Fungus Strain Having Decreased Viscosity

Abstract

The invention relates to a strain of fungus having a reduced viscosity, wherein the ID 78713 (GEL3) gene has been invalidated. The invention also relates to the different uses of this strain, as well as to the method of genetic modification.

Claims

1-16. (canceled)

17. A strain of fungus wherein the ID78713 gene has been invalidated.

18. The fungal strain according to claim 17, said strain having a reduced viscosity compared to a parent strain wherein the ID78713 (GEL3) gene has not been invalidated.

19. The fungal strain according to claim 17, wherein the ID78713 gene has been invalidated by mutagenesis or by homologous recombination, particularly by using an invalidation cassette represented by SEQ ID NO: 1.

20. The fungal strain according to claim 17, wherein the fungus is a filamentous fungus.

21. The fungal strain according to claim 20, wherein the filamentous fungus belongs to the class of sordariomycetes.

22. The fungal strain according to claim 20, wherein the filamentous fungus belongs to the genus Trichoderma.

23. The fungal strain according to claim 20, wherein the filamentous fungus belongs to the species Trichoderma reesei.

24. The fungal strain according to claim 17, wherein the ID78713 gene corresponds to a gene represented by SEQ ID NO: 2 or a gene having at least 80% identity with the gene of SEQ ID NO: 2, particularly at least 90%, and more specifically at least 95%.

25. A method of genetically modifying a fungal strain according to claim 17, comprising a step of invalidating the ID78713 gene.

26. The method of genetically modifying a fungal strain according to claim 25, wherein the step of invalidating the ID78713 gene is carried out by mutagenesis, by homologous recombination, or more preferentially by using an invalidation cassette represented by SEQ ID NO: 1.

27. A method of producing a fungal biomass, comprising a step of culturing a fungal strain according to claim 17 in a culture medium comprising an appropriate substrate.

28. A method of producing proteins of interest, particularly enzymes, comprising a step of culturing a fungal strain according to claim 17 in a culture medium comprising an appropriate substrate.

29. A method of producing bio-sourced products from cellulosic or lignocellulosic substrates, comprising a step of incubating the fungal strain according to claim 17 under suitable conditions to produce cellulolytic enzymes.

30. A method of producing a biofuel from cellulosic or lignocellulosic substrates, comprising a step of using the fungal strain according to claim 17 to produce cellulolytic enzymes.

31. The method of producing a biofuel from cellulosic or lignocellulosic substrates comprising: (i) a step of pretreating a cellulosic or lignocellulosic substrate in order to obtain a pretreated substrate, (ii) a step of using the strain according to claim 1 to produce cellulolytic enzymes, (iii) a step of enzymatically hydrolyzing the pretreated substrate, in the presence of the cellulolytic enzymes obtained in step (ii) and an appropriate substrate, in order to obtain a hydrolysate, (iv) a step of alcoholic fermentation of the hydrolysate obtained, step (iv) being optionally carried out simultaneously with step (iii). (v) a separation step, optionally by distillation.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0094] Other features, details and advantages of the invention will appear upon reading the attached Figures.

[0095] FIG. 1

[0096] FIG. 1 represents the apparent viscosities measured at a shear rate of 5 s.sup.−1 for different strains cultivated in shake flasks.

[0097] FIG. 2

[0098] FIG. 2 represents the apparent viscosities measured at a shear rate of 5 s.sup.−1 for different strains cultivated in a bioreactor (RutC30 and TR3126-ΔGEL3)

[0099] FIG. 3

[0100] FIG. 3 represents the apparent viscosities measured at a shear rate of 5 s.sup.−1 for different strains cultivated in a bioreactor (CL847 and CL847-ΔGEL3)

SEQUENCES OF THE PRESENT INVENTION

[0101]

TABLE-US-00001 TABLE 1 Name of the se- quence Sequence SEQ ID gactaagaga gagagagagg gagcattggc ctctatcgtg atgcctcggt gaatagagag agtgagtgtg tgtgtggtat NO: 1 ttcccactgc tgcgagcttc aattgagagc aatggccatg atattacaca aggttcctac ctaggtaagg taatgcaagg tactacagcg aggtacagta atactagcac gcaaacaaac atgggccgca acacgagagt cgtaaatccc atcgtctacc tactgcctac gagtacatgt agcctcgtga ccaactcccc tccccccctt gtcctacgag gctctttgtg cctggacagc acaggtttcc aaccgtcgca cctcgagcca agaaatccga gccgtttagt agccaattga tgccggatcc tcttcatcct cgtctggggc actttccacc cacccctgtt tttttttctc ttttcttttt tgcgagctac tgctagtcgc attctggcag agatggggaa attgaaaggc tagacaggac gatatccttg attttccctg ttcgtatcgt atctggcttg aaaagatggg ctaacggcaa gggggtctga agcggaacta gtaggacagg gcaggacggg acaggacagg acagggtcaa ggccaaggcc gaggtcgtgg tgcatgctgg tgtacaagta cgatgccata acaatctcat catcgcgctt ctcttgctgg tggacgggat ctgcaggttt cctcctcttt tctcttacca ttcttttgac tctctcggtg cctctgcagc cgctctttct tgcattcgcc ggcttcaatc gcggttgctg gggttattca ctgttggatc caatcactcg ctctttagat aaaacccact ggcttgccag ctgtcgaggc ccgtccgtcc accgctctcg cttcctctcg tctctctcaa ccctcgctcg ctcgctcgct ctctctctgg gtcacgttgc gctcactttc ttggttttcc cccctttccg cctctggggg acaatctgtg gccaaacaat tcttgggttg ggacgagaaa aaaatccgcc ttgtgctcgg ttccctcttt cttttacctt tctctcagtt tcggttttga acccattgac gagcttctct tcttggttgg tttggatctg ctcgaccaca tcgcgagccc ttcattcatc ccatctttct tttttttttt ctcttgttcg ccaagagttt tggaacagtg aacagaattc gatttaccgc cgcttccctt tggaaccaac agcttacctg catttcgact gtgtgacaca cgcaagtgta cctgtgcatt ctgggtaaac gactcatagg agagttgtaa aaaagtttcg gccggcgtat tgggtgttac ggagcattca ctaggcaacc atgcatcctt actattgtat accatcttag taggaatgat ttcgaggttt atacctacga tgaatgtgtg tcctgtaggc ttgagagttc aaggaagaaa catgcaatta tctttgcgaa cccagggctg gtgacggaat tttcatagtc aagctatcag agtaaagaag aggagcatgt caaagtacaa ttagagacaa atatatagtc gcgtggagcc aagagcggat tcctcagtct cgtaggtctc ttgacgaccg ttgatctgct tgatctcgtc tcccgaaaat gaaaatagct ctgctaagct attcttctct tcgccggagc ctgaaggcgt tactaggttg cagtcaatgc attaatgcat tgcagatgag ctgtatctgg aagaggtaaa cccgaaaacg cgttttattc ttgttgacat ggagctatta aatcactaga aggcactctt tgctgcttgg acaaatgaac gtatcttatc gagatcctga acaccatttg tctcaactcc ggctagcgaa ttctcgactc attcctttgc cctcggacga gtgctggggc gtcggtttcc actatcggcg agtacttcta cacagccatc ggtccagacg gccgcgcttc tgcgggcgat ttgtgtacgc ccgacagtcc cggctccgga tcggacgatt gcgtcgcatc gaccctgcgc ccaagctgca tcatcgaaat tgccgtcaac caagctctga tagagttggt caagaccaat gcggagcata tacgcccgga gtcgtggcga tcctgcaagc tccggatgcc tccgctcgaa gtagcgcgtc tgctgctcca tacaagccaa ccacggcctc cagaagaaga tgttggcgac ctcgtattgg gaatccccga acatcgcctc gctccagtca atgaccgctg ttatgcggcc attgtccgtc aggacattgt tggagccgaa atccgcgtgc acgaggtgcc ggacttcggg gcagtcctcg gcccaaagca tcagctcatc gagagcctgc gcgacggacg cactgacggt gtcgtccatc acagtttgcc agtgatacac atggggatca gcaatcgcgc atatgaaatc acgccatgta gtgtattgac cgattccttg cggtccgaat gggccgaacc cgctcgtctg gctaagatcg gccgcagcga tcgcatccat agcctccgcg accggttgta gaacagcggg cagttcggtt tcaggcaggt cttgcaacgt gacaccctgt gcacggcggg agatgcaata ggtcaggctc tcgctaaact ccccaatgtc aagcacttcc ggaatcggga gcgcggccga tgcaaagtgc cgataaacat aacgatcttt gtagaaacca tcggcgcagc tatttacccg caggacatat ccacgccctc ctacatcgaa gctgaaagca cgagattctt cgccctccga gagctgcatc aggtcggaga cgctgtcgaa cttttcgatc agaaacttct cgacagacgt cgcggtgagt tcaggctttt tcatgatggc cctcctaccg gtgatctcag ctgtaggaaa gagaagaagg ttagtagtcg acatggtggc cctcctatag tgagtcgtat tatactatgc cgatatacta tgccgatgat taattgtcaa cactaggcgc cggtcacaac tagtagatat cacttacgtg ttgagaggcg gcatgcgata agaggtgtaa ttacctgaga acatcttgtt gccctgcttt ccgtgcgaaa tactaccggt acttttggga aacaagggaa caggagggcg ctgctgtgcg cggttctgag tgttcaggat tgaagctgaa gaaggtgctg aggaagcgta gaactgttgc ggacgcgagt tctgagaaga gctgtaccga ttggtgaaag ccgaagaagt gagttggtgc cctgttgcct ggataatgtt tgcaactcgc tggttctgca gagacggaga caaatgctgg ctacgatgtt gctgattcag gttgatacct cggtcgagac actgttttgg tttgataggg tggatttggt tgcagagaag agaaaggaag gtcaaagagg gaaaactggg cggagggaag gattttgtat caggcagcaa actgccactg cagtggccct ggcagtgccg ggcgaggcac ccacgcacgg ccgcgcaacc ggttggtcct tgcccaccac gaaacccttc tgaaaggtca gatggaagtg tgcgacagtg cgcgtcccca agccaatgca ggcgccatgc actccccacc cgcaagattc actgtgcgtt cttattggtt gccgcaaggc cagccaaagg gggaagtatg agtcacagca ccgatacaag aaaattgcag aactaacata tggatgcgcg cgctattctg tagagctctg ggcaaagcac caatcctgcg ggtcggtaca cacactagca ctgccattct ggccgtcaag accgaaactg tcagcgtggc caccgagacc ggtaccagca cggcggagag caccgcagac agcacctctg gcgacagcac gggtgctgcc acgacttcta ccccgaaatc atcatcttcc accacctcta gcgccgaaag cacggagacg tccaacggca gccccttgga caagcgcgtc actgcgtttg gtgccggcct ggttggcgtc gttttgggtg ctgcggtctt gttgtaagat ggattcaagt tggttcgttg gatttgtatc gtcaaatcag tatcagatac ccatctactt ctcgccatgc ttttctgctc tttgtttctt ttttttgatg ttaccaaggg gcttgagctt gttggcctaa attcccccct aatgctcttt gtatgatggc tgaacgtttg gagaggggat ggttttgttc tatgacattt gccgcgtaac gaggcatgac tttattatat taatgcatga ggatatgttg tacatgaggt gtgaagagcc cttgcttatt tatggtgtgg tgtggtctga cgccgaccaa ccctgtcatc gtgttcttgt tattatgcca cgcgctatcc tagctgaaat acgggaaccg ataattgccc tcgcgttgtg aaccctcgcg ttgtgaaccc ttgcgttgtg aaccctcgaa aaccgtgttt cccccttttc atatatgctt tttcatcttc tttctatcca atatctgcgg ttttggtttc atctagtgga actgtaatcg taggtgagag tagtgttttt aggccagtaa tggttatagt tgtgattgtt tctgaagtat tctgattcag cctggtggca ctgatattcg tcgtcgacaa aggtatctcg gtcgacaaag gtatgctggt cgaggcggtt gtatcgtcgt cgtcagagga ggaagaggac gagtaggtcc tgacggccga gttgccgacg aagacgaggc tgacggtgaa gatggcgagg acggaggcac agaggaggcc catggccagg tagcggatgc gcgggtcgta gcggtggggg gaagagcccc agggggtgcg ggtgtgggtt tggcattgga ggttgtcgat catttatacg gttttttttc tcttgttttg attttgtagg ttgtttgagc ttgattgatg ttgctgaagt atgtggttga gaatt SEQ ID ATGCGTTGGTCTTCGGTTGCCGTTGCGCTGGCGAGCGCCAAATCCTTTGCCGTGGCGCTG NO: 2 GATCCCGTCTCCGTCGTGGGAAACAAGTTCTTCAACAAGGACGGCTCGCAGTTCTTTATCA AGGGCATTGCGTACCAGCTTGTTCCGCAGGACCCCCTCGTGGACACTGACCAGTGCAAAC GCGATGCTAAGCTCATGGCCGAGCTCGGAACAAACACCATCCGCGTCTATCACGTCGACC CGGACGCCGACCATGACGGCTGCATGAGTGCGTTTGACGACGCCGGCATCTACGTCCTTG CTGATCTGGATACCTTTGATACCTACATTATTCCCCAAAATAACTACTGGAATAAGACAAAGT TCGACAGGTATGCCGAGGTCCTGGACACGTTCCAAAAGTACGACAACCTCCTGGGCGTCT TCGTCGGAAACGAGAACATCGCTACTAAAGATGACTCGCCCACGGCGCCCTACCTCAAGG CTGCTGCCCGCGACATGAAGGCGTACCGCGACGCCCAGGGCTACCGCGAGATCCCCGTC GGCTACTCGGCCGCCGATATCTTGCAGCTTCGCCCCATGCTCCAGGACTACCTGACCTGC GGCGGCAACTCGTCCGAGACAGTCGACTTCTTCGCCCTCAACTCGTACTCGTGGTGCGAC CCCAGCACGTACAAGGAGTCCACCTACGACCAGCTTGAGGCCTACGCCAAGAAATTCCCC GTGCCCATCTTCCTCTCCGAGACAGGCTGCATCGTTCCCGGCCCTCGCCAGTTCGACGAC CAGGACGCCATCTTTGGCCCTGAGATGGTCAATGACTGGAGCGGCGCCATCATCTACGAG TGGATTCAGGAGGAAAACGGCTACGGAATTATAACATATGCCCCAGCCGGCCAAGCTGCT GGACCCAACGTCGAGGGTGGCTTCCTTCGCAAGGGCACTCCCACGCCAAAGCTGCCCGA CTTCACCGCGCTCAAGTCCAAGTGGGCGACCAACACCCCTACCGGCGTCAGCCGAGACGA CTACGACGCAAAGGACGTGTCGACCCGTGCGTGTCCTTCGTCCACTGCCGGCGGCTGGTG GCAGGTGGATGGCGATGCCAAATTGCCCACCCTGGGCCAG SEQ ID MRWSSVAVALASAKSFAVALDPVSVVGNKFFNKDGSQFFIKGIAYQLVPQDPLVDTDQCKRDA NO: 3 KLMAELGTNTIRVYHVDPDADHDGCMSAFDDAGIYVLADLDTFDTYIIPQNNYWNKTKFDRYAE VLDTFQKYDNLLGVFVGNENIATKDDSPTAPYLKAAARDMKAYRDAQGYREIPVGYSAADILQL RPMLQDYLTCGGNSSETVDFFALNSYSWCDPSTYKESTYDQLEAYAKKFPVPIFLSETGCIVPG PRQFDDQDAIFGPEMVNDWSGAIIYEWIQEENGYGIITYAPAGQAAGPNVEGGFLRKGTPTPKL PDFTALKSKWATNTPTGVSRDDYDAKDVSTRACPSSTAGGWWQVDGDAKLPTLGQ SEQ ID GACTAAGAGAGAGAGAGAGGGAGC NO: 4 SEQ ID AATTCTCAACCACATACTTCAGCA NO: 5 SEQ ID AACACCCAATACGCCGGC NO: 6 SEQ ID GCTCTGGGCAAAGCACCAA NO: 7 SEQ ID GTGCGGGATGCATGAAGACG NO: 8 SEQ ID CCACCCTACGATTCTGCAACC NO: 9 SEQ ID gactaagaga gagagagagg gagcattggc ctctatcgtg atgcctcggt gaatagagag agtgagtgtg NO: 10 tgtgtggtat ttcccactgc tgcgagcttc aattgagagc aatggccatg atattacaca aggttcctac ctaggtaagg taatgcaagg tactacagcg aggtacagta atactagcac gcaaacaaac atgggccgca acacgagagt cgtaaatccc atcgtctacc tactgcctac gagtacatgt agcctcgtga ccaactcccc tccccccctt gtcctacgag gctctttgtg cctggacagc acaggtttcc aaccgtcgca cctcgagcca agaaatccga gccgtttagt agccaattga tgccggatcc tcttcatcct cgtctggggc actttccacc cacccctgtt tttttttctc ttttcttttt tgcgagctac tgctagtcgc attctggcag ctaacggcaa agatggggaa attgaaaggc tagacaggac gatatccttg attttccctg ttcgtatcgt atctggcttg aaaagatggg gggggtctga agcggaacta gtaggacagg gcaggacggg acaggacagg acagggtcaa ggccaaggcc gaggtcgtgg tgcatgctgg tgtacaagta cgatgccata acaatctcat catcgcgctt ctcttgctgg tggacgggat ctgcaggttt cctcctcttt tctcttacca ttcttttgac tctctcggtg cctctgcagc cgctctttct tgcattcgcc ggcttcaatc gcggttgctg gggttattca ctgttggatc caatcactcg ctctttagat aaaacccact ggcttgccag ctgtcgaggc ccgtccgtcc accgctctcg cttcctctcg tctctctcaa ccctcgctcg ctcgctcgct ctctctctgg gtcacgttgc gctcactttc ttggttttcc cccctttccg cctctggggg acaatctgtg gccaaacaat tcttgggttg ggacgagaaa aaaatccgcc ttgtgctcgg ttccctcttt cttttacctt tctctcagtt tcggttttga acccattgac gagcttctct tcttggttgg tttggatctg ctcgaccaca tcgcgagccc ttcattcatc ccatctttct tttttttttt ctcttgttcg ccaagagttt tggaacagtg aacagaattc gatttaccgc cgcttccctt tggaaccaac agcttacctg catttcgact gtgtgacaca cg SEQ ID caagtgta cctgtgcatt ctgggtaaac gactcatagg agagttgtaa aaaagtttcg geeggegtat tgggtgttac NO: 11 ggagcattca ctaggcaacc atgcatcctt actattgtat accatcttag taggaatgat ttcgaggttt atacctacga tgaatgtgtg teetgtagge ttgagagttc aaggaagaaa catgcaatta tctttgcgaa cccagggctg gtgacggaat tttcatagtc aagctatcag agtaaagaag aggagcatgt caaagtacaa ttagagacaa atatatagtc gcgtggagcc aagagcggat tcctcagtct cgtaggtctc ttgacgaccg ttgatctgct tgatctcgtc tcccgaaaat gaaaatagct ctgctaagct attcttctct tcgccggagc ctgaaggcgt tactaggttg cagtcaatgc attaatgcat tgcagatgag ctgtatctgg aagaggtaaa cccgaaaacg cgttttattc ttgttgacat ggagctatta aatcactaga aggcactctt tgctgcttgg acaaatgaac gtatcttatc gagatcctga acaccatttg tctcaactcc ggctagcgaa ttctcgactc attcctttgc cctcggacga gtgctggggc gtcggtttcc actatcggcg agtacttcta cacagccatc ggtccagacg gccgcgcttc tgcgggcgat ttgtgtacgc ccgacagtcc cggctccgga tcggacgatt gcgtcgcatc gaccctgcgc ccaagctgca tcatcgaaat tgccgtcaac caagctctga tagagttggt caagaccaat gcggagcata tacgcccgga gtcgtggcga tcctgcaagc tccggatgcc tccgctcgaa gtagcgcgtc tgctgctcca tacaagccaa ccacggcctc cagaagaaga tgttggcgac ctcgtattgg gaatccccga acatcgcctc gctccagtca atgaccgctg ttatgcggcc attgtccgtc aggacattgt tggagccgaa atccgcgtgc acgaggtgcc ggacttcggg gcagtcctcg gcccaaagca tcagctcatc gagagcctgc gcgacggacg cactgacggt gtcgtccatc acagtttgcc agtgatacac atggggatca gcaatcgcgc atatgaaatc acgccatgta gtgtattgac cgattccttg cggtccgaat gggccgaacc cgctcgtctg gctaagatcg gccgcagcga tcgcatccat agcctccgcg accggttgta gaacagcggg cagttcggtt tcaggcaggt cttgcaacgt gacaccctgt gcacggcggg agatgcaata ggtcaggctc tcgctaaact ccccaatgtc aagcacttcc ggaatcggga gcgcggccga tgcaaagtgc cgataaacat aacgatcttt gtagaaacca tcggcgcagc tatttacccg caggacatat ccacgccctc ctacatcgaa gctgaaagca cgagattctt cgccctccga gagctgcatc aggtcggaga cgctgtcgaa cttttcgatc agaaacttct cgacagacgt cgcggtgagt tcaggctttt tcatgatggc cctcctaccg gtgatctcag ctgtaggaaa gagaagaagg ttagtagtcg acatggtggc cctcctatag tgagtcgtat tatactatgc cgatatacta tgccgatgat taattgtcaa cactaggcgc cggtcacaac tagtagatat cacttacgtg ttgagaggcg gcatgcgata agaggtgtaa ttacctgaga acatcttgtt gccctgcttt ccgtgcgaaa tactaccggt acttttggga aacaagggaa caggagggcg ctgctgtgcg cggttctgag tgttcaggat tgaagctgaa gaaggtgctg aggaagcgta gaactgttgc ggacgcgagt tetgagaaga gctgtaccga ttggtgaaag ccgaagaagt gagttggtgc cctgttgcct ggataatgtt tgcaactcgc tggttctgca gagacggaga caaatgctgg ctacgatgtt gctgattcag gttgatacct cggtcgagac actgttttgg tttgataggg tggatttggt tgcagagaag agaaaggaag gtcaaagagg gaaaactggg cggagggaag gattttgtat caggcagcaa actgccactg cagtggccct ggcagtgccg ggcgaggcac ccacgcacgg ccgcgcaacc ggttggtcct tgcccaccac gaaacccttc tgaaaggtca gatggaagtg tgcgacagtg cgcgtcccca agccaatgca ggcgccatgc actccccacc cgcaagattc actgtgcgtt cttattggtt gccgcaaggc cagccaaagg gggaagtatg agtcacagca ccgatacaag aaaattgcag aactaacata tggatgcgcg cgctattctg tagagctctg ggcaaagcac caatcctgcg ggtcggtaca cacactagca ctgcc SEQ ID attct ggccgtcaag accgaaactg tcagcgtggc caccgagacc ggtaccagca cggcggagag NO: 12 caccgcagac agcacctctg gcgacagcac gggtgctgcc acgacttcta ccccgaaatc atcatcttcc accacctcta gcgccgaaag cacggagacg tccaacggca gccccttgga caagcgcgtc actgcgtttg gtgccggcct ggttggcgtc gttttgggtg ctgcggtctt gttgtaagat ggattcaagt tggttcgttg gatttgtatc gtcaaatcag tatcagatac ccatctactt ctcgccatgc ttttctgctc tttgtttctt ttttttgatg ttaccaaggg gcttgagctt gttggcctaa attcccccct aatgctcttt gtatgatggc tgaacgtttg gagaggggat ggttttgttc tatgacattt gccgcgtaac gaggcatgac tttattatat taatgcatga ggatatgttg tacatgaggt gtgaagagcc cttgcttatt tatggtgtgg tgtggtctga cgccgaccaa ccctgtcatc gtgttcttgt tattatgcca cgcgctatcc tagctgaaat acgggaaccg ataattgccc tcgcgttgtg aaccctcgcg ttgtgaaccc ttgcgttgtg aaccctcgaa aaccgtgttt cccccttttc atatatgctt tttcatcttc tttctatcca atatctgcgg ttttggtttc atctagtgga actgtaatcg taggtgagag tagtgttttt aggccagtaa tggttatagt tgtgattgtt tctgaagtat tctgattcag cctggtggca ctgatattcg tcgtcgacaa aggtatctcg gtcgacaaag gtatgctggt cgaggcggtt gtatcgtcgt cgtcagagga ggaagaggac gagtaggtcc tgacggccga gttgccgacg aagacgaggc tgacggtgaa gatggcgagg acggaggcac agaggaggcc catggccagg tagcggatgc gcgggtcgta gcggtggggg gaagagcccc agggggtgcg ggtgtgggtt tggcattgga ggttgtcgat catttatacg gttttttttc tcttgttttg attttgtagg ttgtttgagc ttgattgatg ttgctgaagt atgtggttga gaatt

LIST OF REFERENCES CITED IN THE PRESENT PATENT APPLICATION

[0102] Durand H, Clanet M, Tiraby G. Genetic improvement of Trichoderma reesei for large scale cellulase production. Enzyme Microb Technol 1988, 10:341-346. [0103] Guangtao Z, Hartl L, Schuster A, Polak S, Schmoll M, Wang T, Seidl V, Seiboth B., (2009). Gene targeting in a nonhomologous end joining deficient Hypocrea jecorina. J Biotechnol. 139(2):146-51. [0104] Montenecourt, B. S.; Eveleigh, D. E. (1977) Semiquantitative Plate Assay for Determination of Cellulase Production by Trichoderma viride. In: Applied and environmental microbiology, vol. 33, No. 1, p. 178-183 [0105] Penttila M, Nevalainen H, Rättö M, Salminen E, Knowles J., (1987). A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei. Gene. 61(2):155-64. [0106] Punt, P. J.; van den Hondel, C. A., (1992) Transformation of filamentous fungi based on hygromycin B and phleomycin resistance markers. In: Methods in enzymology, vol. 216, p. 447-457. [0107] Te'o V S, Bergquist P L, Nevalainen K M., (2002). Biolistic transformation of Trichoderma reesei using the Bio-Rad seven barrels Hepta Adaptor system. J Microbiol Methods. 51(3):393-9.

EXAMPLES

Example 1a: Invalidation of the ID78713 (GEL3) Gene in a Hyper-Producing Strain

[0108] The invalidation cassette for ID78713 (GEL3) is composed of the hygromycin antibiotic resistance gene hph placed under the control of the GPDa promoter and the TRPc terminator (Punt and van den Hondel, 1992), with 5′ and 3′ flanking regions upstream and downstream from the ID78713 (GEL3) gene. This sequence is represented in SEQ ID NO: 1. The DNA cassette was synthesized and the cassette was inserted into a pEX-A plasmid (available, for example, from Addgene). After amplification and extraction of the plasmid, the invalidation cassette was amplified by PCR (Polymerase Chain Reaction) using primers p61 and p62 (see Table 2 below).

TABLE-US-00002 TABLE 2 Primer name Sequences corresponding to the primer P61 SEQ ID NO: 4 P62 SEQ ID NO: 5 P78 SEQ ID NO: 6 P79 SEQ ID NO: 7 P91 SEQ ID NO: 8 P92 SEQ ID NO: 9

[0109] Primer Sequences of the Present Invention

[0110] The strain used for the transformation is the hyper-producing strain RutC30 (Montenecourt and Eveleigh, 1977) in which the gene KU70 (ID 63200) was invalidated by replacing the coding sequence by the gene encoding the selection marker AmdS (Pentillä et al., 1987). Invalidation of this gene promotes homologous recombination (Guangtao et al., 2009). This strain is called TR3126.

[0111] Transformations of the TR3126 strain with the cassette represented by SEQ ID NO: 1 were carried out by Biolistique (Te'o et al., 2002) by using 5 μg of purified cassette. Integration at the locus of the invalidation cassette was verified by PCR with a primer (p91) upstream from the cassette and a primer (p78) in the hph gene (5′ verification) and a primer (p92) downstream from the cassette and a primer (p79) in the hph gene (3′ verification). The strains invalidated by the ID78713 (GEL3) gene thus obtained are called TR3126-ΔGEL3.

Example 1b: Invalidation of the ID78713 (GEL3) Gene in a Hyper-Producing Strain

[0112] A second strain was tested: the CL847 strain (Durand H et al.).

[0113] Transformation of the CL847 strain was carried out by the protoplast method (Penttila M et al., doi: 10.1016/0378-1119(87)90110-7). Similarly to example 1a, integration at the locus of the invalidation cassette was verified. The strains invalidated by the ID78713 (GEL3) gene thus obtained are called CL847-ΔGEL3.

Example 2: Method of Measuring Viscosity

[0114] For the rheological measurement, the shaft (rotor) used is a large impeller of stainless steel with a diameter of 38 mm, a height of 32 mm, a pitch of 29 mm, and a ribbon 8 mm in width. This impeller is used with a cup (stator) with an inner diameter of 45 mm and a vertical space between the rotor and the stator of 500 μm. After calibration, the impeller is similar to a Couette cylinder with a radius of 14 mm.

[0115] The cup is filled with 70 mL of fermentation must collected from a reactor (shake flask, for example as indicated in Example 3, or bioreactor). The viscosity measurements are carried out by logarithmic shear rate sweeps of between 4 s.sup.−1 and 100 s.sup.−1, at a temperature of 27° C. This range corresponds to the average shear rates expected on an industrial scale. The sweeps are two-way sweeps (from 4 s.sup.−1 to 100 s.sup.−1 and then from 100 s.sup.−1 to 4 s.sup.−1). The rheological measurements have been carried out in duplicate.

[0116] All measurements are carried out on the TA Instruments AR 2000 rheometer.

Example 3: Culture Protocol in Shake Flasks

[0117] Shake flask culturings are carried out in Fernbach flasks with a diameter of 19 cm, containing 400 mL of culture medium, seeded with spores of different strains from cryotubes, and incubated at 150 rpm and 30° C. in an Infors Multitron incubator.

[0118] The culture medium has the following final composition: [0119] 5.6 g/L of (NH.sub.4).sub.2SO.sub.4 [0120] 4.4 g/L of K.sub.2HPO.sub.4 [0121] 0.3 g/L of MgSO.sub.4, 7H.sub.2O [0122] 0.15 g/L of CaCl.sub.2, 2H.sub.2O [0123] 1 mL/L of trace element solution (FeSO.sub.4: 5 g/L, MnSO.sub.4: 1.4 g/L, ZnSO.sub.4: 1.4 g/L, CoCl.sub.2: 3.7 g/I) [0124] 5.85 g/L of BTCA (butane tetracarboxylic acid) [0125] 3.0 g/L of KOH in crystals [0126] 1.5 g/L of corn steep liquor (for example Roquette Solulys®) [0127] 30 g/L of glucose

[0128] The pH of the culture medium is adjusted to 6.0 with 30% sodium hydroxide.

[0129] The compounds are sterilized for 20 minutes at 121° C. (the glucose is sterilized separately from the other compounds).

[0130] Regular 2 mL samples are taken to monitor the residual glucose. Then when the residual glucose is less than 5 g/L (which corresponds to a fungal concentration on the order of 10 g/L), a 100 mL sample is taken to measure precisely the fungal concentration (by filtration and then drying on 1.2 μm filters) and the must viscosity (according to the method described in example 2).

Example 4: Comparison of Viscosities in Shake Flask Cultures

[0131] Two strains were cultivated in duplicate according to the method described in Example 3, and then the viscosity of the fermentation must was characterized according to the method described in Example 2:

[0132] The two strains tested are: [0133] The TR3126-ΔGEL3 strain presenting invalidation of the ID78713 (GEL3) gene; [0134] The parent strain TR3126 used as a high-viscosity control

[0135] Fungal concentration measurements show that the concentrations are of the same order in all culturing carried out, around 10 g/L of fungus in suspension (see Table 3 below).

TABLE-US-00003 TABLE 3 Fungal concentration (g/L) Strains Replicate 1 Replicate 2 TR3126 9.4 8.7 TR3126-ΔGEL3 8.6 11.6

[0136] Fungal concentration for the strains tested (experiments carried out in duplicate)

[0137] The viscosity measurements (presented in FIG. 1) show that invalidation of the ID78713 (GEL3) gene led to a drastic lowering of viscosity. At a shear rate of 5 s.sup.−1, the viscosity obtained for the TR3126-ΔGEL3 strain is approximately 8 to 10 times lower than that of the viscous control (TR3126).

Example 5: Culture Protocol in Bioreactor

[0138] Bioreactor culturings are carried out in fermenters with a diameter of 16 cm, containing 2 L of culture medium, seeded at 10% v/v from a pre-culture done according to the protocol described in Example 3. Agitation is performed by a Rayneri turbine with a diameter of 8 cm at a fixed speed of 1000 rpm. The temperature is controlled at 27° C., and the pH is controlled at 4.8 by the automatic addition of 5N ammonia solution.

[0139] The culture medium has the following final composition: [0140] 3 mL/L of orthophosphoric acid at 85% [0141] 0.25 mL/L of sulfuric acid at 96% [0142] 1.66 g/L of potassium hydroxide KOH in crystals [0143] 2.8 g/L of (NH.sub.4).sub.2SO.sub.4 [0144] 0.6 g/L of MgSO.sub.4, 7H.sub.2O [0145] 0.6 g/L of CaCl.sub.2, 2H.sub.2O [0146] 0.12 g/L of Na.sub.2HPO.sub.4, 12H.sub.2O [0147] 1 mL/L of trace element solution (FeSO.sub.4: 5 g/L, MnSO.sub.4: 1.4 g/L, ZnSO.sub.4: 1.4 g/L, CoCl.sub.2: 3.7 g/I) [0148] 1 g/L of corn steep liquor (for example Roquette Solulys®) [0149] 80 g/L of glucose

[0150] The compounds are sterilized for 20 minutes at 121° C. (the glucose is sterilized separately from the other compounds).

[0151] The pH of the culture medium is adjusted and then controlled at 4.8 with the ammonia solution used to check the pH.

[0152] Regular samples of approximately 100 mL are taken to: (i) monitor the residual glucose, (ii) precisely measure the fungal concentration (by filtration and then drying on 1.2 μm filters) and (iii) measure the viscosity of the must (according to the method described in Example 2).

Example 6: Comparison of Viscosities in Bioreactor Cultures

[0153] Two strains were cultivated in duplicate according to the method described in Example 5, and the viscosity of the fermentation must was characterized (for different concentrations of fungus in the must) according to the method described in Example 2: [0154] The TR3126-Δ GEL3 strain presenting invalidation of the ID78713 (GEL3) gene [0155] The reference strain Rut-C30 (used as a high-viscosity control)

[0156] The characterization of viscosity at different fungal concentrations shows a real advantage conferred by invalidation of the ID78713 (GEL3) gene (see FIG. 2) with: [0157] as in shake flasks, a viscosity approximately 10 times lower when the fungal concentration is on the order of 10 g/L; [0158] a viscosity approximately 3 times lower when the concentration is on the order of 25 g/L; [0159] the viscosity of the strain invalidated for ID78713 (GEL3) (TR3126-ΔGEL3) at 35 g/L of the same order as the viscosity of the wild-type strain (Rut-C30) at 15 g/L.

[0160] Thus, a culture of the strain invalidated for ID78713 (GEL3) at 35 g/L does not require more energy for agitation than a culture of the wild-type strain at 15 g/L, which enables the productivity of the culture to be increased at the same energy expenditure.

Example 7: Comparison of Viscosities of Two Other Strains in Bioreactor Cultures

[0161] Two strains were cultivated according to the method described in Example 5, and the viscosity of the fermentation must was characterized (for different concentrations of fungus in the must) according to the method described in Example 2: [0162] The CL847-ΔGEL3 strain presenting invalidation of the ID78713 (GEL3) gene [0163] The parent strain CL847 (used as a control)

[0164] The characterization of the viscosity at different fungal concentrations again shows in the CL847 strain an advantage conferred by invalidation of the ID78713 (GEL3) gene (see FIG. 3) with a viscosity approximately 3 times lower when the fungal concentration is on the order of 12 to 14 g/L.