CA2268004A1 - Candida utilis transformation system - Google Patents
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Abstract
The present invention discloses a transformation system useful to express heterologous proteins in the Candida utilis yeast, based on obtaining auxotrophic mutants of said species as well as the isolation of different genes, from a genomic library, which complement said auxotrophies. The transformation system uses as hosts new auxotrophic mutants obtained from the yeast NRRL Y-1084 of Candida utilis which are defective mainly in the biosynthetic ways of uracyl and histidine, which are transformed with plasmids containing as selection markers the genes URA3 and HIS3 of Candida utilis.
Another aspect of the invention is the isolation of the gene coding for the enzyme sucrose invertase or .beta.-fructofuranosidase of Candida utilis, as well as the identification of sequences for promoting, secretion signalling and termination of said gene INV1. These sequences are useful to obtain the expression of heterologous proteins in said yeast.
Another aspect of the invention is the isolation of the gene coding for the enzyme sucrose invertase or .beta.-fructofuranosidase of Candida utilis, as well as the identification of sequences for promoting, secretion signalling and termination of said gene INV1. These sequences are useful to obtain the expression of heterologous proteins in said yeast.
Description
TRANSFORMATION SYSTEM I:N CANDIDA UTILrB.
Tachnieal Sector.
The present invention is related ~CO the field of the genetic engineering and biotechnology, and in particular with the development of a system host-vector for the genetic transformation of the yeast Candicfa utilis which permits the expression and secretion of hete:rologous proteins in this yeast, which can be further used with several purposes.
Prior Art.
to The genetic engineering and biotechnology have opened an unprecedented goal in the production of many interest proteins with medical, nutritious or industrial purpose, which report excellent benefits.
The bacteria Escherichia coli has been the moat employed microorganism with these purpoeee~ by diverse biotechnology companies, due to the knowledge of their genetics) its easy manipulation and their systems of cultivation at high densities.
however, the hopes of the production of proteins of inrerest in this microorganism are affected for diverse factors, First of a11 the pirogenic and toxic compounds in the cell wall of Escherichia coli have provoked regulations limiting its use when the products obtained are intended to be used as medicaments or food in humane. In addition, the proteins that are over-expressed in Escherichia noli generally appear in an insoluble form that could not be secreted, On the other hand, the mechanisms of transcription, translation and postranslation modifications differ from the eukaryotic systems, resulting in recombinant proteins that in a certain way differ from that of the natura7_ sources.
The possibility of producing 'heterologous proteins in eukaryotic systems, such as the ye~iSL, has some advantages in relation to the prokaryotic systems. Among these, they could mention the capacity to grow to k~.igh cellular densities and the possibility of adapting their cultivation to continuous systems _ Also the yeast are able to secrete proteins to the culture medium in considerable bigger amounts, in comparison with Eacherichia coli, also the c(rowth medium used for the growth of yeast are more economic ~~han those used in bacteria (Lemoine, Y., 1988. Heterologous expression in yeast. 8th International Biotechnology Symposium, Paris, July 17-22).
Also, these systems can carry out other postraductional modifications as it is the case of the glycosylation, which is absent in the bacterial systems (Fiers) W., 1988, Engineering Maximal Expression of Heterologous Gene in Microorganism. 8th International Biotechnology symposium, Paris, July 17-221. In addition, these systems generally have certain preference for the same codon use that the higher eukaryotic systems (Kigsman, S.M. et al., 1990. Heterologous Gene Expression in Saccharomyces cerevisiae, Biotechnology &
Genetic Engineering Reviews, 3, Ed. G.E. Russell).
A11 this has led to the development and dissemination of new eukaryotic transformation systerns and with particular interest in yeast) firstly described for species of the Saccharomyces genus, with special emphasis in Saccharomyces cerevisiae. However, the expression of proteins in Saccharomyces has confronted problems with the expression levels obtained using their homologous promoters as well as the hyperglycosylation observed in the proteins secreted to the medium. That is the main reasons that in the last years it has been intensified the search of non-conventional yeast for their use in the expression of heterologous proteins.
3o With the development of transformation system in other non-Saccharomyces yeast, like Hanse~nula polymorpha, Pichia pastoris, and yeast of the Kluyveromyces genus (Sudbery, P., 1994. Yeast 10; 1707-17z6) has permitted a quick advance in the knowledge and development of these systems, as well as increased the number of foreign proteins expressed with vaccination, diagnoses and industrial purposes in these system.
Also inside the genus Candida E~everal transformation and expression systems have been reported, including Candida tropicalis, Candida boidiini, c'andida glabrata, Candida parapsilosis, Candida maltosa and Candida albicans all with a marked medical interest, because many of these species are the causing of opportunists illnesses in humans.
Candida utilie, in the Candida genus, has special interest due to its particular characteristics. First of a11, Candida utilis uses a great variety of inexpensive carbon sources such as xylose, sucrose and maltose among other. Another interesting feature is that it is possible produce efficiently a great amount of cells in continuous cultures.
Also Candida utilis, as well as Saccharomyces cerevisiae and Kluyveromyces lactie, have been authorized for the FDA (Food and Drug Administration) like s~~fe sources in foodstuff.
Resides) Candida utilis has been used in the industry for the production of L-glutamina, ~etil acetate and invertase among other products.
A preliminary system for a transformation system in Candida utilis has been described by 130, I. et. al., 1984, (Biotechnology and Bioengineering Symp. 14: 295-301). This report is incomplete because tl;~e presence of the drug resistance marker and direct evidence of the transformation process are not disclosed. Recently, a novel strategy concerning a transformation syete:m for Candida utilis has been reported by Kondo, K. et al . , 1995, (.T. Bacteriol . 177 7171-7177). They obtained cycloheximide (CYH) resistant transformants by using a marker gene containing a mutated form of the ribosomal protein L41, which conferred resistance, and also used ribosomal DNA (rDNA) fragment as a multicopy target for plaemid integration because the marker needs to be present in multiple copies for selection of CHY-resistant transformants.
Many attempts has been done to use Cazidida utilis as host for heterologoue gene expression, nevE:rtheless, a transformation procedure in Candida utilis using auxotrophyc mutants has not been developed up to now.
If it is taken into account the knowledge obtained in the to industrial exploitation of Candids~ utilis and the novelty of its genetics, it could be ~=onsidered an attractive microorganism for its commercial utilization as expression system of heterologous proteins.
Disclosure of the Invention.
The object of the present invention has been to provide a transformation system useful to ex;presa heterologous proteins in the yeast Candida ucilis, based on the obtainment of axotrophyc mutants of this specie ~~s well as m the isolation of different genes from a genomic library which complement said auxotrophies.
The transformation process described herein provides means to introduce DNA fragments or sequenc~ae into Candida util.is host cells and allows Candida utilis to be used as a host system for gene expression and protein prnduction_ Furthermore transformed yeast ce7_ls can be identified and selected by the methods describe in the present invention.
Novel strains of Candida utilis, vectors and subclones are provided. Novel yeast strains are used as hosts for introduction of recombinant DNA fragments.
The invention further relates to stable transformation and maintenance of DNA in host cells, where the marker is homologous integrated in the genome of the yeast.
S
Concretely the present invention consists in a transformation system in the yeast Candida utilis, which uses as hosts new auxotrophyc mutants isolated from the strain NRRL Y-1o84 of said yeast. These mutants are defective in the enzyme orotidin-5' phosphate decarboxylase of the biosynthetic pathway of the uracil or in the biosynthetic pathway of the hietidine, and were obtained by classical mutagenesis using both UV and NTG as mutagenic agents known from the prior art (Sherman, F. et al., 1986_ Laboratory course: Manual for methods in yeast genetics. Cold Spring Harbor Laboratory Press, NY). These mutants present a high stability (frequency of reversion approximately of 1o'a) and can be efficiently transformed with the procedure described in this invention.
In addition, it was isolated as selection markers for the mutants of Candida utilis the gene URA3, encoding for the orotidin 5'-phosphate decarboxylaeEa enzyme and HISS encoding for the Imidazol-glycerol-phosphate dehydratase enzyme which were isolated from a gene library of Candida utilis in PUC19 and identified by complementation of the pyrF and hisb463 mutations respectively in the stra~Ln Escherichia coli MC1o66.
Similarly the mutation ura3 of Saccharomyces cerevisiae strain SEY 2202 was used to identify this gene came from C.
utilis, The complete sequence of 'these genes was determined and the predicted amino-acid sequences show high similarities with that of the same gene from other yeast and fungi.
The vectors used in the transformation system were the plasmids pURAS and pUREC3 which comprise the URA3 gene, capable of being integrated into the homologous locus of the Candida utilis mutant host by homo7.ogous recombination.
The present invention also provides a set of plasmids based on those described formerly, which are used for the transformation of the mutants isolated from C. utilis in order to obtain heterologous proteins.
The transformation system of the present invention uses as hosts new auxotrophyc mutants obtained from the strain NRRL
Y-1084 of Candida ut~l.is, which are defective mainly in the uracil and histidine pathways, and among them were selected for their characteristics the mutant CUT-35 (ura') and the mutant TNll~1-3 (his') .
EXA?~LES
Example 1: Mutsgenesia of Caadida t~tilie To develop a transformation system in a microorganism they are generally required three elements:
(1) a marker for the selection o.E the tranaformants, that could be an auxotrophyc or a dominant marker, (2) a mutant or appropriate host for this selection and (3) a method to reproducible introduce the extranuclear DNA
in the host in an efficient form.
In order to achieving the second objective, it was carried out a classical mutagenesis in t:he yeast Candida utilis.
Cultures of the of the selected yeast strain (NRRL Y-1084) were inoculated in 100 ml of YPG medium (Yeast extract 1%) 2o peptone 2%, glucose 2%) and they were incubated in a shaker at 30~C for l0-20 hours. 50 ml of the culture was centrifuged to 3000 rpm for 5 minutes. Later the cells were washed 2 times with citrate buffer 0.1M' (pH 5.5) sterile and resuspended in 50 ml of the same buffer. After, 10 m1 of this suspension was incubated with a solution of NTG to a final concentration of 50 mg/ml. The su=;pension was incubated for minutes at 30~C in repose.
The NTG was removed of the suspension washing 2 times with distilled water. The cells were resuspended in 50 ml of YPG
30 and then they were transferred to an erlenmeyer with 100 ml of YPG. This culture of mutant cells was incubated at 30~C
for 48 hours.
Eltrichment with nyatatin Approximately 5 ml of the 48 hours YPG expressed culture was used to inoculate 100 ml of minimum medium. The minimum medium (YNB, Yeast Nitrogen Base) used for the enrichment Kith the antibiotic was not supplement with the metabolic produced by the biosynthetic via in which the defect is looked for. For example, for the isolation of auxotrophics mutants for uracil, it is not added to the medium.
The incubation was continued until the optical density (oD) of the culture reached 20 to 30% of the initial OD. When the culture reached the desire OD, th.e cellular suspension was treated with 25 units/ml a solution of nyetatin. The solution with the antibiotic was incubated at 30~C for 30 minutes without agitation, The nystatin waos eliminated of the medium washing the cellular suspension wi~~h distilled water 2 times and later the cells were resuspende:d in an appropriate volume in order to obtain 150 to 200 colonies per plate, Screening and selection.
The plates containing the mutageni.zed colonies according to the example 1, were plated in YNB mediums with and without between uracil. The colonies that ~iid not grow in absence of uracil were taken for further analysis.
Specifically in order to identify the presence ura3 or ura5 mutants, the cells were grown in presence of 5-fluorotic acid (SFOA). The resistant colonies were selected as ura3 or ura5 like mutants.
Example 2: Isolation of ura3 mutants.
After the nystatin enrichment the culture was washed twice with distilled water and plate directly on YNB plates containing 0.75 ~g/ml of 5-FOA (5-fluoro-orotic acid, Fluka) and 40 ~g/ml of uracil , The plates were incubated four days and the colonies that grew were analyzed in order to check the ura- phenotype. From the 4x10 viable cells, after the nystatin enrichment) 79 colonies showed resistance to the 5-r FOA. These colonies could be ura3, uraS, or simply resistant to the 5-FOA. To confirm the uracil auxotrophy the supposed mutants were plated in YPG medium incubated 48 hours to 30~C
and replicated in YN$ plates with a.nd without uracil. A total of 67 colonies were unable to grow in YNB without uracil, shown a ura' phenotype.
The frequency of reversion of all these mutants were determined standing out a group of 23 mutants by presenting a frequency of reversion in the order of 10-~, what confers them certain stability to be used as a host for transformation system.
The orotidin 5'-monophosphate decarboxylase (ODCase) activity of a11 the uracil auxotrophyc mutants was determined by the method of Yoshimoto et. al., 197A (Methods Enzymol. 51: 74-79), as well as it was determined their growth conditions.
These results are shown in Table 1.
Table 1. Summary of the characteristics of more significant ura3 mutants.
Name Reversion OMF>DCase Growth Frequency Activity CUT35 c SxlO-' - +++
CUT43 c 1x10-' - +++
CUT61 < 1x10'8 - +++
CUT65 c 1x10-~ - ++
CUT70 < 1x10'~ - +
CUT86 < 7x10-' - +++
CUT93 1x10-~ - +++
CUT166 6x10-~ - +++
Example 3: Isolation of other mutants different of the phenotype ura- .
With the objective of having a vari~Cty of auxotrophyc mutants different than uracil, the cellular suspension obtained according to the nystatin enrichment were plated in YPG and incubated at 30~C for 50 hours. Later the colonies contained in the YPG plates were replicated on plates containing YNB
medium arid incubated at 30~C for 48 hours. The colonies unable to grow in the YNB plates were taken for further analysis.
Around 2411 colonies were screened and consequently was obtained a 2% of appearance of auxotrophyc mutants. These mutants were checked using the Holliday and the Finchan tests. It was obtained 90~ of his mutants, 2% responded to to the phenotype lys', 1% to the p.'henotype leu-, 1% to the phenotype met-, 1% to the phenotype ads- and the 5% did not show a simple auxotrophyc phenotype (Naa).
The mutants having frequency of reversion between 10-~ and l0'~ were selected for further analysis (Table 2) .
Table 2:
Name Phenotype Reversion Frequency TN~13 his- 1x10-Tt4r131 his- ixlo-Th~164 his' 1x10-Tt4rT9 h1s- 4xlo-' TMN12 his- 5x10'' T1113 his' 2 . SxlO-' Tt4r162 his- 8x10'' Tt~174 his' 2x10 ~
TI~178 his' 2xlo'' TMN4 S lys' 8x10'6 T1~T71 his- 2x10-6 TI4N82 Naa 2x10'6 Example 4: Conatructioa Caadidfa utilis geaaaric library.
of s The chromosomal DNA extractedfrom Candida utilis NRRL Y-1084 was partially the e:nxyme Sau3A and fragments digested with with sixes b etween 6 and 9 kb were isolated by zo electrophoresisin law point gel temperature agarose (LGT).
Theee fragments were ligated in the pUCl9 vector previously digested with HamHI and treated ~.rith alkaline phosphatase.
This ligation were transformed in Escherichia coli MC 1066 (F', D Lac x74, hsr, hsm, rpsl, galU, galK, trip C 9030F, 5 leuB, pyrF::tnS) strain. Aproximately 95% of recombinants were obtained in the genomic library.
Example 5: ZBOlation of the QRA3 gE~ne fram Candida utilis.
As a marker for transformation of the Candida utilis ura3 host, the URA3 gene from Candid~t utilis was isolated and 10 characterised. DNA fragments which contained the Candida utilis URA3 gene were isolated from a Candlda utilis pUCl9 genomic library by the ability to complement Escherichia coli pyrF mutation, taking into account that URA3 gene from Saccharomyces cerevisiae complements the pyrF mutation of E.
coli, using fortuitous promoter aci:ivity in Escherichia coli.
When this library was spread on uracil-deficient medium, 12 independent pyrF+ colonies were isolated. Two of these clones (AURA-2 and pURA-5) had the same 2.6-kb genomic Candida utilis insert DNA on pUCl9 using HindIII and EcoRI
restriction digestions. DNA from both plasmids transformed Escherichla coli MC1066 to Ura' at a high fx'equency. The map of one of the Candida utilis UFtA3 gene-pUCl9 recombinant plasmid (AURA-5) is shown in Figure 1. This plasmid was used for further complementation and sequence analysis.
Example 6: Demarcation sad sequence analysis of Candida utilia URA3 gene.
The plaemid pURAS was digested with several restriction enzymes. The fragments corresponding to the EcoRi digestions (1,9 kb), HincII (1,3 kb), SacI i;l,l kb) were subcloned in pBluescript SK (+) giving rise the plasmids pUREc-3, pURHinc-1 pURSac-4, respectively. Fragment corresponding to the plasmid pURSac-4 was not able to complement the pyrF mutation of Escher.ichia coli (Figure 2)_ The 1,9 kb EcoRI fragment of (pilRhc-3, Figure 3) containing the URA3 gene of Candida utilis arse completely double strand sequenced by the method of Sanger et. al. (1977, Proc. Natl.
Acad. Sci USA 74: 5463-5467).
With this end, the universal oligonucleotides of the series Ml3mp/pUC, ae well as internal o7.igonucleotides derived of the eequence were used. The complete sequence of 1179 by of the EcoRI fragment ie shown in Figure 4 (Seq. Id. No.: 1, 2).
This fragment contains an open reading frame of of 80o by (266 codons). The Candida uti3i:: URA3 gene codes for a protein with theoretical moleculaa~ mass of 29 436 Da. The nucleotide sequence flanking to the ATG initiation codon (GAAAATG) corresponds well with the: consent reported in yeast (A/YAA/YAATG), by Cigan y Donehue, l997 (Gene 59; 1-18).
The 3'-non translated region contains a putative polyadenilation site (TATAAAA, consensus AATAAAA) present in the 3' terminal region in most of the eukaryotic genes (Guo, Z y Sherman, F., 1995. Mol. Cell. f~iol. 15: 59A3-5990).
.>rxample 7: Complementation sui~lysis in Saccharomyees cerevissse.
With the objective to verify that the fragment cloned, correspond to the Candida utilis URA3 gene and not a DNA
fragment with supprea8or activity, the 2.8 kb Kpnl/Xbal fragment of the pURA5 plasmid was cloned in a pHR322 derivative vector (pHSARTR-3). The pBSARTR-3 vector posses an autonomous replicating sequence ~;ARS1) and the TRP1 gene selection marker both from t3acchaZ-omyces cez~evieiae.
Consequently, the plasmid pUT64 (figure 5) was obtained and used to transform the SaccharotnyceFr cerevisiae strain SEY2202 (ura3-52-, leu2-112) hie3) using the lithium acetate method, previously reported by Ito. et. al., 1983 (J. Bacteriol. l53:
163-168).
The transformants were obtained 4B hours after the transformation. The presence of t:he replicative plasmid was checked using both colony hybricLization and southern-blot experiments.
The frequency of transformation obtained (2-5 x l02 transf/mg) is in agreement with that of reported in the literature for other auxotrophic ttu3rkere from other yeast.
Consequently, it was demonstrated that the gene URA3 from Candida utilis is able to complement the ura3 mutation of Saccharomyces cerevieiae .
,.,;._ ,r~''. ..,< .., ~a Example 8: Transformation of fandida utilis CUT35 with the plasmids pURAS and pUCURA3 using the LiAc method.
_~:e ~_a3 mutant strain of Canc'ida util-s C:,'T 33, ~henc~ype and deocsvted with access'cn cumber CBS 100C83 at .el'l~raai~'Jurea',: VOCr ~C,h'_.ili'Ttej.Cll~'_:reS On OCtODer ! , 1Q~ , waS
'ransformed using the method cf 1 i thium acetate rapcr~ed by Tto et. al. (1983), and using the previously _sclated UR~3 g ene _ r .''.IiW.a.'l~i..Ja U L_' ~i S aS a ~ie l eC t'_on mar ker . ='='~~e VeC'~"r j ~~=~_.0 and ~'..'C.::W3 ) , used _._ t'-.e _r ans formatlcn S'~-S tem wer a deS'~gne~~ C.'', ~ r2Ci.l'v' 'integrate 1:'_CO t.~e hOIt101 OgcLlS lOC'.',..
.:f '_?2 (JdIlC,t'.~."'.a a ~_' ~=S ml.'.=an t S i.raln : y 1:.~,Iiu;~lOgOL:S r 2C
:i(L.~~:la r_Cn.
~'re plasmid ~;UC~~A3 was obtainec. by cloning the 1, ~ kb ~c.oRI
__ :gale-:t .._ t:'le ~c~l:_a ;.'~~_~S i:a~_? gei'le In L,'?e CQrreS~Oi::_ug ~_~e Cf the VeCt;,_ NL.T'~1~ , ~~l:~re b) . ~reV:'C',iS~y tile ~_~._..__,,r:ClaC~ ~:? '"~rOCe~.ure, COth (,._:.~S:LI~QS Were '_~2S~eu ~._ ?'~,~_ ~~rh,_c'_: is loc~_ed in the ' :rime of t.~:e s~_~c~'ara~
gene. T':e '~ i~eari~at_on cf the plasmids favors ~~e homologous ___CeCrat~~~,r! '~:'1 C_~.e G'e?'lOiTllC iCC'~.';S.
Tne ~_ar_sfor:~a_icn procedure was performed as ~reVvousl~;
descri'oed by I~c. 2s. ai, a~83 ~:~r lithium aceta~e procedure, exoe:~t ~~ha~. she concentration used for LiAc was 3~ mM in the present case. In order tc carry our this method, after ~:~:e ,~~'~,ony growth cn YPD plate, the selected s tra~_-~ is ~.,~1 ~;~ra.d with sharing in 5 m'~ of YPD liqui d medium at 30~C for a~No',.:L 8 hours, it is inoculated in i00 ml cf YPD liquid medium a_ a '~~'~' ~~
concen~~a_ior_ o. OD:: =0.003 and cultured with s~nak_ng ~~ 3~' for about l~ hears. After the cells have grown to logarit=.mlc phase (DO;,,_,=0. 6) , they are collected by centrifugation at 3000 rpm for 5 minutes. The cells were washed once with 3 m1 ,._ sterilised water. After, Lhe cells are suspended into 1 m1 ~_ J0 mu LiAC. Afterward they are incubated wit:: agitation at 30~C for 1 hour. Consequently 100 ~1 of cell s faere al iqucted in microfuge tube and S ~g of DhA was added. TL:en the mix ~a b ~~-~_Si DNT1 ~~:?C.l~lua'tC-C3C~~l:~i" J ~ :il~:=lCsJ.,_:Sa ..,~.-.vas a t . ' the cells/DNA treated wit: 0.7ml of 40=~ c~
?E
:nl~ of ~,_AC re vr~wa'Natedat 3C''C for 1 ncva____ __rwar:~
we . ~.
':eat s:~ocri .1~. i;.r .. mi::was app~~ie~~__ ware= :~at_..
at immediate~~y _.._ 30 secor_ds a~~ _~e t_~_e mix was _ in T
ce isi.~NA was was:.e~~ twicei:__ris 1C:~,M, _~ __ -~n~M ~ri _ "i_.a~ , .:~, s eIP"~Ve~, r!:e:":_ ~__-S _._ t::= ~ Cer~_~a::= r ___ _ _ suspended _.. __~ u'~ : = ~'r~.s i0 .-nM pri ~, DDTA _ m,:~: _ __ .. s;: as tri2 Zl::dl VCi'~a?e .
1~ m~c se~!eCtlO:: .._ t_'?e __.i?S~O~iTla:itS waS Carr'~e~=
__';W','~:'~~:T,LlI:l ITLed'~'ui~t ~'-aClC'~=':~ L:~~C11. i'::e :Tl~~'_~.~_.. sta~~_~- _._ :'!2S2 ~~ai:S=C~~?'La=':t_, i~.ia~ ~ JIl, Clle t0 t.'~e rileCaai iS:'il : -'~Jr;;,~~~ OQOllS
_ :taC~.rat~0=:. _':=~e t_ ,.mss=S'=:.",a't~OI? 'LreC['t'e'."':C;V
CCi~C_~~_niit_'? _:"':at .. _ r eCOr =ed _ J _ .~.~~.,,.._~.~ Oil.';,-;: eS ~~2~ e'v 1 S=.a.~ ailC ~
_.':c= __..._ ~or.:rer_=ior:a'_ _;ea~_ ~..._. ~_.te~~atiVe sector '~ _ %
_ _ s ; ~.a.~l=
_ 13_ ., Example 9: ~ Tranefosmation of Caacfida utjlis CQT'35 with the plasmids p~RAS and pUC0RA3 using electroporation method.
The ura3 mutant strain of Candida utilis CUT35 was transformed using the electroporation method reported by Kondo, K. et al., 1995, (J. Bacteriol. 177: 7l71-7177), using l0 the previously isolated tlR~~3 genes from Candida utilis as a selection marker. The vectors (p~lRAS and pUCURA3), used in the transformation system were designed to directly integrate into the homologous locus of the Candida utilis mutant strain by homologous recombination, The procedure used is based on tine treatment of the intact cell yeast with an electric field,. The following conditions were used: 0.7 kV (3,5 kV/cm) ae a pulse, a resistance of , 800 S2 and a capacitance of 25 NSF.
Previously to the transformation procedure, both plasmids 2o were digested with XhoI which is located in the 5' prime of the structural gene facilitating the homologous integration in the genomic locus.
The selection of the transformant:e was done in YNB minimal medium without uracil.
The frequency of transformation ufaing both pURAS and pUCURA3 depended of the plasmid concentration. A comparison of both methods (LiAc and electroporation) is shown in Table 3.
Table :3 Transformation frequency Vector DNA Concentration (# tranef./~g) (ug) LiAc Electroporation pUCURA-3 0.1 - 70-90 0.5 - 640 3.0 22 -pURA-5 0.1 - 40-50 0.5 - 670 3.0 21 -The mitotic stability of these tra:nsformante was high, due to the mechanism of integration.
The frequencies of transformation coincide with that of S reported for Saccharomyces cerevisise and other non-conventional yeast using integrative vectors.
In figure 7 is shown the outline of the possible integration events in the genome of Candid.n utilis as well as the Southern-blot of some traneformants.
Example 10: Zaolatlon of the HIS3 3~ene of Cand3da utilis.
The HISS gene from Candida utilis was isolated and characterized from the library previously described in the Example 4. DNA fragments, which contained the Candida utilis HISS gene, were isolated from a Candida utilis genomic library by the ability to complement the hisb463 mutation in the Escherichia coli KC8 (hsd, his13463, leuB6) pyrF::TnS Kmr, trp (983o (lact YA), stm, galu, gal), taking into account that HZS3 gene from Saccharomyces cerevis~ae complements the hisb463 mutation of Escherichia coli, using fortuitous promoter activity in Escherichia coli.
In order to isolate the HZS3 gene, 105 cells were spread on minimum medium (M9) supplemented with uracil, tryptophan and leucine. Plasmid DNA was extracted from colonies able to growth in this medium and consequently capable to complement the hisb463 mutation in the Escherichia coli KC8 mutant strain. The plasmid DNAs isolated were used to retransform the Escherichia coli KCe mutant strain. A11 the plasmid able to supplement the histidine requirement of the mutant strain were denominated pHCU. In order to confirm that the his' colonies contained the HISS gene of Candida utilis and not a 5 fragment of ADN with auppressor activity, two of the plasmids obtained from the his' transformanta (pRCU37 pHCU40) were subjected to a PCR reaction. Two degenerate oligonucleotides from two regions highly conserved in five IGPDasae sequences from yeast and fungi were used. '.Che oligonucleotide as well 10 as amino-acid sequences of the degenerate oligonuclcotides are shown in the figure 8.
Approximately a 50o-by PCR band corresponding to the coding sequence of the HIS3 gene from Candida utiZis was amplified.
The approximately 500 by PCR fragment, which was shown by 15 Southern blot to hybridize the Candida utilis genomic DNA, was cloned in T-Vector (pMOSBLUE, Amershan) and the predicted amino-acid translation of its sequence was shown to be highly identical to His3p from other yeast and fungi. The plasmid pHCU37 (Figure 9) was used for the determination of the entire sequence of the HIS3 gene from Candida utilis.
Example 11: Sequencing of the XI83 gene of Candjda util3s.
The HISS gene from Candida uti_Lis was completely double strand sequenced using the method of Sanger et. al. (1977) .
Oligonucleotides of the universal series Ml3mp/pUC were used.
Primers taken from the PCR fragment were used to initiate sequencing of the entire gene. A total 1190 by of the pHCU37 was sequenced. The entire sequence HISS from Candida utilis is shown in Figure 10 (Seq. Id. No.: 5, 6).
This fragment contains an open residing frame of 210 codons .
The Candida utilis HI53 gene code for a protein with theoretical molecular mass of z4 5.L8 Da.
Example I2; Isolation of the INV1 gene that cod.ifiea for the invertase of Candida utilis.
In order to isolate the INV1 gene that codifies for the enzyme invertase in Candida ucilis, it was taken advantage from the fact that the amino acid sequence of this enzyme presents regions highly preserved between different species, thus the sequences of p-fructofw=anosidase from yeasts were aligned. Two degenerated oligonu~~leotides used in said PCR
were designed according to the codons usage in Candida l0 utilis. The polypeptide sequences, as well as the degenerated vligonucleotides are shown in Figure 11.
PCR generated ~a band of 417pb, which was subcloned in the Vector-T (pMOBlue, Amereham), Said band was completely sequenced and the tranelation~ of said DNA fragment corroborated the presence of consensus regions and also a high homology among the enzymes invertases reported in the literature. This demonstrated that the fragment isolated belonged to the INV1 gene, which codifies for this enzyme in candida utilis. This fragment was used as probe for isolating INV1 gene from Candida uti3is.
After the search of the Candida u~til.is library, a total of 6 clones having the iPNl gene were isolated. Two of these clones were selected for their siae for sequencing (pCI-6 and PCI-12), using the oligonucleotides of the previous step for PCR. These oligonucleotides were used to start the complete sequence of the gene from both chains belonging to plasmid pCI-6.
Example 13: Sequencing of the INV;L gene of Caadida utilie.
A total of Z607 by of the clone: pCI-6 containing the INV1 gene codifying for the inverta,se of Candida uti.tis was completely sequenced by the method of Sanger et la., (1977), and for this end were used universal oligonucleotides belonging to the series Ml3mpfpUC, ae well as internal oligonucleotidea derived from t:he sequence. The complete sequence of the 2607 by fragment is shown in Figure 12 (Seq.
Id. No.: 5, 6). Said fragment contains an open reading frame of 1602 by (S34 codone). The IrfVl gene of Candida utilis codifies for a protein of theoretic molecular weight of 60 703 Da.
Considering that the invertase in Candida utilis is a periplasmic enzyme, it should hare in its N-terminal end a signal peptide. Analyzing the sequence up to 5'-end of the gene) two codons ATG are observed (ATG1 and ATGz in Figure 12) giving place to ORF codifying for proteins which differ only the size of their N-terminal ends. Applying the von Heijne's algorithm (1986, Nucl. Acids Res. 14: 4683-4690) to predict the restriction site for the peptidase signal of the mature protein derived from both ~4TG, it is reveled that the restriction sites for both casein are located between the residues S39 and s40 for ATGl and between residues S26 and S27 for ATGi. This gives place tc~ signal peptides of 39 and 26 amino acids respectively. Taking in to account the average size for signal sequences in yeast (estimated as about 20 residues) , it can be suggested that the start codon for the INVl gene is the second ATG.
Eleven potential sites of N-glycoeilation, according to the general rule N-X-T/S, were found in the asparagines of the positions 40, 88, 141, 187, 245, 277, 344, 348, 365, 373, 379 and 399 of the sequence of the mature protein.
The region 5'-non translated sho~Na two possible TATA boxes (consensus TATAA)) in the regions -18 to -14 and -212 to -208, and also various possible union sites for the repressor Migl (consensus SYGGRG).
BRIEF DESCRIPTION OF THE FIGURES:
Figure 1. Plasmid pURAS, obtair,,ed in the Candida utilis genomic library by complementation of the Escherichia coli MC1066 pyrF SaCCharomyces cere~isiae SEY 2202 ura3 mutations.
Figure 2. Restriction enzyme map, sequence strategy and complementation analysis of the URA3 gene from Candida util.is.
Figure 3. Plasmid pUREC3 obtained by cloning of the 1.9 kb EcoRI fragment of the plasmid pU12A.5 in the pBLUESCRIPT SK(+).
Figure 4. Amino acid sequence deduced from the DNA sequence of the URA3 gene, and the DNA sequence of the DNA encoding it.
Figure 5. Plasmid pUT64 obtained for the complementation experiment in the Saccharomyce,s cerevisfae ura3 mutant strain.
Figure 6. Plasmid pUCURA3 used in the Candida utilis transformation experiments.
z5 Figure 7. (A) Predicted arrangements of the vector DNA
integrated at the UFA3 locus by homologous recombination.
(B? DNA-blot hybridization of genomic DNA from some transformante.
Figure 8. DNA sequence of the primers used in the isolation of the HISS gene) and the deduced amino acid sequence encoding it.
Figure 9. Plasmid pHCU37, obtained in the Candida utilis genomic library by complementation of the Escherichia coli KC8 hiab463 mutation.
Figure 10. Amino acid sequence deduced from the DNA sequence of the HIS3 gene, and the DNA sequence of the DNA encoding it.
Figure 11. Amino acid sequence and corresponding DNA sequence of the oligonucleotides used in the PCR for the isolation of l0 the INVl gene of Candida utilis.
Figure 12: DNA sequence corre;Bponding to the fragment containing the INVl gene of Candid!a utilis.
SEQUENCE LISTING
(1) GENERAL
INFORtIATION:
(i) APPLICANT:
(A) NAME: CENTRO DE INOENIERIA GENETICA Y HIOTECNOLOGIA
(B) STREET: AVE. 31 ENTRH 15B 5' 190, CLTHANACAN, PLAYA.
(C) CITY: CItJDAD DE LA HABANA
10 (D) STATE: CIUDAD DE LA HAHANA
(E) COUNTRY: CUBA
(F) POSTAL CODE (ZIP): 12100 (G) TELEPHONE: 53 7 216013 (H) TELEFAX: 53 7 336008 (ii) TITLE OF INVENTION: TRANSFORMAT1:ON SYSTEM
IN CAUDIDA UTILIS.
(iii) NUMBER OF SEQUENCES: 6 2 (iv) COMPUTER READR8L8 FORM:
(A) MEDIUM TYPE: Floppy disk (a) COMPUTER: IHM PC compatible (C) OPERATINQ SYSTEM: PC-DOS/MS'-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUhfl3ER: A2/96 (B) FILING DATE: 03-OCT-1996 (2) INFORMATION
FOR
SEQ
ID
NO.
1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11'19 base pairs 3 (B) TYPE: nucleic acid S
(C) STRANDEDNESS: siagle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (i11) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
45(vi) ORIGINAL SOURCE:
(A) ORGANISM: Candida tltilia (B) STRAIN: NRRL Y-1084 (ix) FEATURE:
SO (A) NAME/KEY: mat_pcptide (B) LOCATION:1..1179 (D) OTHER iNfORMATION:/produet- "Enzyme orotidin-5-phosphate decarboxylaee "
/gene= "URA3"
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
GTTGAATTGG TCCATCCTTG CTACTTTTCC GCCTAGT'.CTC GATTCCGATT CTGATAGAGA 120 AGCCC:AGCTA TC~AATGGAAG AAATTTTTCA CTTTTOT1~TG TCCTTTTTTT CACGCTTCGT 1A0 TGCTTCGGAC AAAAAAATAG TOGAGGCACT C'GGTOGAL9GG AAGCTATCCT CGAGATGAAA 240 AATTTCAAGC TCATCTCATC GTCCAAGTGC3 GAGAGCAAGC TGAdGCTTCT GAAGAGOTTG 300 AGGAAAATGG TCACC'.ACGTT ATCOTACACA GAGAGO(1<:AT CGCACCCTTC GCCACTTGCT 360 AAGCGTCTGT TTTCGCTTAT GGAGTCCAAG AAGACGAi~CC TGTGTGCCAG TG?CGATGTT 4Z0 CGTACCAC:AG AGGAGTTGCT CAAGC?CGTT GATACGC7.'TG GTCCTTATAT CTGTCTGT?G 480 GAGCTTTCAA AAGAGC.'ACAA TTTCCTCATC TTTGAGCiF~CC GTAAGTTTGC TGATATCGGC 600 AACACCGTCA AGGC,'ACAGTA CGCCGOTGGT GCGTTCAAGA TTGCACAATG GGCAGACATC 660 ACCAACGCCC ACGGTGTCAC CGGTCGAGGT ATCGTCAFvGG GGTTCiAAGGA GGCTGCACAG 720 2 5 TTCGCTCACG GGACA2ATAC CGAGGAGACC OTGGAGA7.'TCi CCAAAACTGA TAAC'sG'ACTT'I
TGTATTGGAT TCATCGCACA GAGAGACATG GGTGGCACiAG AAGATGGGTT CGACTGGATC 900 ATCATGACAC CAGGCGTGGG ACTCGACGAT AAGGGCGF~CT CCCTGGGCCA ACAGTACAGA 960 ACTGTCGATG AGGTTGTCAG TGGTGGCTGT CJACATCA7.'CA TCGTTGGTAG AGGCTTGTTT 1020 GGAAAGGGAA GAGATCCAAC AGTGGAACGT GAGCGTTF~TA GAAAACiCAGG CTGGGATGCT 1080 TATCTCAAGA GATACTCAGC TCAATAAACG TTOAGCTC.'rG OCTTGTATAG GTTCACT"TGT 1140 (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: Q66 amino acida (B) TYPE: amino acid (C) STRANDEDNESS: eiagle 4 5 (D) TOPOLOGY: linear (ii) MOLECULE TYpE: proteia (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Car~dsdz utili9 5 5 (s) sTRAIN: NRRL Y-loe4 (ix) FEATURE:
(A) NAME/1CEY: Protein (8) LOCATION:1..266 (zi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Met Val Thr Thr Leu &er Tyr Thr Glu Arg AIa Ser Hie Pro Ser Pro Leu Ala Lye Arg Leu Phe Ser Leu Met Glu Ser Lys Lys Thr Aea Leu Cys Ala Ser Val Asp Val Arg Thr Thr Glu Glu Leu Leu Lys Leu Val 35 40 i5 Aap Thr Leu Gly Pro Tyr Ile Cys Leu Leu Lye Thr Hia Ile Acp Ile Ile Asp Asp Phe Ser Met Glu Ser Thr Val Ala pro Leu Leu Glu Leu 5er Lye Glu His Asn Phe Leu Ile Phe Glu Asp Arg Lys Phe Ala Aep 8$ 90 95 Ile Gly Aan Thr Val Lye Ala Gln Tyr Ala Gly Gly Ala Phe Lye Ile Ala Gln Trp Ala Asp Ile Thr Aen Ala His Gly vat Thr Gly Arg Gly Ile val Lye Gly Leu Lye Olu Ala Ala Gln Glu Thr Thr Aep Glu Pro Arg Gly Leu Leu Met Leu Ala Glu Leu &er Ser Lys Gly Ser Phe Ala His Gly Thr Tyr Thr Glu Glu Thr Val 01u Ile Ala Lys Thr Asp Lye Asp Phe Cys Ile Oly Phe Ile Ala Gln Arg Aep Met Gly Gly Arg Glu Asp Gly Phe Asp Trp Ile Ile Met Thr Pro Gly Val Gly Leu Asp Asp 195 200 2fl5 Lys Gly Asp Ser Leu Gly Oln Gln Tyz .Azg Thr val Asp Glu Val Val 4 5 Ser Gly Gly CYe Asp Ile Ile Ile Val Gly Arg aly Leu Phe Gly Lys Gly Arg Aap Pro Thr Val Glu Gly Glu .Arg Tyr Arg Lys Ala Gly Trp 2f5 250 Z55 Asp Ala Tyr Leu Lys Arg Tyr Ser Ala Gln (2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICSs (A) LENGTH: 1190 base pairs (8) TYPE: nucleic acid (c) sTRANDEDNESS: oingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iw) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISH: Candida utilts (a) STRAIN: NRRL~ Y-1084 (ix) FEATURE:
(A) NAME/I~Y: mat~eptide (H) LOCATION:1..1190 (D) OTHER INFORMATION:/product= "Enzyme imidazol-glycerol phosphate dehydratase ~
/gene= "HISS"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 3:
AAAAGAGTCG AAGAACRACA GTGCGCCRAA AAARAAAC'iC CGGACCGCAC ACGACTCATC 120 GCTCTCGGAA TATCCCTCGG AATGCGCCAC TTCCGGCiT~:~C GTGGCCATCG GAAGAGCGAA 180 GAO?CATCAC CATCGTACTT TAACGACTTA CTATTCTCAT TGAGTATTGA GAAGAAGGAT Q40 AGAGAAATGG CTGAACGAAC GGTGAAACCC CAGAGRAG.AG CTCTTGTGAA TCGTACAACA 300 AACGAAACGA AGATCCAGAT TTCCTTGAGT TTGGATGG'TG GATACGTAAC GGTTCCGGAG 360 TCAATCTTCA AGGATAAGAA GTACGACGAT GCTACTCA~AO TCACCTCTTC TCAGGTGATT 420 4 5 GGGTGGAGTT TGAT?GTGGA GTGTATTGGT GATTTGCACA TTGACGACCA CCACACCACC 540 GACGACGTTG GTATTGCGCT GGGAGACGCC GTCAAGGA~3G CCTTGGCATA TAGAGGTGTC 600 AAGAGATTTG GTAGCGGGTT TGCTCCATTG GACGAOGC'TC TGAGCAGAGC CGTTGTTGAT 660 TTGTCATGTG AGATGATTCC TCACTTCTTG GAGAGTTT'TG CCCAAGCAGC TCATATCACG 780 ATGCATGTTG ACTGTTTGAG AOGCTTCAAC GACCATCAi~A GAGCTGAATC CGCATTCAAG e40 GCCCTGGCRG TCGCCATTAA GGAATCCATC TCCAO?AACG GCACCAATGA TGTTCCCTCA 900 ACAAAGGGTG TTTTGTTCTA GATAGCAGTC TTTCTGTC'TC TCTATTTATT CGATAAATAA 96o CAAGGACTCT ACGACCACTG GTOOCTTTGA TATGATT'CCC T~3CCAGTACT TGTAACAGOT 1140 GCAACGTCAA TGGAAACOOC ACCGTTAGCC TTGATCiO'.CIG CACGGGTAGG 1190 (2) INPORMATZON FOR SEQ ID NO: 4:
(i) SfiQUENCE CHARACTERISTICS:
(A) LENGTH: 210 amino acids (H) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSfi: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Candida utilis 2 5 (H) STRAIN: NRRL Y-1084 (ix) FEATURE:
(A) NAME/KEY: PZOtein (H) LOCATION:1..210 (xi) SEQUENCE DESCRIPTION: 6EQ ID NO: 4:
Met Ala Glu Arg Thr Val Lys Pro Gln A.rg Arg Ala Leu Val Aen Arg 1 5 to is Thr Thr Asn Glu Thr Lya Ile Gln Ile Ser Leu Ser Leu Aep G1y Gly 4 0 Tyr Val Thr Val Pro Glu Ser Ile Phe Lys Asp Lys Lys Tyr Asp Asp Ala Thz Gln Val Thr Ser Ser Gln Val Ile Ser Ile Asn Thr Gly Val Gly Phe Leu Asp His Met Ile His Ala Leu Ala Lys His Gly Gly Trp Ser Leu Ile Val Glu Cys Ile Gly Asp Leu Hie Ile Asp Aep His Hie Thr Thr Glu Aep Val Gly xle Ala Leu Gly Aep Ala Val Lys Glu Ala Leu Ala Tyr Arg Gly Val Lys Arg Phe Gly Ser Gly Phe Ala Pro Leu 115 l20 125 Asp Glu Ala Leu Ser Arg Ala Val Val Asp Leu Ser Asn Arg Pro Phe Ala Val Val Glu Leu Gly Leu Lye Arg Glu Lys Ile Gly Aap Leu Ser 14S l50 155 160 Cys Glu MeL Ile Fro Hie Phe Leu Glu :Ser Phe Ala Gln Ala Ala His 5 165 :170 175 Ile Thr Het His Val Asp Cye Leu Arg G1y Phe Aen Asp Hie H1c Arg 10 Ala Glu Ser Ala Phe Lye Ala Leu Ala Val Ala Ile Lye Glu Ser Ile ser Ser zlo l2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARAC?ERISTICS:
2 0 (A) LENGTH: 26o7 bxae pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single lD) TOPOLOGY: linear 2 5 (ii) MOLfiCULE TYPE= DNA (genomic) (iii) HYPOTHETICAL: NO
(iV) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Candiaa utilic (H) STRAIN: NRRL Y-10B4 3 5 ( i x ) FEATURE :
(A) NAME/KEY: mat-peptide (H) LOCATION:1..2607 (D) OTHER INFORMATION=/product~ "Eazima invertaca (beta-fructofuranoeidaaa)"
4 0 /gcne= "INV1"
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: S:
ATTGGCAGCT CCGGAGCACA CTCAATTGGCi ACTAAAAGF,A GTAACATTTa TACTACAATG 120 AGTCGTATAG AGTCATGTAT AAGAAGAACA GCAAGAAAA,G AAAATATTGG TGCAGAATTC 180 AACAGCTTCT GAGATCGTAA GAACAGCCAA TCATTTACC:G GAATTCATTA TGATACCTAT 240 AGRAAGACAC AAATTGTTGG GTAAAACAAC AGAACATAC!C TGTATAGGGG TTTATACGAG 3D0 AATTTTCTTA GACGTCTCCC CCAGTGTCCG CC,'AAAGCAA,C TTACATGTGG AOTTTGAATT 360 Ci0.ATTTATCGACGTTATGCC TTGTCAGACC ATCGTCGTGA 540 AC?TTTCTAA ACCGGATAAA
CTCTCGCACGOATTATAACG TGCGTCTGTG ATATGCAC'CCCCCCGTGGAG600 CGGRAAAAF.C
AAGTGAAGCGGCCACCTGTG GAGCAGRAAT TTCGATCGi~CCAAATGGTTT660 OTTTCAAGTT
CCTGTTGTCAAAGGOCTTGA GATTTACCAC TTGACCAT'CTTTCGGAGAGC720 GTGCTCAGAA
CAGGGATGTC
GATGCCTCAGAGGACCAAGA AGACATCAAG AGTCTt:ACGATTTAGTTGAT840 TGAACACTA(i TGGGGTGGAT
AATGGTCTCT?CTACGATTC ATCTGAATCT ACTTACCA'CGATACAACCCA960 TGTACTACCA
AACGATACGATTTGGGGATT GCCTCTATAT TGGGGAt:Jt.'.CGTGATTTGTTA1020 CCACCTCTGA
ACGTGGGACCACCATGCGCC TOCAATTGGA CCTGAGAA'CGTATTTACTCT108O
ATGATGAGGG
GGATCTATAGTCATAGACTA CGATAATACC TCAGGGT"Tt~2AACAAGACCA1140 TTGACOA'rTC
GAACAGAGAATCGTTGCCAT TTATACCAAT AACTTACCi~GGCAAGACATT1200 ATGTCGAGAC
2 GCCTATTCCACGGACGGTGG TTATACTTTC GAAAAGTA'CGAGTTATAGAC1260 GTCiaATTCGACCCAATTTAG GGATCCGAAG GTGATTTGL3TTGAACAATGG1320 ATGAGGAAAC
GTCATGACTGTGGCRAAGAG TCi4AGAGTAC AAGATCCAc3ATGACAATTTG1380 TTTACACCTC
AAAGACTGGAGTTTGGCCTC GAATTTCTCA ACCAAGGG',CTTCAGTATGAA1440 ATOTTGGTTA
TGTCt:AGGTCTATTCGAAGC CACTATTGAA AACCCAAAGA AGAGAAGAAA1500 GTGGTGACCC
3 TGGGTTATGGTCTTAGCAAT CAATCCAGGC TCACCTCT'CGAAATGAATAC1560 TTTGTTGGTGATTTCAACGG TACTGAATTC ATTCCAGA'CGAAGATTTATG1620 ATGACGCTAC
ATGCACCGGA
AGG?TCCGGA
TA?AGAAGCTCCATGTCATC AATCAGAGAG TACACTCTt3ATACGAATCCA1800 GATATOTCAG
GAATCTGAACAGTTGATCCT TTGTCAAAAA CCATTCTT'CGAQACTTGAAG1860 TGAACGAGAC
GTGGTTGAAGAGTACAAGGT TTCAAACAG? TCTTTQACCG 0?TTGGAAG?1920 TGGACCACAC
AGCTTTGCAAACTCCAACAC CACTGGACTG TTGGATTTt:a1CACGGTTAAC1980 ACATGACTTT
GGTACAACTGACGTTACGCA GAAOGACTCC GTCACCTT'CCiCAAATCTAAC2040 AGCTCAGAAT
ACGAGCRATT
TCCAGGAGAG
TACGTTCAGCCTCTCACAAT CACCGAATCT GGTGATRAa0.CCTACGGATTG2220 AGTACCAGCT
GT'TGATAACAACATCCTTGA GTTGTACTTC AACGACGG~:~(3CACAAACACC2 TTGACGAATA
AGCTATATAA GGGATCACGT GGTCTAGCCA CCCCAGTC'.CACAAACCGCCA2460 AAAOCTTCAG
CTATATAAAC AGACAGGTTT GTCACTTTTC AACAAAAGiA CTTTTACCCT2520 ATATCTTCTT
TCAGAGTAGT TTGTACGAGT GCTTTTTTCA ATTATATA'.CAAGCTGCCTTT25A0 CAACAACGTG
(2) INFORhIATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERI&TIC9:
(A) LENGTH: 533 amino acids (B) TYPE: amino acid (C) STRANDEDNES6: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTF~TICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Candida utilis 3 (H) STRAIN: NRRL Y-10B4 (ix) FEATURE:
(A) NAME/KEY: Protein (H) LOCATION:1..533 (xi) SEQDENCE DESCRIPTION: SEQ ID N0: 6:
Met 5er Leu Thr Lye Aap Ala Ser Glu Asp Gln Glu Asp Ile Lya Ser 1 5 7.0 15 Leu Thr Met Aan Thr Ser Leu Val Asp :~eX Ser Ile Tyr Arg pro Leu Val His Leu Thr Pro Pro Val Gly Trp rtet Asn Asp Pro Asn Gly Leu Phe Tyr Asp Ser Ser Glu her Thr Tyr Ftis Val Tyr Tyr Gln Tyr Asn ?ro Asn Aap Thr Ile Trp Gly Leu Pro Leu Tyr Trp Gly Hia Ala Thr Ser Aep Aap Leu Leu Thr Trp Asp His Ftis Ala Pro Ala Ile Gly Pro 85 .'~0 95 Glu Asn Asp Asp Glu Gly Ile Tyr Ser cily Ser Ile Val Ile Asp Tyr 1o0 1o5 110 Asp Asn Thr 6ez Gly Phe Phe Aep Asp Ser Thr Arg Pro Glu Gln Arg Ile Val Ala Ile Tyr Thr Aen Aen Leu FaroThr Gln Asp Val Glu Asp Ile Ala Tyr Ser Thr Asp Gly Gly Tyr 7~hrTyr Glu Phe Glu Lys Asn 14S l50 155 160 Asn Pro Val Ile Asp Val Asn Ser Tht CflnPro Lys Phe Arg Asp Val 165 1.70 175 Ile Trp Tyr Glu Glu Thr Glu Gln Trp Val Ala Lye Met Thr Val Ser Gln Glu Tyr Lye Ile Gln Ile Tyr Thr f~erLys Asp Asp Asn Leu Trp Set Leu Ala Ser Asn Phe 5er Thr Lys GflyTyr Gln Tyz Val Gly Tyr 21o 2l5 zzo Glu Cys Pro Gly Leu Phe Glu Ala Thr 1:1eLye Ser Glu Aan Pro Gly 2 Asp Pro Glu Lye Lys Trp Val Met Val heu Pro Gly 5 Ala Ile Asn Ser Z45 i'.50 2S5 Pro Leu Gly Gly Ser Ile Asn Glu Tyr F~hePhe Aan val Gly Asp Gly 260 265 27o Thr Glu Phe Ile Pro Aep Asp Rsp Ala Thr Asp Thr Arg Phe Met Gly Lys Aep Phe Ty= Ala Phe Gln Ala Phe F~heGlu A9n Asn Ala Pro Arg Ser Ile Gly Val Ala Trp her 8er Asn Trp Asn Gln Gln Tyr Ser Val Pro Asp Pro Asp 01y Tyr Arg Set Ser Met Arg Glu Ser Ser Ile Tyr Thr Leu Arg Tyr Val Ser Thr Aen Pro GlluLeu Ile Ser Glu Glri Leu C'ys Glri Lye Pro Phe Phe Val Asn Glu Val Val 1'hr Aap Leu Lys Glu Glu Tyr Lye Val Ser Aen Ser Ser Leu Z'hrThr Phe Val Asp His Gly Ser Ser Phe Ala Asa Ser Asn Thr Thr LilyPhe Jean Leu Leu Aep Met Thr Phi Thr Val Asri Gly Thi' Thr Aep Aep Ser Val Thr Gln Lys val Thr Phe Glu Leu Arg Ile Lys Ser Asn G~lnAla ile Ser Aap Glu Ala Leu Gly Tyr Asp Tyr Asn Asn Glu Gln )?he Tyr Ile Asn Arg Ala Thr Glu Ser Tyr Phe Gln Arg Thr Asn Gln I?he Phe Gln Glu Azg Trp Ser Thz Tyz Val Gln Pro Leu Thr Ile Thr (~lu Ser Gly Aep Lys Gln 2~r Gln heu Tyr Gly Leu Val Asp Aen Aen :Cle Leu Glu Leu Tyr Phe Asn 4B5 !190 495 Aep Gly Ala Phe Thr Ser Thr Asn Thr 1?he Phe Leu Glu Lys Gly Lys Pro Ser AHn Val Aep Ile Val Aln Ser E:er Ser Lys Glu Ala Tyz His Arg Gly Pro Ala Aep
Tachnieal Sector.
The present invention is related ~CO the field of the genetic engineering and biotechnology, and in particular with the development of a system host-vector for the genetic transformation of the yeast Candicfa utilis which permits the expression and secretion of hete:rologous proteins in this yeast, which can be further used with several purposes.
Prior Art.
to The genetic engineering and biotechnology have opened an unprecedented goal in the production of many interest proteins with medical, nutritious or industrial purpose, which report excellent benefits.
The bacteria Escherichia coli has been the moat employed microorganism with these purpoeee~ by diverse biotechnology companies, due to the knowledge of their genetics) its easy manipulation and their systems of cultivation at high densities.
however, the hopes of the production of proteins of inrerest in this microorganism are affected for diverse factors, First of a11 the pirogenic and toxic compounds in the cell wall of Escherichia coli have provoked regulations limiting its use when the products obtained are intended to be used as medicaments or food in humane. In addition, the proteins that are over-expressed in Escherichia noli generally appear in an insoluble form that could not be secreted, On the other hand, the mechanisms of transcription, translation and postranslation modifications differ from the eukaryotic systems, resulting in recombinant proteins that in a certain way differ from that of the natura7_ sources.
The possibility of producing 'heterologous proteins in eukaryotic systems, such as the ye~iSL, has some advantages in relation to the prokaryotic systems. Among these, they could mention the capacity to grow to k~.igh cellular densities and the possibility of adapting their cultivation to continuous systems _ Also the yeast are able to secrete proteins to the culture medium in considerable bigger amounts, in comparison with Eacherichia coli, also the c(rowth medium used for the growth of yeast are more economic ~~han those used in bacteria (Lemoine, Y., 1988. Heterologous expression in yeast. 8th International Biotechnology Symposium, Paris, July 17-22).
Also, these systems can carry out other postraductional modifications as it is the case of the glycosylation, which is absent in the bacterial systems (Fiers) W., 1988, Engineering Maximal Expression of Heterologous Gene in Microorganism. 8th International Biotechnology symposium, Paris, July 17-221. In addition, these systems generally have certain preference for the same codon use that the higher eukaryotic systems (Kigsman, S.M. et al., 1990. Heterologous Gene Expression in Saccharomyces cerevisiae, Biotechnology &
Genetic Engineering Reviews, 3, Ed. G.E. Russell).
A11 this has led to the development and dissemination of new eukaryotic transformation systerns and with particular interest in yeast) firstly described for species of the Saccharomyces genus, with special emphasis in Saccharomyces cerevisiae. However, the expression of proteins in Saccharomyces has confronted problems with the expression levels obtained using their homologous promoters as well as the hyperglycosylation observed in the proteins secreted to the medium. That is the main reasons that in the last years it has been intensified the search of non-conventional yeast for their use in the expression of heterologous proteins.
3o With the development of transformation system in other non-Saccharomyces yeast, like Hanse~nula polymorpha, Pichia pastoris, and yeast of the Kluyveromyces genus (Sudbery, P., 1994. Yeast 10; 1707-17z6) has permitted a quick advance in the knowledge and development of these systems, as well as increased the number of foreign proteins expressed with vaccination, diagnoses and industrial purposes in these system.
Also inside the genus Candida E~everal transformation and expression systems have been reported, including Candida tropicalis, Candida boidiini, c'andida glabrata, Candida parapsilosis, Candida maltosa and Candida albicans all with a marked medical interest, because many of these species are the causing of opportunists illnesses in humans.
Candida utilie, in the Candida genus, has special interest due to its particular characteristics. First of a11, Candida utilis uses a great variety of inexpensive carbon sources such as xylose, sucrose and maltose among other. Another interesting feature is that it is possible produce efficiently a great amount of cells in continuous cultures.
Also Candida utilis, as well as Saccharomyces cerevisiae and Kluyveromyces lactie, have been authorized for the FDA (Food and Drug Administration) like s~~fe sources in foodstuff.
Resides) Candida utilis has been used in the industry for the production of L-glutamina, ~etil acetate and invertase among other products.
A preliminary system for a transformation system in Candida utilis has been described by 130, I. et. al., 1984, (Biotechnology and Bioengineering Symp. 14: 295-301). This report is incomplete because tl;~e presence of the drug resistance marker and direct evidence of the transformation process are not disclosed. Recently, a novel strategy concerning a transformation syete:m for Candida utilis has been reported by Kondo, K. et al . , 1995, (.T. Bacteriol . 177 7171-7177). They obtained cycloheximide (CYH) resistant transformants by using a marker gene containing a mutated form of the ribosomal protein L41, which conferred resistance, and also used ribosomal DNA (rDNA) fragment as a multicopy target for plaemid integration because the marker needs to be present in multiple copies for selection of CHY-resistant transformants.
Many attempts has been done to use Cazidida utilis as host for heterologoue gene expression, nevE:rtheless, a transformation procedure in Candida utilis using auxotrophyc mutants has not been developed up to now.
If it is taken into account the knowledge obtained in the to industrial exploitation of Candids~ utilis and the novelty of its genetics, it could be ~=onsidered an attractive microorganism for its commercial utilization as expression system of heterologous proteins.
Disclosure of the Invention.
The object of the present invention has been to provide a transformation system useful to ex;presa heterologous proteins in the yeast Candida ucilis, based on the obtainment of axotrophyc mutants of this specie ~~s well as m the isolation of different genes from a genomic library which complement said auxotrophies.
The transformation process described herein provides means to introduce DNA fragments or sequenc~ae into Candida util.is host cells and allows Candida utilis to be used as a host system for gene expression and protein prnduction_ Furthermore transformed yeast ce7_ls can be identified and selected by the methods describe in the present invention.
Novel strains of Candida utilis, vectors and subclones are provided. Novel yeast strains are used as hosts for introduction of recombinant DNA fragments.
The invention further relates to stable transformation and maintenance of DNA in host cells, where the marker is homologous integrated in the genome of the yeast.
S
Concretely the present invention consists in a transformation system in the yeast Candida utilis, which uses as hosts new auxotrophyc mutants isolated from the strain NRRL Y-1o84 of said yeast. These mutants are defective in the enzyme orotidin-5' phosphate decarboxylase of the biosynthetic pathway of the uracil or in the biosynthetic pathway of the hietidine, and were obtained by classical mutagenesis using both UV and NTG as mutagenic agents known from the prior art (Sherman, F. et al., 1986_ Laboratory course: Manual for methods in yeast genetics. Cold Spring Harbor Laboratory Press, NY). These mutants present a high stability (frequency of reversion approximately of 1o'a) and can be efficiently transformed with the procedure described in this invention.
In addition, it was isolated as selection markers for the mutants of Candida utilis the gene URA3, encoding for the orotidin 5'-phosphate decarboxylaeEa enzyme and HISS encoding for the Imidazol-glycerol-phosphate dehydratase enzyme which were isolated from a gene library of Candida utilis in PUC19 and identified by complementation of the pyrF and hisb463 mutations respectively in the stra~Ln Escherichia coli MC1o66.
Similarly the mutation ura3 of Saccharomyces cerevisiae strain SEY 2202 was used to identify this gene came from C.
utilis, The complete sequence of 'these genes was determined and the predicted amino-acid sequences show high similarities with that of the same gene from other yeast and fungi.
The vectors used in the transformation system were the plasmids pURAS and pUREC3 which comprise the URA3 gene, capable of being integrated into the homologous locus of the Candida utilis mutant host by homo7.ogous recombination.
The present invention also provides a set of plasmids based on those described formerly, which are used for the transformation of the mutants isolated from C. utilis in order to obtain heterologous proteins.
The transformation system of the present invention uses as hosts new auxotrophyc mutants obtained from the strain NRRL
Y-1084 of Candida ut~l.is, which are defective mainly in the uracil and histidine pathways, and among them were selected for their characteristics the mutant CUT-35 (ura') and the mutant TNll~1-3 (his') .
EXA?~LES
Example 1: Mutsgenesia of Caadida t~tilie To develop a transformation system in a microorganism they are generally required three elements:
(1) a marker for the selection o.E the tranaformants, that could be an auxotrophyc or a dominant marker, (2) a mutant or appropriate host for this selection and (3) a method to reproducible introduce the extranuclear DNA
in the host in an efficient form.
In order to achieving the second objective, it was carried out a classical mutagenesis in t:he yeast Candida utilis.
Cultures of the of the selected yeast strain (NRRL Y-1084) were inoculated in 100 ml of YPG medium (Yeast extract 1%) 2o peptone 2%, glucose 2%) and they were incubated in a shaker at 30~C for l0-20 hours. 50 ml of the culture was centrifuged to 3000 rpm for 5 minutes. Later the cells were washed 2 times with citrate buffer 0.1M' (pH 5.5) sterile and resuspended in 50 ml of the same buffer. After, 10 m1 of this suspension was incubated with a solution of NTG to a final concentration of 50 mg/ml. The su=;pension was incubated for minutes at 30~C in repose.
The NTG was removed of the suspension washing 2 times with distilled water. The cells were resuspended in 50 ml of YPG
30 and then they were transferred to an erlenmeyer with 100 ml of YPG. This culture of mutant cells was incubated at 30~C
for 48 hours.
Eltrichment with nyatatin Approximately 5 ml of the 48 hours YPG expressed culture was used to inoculate 100 ml of minimum medium. The minimum medium (YNB, Yeast Nitrogen Base) used for the enrichment Kith the antibiotic was not supplement with the metabolic produced by the biosynthetic via in which the defect is looked for. For example, for the isolation of auxotrophics mutants for uracil, it is not added to the medium.
The incubation was continued until the optical density (oD) of the culture reached 20 to 30% of the initial OD. When the culture reached the desire OD, th.e cellular suspension was treated with 25 units/ml a solution of nyetatin. The solution with the antibiotic was incubated at 30~C for 30 minutes without agitation, The nystatin waos eliminated of the medium washing the cellular suspension wi~~h distilled water 2 times and later the cells were resuspende:d in an appropriate volume in order to obtain 150 to 200 colonies per plate, Screening and selection.
The plates containing the mutageni.zed colonies according to the example 1, were plated in YNB mediums with and without between uracil. The colonies that ~iid not grow in absence of uracil were taken for further analysis.
Specifically in order to identify the presence ura3 or ura5 mutants, the cells were grown in presence of 5-fluorotic acid (SFOA). The resistant colonies were selected as ura3 or ura5 like mutants.
Example 2: Isolation of ura3 mutants.
After the nystatin enrichment the culture was washed twice with distilled water and plate directly on YNB plates containing 0.75 ~g/ml of 5-FOA (5-fluoro-orotic acid, Fluka) and 40 ~g/ml of uracil , The plates were incubated four days and the colonies that grew were analyzed in order to check the ura- phenotype. From the 4x10 viable cells, after the nystatin enrichment) 79 colonies showed resistance to the 5-r FOA. These colonies could be ura3, uraS, or simply resistant to the 5-FOA. To confirm the uracil auxotrophy the supposed mutants were plated in YPG medium incubated 48 hours to 30~C
and replicated in YN$ plates with a.nd without uracil. A total of 67 colonies were unable to grow in YNB without uracil, shown a ura' phenotype.
The frequency of reversion of all these mutants were determined standing out a group of 23 mutants by presenting a frequency of reversion in the order of 10-~, what confers them certain stability to be used as a host for transformation system.
The orotidin 5'-monophosphate decarboxylase (ODCase) activity of a11 the uracil auxotrophyc mutants was determined by the method of Yoshimoto et. al., 197A (Methods Enzymol. 51: 74-79), as well as it was determined their growth conditions.
These results are shown in Table 1.
Table 1. Summary of the characteristics of more significant ura3 mutants.
Name Reversion OMF>DCase Growth Frequency Activity CUT35 c SxlO-' - +++
CUT43 c 1x10-' - +++
CUT61 < 1x10'8 - +++
CUT65 c 1x10-~ - ++
CUT70 < 1x10'~ - +
CUT86 < 7x10-' - +++
CUT93 1x10-~ - +++
CUT166 6x10-~ - +++
Example 3: Isolation of other mutants different of the phenotype ura- .
With the objective of having a vari~Cty of auxotrophyc mutants different than uracil, the cellular suspension obtained according to the nystatin enrichment were plated in YPG and incubated at 30~C for 50 hours. Later the colonies contained in the YPG plates were replicated on plates containing YNB
medium arid incubated at 30~C for 48 hours. The colonies unable to grow in the YNB plates were taken for further analysis.
Around 2411 colonies were screened and consequently was obtained a 2% of appearance of auxotrophyc mutants. These mutants were checked using the Holliday and the Finchan tests. It was obtained 90~ of his mutants, 2% responded to to the phenotype lys', 1% to the p.'henotype leu-, 1% to the phenotype met-, 1% to the phenotype ads- and the 5% did not show a simple auxotrophyc phenotype (Naa).
The mutants having frequency of reversion between 10-~ and l0'~ were selected for further analysis (Table 2) .
Table 2:
Name Phenotype Reversion Frequency TN~13 his- 1x10-Tt4r131 his- ixlo-Th~164 his' 1x10-Tt4rT9 h1s- 4xlo-' TMN12 his- 5x10'' T1113 his' 2 . SxlO-' Tt4r162 his- 8x10'' Tt~174 his' 2x10 ~
TI~178 his' 2xlo'' TMN4 S lys' 8x10'6 T1~T71 his- 2x10-6 TI4N82 Naa 2x10'6 Example 4: Conatructioa Caadidfa utilis geaaaric library.
of s The chromosomal DNA extractedfrom Candida utilis NRRL Y-1084 was partially the e:nxyme Sau3A and fragments digested with with sixes b etween 6 and 9 kb were isolated by zo electrophoresisin law point gel temperature agarose (LGT).
Theee fragments were ligated in the pUCl9 vector previously digested with HamHI and treated ~.rith alkaline phosphatase.
This ligation were transformed in Escherichia coli MC 1066 (F', D Lac x74, hsr, hsm, rpsl, galU, galK, trip C 9030F, 5 leuB, pyrF::tnS) strain. Aproximately 95% of recombinants were obtained in the genomic library.
Example 5: ZBOlation of the QRA3 gE~ne fram Candida utilis.
As a marker for transformation of the Candida utilis ura3 host, the URA3 gene from Candid~t utilis was isolated and 10 characterised. DNA fragments which contained the Candida utilis URA3 gene were isolated from a Candlda utilis pUCl9 genomic library by the ability to complement Escherichia coli pyrF mutation, taking into account that URA3 gene from Saccharomyces cerevisiae complements the pyrF mutation of E.
coli, using fortuitous promoter aci:ivity in Escherichia coli.
When this library was spread on uracil-deficient medium, 12 independent pyrF+ colonies were isolated. Two of these clones (AURA-2 and pURA-5) had the same 2.6-kb genomic Candida utilis insert DNA on pUCl9 using HindIII and EcoRI
restriction digestions. DNA from both plasmids transformed Escherichla coli MC1066 to Ura' at a high fx'equency. The map of one of the Candida utilis UFtA3 gene-pUCl9 recombinant plasmid (AURA-5) is shown in Figure 1. This plasmid was used for further complementation and sequence analysis.
Example 6: Demarcation sad sequence analysis of Candida utilia URA3 gene.
The plaemid pURAS was digested with several restriction enzymes. The fragments corresponding to the EcoRi digestions (1,9 kb), HincII (1,3 kb), SacI i;l,l kb) were subcloned in pBluescript SK (+) giving rise the plasmids pUREc-3, pURHinc-1 pURSac-4, respectively. Fragment corresponding to the plasmid pURSac-4 was not able to complement the pyrF mutation of Escher.ichia coli (Figure 2)_ The 1,9 kb EcoRI fragment of (pilRhc-3, Figure 3) containing the URA3 gene of Candida utilis arse completely double strand sequenced by the method of Sanger et. al. (1977, Proc. Natl.
Acad. Sci USA 74: 5463-5467).
With this end, the universal oligonucleotides of the series Ml3mp/pUC, ae well as internal o7.igonucleotides derived of the eequence were used. The complete sequence of 1179 by of the EcoRI fragment ie shown in Figure 4 (Seq. Id. No.: 1, 2).
This fragment contains an open reading frame of of 80o by (266 codons). The Candida uti3i:: URA3 gene codes for a protein with theoretical moleculaa~ mass of 29 436 Da. The nucleotide sequence flanking to the ATG initiation codon (GAAAATG) corresponds well with the: consent reported in yeast (A/YAA/YAATG), by Cigan y Donehue, l997 (Gene 59; 1-18).
The 3'-non translated region contains a putative polyadenilation site (TATAAAA, consensus AATAAAA) present in the 3' terminal region in most of the eukaryotic genes (Guo, Z y Sherman, F., 1995. Mol. Cell. f~iol. 15: 59A3-5990).
.>rxample 7: Complementation sui~lysis in Saccharomyees cerevissse.
With the objective to verify that the fragment cloned, correspond to the Candida utilis URA3 gene and not a DNA
fragment with supprea8or activity, the 2.8 kb Kpnl/Xbal fragment of the pURA5 plasmid was cloned in a pHR322 derivative vector (pHSARTR-3). The pBSARTR-3 vector posses an autonomous replicating sequence ~;ARS1) and the TRP1 gene selection marker both from t3acchaZ-omyces cez~evieiae.
Consequently, the plasmid pUT64 (figure 5) was obtained and used to transform the SaccharotnyceFr cerevisiae strain SEY2202 (ura3-52-, leu2-112) hie3) using the lithium acetate method, previously reported by Ito. et. al., 1983 (J. Bacteriol. l53:
163-168).
The transformants were obtained 4B hours after the transformation. The presence of t:he replicative plasmid was checked using both colony hybricLization and southern-blot experiments.
The frequency of transformation obtained (2-5 x l02 transf/mg) is in agreement with that of reported in the literature for other auxotrophic ttu3rkere from other yeast.
Consequently, it was demonstrated that the gene URA3 from Candida utilis is able to complement the ura3 mutation of Saccharomyces cerevieiae .
,.,;._ ,r~''. ..,< .., ~a Example 8: Transformation of fandida utilis CUT35 with the plasmids pURAS and pUCURA3 using the LiAc method.
_~:e ~_a3 mutant strain of Canc'ida util-s C:,'T 33, ~henc~ype and deocsvted with access'cn cumber CBS 100C83 at .el'l~raai~'Jurea',: VOCr ~C,h'_.ili'Ttej.Cll~'_:reS On OCtODer ! , 1Q~ , waS
'ransformed using the method cf 1 i thium acetate rapcr~ed by Tto et. al. (1983), and using the previously _sclated UR~3 g ene _ r .''.IiW.a.'l~i..Ja U L_' ~i S aS a ~ie l eC t'_on mar ker . ='='~~e VeC'~"r j ~~=~_.0 and ~'..'C.::W3 ) , used _._ t'-.e _r ans formatlcn S'~-S tem wer a deS'~gne~~ C.'', ~ r2Ci.l'v' 'integrate 1:'_CO t.~e hOIt101 OgcLlS lOC'.',..
.:f '_?2 (JdIlC,t'.~."'.a a ~_' ~=S ml.'.=an t S i.raln : y 1:.~,Iiu;~lOgOL:S r 2C
:i(L.~~:la r_Cn.
~'re plasmid ~;UC~~A3 was obtainec. by cloning the 1, ~ kb ~c.oRI
__ :gale-:t .._ t:'le ~c~l:_a ;.'~~_~S i:a~_? gei'le In L,'?e CQrreS~Oi::_ug ~_~e Cf the VeCt;,_ NL.T'~1~ , ~~l:~re b) . ~reV:'C',iS~y tile ~_~._..__,,r:ClaC~ ~:? '"~rOCe~.ure, COth (,._:.~S:LI~QS Were '_~2S~eu ~._ ?'~,~_ ~~rh,_c'_: is loc~_ed in the ' :rime of t.~:e s~_~c~'ara~
gene. T':e '~ i~eari~at_on cf the plasmids favors ~~e homologous ___CeCrat~~~,r! '~:'1 C_~.e G'e?'lOiTllC iCC'~.';S.
Tne ~_ar_sfor:~a_icn procedure was performed as ~reVvousl~;
descri'oed by I~c. 2s. ai, a~83 ~:~r lithium aceta~e procedure, exoe:~t ~~ha~. she concentration used for LiAc was 3~ mM in the present case. In order tc carry our this method, after ~:~:e ,~~'~,ony growth cn YPD plate, the selected s tra~_-~ is ~.,~1 ~;~ra.d with sharing in 5 m'~ of YPD liqui d medium at 30~C for a~No',.:L 8 hours, it is inoculated in i00 ml cf YPD liquid medium a_ a '~~'~' ~~
concen~~a_ior_ o. OD:: =0.003 and cultured with s~nak_ng ~~ 3~' for about l~ hears. After the cells have grown to logarit=.mlc phase (DO;,,_,=0. 6) , they are collected by centrifugation at 3000 rpm for 5 minutes. The cells were washed once with 3 m1 ,._ sterilised water. After, Lhe cells are suspended into 1 m1 ~_ J0 mu LiAC. Afterward they are incubated wit:: agitation at 30~C for 1 hour. Consequently 100 ~1 of cell s faere al iqucted in microfuge tube and S ~g of DhA was added. TL:en the mix ~a b ~~-~_Si DNT1 ~~:?C.l~lua'tC-C3C~~l:~i" J ~ :il~:=lCsJ.,_:Sa ..,~.-.vas a t . ' the cells/DNA treated wit: 0.7ml of 40=~ c~
?E
:nl~ of ~,_AC re vr~wa'Natedat 3C''C for 1 ncva____ __rwar:~
we . ~.
':eat s:~ocri .1~. i;.r .. mi::was app~~ie~~__ ware= :~at_..
at immediate~~y _.._ 30 secor_ds a~~ _~e t_~_e mix was _ in T
ce isi.~NA was was:.e~~ twicei:__ris 1C:~,M, _~ __ -~n~M ~ri _ "i_.a~ , .:~, s eIP"~Ve~, r!:e:":_ ~__-S _._ t::= ~ Cer~_~a::= r ___ _ _ suspended _.. __~ u'~ : = ~'r~.s i0 .-nM pri ~, DDTA _ m,:~: _ __ .. s;: as tri2 Zl::dl VCi'~a?e .
1~ m~c se~!eCtlO:: .._ t_'?e __.i?S~O~iTla:itS waS Carr'~e~=
__';W','~:'~~:T,LlI:l ITLed'~'ui~t ~'-aClC'~=':~ L:~~C11. i'::e :Tl~~'_~.~_.. sta~~_~- _._ :'!2S2 ~~ai:S=C~~?'La=':t_, i~.ia~ ~ JIl, Clle t0 t.'~e rileCaai iS:'il : -'~Jr;;,~~~ OQOllS
_ :taC~.rat~0=:. _':=~e t_ ,.mss=S'=:.",a't~OI? 'LreC['t'e'."':C;V
CCi~C_~~_niit_'? _:"':at .. _ r eCOr =ed _ J _ .~.~~.,,.._~.~ Oil.';,-;: eS ~~2~ e'v 1 S=.a.~ ailC ~
_.':c= __..._ ~or.:rer_=ior:a'_ _;ea~_ ~..._. ~_.te~~atiVe sector '~ _ %
_ _ s ; ~.a.~l=
_ 13_ ., Example 9: ~ Tranefosmation of Caacfida utjlis CQT'35 with the plasmids p~RAS and pUC0RA3 using electroporation method.
The ura3 mutant strain of Candida utilis CUT35 was transformed using the electroporation method reported by Kondo, K. et al., 1995, (J. Bacteriol. 177: 7l71-7177), using l0 the previously isolated tlR~~3 genes from Candida utilis as a selection marker. The vectors (p~lRAS and pUCURA3), used in the transformation system were designed to directly integrate into the homologous locus of the Candida utilis mutant strain by homologous recombination, The procedure used is based on tine treatment of the intact cell yeast with an electric field,. The following conditions were used: 0.7 kV (3,5 kV/cm) ae a pulse, a resistance of , 800 S2 and a capacitance of 25 NSF.
Previously to the transformation procedure, both plasmids 2o were digested with XhoI which is located in the 5' prime of the structural gene facilitating the homologous integration in the genomic locus.
The selection of the transformant:e was done in YNB minimal medium without uracil.
The frequency of transformation ufaing both pURAS and pUCURA3 depended of the plasmid concentration. A comparison of both methods (LiAc and electroporation) is shown in Table 3.
Table :3 Transformation frequency Vector DNA Concentration (# tranef./~g) (ug) LiAc Electroporation pUCURA-3 0.1 - 70-90 0.5 - 640 3.0 22 -pURA-5 0.1 - 40-50 0.5 - 670 3.0 21 -The mitotic stability of these tra:nsformante was high, due to the mechanism of integration.
The frequencies of transformation coincide with that of S reported for Saccharomyces cerevisise and other non-conventional yeast using integrative vectors.
In figure 7 is shown the outline of the possible integration events in the genome of Candid.n utilis as well as the Southern-blot of some traneformants.
Example 10: Zaolatlon of the HIS3 3~ene of Cand3da utilis.
The HISS gene from Candida utilis was isolated and characterized from the library previously described in the Example 4. DNA fragments, which contained the Candida utilis HISS gene, were isolated from a Candida utilis genomic library by the ability to complement the hisb463 mutation in the Escherichia coli KC8 (hsd, his13463, leuB6) pyrF::TnS Kmr, trp (983o (lact YA), stm, galu, gal), taking into account that HZS3 gene from Saccharomyces cerevis~ae complements the hisb463 mutation of Escherichia coli, using fortuitous promoter activity in Escherichia coli.
In order to isolate the HZS3 gene, 105 cells were spread on minimum medium (M9) supplemented with uracil, tryptophan and leucine. Plasmid DNA was extracted from colonies able to growth in this medium and consequently capable to complement the hisb463 mutation in the Escherichia coli KC8 mutant strain. The plasmid DNAs isolated were used to retransform the Escherichia coli KCe mutant strain. A11 the plasmid able to supplement the histidine requirement of the mutant strain were denominated pHCU. In order to confirm that the his' colonies contained the HISS gene of Candida utilis and not a 5 fragment of ADN with auppressor activity, two of the plasmids obtained from the his' transformanta (pRCU37 pHCU40) were subjected to a PCR reaction. Two degenerate oligonucleotides from two regions highly conserved in five IGPDasae sequences from yeast and fungi were used. '.Che oligonucleotide as well 10 as amino-acid sequences of the degenerate oligonuclcotides are shown in the figure 8.
Approximately a 50o-by PCR band corresponding to the coding sequence of the HIS3 gene from Candida utiZis was amplified.
The approximately 500 by PCR fragment, which was shown by 15 Southern blot to hybridize the Candida utilis genomic DNA, was cloned in T-Vector (pMOSBLUE, Amershan) and the predicted amino-acid translation of its sequence was shown to be highly identical to His3p from other yeast and fungi. The plasmid pHCU37 (Figure 9) was used for the determination of the entire sequence of the HIS3 gene from Candida utilis.
Example 11: Sequencing of the XI83 gene of Candjda util3s.
The HISS gene from Candida uti_Lis was completely double strand sequenced using the method of Sanger et. al. (1977) .
Oligonucleotides of the universal series Ml3mp/pUC were used.
Primers taken from the PCR fragment were used to initiate sequencing of the entire gene. A total 1190 by of the pHCU37 was sequenced. The entire sequence HISS from Candida utilis is shown in Figure 10 (Seq. Id. No.: 5, 6).
This fragment contains an open residing frame of 210 codons .
The Candida utilis HI53 gene code for a protein with theoretical molecular mass of z4 5.L8 Da.
Example I2; Isolation of the INV1 gene that cod.ifiea for the invertase of Candida utilis.
In order to isolate the INV1 gene that codifies for the enzyme invertase in Candida ucilis, it was taken advantage from the fact that the amino acid sequence of this enzyme presents regions highly preserved between different species, thus the sequences of p-fructofw=anosidase from yeasts were aligned. Two degenerated oligonu~~leotides used in said PCR
were designed according to the codons usage in Candida l0 utilis. The polypeptide sequences, as well as the degenerated vligonucleotides are shown in Figure 11.
PCR generated ~a band of 417pb, which was subcloned in the Vector-T (pMOBlue, Amereham), Said band was completely sequenced and the tranelation~ of said DNA fragment corroborated the presence of consensus regions and also a high homology among the enzymes invertases reported in the literature. This demonstrated that the fragment isolated belonged to the INV1 gene, which codifies for this enzyme in candida utilis. This fragment was used as probe for isolating INV1 gene from Candida uti3is.
After the search of the Candida u~til.is library, a total of 6 clones having the iPNl gene were isolated. Two of these clones were selected for their siae for sequencing (pCI-6 and PCI-12), using the oligonucleotides of the previous step for PCR. These oligonucleotides were used to start the complete sequence of the gene from both chains belonging to plasmid pCI-6.
Example 13: Sequencing of the INV;L gene of Caadida utilie.
A total of Z607 by of the clone: pCI-6 containing the INV1 gene codifying for the inverta,se of Candida uti.tis was completely sequenced by the method of Sanger et la., (1977), and for this end were used universal oligonucleotides belonging to the series Ml3mpfpUC, ae well as internal oligonucleotidea derived from t:he sequence. The complete sequence of the 2607 by fragment is shown in Figure 12 (Seq.
Id. No.: 5, 6). Said fragment contains an open reading frame of 1602 by (S34 codone). The IrfVl gene of Candida utilis codifies for a protein of theoretic molecular weight of 60 703 Da.
Considering that the invertase in Candida utilis is a periplasmic enzyme, it should hare in its N-terminal end a signal peptide. Analyzing the sequence up to 5'-end of the gene) two codons ATG are observed (ATG1 and ATGz in Figure 12) giving place to ORF codifying for proteins which differ only the size of their N-terminal ends. Applying the von Heijne's algorithm (1986, Nucl. Acids Res. 14: 4683-4690) to predict the restriction site for the peptidase signal of the mature protein derived from both ~4TG, it is reveled that the restriction sites for both casein are located between the residues S39 and s40 for ATGl and between residues S26 and S27 for ATGi. This gives place tc~ signal peptides of 39 and 26 amino acids respectively. Taking in to account the average size for signal sequences in yeast (estimated as about 20 residues) , it can be suggested that the start codon for the INVl gene is the second ATG.
Eleven potential sites of N-glycoeilation, according to the general rule N-X-T/S, were found in the asparagines of the positions 40, 88, 141, 187, 245, 277, 344, 348, 365, 373, 379 and 399 of the sequence of the mature protein.
The region 5'-non translated sho~Na two possible TATA boxes (consensus TATAA)) in the regions -18 to -14 and -212 to -208, and also various possible union sites for the repressor Migl (consensus SYGGRG).
BRIEF DESCRIPTION OF THE FIGURES:
Figure 1. Plasmid pURAS, obtair,,ed in the Candida utilis genomic library by complementation of the Escherichia coli MC1066 pyrF SaCCharomyces cere~isiae SEY 2202 ura3 mutations.
Figure 2. Restriction enzyme map, sequence strategy and complementation analysis of the URA3 gene from Candida util.is.
Figure 3. Plasmid pUREC3 obtained by cloning of the 1.9 kb EcoRI fragment of the plasmid pU12A.5 in the pBLUESCRIPT SK(+).
Figure 4. Amino acid sequence deduced from the DNA sequence of the URA3 gene, and the DNA sequence of the DNA encoding it.
Figure 5. Plasmid pUT64 obtained for the complementation experiment in the Saccharomyce,s cerevisfae ura3 mutant strain.
Figure 6. Plasmid pUCURA3 used in the Candida utilis transformation experiments.
z5 Figure 7. (A) Predicted arrangements of the vector DNA
integrated at the UFA3 locus by homologous recombination.
(B? DNA-blot hybridization of genomic DNA from some transformante.
Figure 8. DNA sequence of the primers used in the isolation of the HISS gene) and the deduced amino acid sequence encoding it.
Figure 9. Plasmid pHCU37, obtained in the Candida utilis genomic library by complementation of the Escherichia coli KC8 hiab463 mutation.
Figure 10. Amino acid sequence deduced from the DNA sequence of the HIS3 gene, and the DNA sequence of the DNA encoding it.
Figure 11. Amino acid sequence and corresponding DNA sequence of the oligonucleotides used in the PCR for the isolation of l0 the INVl gene of Candida utilis.
Figure 12: DNA sequence corre;Bponding to the fragment containing the INVl gene of Candid!a utilis.
SEQUENCE LISTING
(1) GENERAL
INFORtIATION:
(i) APPLICANT:
(A) NAME: CENTRO DE INOENIERIA GENETICA Y HIOTECNOLOGIA
(B) STREET: AVE. 31 ENTRH 15B 5' 190, CLTHANACAN, PLAYA.
(C) CITY: CItJDAD DE LA HABANA
10 (D) STATE: CIUDAD DE LA HAHANA
(E) COUNTRY: CUBA
(F) POSTAL CODE (ZIP): 12100 (G) TELEPHONE: 53 7 216013 (H) TELEFAX: 53 7 336008 (ii) TITLE OF INVENTION: TRANSFORMAT1:ON SYSTEM
IN CAUDIDA UTILIS.
(iii) NUMBER OF SEQUENCES: 6 2 (iv) COMPUTER READR8L8 FORM:
(A) MEDIUM TYPE: Floppy disk (a) COMPUTER: IHM PC compatible (C) OPERATINQ SYSTEM: PC-DOS/MS'-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUhfl3ER: A2/96 (B) FILING DATE: 03-OCT-1996 (2) INFORMATION
FOR
SEQ
ID
NO.
1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11'19 base pairs 3 (B) TYPE: nucleic acid S
(C) STRANDEDNESS: siagle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (i11) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
45(vi) ORIGINAL SOURCE:
(A) ORGANISM: Candida tltilia (B) STRAIN: NRRL Y-1084 (ix) FEATURE:
SO (A) NAME/KEY: mat_pcptide (B) LOCATION:1..1179 (D) OTHER iNfORMATION:/produet- "Enzyme orotidin-5-phosphate decarboxylaee "
/gene= "URA3"
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
GTTGAATTGG TCCATCCTTG CTACTTTTCC GCCTAGT'.CTC GATTCCGATT CTGATAGAGA 120 AGCCC:AGCTA TC~AATGGAAG AAATTTTTCA CTTTTOT1~TG TCCTTTTTTT CACGCTTCGT 1A0 TGCTTCGGAC AAAAAAATAG TOGAGGCACT C'GGTOGAL9GG AAGCTATCCT CGAGATGAAA 240 AATTTCAAGC TCATCTCATC GTCCAAGTGC3 GAGAGCAAGC TGAdGCTTCT GAAGAGOTTG 300 AGGAAAATGG TCACC'.ACGTT ATCOTACACA GAGAGO(1<:AT CGCACCCTTC GCCACTTGCT 360 AAGCGTCTGT TTTCGCTTAT GGAGTCCAAG AAGACGAi~CC TGTGTGCCAG TG?CGATGTT 4Z0 CGTACCAC:AG AGGAGTTGCT CAAGC?CGTT GATACGC7.'TG GTCCTTATAT CTGTCTGT?G 480 GAGCTTTCAA AAGAGC.'ACAA TTTCCTCATC TTTGAGCiF~CC GTAAGTTTGC TGATATCGGC 600 AACACCGTCA AGGC,'ACAGTA CGCCGOTGGT GCGTTCAAGA TTGCACAATG GGCAGACATC 660 ACCAACGCCC ACGGTGTCAC CGGTCGAGGT ATCGTCAFvGG GGTTCiAAGGA GGCTGCACAG 720 2 5 TTCGCTCACG GGACA2ATAC CGAGGAGACC OTGGAGA7.'TCi CCAAAACTGA TAAC'sG'ACTT'I
TGTATTGGAT TCATCGCACA GAGAGACATG GGTGGCACiAG AAGATGGGTT CGACTGGATC 900 ATCATGACAC CAGGCGTGGG ACTCGACGAT AAGGGCGF~CT CCCTGGGCCA ACAGTACAGA 960 ACTGTCGATG AGGTTGTCAG TGGTGGCTGT CJACATCA7.'CA TCGTTGGTAG AGGCTTGTTT 1020 GGAAAGGGAA GAGATCCAAC AGTGGAACGT GAGCGTTF~TA GAAAACiCAGG CTGGGATGCT 1080 TATCTCAAGA GATACTCAGC TCAATAAACG TTOAGCTC.'rG OCTTGTATAG GTTCACT"TGT 1140 (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: Q66 amino acida (B) TYPE: amino acid (C) STRANDEDNESS: eiagle 4 5 (D) TOPOLOGY: linear (ii) MOLECULE TYpE: proteia (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Car~dsdz utili9 5 5 (s) sTRAIN: NRRL Y-loe4 (ix) FEATURE:
(A) NAME/1CEY: Protein (8) LOCATION:1..266 (zi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Met Val Thr Thr Leu &er Tyr Thr Glu Arg AIa Ser Hie Pro Ser Pro Leu Ala Lye Arg Leu Phe Ser Leu Met Glu Ser Lys Lys Thr Aea Leu Cys Ala Ser Val Asp Val Arg Thr Thr Glu Glu Leu Leu Lys Leu Val 35 40 i5 Aap Thr Leu Gly Pro Tyr Ile Cys Leu Leu Lye Thr Hia Ile Acp Ile Ile Asp Asp Phe Ser Met Glu Ser Thr Val Ala pro Leu Leu Glu Leu 5er Lye Glu His Asn Phe Leu Ile Phe Glu Asp Arg Lys Phe Ala Aep 8$ 90 95 Ile Gly Aan Thr Val Lye Ala Gln Tyr Ala Gly Gly Ala Phe Lye Ile Ala Gln Trp Ala Asp Ile Thr Aen Ala His Gly vat Thr Gly Arg Gly Ile val Lye Gly Leu Lye Olu Ala Ala Gln Glu Thr Thr Aep Glu Pro Arg Gly Leu Leu Met Leu Ala Glu Leu &er Ser Lys Gly Ser Phe Ala His Gly Thr Tyr Thr Glu Glu Thr Val 01u Ile Ala Lys Thr Asp Lye Asp Phe Cys Ile Oly Phe Ile Ala Gln Arg Aep Met Gly Gly Arg Glu Asp Gly Phe Asp Trp Ile Ile Met Thr Pro Gly Val Gly Leu Asp Asp 195 200 2fl5 Lys Gly Asp Ser Leu Gly Oln Gln Tyz .Azg Thr val Asp Glu Val Val 4 5 Ser Gly Gly CYe Asp Ile Ile Ile Val Gly Arg aly Leu Phe Gly Lys Gly Arg Aap Pro Thr Val Glu Gly Glu .Arg Tyr Arg Lys Ala Gly Trp 2f5 250 Z55 Asp Ala Tyr Leu Lys Arg Tyr Ser Ala Gln (2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICSs (A) LENGTH: 1190 base pairs (8) TYPE: nucleic acid (c) sTRANDEDNESS: oingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iw) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISH: Candida utilts (a) STRAIN: NRRL~ Y-1084 (ix) FEATURE:
(A) NAME/I~Y: mat~eptide (H) LOCATION:1..1190 (D) OTHER INFORMATION:/product= "Enzyme imidazol-glycerol phosphate dehydratase ~
/gene= "HISS"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 3:
AAAAGAGTCG AAGAACRACA GTGCGCCRAA AAARAAAC'iC CGGACCGCAC ACGACTCATC 120 GCTCTCGGAA TATCCCTCGG AATGCGCCAC TTCCGGCiT~:~C GTGGCCATCG GAAGAGCGAA 180 GAO?CATCAC CATCGTACTT TAACGACTTA CTATTCTCAT TGAGTATTGA GAAGAAGGAT Q40 AGAGAAATGG CTGAACGAAC GGTGAAACCC CAGAGRAG.AG CTCTTGTGAA TCGTACAACA 300 AACGAAACGA AGATCCAGAT TTCCTTGAGT TTGGATGG'TG GATACGTAAC GGTTCCGGAG 360 TCAATCTTCA AGGATAAGAA GTACGACGAT GCTACTCA~AO TCACCTCTTC TCAGGTGATT 420 4 5 GGGTGGAGTT TGAT?GTGGA GTGTATTGGT GATTTGCACA TTGACGACCA CCACACCACC 540 GACGACGTTG GTATTGCGCT GGGAGACGCC GTCAAGGA~3G CCTTGGCATA TAGAGGTGTC 600 AAGAGATTTG GTAGCGGGTT TGCTCCATTG GACGAOGC'TC TGAGCAGAGC CGTTGTTGAT 660 TTGTCATGTG AGATGATTCC TCACTTCTTG GAGAGTTT'TG CCCAAGCAGC TCATATCACG 780 ATGCATGTTG ACTGTTTGAG AOGCTTCAAC GACCATCAi~A GAGCTGAATC CGCATTCAAG e40 GCCCTGGCRG TCGCCATTAA GGAATCCATC TCCAO?AACG GCACCAATGA TGTTCCCTCA 900 ACAAAGGGTG TTTTGTTCTA GATAGCAGTC TTTCTGTC'TC TCTATTTATT CGATAAATAA 96o CAAGGACTCT ACGACCACTG GTOOCTTTGA TATGATT'CCC T~3CCAGTACT TGTAACAGOT 1140 GCAACGTCAA TGGAAACOOC ACCGTTAGCC TTGATCiO'.CIG CACGGGTAGG 1190 (2) INPORMATZON FOR SEQ ID NO: 4:
(i) SfiQUENCE CHARACTERISTICS:
(A) LENGTH: 210 amino acids (H) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSfi: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Candida utilis 2 5 (H) STRAIN: NRRL Y-1084 (ix) FEATURE:
(A) NAME/KEY: PZOtein (H) LOCATION:1..210 (xi) SEQUENCE DESCRIPTION: 6EQ ID NO: 4:
Met Ala Glu Arg Thr Val Lys Pro Gln A.rg Arg Ala Leu Val Aen Arg 1 5 to is Thr Thr Asn Glu Thr Lya Ile Gln Ile Ser Leu Ser Leu Aep G1y Gly 4 0 Tyr Val Thr Val Pro Glu Ser Ile Phe Lys Asp Lys Lys Tyr Asp Asp Ala Thz Gln Val Thr Ser Ser Gln Val Ile Ser Ile Asn Thr Gly Val Gly Phe Leu Asp His Met Ile His Ala Leu Ala Lys His Gly Gly Trp Ser Leu Ile Val Glu Cys Ile Gly Asp Leu Hie Ile Asp Aep His Hie Thr Thr Glu Aep Val Gly xle Ala Leu Gly Aep Ala Val Lys Glu Ala Leu Ala Tyr Arg Gly Val Lys Arg Phe Gly Ser Gly Phe Ala Pro Leu 115 l20 125 Asp Glu Ala Leu Ser Arg Ala Val Val Asp Leu Ser Asn Arg Pro Phe Ala Val Val Glu Leu Gly Leu Lye Arg Glu Lys Ile Gly Aap Leu Ser 14S l50 155 160 Cys Glu MeL Ile Fro Hie Phe Leu Glu :Ser Phe Ala Gln Ala Ala His 5 165 :170 175 Ile Thr Het His Val Asp Cye Leu Arg G1y Phe Aen Asp Hie H1c Arg 10 Ala Glu Ser Ala Phe Lye Ala Leu Ala Val Ala Ile Lye Glu Ser Ile ser Ser zlo l2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARAC?ERISTICS:
2 0 (A) LENGTH: 26o7 bxae pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single lD) TOPOLOGY: linear 2 5 (ii) MOLfiCULE TYPE= DNA (genomic) (iii) HYPOTHETICAL: NO
(iV) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Candiaa utilic (H) STRAIN: NRRL Y-10B4 3 5 ( i x ) FEATURE :
(A) NAME/KEY: mat-peptide (H) LOCATION:1..2607 (D) OTHER INFORMATION=/product~ "Eazima invertaca (beta-fructofuranoeidaaa)"
4 0 /gcne= "INV1"
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: S:
ATTGGCAGCT CCGGAGCACA CTCAATTGGCi ACTAAAAGF,A GTAACATTTa TACTACAATG 120 AGTCGTATAG AGTCATGTAT AAGAAGAACA GCAAGAAAA,G AAAATATTGG TGCAGAATTC 180 AACAGCTTCT GAGATCGTAA GAACAGCCAA TCATTTACC:G GAATTCATTA TGATACCTAT 240 AGRAAGACAC AAATTGTTGG GTAAAACAAC AGAACATAC!C TGTATAGGGG TTTATACGAG 3D0 AATTTTCTTA GACGTCTCCC CCAGTGTCCG CC,'AAAGCAA,C TTACATGTGG AOTTTGAATT 360 Ci0.ATTTATCGACGTTATGCC TTGTCAGACC ATCGTCGTGA 540 AC?TTTCTAA ACCGGATAAA
CTCTCGCACGOATTATAACG TGCGTCTGTG ATATGCAC'CCCCCCGTGGAG600 CGGRAAAAF.C
AAGTGAAGCGGCCACCTGTG GAGCAGRAAT TTCGATCGi~CCAAATGGTTT660 OTTTCAAGTT
CCTGTTGTCAAAGGOCTTGA GATTTACCAC TTGACCAT'CTTTCGGAGAGC720 GTGCTCAGAA
CAGGGATGTC
GATGCCTCAGAGGACCAAGA AGACATCAAG AGTCTt:ACGATTTAGTTGAT840 TGAACACTA(i TGGGGTGGAT
AATGGTCTCT?CTACGATTC ATCTGAATCT ACTTACCA'CGATACAACCCA960 TGTACTACCA
AACGATACGATTTGGGGATT GCCTCTATAT TGGGGAt:Jt.'.CGTGATTTGTTA1020 CCACCTCTGA
ACGTGGGACCACCATGCGCC TOCAATTGGA CCTGAGAA'CGTATTTACTCT108O
ATGATGAGGG
GGATCTATAGTCATAGACTA CGATAATACC TCAGGGT"Tt~2AACAAGACCA1140 TTGACOA'rTC
GAACAGAGAATCGTTGCCAT TTATACCAAT AACTTACCi~GGCAAGACATT1200 ATGTCGAGAC
2 GCCTATTCCACGGACGGTGG TTATACTTTC GAAAAGTA'CGAGTTATAGAC1260 GTCiaATTCGACCCAATTTAG GGATCCGAAG GTGATTTGL3TTGAACAATGG1320 ATGAGGAAAC
GTCATGACTGTGGCRAAGAG TCi4AGAGTAC AAGATCCAc3ATGACAATTTG1380 TTTACACCTC
AAAGACTGGAGTTTGGCCTC GAATTTCTCA ACCAAGGG',CTTCAGTATGAA1440 ATOTTGGTTA
TGTCt:AGGTCTATTCGAAGC CACTATTGAA AACCCAAAGA AGAGAAGAAA1500 GTGGTGACCC
3 TGGGTTATGGTCTTAGCAAT CAATCCAGGC TCACCTCT'CGAAATGAATAC1560 TTTGTTGGTGATTTCAACGG TACTGAATTC ATTCCAGA'CGAAGATTTATG1620 ATGACGCTAC
ATGCACCGGA
AGG?TCCGGA
TA?AGAAGCTCCATGTCATC AATCAGAGAG TACACTCTt3ATACGAATCCA1800 GATATOTCAG
GAATCTGAACAGTTGATCCT TTGTCAAAAA CCATTCTT'CGAQACTTGAAG1860 TGAACGAGAC
GTGGTTGAAGAGTACAAGGT TTCAAACAG? TCTTTQACCG 0?TTGGAAG?1920 TGGACCACAC
AGCTTTGCAAACTCCAACAC CACTGGACTG TTGGATTTt:a1CACGGTTAAC1980 ACATGACTTT
GGTACAACTGACGTTACGCA GAAOGACTCC GTCACCTT'CCiCAAATCTAAC2040 AGCTCAGAAT
ACGAGCRATT
TCCAGGAGAG
TACGTTCAGCCTCTCACAAT CACCGAATCT GGTGATRAa0.CCTACGGATTG2220 AGTACCAGCT
GT'TGATAACAACATCCTTGA GTTGTACTTC AACGACGG~:~(3CACAAACACC2 TTGACGAATA
AGCTATATAA GGGATCACGT GGTCTAGCCA CCCCAGTC'.CACAAACCGCCA2460 AAAOCTTCAG
CTATATAAAC AGACAGGTTT GTCACTTTTC AACAAAAGiA CTTTTACCCT2520 ATATCTTCTT
TCAGAGTAGT TTGTACGAGT GCTTTTTTCA ATTATATA'.CAAGCTGCCTTT25A0 CAACAACGTG
(2) INFORhIATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERI&TIC9:
(A) LENGTH: 533 amino acids (B) TYPE: amino acid (C) STRANDEDNES6: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTF~TICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Candida utilis 3 (H) STRAIN: NRRL Y-10B4 (ix) FEATURE:
(A) NAME/KEY: Protein (H) LOCATION:1..533 (xi) SEQDENCE DESCRIPTION: SEQ ID N0: 6:
Met 5er Leu Thr Lye Aap Ala Ser Glu Asp Gln Glu Asp Ile Lya Ser 1 5 7.0 15 Leu Thr Met Aan Thr Ser Leu Val Asp :~eX Ser Ile Tyr Arg pro Leu Val His Leu Thr Pro Pro Val Gly Trp rtet Asn Asp Pro Asn Gly Leu Phe Tyr Asp Ser Ser Glu her Thr Tyr Ftis Val Tyr Tyr Gln Tyr Asn ?ro Asn Aap Thr Ile Trp Gly Leu Pro Leu Tyr Trp Gly Hia Ala Thr Ser Aep Aap Leu Leu Thr Trp Asp His Ftis Ala Pro Ala Ile Gly Pro 85 .'~0 95 Glu Asn Asp Asp Glu Gly Ile Tyr Ser cily Ser Ile Val Ile Asp Tyr 1o0 1o5 110 Asp Asn Thr 6ez Gly Phe Phe Aep Asp Ser Thr Arg Pro Glu Gln Arg Ile Val Ala Ile Tyr Thr Aen Aen Leu FaroThr Gln Asp Val Glu Asp Ile Ala Tyr Ser Thr Asp Gly Gly Tyr 7~hrTyr Glu Phe Glu Lys Asn 14S l50 155 160 Asn Pro Val Ile Asp Val Asn Ser Tht CflnPro Lys Phe Arg Asp Val 165 1.70 175 Ile Trp Tyr Glu Glu Thr Glu Gln Trp Val Ala Lye Met Thr Val Ser Gln Glu Tyr Lye Ile Gln Ile Tyr Thr f~erLys Asp Asp Asn Leu Trp Set Leu Ala Ser Asn Phe 5er Thr Lys GflyTyr Gln Tyz Val Gly Tyr 21o 2l5 zzo Glu Cys Pro Gly Leu Phe Glu Ala Thr 1:1eLye Ser Glu Aan Pro Gly 2 Asp Pro Glu Lye Lys Trp Val Met Val heu Pro Gly 5 Ala Ile Asn Ser Z45 i'.50 2S5 Pro Leu Gly Gly Ser Ile Asn Glu Tyr F~hePhe Aan val Gly Asp Gly 260 265 27o Thr Glu Phe Ile Pro Aep Asp Rsp Ala Thr Asp Thr Arg Phe Met Gly Lys Aep Phe Ty= Ala Phe Gln Ala Phe F~heGlu A9n Asn Ala Pro Arg Ser Ile Gly Val Ala Trp her 8er Asn Trp Asn Gln Gln Tyr Ser Val Pro Asp Pro Asp 01y Tyr Arg Set Ser Met Arg Glu Ser Ser Ile Tyr Thr Leu Arg Tyr Val Ser Thr Aen Pro GlluLeu Ile Ser Glu Glri Leu C'ys Glri Lye Pro Phe Phe Val Asn Glu Val Val 1'hr Aap Leu Lys Glu Glu Tyr Lye Val Ser Aen Ser Ser Leu Z'hrThr Phe Val Asp His Gly Ser Ser Phe Ala Asa Ser Asn Thr Thr LilyPhe Jean Leu Leu Aep Met Thr Phi Thr Val Asri Gly Thi' Thr Aep Aep Ser Val Thr Gln Lys val Thr Phe Glu Leu Arg Ile Lys Ser Asn G~lnAla ile Ser Aap Glu Ala Leu Gly Tyr Asp Tyr Asn Asn Glu Gln )?he Tyr Ile Asn Arg Ala Thr Glu Ser Tyr Phe Gln Arg Thr Asn Gln I?he Phe Gln Glu Azg Trp Ser Thz Tyz Val Gln Pro Leu Thr Ile Thr (~lu Ser Gly Aep Lys Gln 2~r Gln heu Tyr Gly Leu Val Asp Aen Aen :Cle Leu Glu Leu Tyr Phe Asn 4B5 !190 495 Aep Gly Ala Phe Thr Ser Thr Asn Thr 1?he Phe Leu Glu Lys Gly Lys Pro Ser AHn Val Aep Ile Val Aln Ser E:er Ser Lys Glu Ala Tyz His Arg Gly Pro Ala Aep
Claims (27)
1. Candida utilis transformation system wherein it is used a host yeast cell able to be transformed with a recombinant DNA
and this host is defective in at least one biosynthetic pathway.
and this host is defective in at least one biosynthetic pathway.
2. Candida utilis transformation system according to claim 1 wherein it is used a host yeast cell that is defective in at least the biosynthetic pathway of one amino acid.
3. Candida utilis transformation systemaccording to claim 2 wherein it is used a host yeast cell that is defective in the biosynthetic pathway of the uracil.
4. Candida utilis transformation system according to claim 3 wherein said host yeast cell is defective in the activity of the enzyme orotidin-5-phosphate decarbosylase.
5. Candida utilis transformation system according to claim 4 wherein said host yeast cell is Candida utilis NRRL Y-1084 phenotype ura-, deposited with accession number CBS 100085 at Centraalbureau voor Schimmelcutures on October 1, 1997.
6. Candida utilis transformation system according to claim 2 wherein it is used a host yeast cell that is defective in at least the biosynthetic pathway of the histidine.
7. Candida utilis transformation system according to claim 6 wherein the host yeast cell is defective in the activity of the enzyme imidazol-glycerol phosphate dehydratase.
8. Candida utilis transformation system according to claim 7 wherein said host yeast cell is Candida utilis NRRL Y-1084 phenotype his- designated as TMN3.
9. Candida utilis transformation system according to claim 1 wherein the recombinant DNA material contains a functional gene that complements the defect in the biosynthetic pathway in which the host is defective.
10. Candida utilis transformation system according to claim 9 wherein the functional gene is the gene URA3 of Candida utilis.
11. Candida utilis transformation system according to claim wherein said recombinant DNA material are the plasmids pUC
19 with a 2.6 kb fragment containing the URA3 gene from Candida utilis inserted into BamHI site, and pUC19 with a 1.8 kb fragment containing the URA3 gene from Candida utilis into EcoRI site.
19 with a 2.6 kb fragment containing the URA3 gene from Candida utilis inserted into BamHI site, and pUC19 with a 1.8 kb fragment containing the URA3 gene from Candida utilis into EcoRI site.
12. Candida utilis transformation system according to claim 9 wherein this functional gene is the HIS3 gene of Candida utilis.
13. Candida utilis transformation system according to claim 12 wherein the recombinant DNA material are the plasmids pUC19 with a 4.3 kb fragment containing the HIS3 gene from Candida utilis into BamHI site and pUC 19 with a 4.5 kb fragment containing the HIS3 gene from Candida utilis into BamHI site.
14. A yeast host cell of Candida utilis able to be transformed with recombinant DNA wherein said host is defective in at least one biosynthetic pathway.
15. A yeast host cell according to claim 14 wherein said host cell is defective in at least the biosynthetic pathway of one amino acid.
16. A yeast host cell according to claim 15 wherein said host cell is defective in at least the biosynthetic pathway of the uracil.
17. A yeast host cell according to claim 16 wherein this host cell is defective in the activity of the enzyme orotidin-5-phosphate decarboxylase.
18. The yeast host cell according to claim 17 wherein said host cell is Candida utilis NRRL Y-1084 phenotype ura-, deposited with daccession number CBS 100085 at Centraalbureau voor Schimmelcutures on October 1, 1997.
19. A yeast host cell according to claim 15 wherein this host cell is defective in the biosynthetic pathway of the histidine.
20. The yeast host cell according to claim 19 wherein this host cell is defective in the activity of the enzyme imidazol-glycerol phosphate dehydratase.
21. The yeast host cell according to claim 20 wherein said host cell is Candida utilis NRRL Y-1084 phenotype his-, deisgnated as TMN 3.
22. A recombinant DNA material able to transform by integration to its genome a yeast host cell of Candida utilis, wherein said recombinant DNA material contains a functional gene that supplements the defect in the biosynthetic pathway in which the host is defective.
23. A recombinant DNA material according to claim 22 wherein the functional gene is the URA3 gene of Candida utilis.
24. A recombinant DNA material according to claim 23 wherein the recombinant DNA material are the plasmids pUC 19 with a 2.6 kb fragment containing the URA3 gene from Candida utilis inserted into BamHI site, and pUC19 with a 1.8 kb fragment containing the URA3 gene from Candida utilis into EcoRI site.
25. A recombinant DNA material according to claim 22 wherein the functional gene is the HIS3 gene of Candida utilis.
26. A recombinant DNA material according to claim 25 wherein the recombinant DNA material are the plasmids pUC19 with a 4.3 kb fragment containing the HIS3 gene from Candida utilis into BamHI site and pUC 19 with a 4.5 kb fragment containing the HIS3 gene from Candida utilis into BamHI site.
27. A dDNA sequence codifying for the HIS3 gene of Candida utilis (Id. Seq. No. 3).
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CU82/96 | 1996-10-03 | ||
| CU1996082A CU22722A1 (en) | 1996-10-03 | 1996-10-03 | TRANSFORMATION SYSTEM FOR THE EXPRESSION OF HETEROLOGICAL GENES IN CANDIDA UTILIS YEAST |
| PCT/CU1997/000005 WO1998014600A1 (en) | 1996-10-03 | 1997-10-03 | Candida utilis transformation system |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2268004A1 true CA2268004A1 (en) | 1998-04-09 |
Family
ID=5459318
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002268004A Abandoned CA2268004A1 (en) | 1996-10-03 | 1997-10-03 | Candida utilis transformation system |
Country Status (9)
| Country | Link |
|---|---|
| EP (1) | EP0956356A1 (en) |
| JP (1) | JP2001501475A (en) |
| CN (1) | CN1237208A (en) |
| AU (1) | AU744698B2 (en) |
| BR (1) | BR9713313A (en) |
| CA (1) | CA2268004A1 (en) |
| CU (1) | CU22722A1 (en) |
| RU (1) | RU2235127C2 (en) |
| WO (1) | WO1998014600A1 (en) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003047471A (en) * | 2001-08-01 | 2003-02-18 | Kirin Brewery Co Ltd | Modified yeast having excellent secretory production of foreign protein and method for producing foreign protein by using the yeast |
| MY142328A (en) | 2002-03-26 | 2010-11-15 | Ajinomoto Kk | CANDIDA UTILIS CONTAINING y-GLUTAMYLCYSTEINE |
| ATE420198T1 (en) * | 2002-04-26 | 2009-01-15 | Kirin Pharma Kk | METHYLOTROPHIC YEAST THAT PRODUCES A MAMMAL SUGAR CHAIN |
| JP2005073638A (en) * | 2003-09-02 | 2005-03-24 | Ajinomoto Co Inc | Glutathione synthetase-coding gene of candida utilis |
| CN100347287C (en) * | 2004-09-30 | 2007-11-07 | 汪和睦 | Recombinated multi shape ttansenula yeast, its structural method and application |
| WO2012046731A1 (en) | 2010-10-05 | 2012-04-12 | 味の素株式会社 | YEAST AND YEAST EXTRACT CONTAINING γ-Glu-Abu, AND METHOD FOR PRODUCING THE YEAST OR YEAST EXTRACT CONTAINING γ-Glu-Abu |
| CN114657079A (en) | 2013-07-12 | 2022-06-24 | 味之素株式会社 | Yeast with high content of Abu, gamma-Glu-Abu and/or gamma-Glu-Abu-Gly |
| CN103468594B (en) * | 2013-07-31 | 2015-06-17 | 浙江科峰生物技术有限公司 | Candidautilis strain and application thereof |
| JPWO2022201917A1 (en) | 2021-03-22 | 2022-09-29 | ||
| JPWO2022210308A1 (en) | 2021-03-29 | 2022-10-06 | ||
| KR102556901B1 (en) * | 2021-05-25 | 2023-07-17 | 국립낙동강생물자원관 | Edenia sp. NNIBRFG15114 strain isolated from freshwater having antifungal activity and plant growth promotion and uses thereof |
| JPWO2023286629A1 (en) | 2021-07-16 | 2023-01-19 | ||
| CN115838645B (en) * | 2022-09-15 | 2024-03-08 | 天津大学 | Yeast strain for high production of orotic acid and application thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59162884A (en) * | 1983-03-09 | 1984-09-13 | Kohjin Co Ltd | Novel hybrid plasmid and microorganism containing the same |
| NZ207925A (en) * | 1983-04-25 | 1988-05-30 | Genentech Inc | Yeast expression vehicle consisting of a yeast promoter and signal peptide encoding region linked to a heterologus peptide coding region; expression and culture |
| US5204252A (en) * | 1989-02-08 | 1993-04-20 | Henkel Research Corporation | Candida tropicalis transformation system |
| JPH08173170A (en) * | 1994-05-25 | 1996-07-09 | Kirin Brewery Co Ltd | Transformation system of candida utilis yeast and expression of heterologous gene therewith |
-
1996
- 1996-10-03 CU CU1996082A patent/CU22722A1/en not_active IP Right Cessation
-
1997
- 1997-10-03 BR BR9713313-2A patent/BR9713313A/en not_active IP Right Cessation
- 1997-10-03 CA CA002268004A patent/CA2268004A1/en not_active Abandoned
- 1997-10-03 AU AU45485/97A patent/AU744698B2/en not_active Ceased
- 1997-10-03 WO PCT/CU1997/000005 patent/WO1998014600A1/en not_active Application Discontinuation
- 1997-10-03 JP JP10516085A patent/JP2001501475A/en not_active Ceased
- 1997-10-03 CN CN 97199610 patent/CN1237208A/en active Pending
- 1997-10-03 RU RU99109095/13A patent/RU2235127C2/en not_active IP Right Cessation
- 1997-10-03 EP EP97943729A patent/EP0956356A1/en not_active Withdrawn
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|---|---|
| CU22722A1 (en) | 2002-02-28 |
| JP2001501475A (en) | 2001-02-06 |
| EP0956356A1 (en) | 1999-11-17 |
| CN1237208A (en) | 1999-12-01 |
| BR9713313A (en) | 2000-10-24 |
| RU2235127C2 (en) | 2004-08-27 |
| WO1998014600A1 (en) | 1998-04-09 |
| AU744698B2 (en) | 2002-02-28 |
| AU4548597A (en) | 1998-04-24 |
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