Patent Application: US-54399403-A

Abstract:
there are provided genetically modified yeasts with the capability to form a biofilm at the liquid / air interphase , as well as processes for obtaining fungi and yeasts with the capability to form a biofilm with those features , and uses thereof .

Description:
the invention is based on the authors &# 39 ; discovery that the capability to form a biofilm on the surface of the wine , known as flor in the field of biologically aged sherry wine production , depends on modifications in the flo11 gene of yeasts , and that , therefore , this feature of growing and remaining on the liquid surface can be controlled with modifications in the corresponding flo11 p protein ( fig2 ). the main modifications allowing the inventors to induce the formation of a biofilm at the liquid - air interphase are of three types : 1 ) removal of proteins competing for the same actuations areas as flo11p in yeast , such as the homologous flo1p protein or the flo1 gene encoding it . 2 ) increasing the amount of flo11p protein that the cell has , which is achieved , among other ways , by means of the expression with a very powerful promoter , or by means of deletion of the elements limiting or inhibiting expression of its own promoter . 3 ) flo11 p is formed by repetitions of two types of peptides : type a ), represented by amino acid sequence p - v / a - p - t - p - s - s - s - t - t - e - s - s - s - a and analogous sequences , and type b ) represented by sequence p - v / p - t / s / f - s - s - t / s - t - e - s - s - s / v - a / v and analogous sequences . growth at the liquid - air interphase is also achieved by expressing variants of flo11 p protein which alternate these two basic repetition types , preventing the existence of more than two repetitions of the same type together , as well as increasing the number of repetitions of the protein . each one of these elements alone or combined allows a yeast to form a biofilm at the liquid - air interphase . the experiments listed below are described as support of particular aspects of the invention , and by no means to limit the scope thereof . said experiments were developed in the steps detailed below : 1 . 1 to carry out the disruption of the flo1 gene , which encodes pflo1 , a protein having a cell wall being highly homologous to flo11 , flo1 - 13 (− 222 to 245 with regard to + 1 of orf ) and flo1 - 42 ( 3135 to 3551 ) fragments corresponding to ends 5 ′ and 3 ′, respectively , of said gene were cloned into the pbssk vector by pcr . to do this , the flo1 - 13 fragment is amplified by pcr with taq using flo1 - 1 and flo1 - 3 primers , and the following program : 95 ° c . 5 min . 95 ° c . 30 sec . 53 ° c . 30 sec . { close oversize bracket } × 30 cycles 72 ° c . 4 min . 72 ° c . 10 min . 4 ° c . 5 min . flo1 - 13 was cloned into the ecorv ( kpni - saci ) site of pbssk vector , generating plasmid pbsflo1 - 13 ( fig1 ). 1 . 2 . the flo1 - 42 fragment is amplified by pcr with taq using the flo1 - 2 and it was cut with ecori and bamhi and cloned into pbssk at ecori / bamhi sites , generating plasmid pbsflo1 - 42 ( fig2 ). 1 . 3 . then , the flo1 - 42 fragment was extracted from plasmid pbsflo1 - 42 with ecori and xbai enzymes , and it was cloned into plasmid pbsflo1 - 1342 at the ecori and xbai sites , generating plasmid pbsflo1 - 1342 ( fig3 ). 1 . 4 . the fragment corresponding to the kan gene ( confers dominant geneticin resistance ) flanked by the loxp sequences originating from plasmid pug6 ( guldener et al 1996 ), cut with ecorrv and pvuii , was cloned into plasmid pbsflo1 - 1342 between the flo1 - 13 and flo1 - 42 regions , at the ecorv site , generating the plasmid called pflo1 - 1342 - kanioxp ( fig4 ). guldener u , heck s , fielder t , beinhauer u , and hegemann j h . ( 1996 ). a new efficient gene disruption cassette for repeated use in budding yeast . nucleic acids res . 24 : 2519 - 24 . 1 . 5 digestion of the previous construct with bamhi generates a 2500 - base pair fragment . this fragment was transformed into the my138 strain by the lithium acetate method ( ito et al 1983 ), and geneticin resistant transformants were selected on rich medium plates containing 200 μg / ml of geneticin . several selected transformants were re - checked for the geneticin resistance phenotype , and a pcr reaction was subsequently carried out using the flo1 - 1 and flo1 - 2 primers and the previously described cycles and a southern blot using the flo1 - 13 and flo1 - 42 fragments as a probe to check if the transformed fragment had been integrated and , therefore , disrupted only the flo1 gene . 1 . 6 . to remove the dna fragments not pertaining to the yeast and to thus achieve a gras strain , a disruptant selected with plasmid yep32cre - cyh ( ito h ., fukada y ., murata k ., and kimura a . ( 1983 ). j . bacteriol . 153 , 163 ), which carries the sequence corresponding to cre recombinase under the control of the gall promoter , was transformed . once the transformants are selected by means of growth on ynb medium plates containing 200 μg / ml of cycloheximide , these were grown in medium containing galactose as a carbon source to induce cre recombinase expression . cycloheximide - sensitive ( they have lost plasmid yep32cre - cyh ) and geneticin - sensitive ( they have lost the kan gene of the fragment used for flo1 disruption ) colonies were subsequently selected . the loss of said elements was subsequently checked by means of pcr reactions using the flo1 - 1 and flo1 - 2 primers as was done previously , and by means of southern blot using fragments corresponding to the kan , ura3 gene and the flo1 - 13 fragment as a probe . once a strain was selected complying with all those conditions , the process was repeated to remove the second copy of the flo1 gene which the my138 strain has , thus obtaining a gras strain with double disruption of the flo1 gene , which is called osb101 . application of the disruption of the flo1 gene is the improvement of the capability to form flor of strains already having said capability . 2 ) and 3 ) construction of plasmid pflt - flo11 - f conferring the capability to form a biofilm at the liquid - air interphase to those strains not having this capability by means of the increase of the protein amount and production of a variant of this flo11 - fp protein present in strain my138 forming the biofilm at the liquid - air interphase . 2 . 1 . for the construction of plasmid pflt - flo11 - f , the promoter of the flo11 - f gene ( sequence in attached document ) was amplified by pcr using the pfu enzyme , genomic dna extracted from the 133d strain by means of the method disclosed by hoffman & amp ; winston , gene 57 , 267 - 272 ( 1987 ), and the flo11 - p5 and flo11 - p6 primers . 94 ° c . 2 min . 94 ° c . 45 sec . 56 ° c . 30 sec . { close oversize bracket } × 30 cycles 72 ° c . 7 min . 72 ° c . 7 min . 2 . 2 the 3 . 1 kb fragment obtained was digested with the ecori enzyme and was cloned into the prs316 vector . the resulting plasmid is called prspflo11 . then , the tadh terminator of plasmid pdbgal1 ( fig5 ) was cloned by means of hindiii - xbai digestion and blunt - end reaction into plasmid prspflo11 . 2 . 3 . the fragment corresponding to the flo11 - f gene ( 5 kb ) previously amplified by pcr using the pfu enzyme , genomic dna of the 133d strain and the flo11 - end5 and flo11 - end3 primers , was cloned into vector pbssk +. 94 ° c . 2 min . 94 ° c . 45 sec . 53 ° c . 30 sec . ( increase 0 . 5 ° c ./ cycle ) 72 ° c . 9 min . number of cycles : 10 94 ° c . 45 sec . 58 ° c . 30 sec . 72 ° c . 9 min . ( increase 5 sec ./ cycle ) number of cycles : 20 72 ° c . 7 min . 2 . 4 . the flo11 fragment was finally extracted from plasmid pflo11 - c by means of digestion with apai ( and reaction to make blunt ends ) and noti , and it was cloned into plasmid pflt at smai - noti . the resulting plasmid is called pflt - flo11 ( fig7 ). application of the pflt - flo11 construct is to confer the capability to form a biofilm at the liquid - air interphase to strains lacking said capability , and to improve this capability in those already having it . fig8 shows the different steps in producing compounds using fungi and yeasts , taking advantage of the capability to form a biofilm . in said figure : 1 represents the growth of the culture , 2 the transfer of the metabolized medium or substrate , 3 the strained biofilm , and 4 the filling with new medium or substrate . fig9 allows seeing the effect of the deletion of the flo11 gene in a strain with the capability to form a biofilm at the liquid - air interphase , 5 corresponding to the 133d strain and 6 to 133dδflo11 strain . gene sequences used flo1 - 1000 until the end of the open reading frame aaaaaaaagtgcatttatttaggtaagtctcattacctaaacgccagttt gtttcacgtaattggtaacgatgagggaaccgcagtagaaaaaactttc attcacaaacgattaaagtgttatgctagccagtttcaggctttttgttt tatgcaagagaacattcgactagatgtccagttaagtgtgcgtcactttt cctacggtgcctcgcacatgaatgttatccggcgcacgatacttatcacc gaaaaaccttattctacggaaaaccttatttacattaaagttggaaaaat ttcctctttttcctaataaggtggagcttttggcttccagtatgctttca cggaattatttctcatgtacatttagctccatttccagtgcctccgatag ggaggcatcatggtactaccgtgacggagaatacgtaggctgactttttc gtcagtttgttgtccgtttacaaaattggtgaatgaattctagccttcct ctgctcattaattgccctcacaagaatttggaagtgcgtagacaggtaaa agattgtactacagaggtattgtggaaccttctacagtacttcggaatac acctaaaaggttgttggatgctaaatttagcaaaagtcttttttagctca ctattaggcttgttaaagtctgaaattgttgaaaggcactcaaaaagata aatcaacaatcagcattaacggcacagttgaaagagtcacccacttgaaa ttagctcggttatcaaatataattatctctggtaaagagctctgcagcag ggttaatctattcgcatacttacgctgtaggaacattttattattaggat ccgactactgcctacatatttattcggaaggcatgatgtcgaaaattttt gagcttataaaaggaacatatttcactcttgctcgttgatgtaagctctc ttccgggttcttatttttaattcttgtcaccagtaaacagaacatccaaa aatgacaatgcctcatcgctatatgtttttggcagtctttacacttctgg cactaactagtgtggcctcaggagccacagaggcgtgcttaccagcaggc cagaggaaaagtgggatgaatataaatttttaccagtattcattgaaaga ttcctccacatattcgaatgcagcatatatgggttatggatatgcctcaa aaaccaaactaggttctgtcggaggacaaactgatatctcgattgattat aatattccctgtgttagttcatcaggcacatttccttgtcctcaagaaga ttcctatggaaactggggatgcaaaggaatgggtgcttgttctaatagtc aaggaattgcatactggagtactgatttatttggtttctatactacccca acaaacgtaaccctagaaatgacaggttattttttaccaccacagacggg ttcttacacattcaagtttgctacagttgacgactctgcaattctatcag taggtggtgcaaccgcgttcaactgttgtgctcaacagcaaccgccgatc acatcaacgaactttaccattgacggtatcaagccatggggtggaagttt gccacctaatatcgaaggaaccgtctatatgtacgctggctactattatc caatgaaggttgtttactcgaacgctgtttcttggggtacacttccaatt agtgtgacacttccagatggtaccactgtaagtgatgacttcgaagggta cgtctattcctttgacgatgacctaagtcaatctaactgtactgtccctg acccttcaaattatgctgtcagtaccactacaactacaacggaaccatgg accggtactttcacttctacatctactgaaatgaccaccgtcaccggtac caacggcgttccaactgacgaaaccgtcattgtcatcagaactccaagaa ctgctagcaccatcataactacaactgagccatggaacagcacttttacc tctacttctaccgaattgaccacagtcactggcaccaatggtgtacgaac tgacgaaaccatcattgtaatcagaacaccaacaacagccactactgcca taactacaactgagccatggaacagcacttttacctctacttctaccgaa ttgaccacagtcaccggtaccaatggtttgccaactgatgagaccatcat tgtcatcagaacaccaacaacagccactactgccatgactacaactcagc catggaacgacacttttacctctacttctaccgaattgaccacagtcacc ggtaccaatggtttgccaactgatgagaccatcattgtcatcagaacacc aacaacagccactactgccatgactacaactcagccatggaacgacactt ttacctctacttctaccgaattgaccacagtcaccggtaccaatggtttg ccaactgatgagaccatcattgtcatcagaacaccaacaacagccactac tgccatgactacaactcagccatggaacgacacttttacctctacatcca ctgaaatcaccaccgtcaccggtaccaatggtttgccaactgatgagacc atcattgtcatcagaacaccaacaacagccactactgccatgactacacc tcagccatgaacgacacttttacctctacatccactgaaatgaccaccgt 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cagcttactggccggtagtggtttaagtgtcttcattgcgtccttattgc tggcaattatttaa flo11 promoter of flor yeast gaattcctaaattctttacagatgacatctgggtctagggagatcatgca aaaaaataagacatcatttataactttcaaccttccgctcacaggactaa ttctcgaagacgctacgaaaagatgctgagcttgatcataattcttcagg ctcccaattcaagaatacaattacttagtgtgggtgcttctgctcagtct tcgtttcctatctccacatacgaatcactcgcttgttactaattcgaata atttactgctgtaaggagtcgtaccgccaactaaatctgaataacaattt ggctgctagaagtcaaaaagtaggcgctgcaatgattatgtggtatgatc agattgtgtcgcaacattcagcggggttttgattcaatgggaccgctgct aataaagtgtttaactagcgaattttggaacacacccgttgacaaaagta taaggagtctctttccgtgagttcccctcttcctctcactgcacttcaac tatgccttatagcaaccaagaagctagaaaatgccaactattaaaaagat aacctctttggaattaaataaaacggagattatcttgggatctatttcac aaatttacggctaattggaatgaacagcgccaagtagcactggacaaact gcagagagtattagaacttgctttattccgtatatcgtactttaggtcgt cagttttaccacttcggtacgcaaatgaaaataaaaagctcattcacaac ttagacacaatttcgccgtttacaagtagctgaaaagtccatctatcgaa ttatgaatgatactattttaaatatcgtcgttactgcactcgttttccac 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gcctacgccagccccagagtatgttctcacagctgtaattcctagtgatc ttttcctggctccaataggaacgccggtaggcaaattaaggtttttttct tctgttttcttgacaagaaaatgtcgcccaaagagtttcggccgttattt tcctatcgaagtgggtcctttttgtctttagtccttctctggggctagcg atcactgcaaaattaggcttcactggtacgagttaactttttttcttttt tttttttgtcatccttttctttggggctaagaatggacttcccttttctt tttctttgttgcagcagtggcttcaaagaactgctgattgctcaaggcaa tcagtccgagcgtttagaaggtgattgtaggcagaaattaactttgcggt aaaagaatgacattctttcttaaaagaaaaattagctttttttttgtcag gcattgcacaaacttttttatttctgcctatactcttaaacagatcagtc attcatgttgtctttttaacggtcgtactgggacatcacatgccttggga ttccgtaattaggtgcaacaatacgggcacaactcattctgcgctatctt cacggacagaacttctattgcctatcggtggtgtgattaacaattggagg cgcagagcttggaatggatttccaattcaatggatttggaggtattcgtt tgtttactaatatttactttgaggacattgcccaaccctaaaagtgcctg ttccagaatagaataacattatgatacgttttcttgaccgctgagcaatt taaagcgattattagggtacgattgtttctagagaaatgtgggtcatctt tttaggtccgttctcttctgatgaggtaacctttacaaaaatgtcataga gttaccaattgggattcaaggcatcatcgcaatatacttcgttcttttac ggagaaattaagctctttctactttgaattaactgttagacttgtcttat ctgaggaatgtccgtgttcaaattaaataaaaatttagggcagttttatt taccttaacaaatatgttcaagcatgtacgttactgcgctctcttctagt tcaagaacggataactcatagacttaccagtacaagttgttgaagggttc ccaattgataaaaaaggatcttttgcttcctgaaataaacgtataaaaag caccctattcatcagttatactccctcatcatgttgtggttctaattaag aatatacttttgtaggcctcaaaatccatatacgcacactatccaaagac gaattc flo11 open reading frame of flor yeast atgcaaagaccatttctactcgcttatttggtcctttcgcttctatttaa ctcagctttgggttttccaactgcactagttcctagaggatcctccgaag gaactagctgtaattctatcgttaatggctgtcccaacttagacttcaat tggcacatgaaccaacaaactatcatgcagtatactttggatgtgacttc cgtttcttgggttcaagacaacacataccaaatcactattcatgtcaaag gtaaagaaaacattgacctaaaatatctacggtctttgaaaatcattggt gtcactggtccaaaaggtaccgtccaactatacggttacaacgaaaatac ctatttgattgacaacccaactgatttcacagccacttttgaagtctatg ccacacaagatgtcaacagctgtcaggtgtggatgcctaacttccaaatt caattcgagtatttgcaaggtagtgccgctcaatatgcaagctcttggaa atggggaactacatctttttatttgtctactggttgtaacaactatgaca atcaaggccactctcaaacagatttcccaggcttctattggaacatagat tgggacaacaattgtggcggtacgaagtcatctaccactacatcaactag tacttccgagtcatctaccactacatctagcacttccgagtcatctacca ctacatcaactaccacttcagaatcatctacatcatcatcaaccaccgct cctgctacaccaaccactacctcatgcactaagaagaaaaccaccagatc taagacatgcactaagaagactactactccagtaccaaccccatcaagct ctactactgaaagttcttctactccagtaaccagctccaccactgaaagc tcttctgctccagtaccaactccatccagctccaccactgaaagctcttc tgctccagctccaaccccatcaagctctactactgaaagctcttctgctc cagtatccagctctactactgaaagctcttctgctccagtaccaactcca tccagctctactaccgaaagctcttctactccagtaaccagctccaccac tgaaagctcttctgctccagccccaaccccatcaagctctactactgaaa gctcctctgctccagtaaccagctctactactgaaagctcttctgctcca gctccaaccccatcaagctctactactgaaagctcctctgctccagtatc cagctctactactgaaagctcttctgctccagtaccaactccatccagct ctactaccgaaagctcttctactccagtaaccagctccaccactgaaagc tcttctgctccagctccaaccccatcaagctctactactgaaagctcctc tgctccagtaaccagctctactactgaaagctcttctgctccagtatcca gctccaccactgaaagctctgtagcaccagtaccaaccccatcaagctct actactgaaagctcttctgctccagtaccaactccatccagctctactac cgaaagctcttctactccagtaaccagctccaccactgaaagctcttctg ctccagctccaaccccatcaagctctactactgaaagctcctctgctcca gtaaccagctctactactgaaagctcttctgctccagtaccaactccatc cagctctactactgaaagctcctctgctccagtatccagctctactactg aaagctcttctgctccagtaccaactccatccagctctactaccgaaagc tcttctactccagtaaccagctccaccactgaaagctcttctgctccagc tccaaccccatcaagctctactactgaaagctcctctgctccagtaacca gctctactactgaaagctcttctgctccagtatccagctccaccactgaa agctctgtagcaccagtaccaaccccatcaagctctactactgaaagctc ttctgctccagtaccaactccatccagctctactaccgaaagctcttcta ctccagtaaccagctccaccactgaaagctcttctgctccagctccaacc ccatcaagctctactactgaaagctcctctgctccagtaaccagctctac tactgaaagctcttctgctccagtaccaactccatccagctctactactg aaagctcttctactccagtaaccagctccaccactgaaagctcttctgct ccagctccaaccccatcaagctctactactgaaagctcctctgctccagt aaccagctctactactgaaagctcttctgctccagtaccaactccatcca gctctactactgaaagctcttctactccagtaaccagctccaccactgaa agctcttctgctccagctccaaccccatcaagctctactactgaaagctc ctctgctccagtaaccagctctactactgaaagctcttctgctccagctc caaccccatcaagctctactactgaaagctcctctgctccagtaaccagc tctactactgaaagctcttctgctccagtaccaactccatccagctctac tactgaaagctcttctgctccagtatccagctccaccactgaaagctctg tagcaccagtaccaaccccatcaagctctactactgaaagctcttctgct ccagtaccaactccatccagctctactaccgaaagctcttctactccagt aaccagctccaccactgaaagctcttctgctccagctccaaccccatcaa gctctactactgaaagctcctctgctccagtaaccagctctactactgaa agctcttctgctcaagtaccaactccatccagctctactactgaaagctc ttctgctccagtatccagctccaccactgaaagctctgtagcaccagtac caaccccatcaagctctactactgaaagctcttctgctccagtaccaact ccatccagctctactaccgaaagctcttctactccagtaaccagctccac cactgaaagctcctctgctccagctccaactccatccagctctactactg aaagctcttctgctccagtatccagctccaccactgaaagctctgtagca ccagtaccaaccccatcaagctctactactgaaagctcttctgctccagt accaactccatccagctctactaccgaaagctcttctactccagtaacca gctccaccactgaaagctctgtagcaccagtaccaaccccatcaagctct actactgaaagctcttctgctccagtatccagctctactactgaaagctc tgtagcaccagtaccaaccccatcttcctctagcaacatcacttcctctg ctccatcttcaactccattcagctctagcactgaaagctctgtagcacca gtaccaaccccatccagctctactactgaaagctcttctgctccagtatc cagctccaccactgaaagctctgtagcaccagtaccaaccccatcttcct ctagcaacatcacttcctccgctccatcttcaactccattcagctctact actgaaagcttttctactggcactactgtcactccatcatcatccaaata ccctggcagtcaaacagaaacctctgtttcttctacaaccgaaactacca ttgttccaactacaactacgacttctgtcactacatcatcaacaaccact attactactacagtttgctctacaggaacaaactctgccggtgaaactac ttctggatgctctccaaagaccattacaactactgttccatgttcaacca gtccaagcgaaaccgcctcggaatcaacaaccacttcacctaccacacct gtaactacagttgtctcaaccaccgtcgttactgctgagtattctactag tacaaaaccaggtggtgaaattacaactacatttgtcaccaaaaacattc caaccacttacctaaccgcaattgctccaactccatcagtcactacggtt accaatttcaccccaaccactattactactacggtttgctctacaggtac aaactctgccggtgaaactacctctggatgctctccaaagactgtcacaa ccactgttccttgttcaactggtactggcgaatacactactgaagctacc acccctgttacaacagctgtcacaaccaccgttgttaccactgaatcctc tacgggtactaactccgctggtaagacgacaactggtcacacaacaaagt ctgtaccaaccacctatgtaaccactttggctccaagtgcaccagtaact cctgccactaatgccataccaactac