon
of w-hydroxylase
in C
Sanglard. D and Loper. Chew C, Turi, T G lsolatlon of the Candida trop~calts gene for lanosterol demethylase and Its expresslo” Saccharomyces cerevwae Blochem Blophys Comm. 146, 1311.1317(1987).
segment of this as this
Publications
1.
describe
research
supported
by
Lope,.J C..Chen. C. andDey, C.R. Geneengmeer~ng in yeast for bmdegradatlonimmunological crossreactwty among cytochrome P-450 system protems of S cerewsiae and C rropicak Hazardous Waste Hazard. Mat. 2.131-141, 7985
)
J C. P450 I” Res
Chew C Dey. C R., Kalb. V F, Sanglard, D Sutter. T R.. and Lope,, J C Englneerlng P450 genes I” yeast ,n Land Disposal Remedlal Actton. lnclneration and Treatment of Hazardous Waste Proceedings of the thirteenth annual research symposium US EPA, EPA/600/9-87/015. July 1987. pp 403.410
Expression products, production
has been isolated as part of the C. trop!ca/,s DNA contalnmg the w-hydroxylase gene(6). Sequencing promote, was I” progress I” this laboratory Cooperative Agreement ended
followng publlcatlons Cooperatwe Agreement
1986
Sanglard. 0.. Chen, C and Loper. J. C lsolatlon of the alkane lnduclble cytochrome P~450 (P450alk) g,?ne from the yeast CandIda rroprcal~ Blochem Blophys Res Comm 144, 261.257 (19871
rrop~ca/is
of Intracellular organelles Involved m catabohsm of the alkanes. The result IS a yeast extensrvely adapted for utllwatlon 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 monooxygenation of specifvz, hazardous hydrocarbons Into such Yeasts. with the inserted P-450 genes under the unductlve control of the w-hydroxylase P-450 gene promoter. This promote,
The this
September
Kalb. V F and Loper, J. C A segmented region of sequence slmllarlty found I” e!ght different eukaiyolrc P450 famtlies (Manuscript submItted for review
are of particular Interest for gene eng~neerlng the biodegradation of toxic hydrophobic compounds This work lndlcates that transcrlptlon of this enzyme is Induced one hundred-fold or more as the cells adapt to asslmllation of mIneral oil alkanes It appears that over 24 addttronal genes are involved in this asslmllation process. of these genes prcwdes for changes I” surfactant llpophllnc changes !n cell wall structure and
EPA/600/9-85/02B.
Kalb, V F, Woods. C W, Turi. T. G, Dey. C A Sutte,. T R and Looer. J C Prlmarv structure of the P450 lanoste,ol demethylax gene from Sack charomyces cerevr+,ae DNA. 616) 529.537 (1987)
in Yeast
(3).
signals
Waste, 112-119
Annual Research and Treatment of
Woods, C W Kalb. V F, Loper, J C, Dey. C R and Sutter. T R lsolatlon of a cytochrome P-450 structuial gene from S cerevrsrae Gene, 45(3J 237.
UnderstandIng and gene eng,neer,ng of regulatory slgrals are important for rhe controlled expresslo” of a P-450 gene for degradatjon reactions Considerable progress has been made on several aspects of this questIon For S. cerevis~ae, the DNA sequence analysis has Included over 800 nucleotide bases proximal to the structural gene codtng region. Deletion analysis of this region has shown that the major upstream porno” can be removed wthout preventing gene expression (9) Also, addItIonal genes can alter the level of lanosterol demethylase maIntanned in th!so,ganlsm. Recombinant st,alns have beenconstructed that provide for stable high cytoplasmlc levels of this enzyme
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Agreement
Loper, J C, Kalb, V. F and Chen. C Cvtochrome P-450 genes I” CandIda frop/calrs. 6th lnternatlonal Svmpos~um Mlcrosomes and Drug Owldarlons. B,ighton. England, 5-10 August. 1984 Chen. C. Dey, C R and Loper, J. C Multtple cytochrome P-450 prore~ns from S cerevrs,ae and C trop~airs lmmunologlcal comparnsons. American Socrety for Mlcroblologv Genetlcs and Molecular Biology of Industrial MIcroorganIsms Conference. Bioomlngton. IN. Sept 3O~Oct 3. 1984 Sutter. T R Kalb, V. F Woods, C Looer. J. C Yeast cvtochrome P-450 Annual Meeting Society of Tox+xlogy. LA. March. 1986
Dey. C R and oenes 1986 New Orleans.
326
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Kalb. V K.. Woods. C. W.. Dey. C. R Sutter. T R. and Loper, J. C lsolaf~on and sequence of a cytochrome P-450 structural gene from Saccharomyces cerevisiae 55th Annuef Meeting Genetics Society of America. Urbana, IL, June. 1986.
5.
Sanglard, D Chen, C. and Loper. J. C. lsolatlon of the genes for the cytochrome P-450 w-hydroxylase system of the alkane asslmllating yeast Candida 55th Annual Meeting Genetics Soclety of America. Urbana. IL, June. 1966.
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