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WO2001025468A1 - Criblage a rendement eleve de banques d'adn exprimees, dans des champignons filamenteux - Google Patents

Criblage a rendement eleve de banques d'adn exprimees, dans des champignons filamenteux Download PDF

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Publication number
WO2001025468A1
WO2001025468A1 PCT/US2000/010199 US0010199W WO0125468A1 WO 2001025468 A1 WO2001025468 A1 WO 2001025468A1 US 0010199 W US0010199 W US 0010199W WO 0125468 A1 WO0125468 A1 WO 0125468A1
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Prior art keywords
protein
fungus
expression
clonal
library
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PCT/US2000/010199
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English (en)
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Mark Aaron Emalfarb
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Mark Aaron Emalfarb
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Priority claimed from PCT/NL1999/000618 external-priority patent/WO2000020555A2/fr
Application filed by Mark Aaron Emalfarb filed Critical Mark Aaron Emalfarb
Priority to AU43527/00A priority Critical patent/AU4352700A/en
Publication of WO2001025468A1 publication Critical patent/WO2001025468A1/fr
Priority to IL14693501A priority patent/IL146935A0/xx
Priority to AU53544/01A priority patent/AU5354401A/en
Priority to BR0105795-2A priority patent/BR0105795A/pt
Priority to DE60138947T priority patent/DE60138947D1/de
Priority to EA200200035A priority patent/EA006873B1/ru
Priority to CN01801513.1A priority patent/CN1380905A/zh
Priority to US09/834,434 priority patent/US7122330B2/en
Priority to ES01927056T priority patent/ES2328011T3/es
Priority to CA002376552A priority patent/CA2376552A1/fr
Priority to MXPA01012905A priority patent/MXPA01012905A/es
Priority to DK01927056T priority patent/DK1272669T3/da
Priority to EP01927056A priority patent/EP1272669B1/fr
Priority to CN2010106218825A priority patent/CN102174551A/zh
Priority to PCT/US2001/012335 priority patent/WO2001079558A1/fr
Priority to KR1020017016040A priority patent/KR20020026456A/ko
Priority to JP2001576942A priority patent/JP5138855B2/ja
Priority to AT01927056T priority patent/ATE433486T1/de
Priority to US11/490,761 priority patent/US7794962B2/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries

Definitions

  • the invention provides a method for the the expression and subsequent screening of synthetic, genomic, and cDNA libraries in filamentous fungal hosts.
  • the system employs transformed or transfected mutant fungal strains which exhibit a morphology that minimizes or eliminates the formation of entangled mycelia.
  • the fungal strains are also capable of efficient sporulation under submerged growth conditions, and are capable of expressing isolatable quantities of exogenous proteins for evaluation.
  • the mutant fungal strains of the invention are particularly well-suited for high-throughput screening techniques due to their unique morphology and the very low viscosity of their cultures.
  • RNA transc ⁇ pts any genes not actively being transc ⁇ bed will not be represented in the library.
  • Many desirable proteins are expressed only under specific conditions (e g , virulence factors in pathogenic fungi) and these conditions may not exist at the time the mRNA is harvested.
  • virulence factors in pathogenic fungi virulence factors in pathogenic fungi
  • E coli is incapable of secretion of many proteins, and thus is undesirable as a host cell for screening purposes where the screening relies upon secretion of the gene product.
  • yeast tend to hyper-glycosylate exogenous proteins (Bretthauer and Castellino, 1999, Biotechnol. Appl. Biochem. 30:193-200), and although yeast are capable of coping with a limited number of introns they are not generally capable of handling complex genes from higher species such as vertebrates. Even genes from filamentous fungi are usually too complex for yeast to transcribe efficiently, and this problem is compounded by differences in expression and splicing sequences between yeast and filamentous fungi (e.g., see M. Innis et al, Science (1985) 228:21-26).
  • yeast expression systems have been developed which are used to screen for naturally secreted and membrane proteins of mammalian origin (Klein, et al, 1996 Proc. Natl. Acad. Sci. USA 93:7108-7113; Treco, U.S. patent 5,783,385), and for heterologous fungal proteins (Dalboge and Heldt-Hansen, Mol. Gen. Genet. 243:253- 260 (1994)) and mammalian proteins (Tekamp-Olson and Meryweather, U.S. patent 6,017,731).
  • yeast as used in the context of yeast expression systems generally refers to organisms of the order Saccharomycetales, such as S. cerevisiae and Pichiapastoris.
  • fungi and “fungal” should be understood to refer to Basidiomycetes, Zygomycetes, Oomycetes, and Chythridiomycetes, and Ascomycetes of the class Euascomycetes, which are not of the order Saccharomycetales.
  • filamentous fungi superior hosts for screening genomic DNA from soil samples. It also makes them excellent hosts for the production of fungal enzymes of commercial interest, such as proteases, cellulases, and amylases. It has also been found that filamentous fungi are capable of transcribing, translating, processing, and secreting the products of other eucaryotic genes, including mammalian genes. The latter property makes filamentous fungi attractive hosts for the production of proteins of biomedical interest.
  • Yelton et al U.S. Pat. No. 4,816,405, discloses the modification of filamentous Ascomycetes to produce and secrete heterologous proteins.
  • Buxton et al in U.S. Pat. No. 4,885,249, and in Buxton and Radford, Mol. Gen. Genet. 196:339-344 (1984), discloses the transformation of Aspergillus niger by a DNA vector that contains a selectable marker capable of being incorporated mto the host cells.
  • McKnight et al U.S. patent. 4,935,349, and Boel, in U.S.
  • patent 5,536,661 disclose methods for expressing eukaryotic genes in Aspergillus involving promoters capable of directing the expression of heterologous genes in Aspergillus and other filamentous fungi.
  • Conneely et al in U.S. patent 5,955,316, discloses plasmid constructs suitable for the expression and production of lactofer ⁇ n in Aspergillus. Cladosporium glucose oxidase had been expressed in Aspergillus, see U.S. patent 5,879,921.
  • desc ⁇ be a transformation system for Rhizopus in U.S. patent 5,436,158.
  • Sismega-Barroso et al. desc ⁇ be a transformation system for filamentous fungi in WO 99/51756, which employs promoters of the glutamate dehydrogenase genes from Aspergillus awamori. Dantas-Barbosa et al, in FEMS Microbiol. Lett. 169:185-90 (1998), desc ⁇ be transformation of Humicola grisea var. thermoidea to hygromycin B resistance, using either the lithium acetate method or electroporation.
  • filamentous fungi which tend to form entangled mats of myceha and highly viscous suspension (submerged) cultures. They are not amenable to micropipetting of suspension cultures into microtiter plates, and are not easily separated into separate clones on a large scale, as would be required in a high-throughput assay system.
  • filamentous fungi also cause some problems in the mdust ⁇ al production of enzymes in fungal host cells. For example, high viscosity and/or the local formation of dense aggregates of mycelium, leads to difficulties m agitation, aeration, and nut ⁇ ent diffusion.
  • the method comp ⁇ ses screenmg mutants of a parent fungal cell lme, rather than wild-type strains, to find a specific altered morphology, transforming the mutant, and assessing whether a culture of the transformed mutant produces more heterologous protem than the parent cell lme.
  • Mutants with at least 10% greater hyphal branchmg are particulary claimed. The method is illustrated for strains of Fusanum and Aspergillus, and is suggested to be applicable to numerous other genera. The effect of branchmg frequency on culture viscosity of Aspergillus oryzae mutants was examined by Bocking et al, Biotechnol Bioeng. 65:638-648 (1999); more branched strains exhibited lower viscosity in this study.
  • the method mvolves transforming the microorganisms with the SsgA gene of Streptomyces gnseus.
  • the method is demonstrated in filamentous bacte ⁇ a of the order Adinomycetales, but is stated to be applicable to filamentous fungi.
  • a fungal host cell suitable for expression of a DNA library are different in many respects from the characteristics of hosts suitable for industrial protein manufacture.
  • a suitable fungal host for high-throughput screening should meet numerous criteria: The host must be transformed with high efficiency.
  • the host must process intron-containing genes and carry out any necessary splicing.
  • the host must post-translationally process the expressed protein so that it is produced in an active form.
  • the host should be capable of secretion of the protein.
  • the host must produce the protein in high enough yield for detection by the assay.
  • the host should accept a variety of expression regulatory elements, for ease of use and versatility.
  • the host should permit the use of easily-selectable markers.
  • the host cell cultures should be of low viscosity.
  • the host should be deficient in proteases and/or be anemable to suppression of protease expression.
  • the host must permit screens for a wide variety of exogenous protein activities or properties.
  • the host should secrete only the exogenous protein.
  • the hyphae in a culture of the host fungus should not be so entangled as to prevent the isolation of single clones, and should not be so entangled as to raise the viscosity to the point of preventing efficient transfer and replication in a miniaturized high throughput screening format (e.g. by micropipeting), and/or
  • the host should allow the efficient production of spores or other propagules under the growth conditions provided in the high throughput screen. It would be particularly advantageous if the host also expressed enough heterologous protem to enable isolation and pu ⁇ fication of the protem. A host cell with this characte ⁇ stic would make it possible to further characte ⁇ ze all heterologous protems of interest merely by culturing the host cells, without time- consuming molecular biological manipulations. It would also be advantageous if the host cell were amenable to ready isolation of the heterologous DNA, so that furthe studies and modifications of the gene itself may be earned out.
  • the transformation system should also exhibit certrain characte ⁇ stics.
  • the transformation frequency should be sufficiently high to generate the numbers of transformants required for meaningful screens.
  • expression of the exogenous protem will be mduced by a smgle inducer, by a smgle pathway, acting on a smgle promoter.
  • the present mvention takes advantage of the properties of the transformation system disclosed m mtemational patent applications PCT/NL99/00618 and PCT/EP99/202516. These applications desc ⁇ be an efficient transformation system for filamentous fungal hosts such as the genus Chrysosporium and Aspergillus sojae. These applications also disclose that mutant strams are readily prepared which retain all the advantages of the wild-type host cells, but which have partially lost their filamentous phenotype.
  • the present mvention employs mutant filamentous fungi which do exhibit a less pronounced filamentous phenotype and compact growth morphology, and which produce low-viscosity cultures that are suitable for the physical manipulations mvolved in high-throughput DNA library screenmg.
  • the mvention also provides a transformation system that exhibits high yields of transformants.
  • the mvention also provides libra ⁇ es of transformant fungi which efficiently express the protem products of heterologous cDNA inserts, and especially genomic DNA inserts.
  • the hbranes of transformed fungi may be used m screenmg for activities or properties of the heterologous protems, or in screenmg for metabolites produced by the transformed fungi as a consequence of exogenous protem activities, or m screenmg for the heterologous DNA or for RNA transcnpts denved therefrom. It will be appreciated that the present mvention also enables high-throughput screenmg for metabolites of non- transformed low-viscosity mutant strams.
  • the hbranes of transformed fungi may be screened for useful properties of the fungi themselves, such as for example high levels of production of a particular expressed protem. This aspect of the mvention is illustrated by a quantitative assay for the expressed protem of mterest, where the particular transformant having the most favorable combination of protem production, protem processmg, and protem secretion would be detected.
  • Figure 1 is a Western blot as descnbed m the Examples
  • Figure 2 is a pUT720 map
  • Figure 3 is a pUT970G map
  • Figure 4 is a pUT1064 map
  • Figure 5 is a pUT1065 map
  • Figure 6 is a pF6g map
  • Figure 7 is a pUTl 150 map
  • Figure 8 is a pUTl 152 map
  • Figure 9 is a pUTl 155 map
  • Figure 10 is a pUTl 160 map
  • Figure 11 is a pUTl 162 map
  • Figure 12 is the schematic structure of the pclA protein
  • Figure 13A is a photomicrograph of wildtype Aspergillus sojae
  • Figure 13B is a photomicrobraph of the Aspergillus sojae pclA mutant
  • One aspect of the present mvention is directed at a library of low- viscosity filamentous fungi compnsmg nucleic acid sequences, each nucleic acid sequence encodmg a heterologous protem, each of said nucleic acid sequences bemg operably linked to an expression regulating region and optionally a secretion signal encodmg sequence and/or a earner protem encodmg sequence.
  • a recombinant strain accordmg to the invention will secrete the heterologous protem.
  • the filamentous fungi of the mvention are charactenzed by the low viscosity of the culture medium.
  • a typical lndust ⁇ al-grade filamentous fungus will produce cultures with viscosities well over 200 centipoise (cP) and usually over 1,000 cP, and can reach 10,000 cP
  • the fungi of this mvention exhibit a culture viscosity of less than 200 cP, preferably less than 100 cP, more preferably less than 60 cP, and most preferably less than 10 cP after 48 or more hours of culturing m the presence of adequate nut ⁇ ents under optimal or near-optimal growth conditions.
  • the filamentous fungi of the mvention usually exhibit a morphology charactenzed by short, discrete, non-entangled hyphae or micropellets.
  • Micropellets are slightly entangled or non-entangled collections of hyphae ansmg from a smgle clone, as distinct from pellets which are much larger and are denved from multiple entangled clones.
  • the mutant UV 18-25 Chrysosporium lucknowense strain ( viscosity ⁇ 10 cP ) and the morphologically similar mutant Trichoderma longibrachiatum X-252 strain (viscosity ⁇ 60 cP ) are characterised by the presence of short, distinct, non en tangled hyphae between 100 and 200 microns in length, and the low viscosity engineered mutant Aspergillus sojae pclA is characterized by a compact form with considerable branching and short hyphae.
  • filamentous fungi are the Chrysosporium, Thielavia, Neurospora, Acremonium, Tolypocladium, Scytalidium, Sporotrichum, Myceliophthora, Mucor, Aspegillus, Fusarium, Humicola, and Trichoderma, and teleomorphs thereof. More preferred are Chrysosporium, Trichoderma, Aspergillus, and Fusarium. Most preferred are Trichoderma and Chrysosporium.
  • the genus and species of fungi can be defined by morphology consistent with that disclosed in Barnett and Hunter 1972, Illustrated Genera of Imperfect Fungi, 3rd Edition, Burgess Publishing Company.
  • a source providing details concerning classification of fungi of the genus Chrysosporium is Van Oorschot, C.A.N. (1980) "A revision of Chrysosporium and allied genera" in Studies in Mycology No. 20, Centraal Bureau voor Schimmelcultures (CBS), Baarn, The Netherlands, pp. 1-36. According to these teachings the genus Chrysosporium falls within the family Moniliaceae which belongs to the order Hyphomycetales.
  • Chrysosporium includes but is not limited to these strains: C. botryoides, C. carmichaelii, C. crassitunicatum, C. europae, C. evolceannui, C. farinicola, C.fastidium, C.filiforme, C. georgiae, C. globiferum, C. globiferum var. articulatum, C. globiferum var. niveum, C. hirundo, C. hispanicum, C. holmii, C. indicum, C. inops, C. keratinophilum, C. nikelii, C. kuzurovianum, C.
  • C. lucknowense forms one of the species of Chrysosporium that have raised particular interest as it has provided a natural high producer of cellulase proteins (international applications WO 98/15633, PCT/NL99/00618, and U.S. patents 5,811,381 and 6,015,707).
  • Strams with mtemational depository accession numbers ATCC 44006, CBS 251.72, CBS 143.77, CBS 272.77, and VKM F-3500D are examples of Chrysosporium lucknowense strams.
  • Also mcluded within the definition of Chrysosporium are strams de ⁇ ved from Chrysosporium predecessors including those that have mutated either naturally or by mduced mutagenesis.
  • the methods of the mvention employ mutants of Chrysosporium, obtained by a combination of irradiation and chemically-induced mutagenesis, that exhibit a morphology charactenzed by short discrete, non entangled hyphae, and a phenotype characterized by reduced viscosity of the fermentation medium when grown m suspension.
  • the mvention employs phenotypically similar mutants of Trichoderma.
  • the mvention employs phenotypically similar mutants of Aspergillus sojae.
  • VKM F-3500D (“strain CI”) was mutagemsed by subjecting it to ultraviolet light to generate strain UV13-6. This strain was subsequently further mutated with N-methyl-N'-nitro-N- mtrosoguanidine to generate strain NG7C-19. The latter strain m turn was subjected to mutation by ultraviolet light resulting m strain UV18-25 (VKM F-363 ID). During this mutation process the morphological characte ⁇ stics va ⁇ ed somewhat m culture m liquid or on plates as well as under the microscope.
  • anamorph form of Chrysosporium has been found to be suited for the screenmg application accordmg to the mvention.
  • the metabolism of the anamorph renders it extremely suitable for a high degree of expression.
  • a teleomorph should also be suitable as the genetic make-up of the anamorphs and teleomorphs is identical. The difference between anamorph and teleomorph is that one is the asexual state and the other is the sexual state; the two states exhibit different morphology under certam conditions.
  • Another example embodies genetically engeneered mutant strams of Aspergillus sojae.
  • a specific endoprotease encodmg gene was disrupted. This resulted m a compact growth phenotype exhibiting enhanced branchmg and short hyphae, and the formation of micropellets m submerged cultivation.
  • the Aspergillus sojae referred to m this application displays efficient sporulation under specific submerged cultivation conditions, which is a further advantage for its use m a high throughput screenmg system.
  • non-toxigenic and non-pathogenic fungal strams of which a number are known m the art, as this will reduce nsks to the operators and will simplify the overall screenmg process.
  • the fungi will also be protease deficient, so as to minimize degradation of the exogenous protems, and or amenable to suppression of protease production.
  • protease defidient strams as expression hosts is well known, see for example PCT application W096/29391.
  • Protease deficient strams may be produced by screenmg of mutants, or the protease gene(s) may be "knocked out" or otherwise inactivated by methods known m the art.
  • genes m the host filamentous fungus such as for example those encodmg cellulases and other heavily secreted protems, m order to minimize interference m the assay by host protems.
  • the genes encodmg secreted proteins may be deleted or mutated, or alternatively genes controlling the mduction system or other pathways mvolved in the expession of unwanted protems may be modified m such a way as to reduce such expression.
  • a homologous expression-regulating region enablmg high expression m the selected host is employed.
  • High expression-regulating regions de ⁇ ved from a heterologous host such as from Trichoderma or Aspergillus, are well known m the art, can also be used.
  • examples of protems known to be expressed m large quantities and thus providing suitable expression regulating sequences for use m the present mvention are hydrophobm, protease, amylase, xylanase, pectinase, esterase, beta-galactosidase, cellulase (e g. endo-glucanase, cellobiohydrolase) and polygalacturonase.
  • An expression-regulating region comp ⁇ ses a promoter sequence operably linked to a nucleic acid sequence encodmg the protem to be expressed.
  • the promoter is linked such that the positioning vis-a-vis the initiation codon of the sequence to be expressed allows expression.
  • the promoter sequence can be constitutive but preferably is inducible. Use of an inducible promoter and approp ⁇ ate mduction media favors expression of genes operably linked to the promoter. Any expression regulating sequence from a homologous species, or from a heterologous strain capable of permitting expression of a protem, is envisaged.
  • the expression regulating sequence is suitably a fungal expression-regulating region, e g. an ascomycete regulating region.
  • the ascomycete expression regulating region is a regulating region from any of the following genera: Aspergillus, Trichoderma, Chrysosporium.Humicola, Neurospora, Tolypocladium, Fusarium, Penicillium, Talaromyces, or alternative sexual forms thereof such as Emericela and Hypocrea.
  • a low-viscosity mutant Trichoderma strain designated X-252 was obtained after two rounds of irradiation of Trichoderma longibrachiatum 18.2KK, which in turn was derived by mutation of the QM 9414 strain of T. longibrachiatum (ATCC 26921).
  • the invention employs phenotypically similar mutants of Aspergillus sojae.
  • a Chrysosporium promoter sequence is applied to ensure good recognition thereof by the host.
  • Certain heterologous expression-regulating sequences also work as efficiently in Chrysosporium as native Chrysosporium sequences. This allows well-known constructs and vectors to be used in transformation of Chrysosporium, and offers numerous other possibilities for constructing vectors enabling good rates of transformation and expression in this host. For example standard Aspergillus transformation techniques can be used as described for example by Christiansen et al in Bio/Technology 6: 1419-1422 (1988). Other documents providing details of Aspergillus transformation vectors, e.g.
  • a nucleic acid construct will preferably comprise a nucleic acid expression regulatory region from Chrysosporium, more preferably from Chrysosporium lucknowense or a derivative thereof, operably linked to a nucleic acid sequence encoding a protein to be expressed.
  • Particularly preferred nucleic acid constructs will comprise an expression regulatory region from Chrysosporium associated with cellulase or xylanase expression, preferably cellobiohydrolase expression, most preferably expression of the 55 kDa cellobiohydrolase (CBH1) described in Table A.
  • Chrysosporium endoglucanases C1-EG6 and C1-EG5; SEQ ID NO: 1 and SEQ ID NO:2, respectively
  • Chrysosporium promoter sequences of hydrophobin, protease, amylase, xylanase, esterase, pectinase, beta-galactosidase, cellulase (e.g. endoglucanase, cellobiohydrolase) and polygalacturonase are also considered to fall within the scope of the invention.
  • promoters or regulatory regions of expression of enzymes disclosed in Table A can be suitably employed.
  • the nucleic acid sequences of these promoters and regulatory regions can readily be obtained from a Chrysosporium strain. Methods by which promoter sequences can be determined are numerous and well known in the art. Promoter sequences are generally found immediately preceding the ATG start codon at the beginning of the relevant gene. For example, promoter sequences can be identified by deleting sequences upstream of the relevant gene, using recombinant DNA techniques, and examining the effects of these deletions on expression of the gene. Also, for example, promoter sequences can often be inferred by comparing the sequence of regions upstream of the relevant gene with concensus promoter sequences.
  • promoter sequences of CI endoglucanases were identified in this manner (see PCT/NL99/00618) by cloning the corresponding genes.
  • Preferred promoters according to the invention are the 55 kDa cellobiohydrolase (CBH1), glyceraldehyde-3-phosphate dehydrogenase A, and the 30 kDa xylanase (XylF) promoters from Chrysosporium, as these enzymes are expressed at high level by their own promoters.
  • the promoters of the carbohydrate-degrading enzymes of Chrysosporium lucknowense in particular, especially C. lucknowense GARG 27K can advantageously be used for expressing libraries of proteins in other fungal host organisms.
  • nucleic acid sequences according to the invention are known for Chrysosporium, Aspergillus and Trichoderma. Promoters for Chrysosporium are described in PCT/NL99/00618.
  • the prior art provides a number of expression regulating regions for use in Aspergillus, e.g. U.S. patents 4,935,349; 5,198,345; 5,252,726; 5,705,358; and 5,965,384; and PCT application WO93/07277.
  • Expression in Trichoderma is disclosed in U.S. patent 6,022,725. The contents of these patents are hereby incorporated by reference in their entirety.
  • the hydrophobin gene is a fungal gene that is highly expressed. It is thus suggested that the promoter sequence of a hydrophobin gene, preferably from Chrysosporium, may be suitably applied as expression regulating sequence in a suitable embodiment of the invention.
  • Trichoderma reesei and Trichoderma harzianum gene sequences for hydrophobin have been disclosed for example in the prior art as well as a gene sequence for Aspergillus fumigatus and Aspergillus nidulans and the relevant sequence information is hereby incorporated by reference (Nakari-Setala et al, Eur. J. Biochem. 1996, 235:248-255; Parta et al , Infect.
  • An expression regulating sequence can also additionally comp ⁇ se an enhancer or silencer. These are also well known m the p ⁇ or art and are usually located some distance away from the promoter.
  • the expression regulating sequences can also comp ⁇ se promoters with activator bmdmg sites and repressor bmdmg sites. In some cases such sites may also be modified to eliminate this type of regulation.
  • filamentous fungal promoters in which creA sites are present have been desc ⁇ bed. The creA sites can be mutated to ensure the glucose repression normally resulting from the presence of the non-mutated sites is eliminated.
  • WO 94/13820 and WO 97/09438 Use of such a promoter enables production of the library of protems encoded by the nucleic acid sequences regulated by the promoter m the presence of glucose.
  • the method is exemplified in WO 94/13820 and WO 97/09438. These promoters can be used either with or without their creA sites. Mutants in which the creA sites have been mutated can be used as expression regulating sequences in a recombmant strain accordmg to the mvention and the library of nucleic acid sequences it regulates can then be expressed m the presence of glucose.
  • Chrysosporium promoters ensure derepression in an analogous manner to that illustrated m WO 97/09438.
  • creA sites The identity of creA sites is known from the p ⁇ or art.
  • Terminator sequences are also expression-regulating sequences and these are operably linked to the 3' termini of the sequences to be expressed.
  • a vanety of known fungal terminators are likely to be functional in the host strams of the mvention. Examples are the A. nidulans trpC terminator, A. niger alpha- glucosidase terminator, A. niger glucoamylase terminator, Mucor miehei carboxyl protease terminator (see US 5,578,463), and the Trichoderma reesei cellobiohydrolase terminator. Chrysosporium terminator sequences, e g the EG6 terminator, will of course function well m Chrysosporium.
  • a suitable transformation vector for use accordmg to the mvention may optionally have the exogenous nucleic acid sequences to be expressed operably linked to a sequence encodmg a signal sequence.
  • a signal sequence is an ammo acid sequence which, when operably linked to the ammo acid sequence of an expressed protem, enables secretion of the protem from the host organism. Such a signal sequence may be one associated with a heterologous protem or it may be one native to the host.
  • the nucleic acid sequence encodmg the signal sequence must be positioned in frame to permit translation of the signal sequence and the heterologous proteins. Signal sequences will be particularly preferred where the invention is being used in conjunction with molecular evolution, and a single, secreted exogenous protein is being evolved.
  • Analysis of the activity of intracellular proteins may be accomplished by pretreating the transformant library with enzymes that convert the fungal cells to protoplasts, followed by lysis.
  • the procedure has been described by van Zeyl et al, J. Biotechnol. 59:221-224 (1997). This procedure has been applied to Chrysosporium to allow colony PCR from Chrysosporium transformants grown in microtiter plates.
  • Any signal sequence capable of permitting secretion of a protein from a Chrysosporium strain is envisaged.
  • a signal sequence is preferably a fungal signal sequence, more preferably an Ascomycete signal sequence.
  • Suitable signal sequences can be derived from eucaryotes generally, preferably from yeasts or from any of the following genera of fungi: Aspergillus, Trichoderma, Chrysosporium, Pichia, Neurospora, Rhizomucor, Hansenula, Humicola, Mucor, Tolypocladium, Fusarium, Penicillium, Saccharomyces, Talaromyces or alternative sexual forms thereof such as Emericella and Hypocrea.
  • Signal sequences that are particularly useful are those natively associated with cellobiohydrolase, endoglucanase, beta-galactosidase, xylanase, pectinase, esterase, hydrophobin, protease or amylase.
  • Examples include amylase or glucoamylase of Aspergillus or Humicola, TAKA amylase of Aspergillus oryzae, ⁇ -amylase of Aspergillus niger, carboxyl peptidase of Mucor (US 5,578,463), a lipase or proteinase from Rhizomucor miehei, cellobiohydrolase of Trichoderma, beta-galactosidase of Penicillium canescens CBH1 from Chrysosporium, and the alpha mating factor of Saccharomyces.
  • the signal sequence can be from an amylase or subtilisin gene of a strain of Bacillus.
  • a signal sequence from the same genus as the host strain is extremely suitable as it is most likely to be specifically adapted to the specific host thus preferably the signal sequence is a signal sequence of Chrysosporium.
  • Chrysosporium strains CI, UV13-6, NG7C-19 and UV18-25 secrete proteins in extremely large amounts, and signal sequences from these strains are of particular interest.
  • Signal sequences from filamentous fungi and yeast are useful, as are signal sequences of non-fungal origin.
  • a transformed recombinant host fungus according to any of the embodiments of the invention can further comprise a selectable marker.
  • a selectable marker permits selection of transformed or transfected cells.
  • a selectable marker often encodes a gene product providing a specific type of resistance foreign to the non-transformed strain. This can be resistance to heavy metals, antibiotics or biocides in general. Prototrophy is also a useful selectable marker of the non-antibiotic va ⁇ ety.
  • Auxotrophic markers generate nutntional deficiencies m the host cells, and genes correcting those deficiencies can be used for selection.
  • Examples of commonly used resistance and auxotrophic selection markers are amdS (acetamidase), hph (hygromycin phosphotransferase), pyrG (orotid ⁇ ne-5 '-phosphate decarboxylase), trpC (anthranilate synthase), argB (ornithme carbamoyltransferase), sC (sulphate adenyltransferase), bar (phosphmothncm acetyltransferase), maD (nitrate reductase), Sh-ble (bleomycin-phleomycin resistance), mutant acetolactate synthase (sulfonylurea resistance), and neomycm phosphotransferase (aminoglycoside resistance). Selection can be earned out by cotransformation where the selection marker is on a separate vector or where the selection marker is on the same nucleic acid fragment as the protem-encoding sequence for
  • a further improvement of the transformation frequency is exemplified by the use of the AMA1 replicator sequence for Aspergillus niger (Verdoes et al , Gene 146:159-165 (1994)).
  • This sequence results in a 10- to 100-fold mcrease m the transformation frequency m a number of different filamentous fungi.
  • the introduced DNA is retained autonomously in the fungal cells without integration mto the fungal genome m a multiple copy fashion.
  • heterologous protem is a protem or polypeptide not normally expressed and secreted by the host strain used for expression accordmg to the mvention.
  • a heterologous protem may be of prokayotic ongm, or it may be denved from a fungus, plant, msect, or higher animal such as mammals.
  • a preferred embodiment will be a host wherem the DNA library is of human ongm. Such embodiments are therefore also considered suitable examples of the mvention.
  • a further embodiment of the mvention mcludes the construction and screenmg of fungal mutant libra ⁇ es, and fungal mutant libra ⁇ es prepared by the methods disclosed herem.
  • the libra ⁇ es may be obtained by transformation of the fungal hosts accordmg to this mvention with any means of lntegrative transformation, usmg methods known to those skilled m the art.
  • This library of fungi based on the preferred host strams may be handled and screened for desired properties or activities of exogenous protems m miniatunzed and/or high-throughput format screenmg methods.
  • property or activity of mterest is meant any physical, physicochemical, chemical, biological, or catalytic property, or any improvement, mcrease, or decrease m such a property, associated with an exogenous protem of a library member.
  • the library may also be screened for a property or activity associated with a metabolite produced as a result of the presence of exogenous and/or endogenous protems
  • the library may also be screened for fungi producmg increased or decreased quantities of such protem or metabolites.
  • the library of transformed fungi may be screened for the presence of fungal metabolites having desirable properties. It is anticipated that multiple genes or gene clusters may be transferred to the host cells of the mvention, and that non-protem products generated by the action of the encoded enzymes may be generated m the host cells. For example, it has been shown that DNA encodmg the protems necessary for production of lovastatin can be transferred to Aspergillus oryzae (U.S. patent 5,362,638; see also U.S. patent 5,849,541).
  • the heterologous DNA may be genomic DNA or cDNA, prepared from biological specimens by methods well known in the art.
  • the biological specimen may be an environmental sample (for example, soil, compost, forest litter, seawater, or fresh water), or an extracted, filtered, or cent ⁇ fuged or otherwise concentrated sample therefrom. Mixed cultures of microorganisms denved from environmental samples may be employed as well.
  • the biological sample may also be denved from any smgle species of organism, such as a cultured microorganism, or plant, msect, or other animal such as a mammal.
  • heterologous DNA may be synthetic or semi-synthetic, for example random DNA sequences or DNA compnsmg naturally-occur ⁇ ng segments which have been shuffled, mutated, or otherwise altered.
  • An example of a semi-synthetic nucleic library is found m Wagner et al, WO 00/0632.
  • DNA from environmental samples (or mixed cultures denved therefrom) will be advantageous for the discovery of novel protems, while the use of DNA from a smgle species will be advantageous m that (1) an approp ⁇ ate vector may be more judiciously chosen, and (2) the practitioner will be directed to related or similar species for further screenmg if a protem of mterest is identified.
  • the vectors of the mvention can comp ⁇ se a promoter sequence denved from a gene encodmg an enzyme, preferably a secreted enzyme.
  • an enzyme preferably a secreted enzyme.
  • suitable enzymes from which promoter sequences may be taken are the carbohydrate-degrading enzymes (e.g., cellulases, xylanases, mannanases, mannosidases, pectinases, amylases, e g.
  • glucoamylases ⁇ -amylases, ⁇ - and ⁇ -galactosidases, ⁇ - and ⁇ - glucosidases , ⁇ -glucanases, chitmases, chitanases), proteases (endoproteases, ammo-proteases, ammo-and carboxy-peptidases), other hydrolases (hpases, esterases, phytases), oxidoreductases (catalases, glucose- oxidases) and transferases (transglycosylases, transglutaminases, isomerases and mvertases).
  • Chrysosporium lucknowense Several examples from Chrysosporium lucknowense are presented in Table A. Table A: Characteristics of selected enzymes from Chrysosporium lucknowense
  • Chrysosporium mutants can be made that have reduced expression of protease, thus making them even more suitable for the production of proteinaceous products, especially if the proteinaceous product is sensitive to protease activity.
  • the invention my also employ a mutant Chrysosporium strain which produces less protease than non-mutant Chrysosporium strain, for example less than C. lucknowense strain CI (VKM F-3500 D).
  • the protease acitivity (other than any selective protease intended to cleave a secreted fusion protem) of such strains is less than half the amount, more preferably less than 30% of the amount, and most preferably less than about 10% the amount produced by the CI strain.
  • the decreased protease activity can be measured by known methods, such as by measuring the halo formed on skim milk plates or by bovine serum albumin (BSA) degradation.
  • BSA bovine serum albumin
  • transformation, expression and secretion rates are exceedingly high when using a Chrysosporium strain exhibiting the mycelial morphology of strain UV18- 25, i.e. short, non-entangled mycelia.
  • a recombmant strain accordmg to the mvention will preferably exhibit such morphology.
  • the invention however also covers non-recombinant strams or otherwise engmeered strams of fungi exhibiting this novel and mventive characte ⁇ stic.
  • Another attractive embodiment of the mvention also covers a recombmant Chrysosporium strain exhibiting a viscosity below that of strain NG7C-19, preferably below that of UV 18-25 under corresponding or identical fermenter conditions.
  • a viscosity of a culture of UV18-25 is below 10 cP as opposed to that of previously known Trichoderma reesei be g of the order 200-600 cP, and with that of traditional Aspergillus niger bemg of the order 1500-2000 cP under optimal culture conditions during the middle to late stages of fermentation.
  • the mvention may employ any engmeered or mutant filamentous fungus exhibiting this low-viscosity charactersistic, such as the Chrysospo ⁇ um UV18-25 (VKM F-3631D) strain, the T ⁇ choderma X 252 strain, or A. sojae pclA (denved from ATCC 11906).
  • VKM F-3631D Chrysospo ⁇ um UV18-25
  • T ⁇ choderma X 252 strain or A. sojae pclA (denved from ATCC 11906).
  • the fluidity of filamentous fungal cultures can vary over a wide range, from nearly solid to a free- flowing liquid. Viscosity can readily be quantitated by Brookfield rotational viscometry, use of kinematic viscosity tubes, falling ball viscometer or cup type viscometer. Fermentation broths are non-Newtonian fluids, and the apparent viscosity will be dependent to some extent upon the shear rate (Goudar et al, Appl. Microbiol. Biotechnol. 51:310-315 (1999)). This effect is however much less pronounced for the low- viscosity cultures employed m the present mvention.
  • the screenmg of an expression library accordmg to the method of the mvention is highly advantageous.
  • the screenmg of DNA hbranes expressed m filamentous fungi has heretofore been limited to relatively slow and labonous methods.
  • fungi once fungi have been transformed (and the transformants optionally selected for), it has been necessary to prepare spores or conidia, or to mechanically disrupt the mycelia, m order to disperse the library of transformed fungi mto individual organisms. This dispersal is necessary so that the separated organisms can be cultured mto clonal colonies or cultures.
  • the spores, conidia, or mycehal fragments are then diluted and "plated out" m standard culture dishes, and the individual colonies are inspected for color, alterations to the substrate, or other detectable indication of the presence of the protem activity or property bemg sought.
  • secreted protems are blotted from the colonies onto a membrane, and the membrane is probed or examined for an indication of the presence of the protem activity or property of interest.
  • membranes has proved useful where proteolytic degradation of exogenous protem is a problem, Asgeirsdottir et al , Appl Environ. Microbiol 1999, 65:2250-2252.
  • high-throughput screenmg refers to any partially or fully automated method that is capable of evaluating the protem expression of about 10,000 or more transformants per day.
  • the automated high-throughput screenmg of a library of transformed fungi accordmg to the present mvention, accordmgly, may be earned out in a number of known ways. Methods that are known to be applicable to bactena or yeast may m general be applied to the low- viscosity fungi of the present mvention.
  • mutant fungi behave very much like mdividual bactena or yeast durmg the mechanical manipulations mvolved m automated high-throughput screenmg. This is m contrast to wild-type fungi, and most indust ⁇ ally-adapted fungi as well, which produce highly entangled mycelia which do not permit the ready separation of the mdividual organisms from one another.
  • a dilute suspension of transformed fungi accordmg to the present mvention may be ahquotted out through a mechanical micropipette mto the wells of a 96-well microplate. It is anticipated that liquid-handling apparatus capable of pipetting mto 384- or 1536- well microplates can also be adapted to the task of automated dispersal of the organisms mto microplates. The concentration of the suspended organisms can be adjusted as desired to control the average number of organisms per well.
  • a cell sorter may be interposed in the fluid path, which is capable of directing the flow of the culture to the wells of the microplate upon the detection of an organism in the detector cell. This embodiment permits the reasonably accurate dispensation of one organism per well.
  • colonies growing on solid media can be picked by a robotic colony picker, and the organisms transferred by the robot to the wells of a microtiter plate.
  • Well-separated colonies will give nse to smgle clones m each well.
  • the dispersed organisms are then permitted to grow mto clonal cultures m the microplate wells.
  • Inducers, nutnents, etc. may be added as desired by the automated fluid dispensmg system.
  • the system may also be used to add any reagents required to enable the detection of the protem activity or property of interest.
  • colorogemc or fluorogenic substrates can be added so as to permit the spectroscopic or fluoromet ⁇ c detection of an enzyme activity.
  • the low viscosity of the cultures m the wells of a microtiter plate permits the rapid diffusion of such reagents mto the culture, greatly enhancing the sensitivity and reliability of the assay.
  • smgle cells are passed through a microfluidic apparatus, and the property or activity of mterest is detected optically (Wada et al , WO 99/67639)
  • Low viscosity is essential to the operation of a microfluidics device, and cultures of the low- viscosity mutant fungi of the present mvention are expected to be amenable to microfluidic manipulation.
  • Another class of high-thoughput screens is by photometnc analysis, by digital imaging spectroscopy, of large numbers of mdividual colonies growmg on a solid substrate. See for example Youvan et al , 1994, Meth Enzymol 246:732-748. In this method, changes m the overall absorption or emission spectra of specialized reagents are indicative of the presence of a heterologous protem activity or property of mterest.
  • the ready dispersal of mdividual organisms attendant upon the use of low- viscosity mutants also enables the use of filamentous fungi m this method
  • the tendency for colonies of the mutant fungi of the mvention to exhibit less lateral growth, and to produce smooth, compact, and well-defined colonies, is also advantageous m such a screenmg system.
  • the supenor expression and secretion charactenstics of fungi as compared to bactena provide greater quantities of protem for spectral analysis.
  • An automated microorganism handlmg tool is descnbed m Japanese patent application publication number 11-304666
  • This device is capable of the transfer of microdroplets contammg mdividual cells, and it is anticipated that the fungal strams of the present mvention, by virtue of their morphology, will be amenable to micromanipulation of mdividual clones with this device.
  • An automated microbiological high-throughput screenmg system is desc ⁇ bed m Beydon et al, J Biomol. Screening 5.13-21 (2000).
  • the robotic system is capable of transferring droplets with a volume of 400 nl to agar plates, and processmg 10,000 screenmg points per hour, and has been used to conduct yeast two-hybnd screens. It is anticipated that the fungal hosts of the present mvention will be as amenable as yeast to high-throughput screenmg with systems of this type.
  • the Chrysosporium strain UV18-25 and the Trichoderma strain X 252 illustrate va ⁇ ous aspects of the mvention exceedmgly well.
  • the mvention may employ other mutant or otherwise engmeered strams of filamentous fungi that exhibit low viscocity m culture.
  • the specific morphology of the fungi may not be c ⁇ tical; the present inventors have observed short, non-entangled mycelia m these two strams but other morphologies, such as close and extensive hyphal branchmg, may also lead to reduced viscosity.
  • Fungal strams accordmg to the mvention are preferred if they exhibit optimal growth conditions at neutral pH and temperatures of 25-43°C.
  • Such screenmg conditions are advantageous for maintaining the activity of exogenous protems, m particular those susceptible to degradation or lnactivation at acidic pH.
  • Most mammalian protems, and human protems in particular have evolved to function at physiological pH and temperature, and screenmg for the normal activity of a human enzyme is best earned out under those conditions. Protems mtended for therapeutic use will have to function under such conditions, which also makes these the preferred screenmg conditions.
  • Chrysosporium strams exhibit precisely this charactenstic, growmg well at neutral pH and 35-40 °C, while other commonly employed fungal host species (e.g. Aspergillus and Trichoderma) grow best at acidic pH and may be less suitable for this reason
  • Another application of the method of the present invention is m the process of "directed evolution," wherem novel protein-encoding DNA sequences are generated, the encoded protems are expressed m a host cell, and those seqences encodmg protems having a desired charactenstic are mutated and expressed agam. The process is repeated for a number of cycles until a protem with the desired charactenstics is obtained.
  • Gene shuffling, protem engineering, error-prone PCR, site-directed mutagenesis, and combmatonal and random mutagenesis are examples of processes through which novel DNA sequences encodmg exogenous protems can be generated.
  • patents 5,223,409, 5,780,279 and 5,770,356 provide teaching of directed evolution. See also Kuchner and Arnold, Trends in Biotechnology, 15:523-530 (1997); Schmidt-Dannert and Arnold, Trends in Biotech , 17.135-136 (1999); Arnold and Volkov, Curr Opin. Chem Biol , 3-54-59 (1999); Zhao et al , Manual of Industrial Microbiology and Biotechnology, 2 nd Ed., (Dema and Davies, eds.) pp. 597-604, ASM Press, Washington DC, 1999; Arnold and Wintrode, Encyclopedia ofBwprocess Technology Fermentation, Biocatalysis, and Bioseparation, (Flickinger and Drew, eds.) pp. 971-987, John Wiley & Sons, New York, 1999; and Minshull and Stemmer, Curr Opin Chem Biol. 3:284-290.
  • the protem activity of mterest is somehow made essential to the survival of the host cells.
  • the activity desired is a cellulase active at pH 8
  • a cellulase gene could be mutated and mtroduced mto the host cells.
  • the transformants are grown with cellulose as the sole carbon source, and the pH raised gradually until only a few survivors remain.
  • the mutanted cellulase gene from the survivors which presumably encodes a cellulase active at relatively high pH, is subjected to another round of mutation, and the process is repeated until transformants that can grow on cellulose at pH 8 are obtained.
  • Thermostable va ⁇ ants of enzymes can likewise be evolved, by cycles of gene mutation and high- temperature culturing of host cells (Liao et al, Proc. Natl. Acad. Sci. USA 83:576-580 (1986); Giver et al, Proc. Natl. Acad. Sci. USA. 95:12809-12813 (1998).
  • the chief advantage of this method is the massively parallel nature of the "survival of the fittest" selection step. Millions, or billions, of unsuccessful mutations are simultaneously eliminated from consideration without the need to evaluate them individually. However, it is not always possible to link an enzyme activity of mterest to the survival of the host. Where the desired protem property is selective bmdmg to a target of mterest, making the bmdmg property essential to survival is especially difficult. Furthermore, survival under forced conditions such as high temperature or extreme pH is likely to be dependent upon multiple factors, and a desirable mutation will not be selected for and will be lost if the host cell is unable to survive for reasons unrelated to the properties of the mutant protem.
  • the screenmg approach has clear advantages over a simple "survival screen," especially if it can be earned out m a high-throughput manner that approaches the throughput of the massively parallel "survival screen” technique.
  • a degree of parallelism has been mtroduced by employmg such measures as digital imaging of the transformed organisms (Joo et al, Chemistry & Biology, 6:699-706 (1999)) or digital spectroscopic evaluation of colonies (Youvan et al, 1994, Meth. Enzymol. 246:732-748).
  • Serial assays can be automated by the use of cell sorting (Fu et al , Nature Biotech , 17:1109-1111 (1999)).
  • a well-established approach to high-thorughput screening involves the automated evaluation of expressed proteins in microtiter plates, using commercially available plate readers, and the method of the present invention is well-suited to the application of this mode of high-throughput screening to directed evolution.
  • a gene encoding a protein of interest is mutated by any known method of generating a plurality of mutants, the mutant protein-encoding DNA is introduced by means of a suitable expression vector into a low-viscosity filamentous fungal host according to the present invention, and the transformants are optionally selected for and cultured.
  • the host cells are then dispersed as described previously into the wells of a microtiter plate, or otherwise spatially separated into resolvable locations, so as to provide individual mono-clonal cultures (or poly-clonal cultures having fewer than about 100 diferent clones).
  • the cells are preferably dispersed into the wells of a micro-titer plate.
  • the protein encodede by the mutant DNA is preferably secreted into the medium in the wells of the microtiter plates. Each of the dispersed cultures is screened for the protein activity of interest, and those most strongly exhibiting the desired property are selected. The gene encoding the protein of interest in the selected cultures is mutated again, the mutant DNA is again introduced into the low- viscosity fungal host, and the transformants are re-screened. The mutating and re-screening process is repeated until the value of the property of interest reaches a desired level.
  • mutant filamentous fungi of the present mvention are excellent overproducers and secretors of exogenous protems, especially when employed with the vectors disclosed herem.
  • Sufficient protem may be isolated not only for purposes of charactenzation, but for evaluation m application tnals. Indeed, the strams used m screenmg may be suitable for mdust ⁇ al production as well, since they possess desirable production properties such as low viscosity and high expression rates.
  • the method further comp ⁇ ses culturing a clonal colony or culture identified accordmg to the method of the mvention, under conditions permitting expression and secretion of the exogenous library protem (or a precursor thereof),and recovermg the subsequently produced protem to obtain the protem of mterest.
  • Expression and secretion of a library protem may be facilitated by creating an m-frame fusion of the cloned gene with the gene for a heterologous protein (or a fragment thereof) with its corresponding signal sequence, or with the signal sequence from a third protem, all operably linked to an expression regulating sequence.
  • this fusion precursor protem may be isolated and recovered usmg punfaction techniques known m the art.
  • the method may optionally compnse subjecting the secreted fusion protem precursor to a cleavage step to generate the library protem of mterest.
  • the cleavage step can be earned out with Kex-2, a Kex-2 like protease, or another selective protease, when the vector is engmeered so that a protease cleavage site links a well-secreted protem earner and the protem of mterest.
  • mutant protems directly from the screening host organism, has not previously been possible with p ⁇ or art screenmg hosts.
  • the present mvention thus provides an advantage, m that the mutant protems deemed of mterest based upon the high-throughput screen can be isolated m sufficient quantities (milligrams) for further charactenzation and even larger quantities (grams to kilograms) for application tnals.
  • This particular embodiment of the mvention thus permits the practitioner to select mutant protems for the next round of directed evolution based upon any number of desirable properties, and not merely upon the one property detected m the high-throughput screen.
  • the more stringent selection cntena made possible by the present mvention should lead to a more efficient and cost-effective directed evolution process.
  • the method of production of a recombmant mutant filamentous fungal strain accordmg to the mvention compnses introducing a library of DNA sequences compnsmg nucleic acid sequences encodmg heterologous protems mto a low- viscosity mutant filamentous fungus accordmg to the mvention, the nucleic acid sequences bemg operably linked to an expression regulating region.
  • the introduction of the DNA sequences may be earned out m any manner known per se for transforming filamentous fungi.
  • the following operating parameter data ranges have been determined for fungal fermentations usmg five different fungal organisms.
  • medium contammg between 20 and 100 g/1 of a carbohydrate carbon source e g., cellulose, lactose, sucrose, xylose, glucose, and the like
  • a carbohydrate carbon source e g., cellulose, lactose, sucrose, xylose, glucose, and the like
  • a growth phase durmg which the carbon source is consumed.
  • Shake flask cultures are shaken at 200 rpm, while one-liter fermentation vessels are stirred with an impeller at 500-1000 rpm. Maximal viscosity typically occurs at or close to the end of the growth phase.
  • the culture is switched to a fed batch mode, wherem a carbon source is fed to the culture at a rate such that the concentration of the carbon source does not nse above about 0.5 g 1.
  • a feed rate of between 1 and 3 g/l/hr is typical.
  • Viscosity is determined on a Brookfield LVF viscometer usmg the small sample adapter and spmdle number 31, operated at 30°C.
  • a fresh sample of fermentation broth (10 ml) is placed m the small sample spmdle.
  • the spmdle speed is adjusted to give a readmg m the range 10-80.
  • After four minutes a readmg is taken from the viscometer scale.
  • the readmg is multiplied by the factor given below to get the viscosity m centipoise (cP).
  • the final viscosity was measured at fermentation end:
  • the protoplast transformation technique was used on Chrysosporium based on the most generally applied fungal transformation technology All spores from one 90mm PDA plate were recovered in 8ml ICl and transferred mto a shake flask of 50ml ICl medium for incubation for 15 hours at 35°C and 200 rpm. After this the culture was cent ⁇ fuged, the pellet was washed in MnP, brought back mto solution m 10ml MnP and lOmg/ml Caylase C 3 and mcubated for 30 mmutes at 35°C with agitation (150 rpm).
  • the homologous protein to be expressed was selected from the group of cellulases produced by Chrysosporium and consisted of endoglucanase 6 which belongs to family 6 (MW 43 kDa) and the heterologous protein was endoglucanase 3 which belongs to family 12 (MW 25 kDa) of Penicillium.
  • pF6g comprises Chrysosporium endoglucanase 6 promoter fragment linked to endoglucanase 6 signal sequence in frame with the endoglucanase 6 open reading frame followed by the endoglucanase 6 terminator sequence. Transformant selection is carried out by using cotransformation with a selectable vector.
  • pUTl 150 comprises Trichoderma reesei cellobiohydrolase promoter linked to endoglucanase 6 signal sequence in frame with the endoglucanase 6 open reading frame followed by the T. reesei cellobiohydrolase terminator sequence.
  • this vector carries a second expression cassette with a selection marker i.e. the phleomycin resistance gene (Sh-ble gene).
  • pUTl 152 comprises Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase A promoter linked to endoglucanase 6 signal sequence in frame with the endoglucanase 6 open reading frame followed by the A.
  • nidulans anthranilate synthase (trpC) terminator sequence carries a second expression cassette with a selection marker, i.e. the phleomycin resistance gene (Sh-ble gene).
  • pUTl 155 comprises A. nidulans glyceraldehyde-3-phosphate dehydrogenase A promoter linked to Trichoderma reesei cellobiohydrolase signal sequence in frame with the carrier protein Sh-ble which in turn is linked in frame to the endoglucanase 6 open reading frame followed by the A. nidulans trpC terminator sequence.
  • pUTl 160 comprises Aspergillus nidulans glyceraldehyde-3 -phosphate dehydrogenase A promoter linked to Trichoderma reesei cellobiohydrolase signal sequence in frame with the carrier protein Sh-ble which in tum is linked in frame to the endoglucanase 3 open reading frame of Penicillium followed by the A. nidulans trpC terminator sequence.
  • pUTl 162 comprises Trichoderma reesei cellobiohydrolase promoter linked to endoglucanase 3 signal sequence in frame with the endoglucanase 3 open reading frame of Penicillium followed by the T. reesei cellobiohydrolase terminator sequence.
  • this vector carries a second expression cassette with the phleomycin resistance gene (Sh-ble gene) asa selection marker.
  • genomic or cDNA can be readily sheared or digested into protein-encoding fragments, and the fragments ligated into vectors such as those lllustrated herem so as to produce a library of expression vectors. It will be further apparent that methods employmg co-transfection are applicable, and that autonomously replicating vectors or integrating vectors may be employed to transfect filamentous fungi with such a library of vectors.
  • Table E shows the results of transformation of both Chrysosporium UV18-25 and Tolypocladium geodes.
  • the transformation protocol used is desc ⁇ bed in the section for heterologous transformation.
  • HETEROLOGOUS AND HOMOLOGOUS EXPRESSION OF CHRYSOSPORIUM TRANSFORMANTS CI strams were tested for their ability to secrete vanous heterologous protems: a bactenal protein (Streptoalloteichus hindustanus phleomycm-resistance protem, Sh-ble), a fungal protem (Trichoderma reesei xylanase II, XYN2) and a human protem (the human lysozyme, HLZ).
  • gpdA glyceraldehyde-3 -phosphate dehydrogenase promoter
  • the vector also carnes the beta-lactamase gene (bla) and E. coli replication ongm from plasmid pUC18 (Ref 6).
  • the detailed plasmid map is provided in figure 2
  • CI protoplasts were transformed accordmg to Durand et al. (ref.7) adapted to CI (media & solutions composition is given elsewhere). All spores from one 90mm PDA plate of untransformed CI stram were recovered m 8ml ICl and transferred mto a shake flask with 50ml ICl medium for mcubation 15 hours at 35°C and 150 rpm.
  • the culture was spun down, the pellet washed m MnP, resolved in 10ml MnP + lOmg/ml Caylase C 3 , and mcubated 30 mm at 35°C with agitation (150 rpm)
  • the solution was filtered and the filtrate was centnfuged 10 mm at 3500 rpm.
  • the pellet was washed with 10ml MnPCa 2+ . This was spun down 10mm at 3500 rpm and the pellet was taken up mto 1ml MnPCa 2+ .
  • lO ⁇ g of pUT720 DNA were added to 200 ⁇ l of protoplast solution and mcubated 10mm at room temperature (ca. 20°C).
  • the Sh-ble production of CI transformants was analysed as follows: Primary transformants were toothpicked to GS+phleomycin (5 ⁇ g/ml) plates and grown for 5 days at 32°C for resistance venfication. Each validated resistant clone was subcloned onto GS plates. Two subclones per transformant were used to moculate PDA plates m order to get spores for liquid culture mitiation. The liquid cultures m ICl were grown 5 days at 27°C (shaking 200 rpm). Then, the cultures were centnfuged (5000g, 10mm.) and 500 ⁇ l of supernatant were collected.
  • the heterologous transcnption/translation signals from pUT720 are functional m Chrysosporium.
  • the heterologous signal sequence of pUT720 is functional m Chrysosporium.
  • Chrysosporium can be used a host for the secretion of heterologous bactenal protems.
  • gpdA glyceraldehyde-3 -phosphate dehydrogenase promoter
  • LGERK linker peptide
  • the vector also car ⁇ es the beta-lactamase gene (bla) and E coli replication ongm from plasmid pUCl 8 6
  • the detailed plasmid map is provided m Figure 3.
  • CI protoplasts were transformed with plasmid pUT970G following the same procedure already descnbed m example 1.
  • the fusion protem (Sh-ble :: GAM hmge :: HLZ) is functional with respect to the phleomycm-resistance thus allowing easy selection of the CI transformants.
  • the level of phleomycm resistance correlates roughly with the level of hlz expression.
  • HLZ production of CI transformants was analysed by lysozyme-activity assay as follow: Primary transformants were toothpicked to GS+phleomycm (5 ⁇ g/ml) plates (resistance venfication) and also on LYSO plates (HLZ activity detection by clearing zone visualisation (refs. 1, 10). Plates were grown for 5 days at 32°C. Each validated clone was subcloned onto LYSO plates. Two subclones per transformant were used to inoculate PDA plates m order to get spores for liquid culture mitiation The liquid cultures in ICl were grown 5 days at 27°C (shaking 180 rpm). Then, the cultures were centnfuged (5000g, lOmin.). From these samples, lysozyme activity was measured accordmg to M ⁇ rsky et al. (ref. 13)
  • Sh-ble is functional m Chrysosporium as resistance marker.
  • Sh-ble is functional m Chrysosporium as earner protem.
  • the KEX2-hke protease cleavage site is functional m Chrysosporium (otherwise HLZ would not be active).
  • Chrysosporium can be used as host for the secretion of heterologous mammalian protems.
  • CI stram UV18-25 was transformed by the plasmids pUT1064 and pUT1065.
  • pUT1064 presents the two following fungal expression cassettes: The first cassette allows the selection of phleomycin-resistant transformants:
  • the second cassette is the xylanase production cassette:
  • the vector also carnes an E coli replication ongm from plasmid pUC19 (ref. 6)
  • the plasmid detailed map is provided m figure 4.
  • pUT1065 presents the following fungal expression cassette:
  • gpdA glyceraldehyde-3 -phosphate dehydrogenase promoter
  • the vector also carnes the beta-lactamase gene (bla) and an E. coli replication ongm from plasmid pUC 18 (Ref. 6).
  • the plasmid detailed map is provided in Figure 5.
  • CI protoplasts were transformed with plasmid pUT1064 or pUT1065 following the same procedure already descnbed m example 1
  • the fusion protem m plasmid pUT1065 (Sh-ble :. XYN2) is functional with respect to the phleomycm-resistance thus allowmg easy selection of the CI transformants.
  • the level of phleomycm resistance correlates roughly with the level of xyn2 expression.
  • xyn2 was cloned with its own signal sequence.
  • CI transformants phleomycin-resistant clones
  • xylanase-activity assay as follow: Pnmary transformants were toothpicked to GS+phleomycm (5 ⁇ g/ml) plates (resistance venfication) and also on XYLAN plates (Ref. 17), where xylanase activity is detected by observation of a clearmg zone. Plates were grown for 5 days at 32°C. Each validated clone was subcloned onto XYLAN plates. Two subclones per transformant were used to moculate PDA plates m order to get spores for liquid culture inoculation.
  • CI can be used as host for the secretion of heterologous fungal protems.
  • Table I shows the results for the plasmids with which transformation of UVl 8-25was earned out.
  • the Table shows expression levels for endoglucanase and cellobiohydrolase usmg heterologous expression regulating sequences and signal sequences and also with homologous expression regulating sequences and signal sequences.
  • the details of the vanous plasmids can be denved elsewhere in the descnption and from the figures.
  • the production occurs at alkaline pH at a temperature of 35°C.
  • Genomic DNA of A sojae was isolated from protoplasts obtained from ATCC 11906 usmg a previously desc ⁇ bed protocol (Punt, van den Hondel, Methods Enzymol 216:447-457 (1992)). After isolation DNA was extracted from the protoplasts usmg the protocol desc ⁇ bed by Kolar et al, Gene 62:127-34 (1988). Subsequently the DNA was partially digested with Mbol to result in a DNA fragment of an average size of 30-50 kb.
  • Vector pAOpyrGcosarpl which was used for the construction of the gene library was constructed by hgation of a 3 kb BamHI-Hmdll fragment from pANsCosl (Osiewacz, Curr Genet. 26:87-90 (1994)) and a 3.2 kb Acc65I-HmdIII fragment from pA04.2 (De Ruiter- Jacobs et al, Curr. Genet. 16-159-63 (1989)) Acc65I-BamHI digested pHELPl (Gems et al , Gene 1991 98:61-67). This cosmid vector carnes the A oryzae pyrG selection marker and is self-replicating in filamentous fungi.
  • Mbol digested genomic DNA was hgated to BamHI-digested pAOpyrGcosarpl, and the hgation mixture was packaged into phage particles usmg the Stratagene Supercosl vector kit (Stratagene Inc., La Jolla CA) resulted in a total of ca. 30,000 individual clones, representing an approximate 30-fold representation of the A. sojae genome. Stocks (in 15% glycerol) of pools of the resulting clones were stored at -80°C for later use.
  • An A. sojae ATCC 11906 pyrG mutant was selected as a fluoroorotic acid-resistant de ⁇ vative from ATCC 11906, as descnbed m PCT/EU99/202516.3.
  • This stram, A. sojae ATCC 11906pyrG was transformed with two vectors carrymg the A. niger pyrG gene.
  • One vector pAB4-l van Hartingsveldt et al, Mol. Gen. Genet. 206:71-75 (1987)
  • pAB4-arpl (Verdoes et al , Gene 146:159-165 (1994)) carnes the pyrG gene and the A.
  • nidulans AMA1 sequence Transformation of ATCC 11906pyrG results m 5-10 transformants per microgram DNA from pAB4-l, whereas with pAB4-arpl frequency were at least 10-100 fold higher. Phenotypic analysis of the transformants revealed that the pyrG phenotype of the pAB4-arpl transformants was maintained only under continuous selection, whereas the pAB4-l transformants were stable with and without selection for the pyrG phenotype. These results confirm autonomous replication of the mtroduced plasmid DNA m pAB4-arpl transformants. Similar results were obtained with alternative fungal transformation vectors carrymg the AMA1 sequence or denvatives thereof, e.g. pAOpyrGcosarpl.
  • This vector results in a high frequency of transformants with freely replicating vector copies.
  • Fungal protoplasts were treated as desc ⁇ bed m Punt and van den Hondel, Methods Enzymol 216:447-457 (1992) with DNA from a cosmid library carrymg genomic fungal DNA clones from A. sojae or Chrysosporium and senal dilutions of the transformed protoplasts were plated on selective agar plates to determine the transformation frequency obtained.
  • the remammg protoplasts were regenerated m selective medium for a few hours and stored at 4°C. Based on the results obtained for the transformation frequency (which dependmg of the experiment will reach values up to several thousand transformants per microgram of cosmid library DNA), limiting dilutions of the regenerated protoplasts were plated m microtiter plates of 96, 248, or alternative well format, resulting m one transformed protoplast per well. Plates were mcubated at 35°C to form fungal biomass. The resulting transformant library is used for further expenments.
  • Vanous patent applications teach that morphological mutants can be isolated by vanous ways of screening.
  • WO9602653 and WO9726330 describe non-defined mutants exhibiting compact morphology. It was found that a proprotem processing mutant of A. sojae had an unexpected aberrant growth phenotype (hyper-branchmg) while no det ⁇ mental effect on protem production was observed. Culture expenments with this strain revealed a very compact growth phenotype with micropellets. The observed charactenstics were not only present in A. sojae but other mutated fungi as well, e.g. A. Niger. (1) Construction of an A. niger proprotein processing mutant
  • PCR was used. Based on the comparison of vanous proprotem convertase genes from vanous yeast species and higher eukaryotes, different PCR pnmers were designed (SEQ. ID Nos 4-12) which are degenerated, respectively, 4, 2, 2, 512, 1152, 4608, 2048 and 49152 times. From the amplification usmg pnmers PE4 and PE6, two individual clones were obtained of which the encoded protem sequence did show significant homology to the S. cerevisiae KEX2 sequence (SEQ. ID No. 13). These clones were used for further expenments.
  • a niger gene was designated pclA (from/?roprotem-convertase-/ ⁇ ke). Southern analysis of genomic digests of A. niger revealed that the pclA gene was a single copy gene with no closely related genes in the A. niger genome, as even at heterologous hybridisation conditions (50°C; washes at 6xSSC) no additional hybndisation signals were evident.
  • a first screening of an EMBL3 genomic library of A. niger N401 van Hartmgsveldt et al, Mol. Gen. Genet.
  • pPCLlA apclA deletion vector, in which a large part of the pclA coding region was replaced for the A oryzaepyrG selection marker, was generated.
  • the 5 kb EcoRI insert fragment was used for transformation of various A niger strams. From these transformations (based on pyrG selection) numerous transformants were obtained. Interestingly, a fraction of the transformants (varying from 1- 50%) displayed a very distinct aberrant phenotype (figure 10).
  • a gene replacement vector was generated following an approach similar to that desc ⁇ bed elsewhere m our examples usmg the reusable pyrG selection marker desc ⁇ bed m PCT EU99/202516.
  • a gene disruption vector was constructed carrying the pyrG selection marker and 5' and 3' truncated fragment from the A sojae pclA gene Both the gene replacement and gene disruption vector were used to generate pclA mutants m ATCC 11906 and ATCC 11906 de ⁇ vatives. Culture expenments with some of the resulting transformants revealed improved morphological charactenstics, in particular compact growth morphology and micropellets.. (3) Isolation of alternative A sojae compact growth mutants
  • Transformation of A sojae ATCC 11906 and denvatives may be earned out with linear DNA fragments carrymg a fungal selection marker. If no specific replicating sequences are provided transformants obtained using this procedure carry the introduced DNA integrated mto the genome of the host stram. As the introduced selection marker is from heterologous ongm (A niger) only heterologous recombination will occur, leadmg to a collection of transformants carrying the marker DNA at vanous positions in the genome. This integration is prone to result in disruption of endogenous A sojae sequences, thus resulting in a collection of A sojae mutant strams.
  • A. sojae GFP Green Fluorescent Protein
  • pGPDGFP GFP
  • Glucoamylase-GFP fusion genes pGPDGLAGFP, derivatives of the vectors described by Gordon et al, Microbiology 146(Pt 2):415-426 (2000), in which the glaA promoter was replaced for the constitutively expressed A. nidulans gpdA promoter
  • pGPDGLAGFP Glucoamylase-GFP
  • Transformation media Appendix to the Examples: Transformation media:
  • MnR Soft MnR with only 7.5 g/1 of agar.
  • Agar 15 g 1 pH should be 6.8 PDA :
  • Potato Dextrose Agar ⁇ (Difco) 39 g/1 pH should be 5.5
  • the regeneration media (MnR) supplemented with 50 ⁇ g/ml phleomycm or 100-150 ⁇ g/ml hygromycm is used to select transformants.
  • GS medium supplemented with 5 ⁇ g/ml phleomycm is used to confirm antibiotic resistance.
  • PDA is a complete medium for fast growth and good sporulation.
  • Liquid media are moculated with l/20th of spore suspension (all spores from one 90mm PDA plate m 5ml 0.1% Tween). Such cultures are grown at 27°C m shake flasks (200 rpm).

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Abstract

Méthode d'expression de banques d'ADN exogènes dans des champignons filamenteux mutants. Lesdits champignons sont capables de traiter des gènes eucaryotes contenant un intron, et peuvent également assurer des phases traitement post-traductionnel, telles que la gylcosylation et le repliement de protéine. L'invention porte également sur l'utilisation de champignons à morphologie modifiée, permettant le criblage à rendement élevé et l'évolution moléculaire dirigée de protéines exprimées. Lesdits champignons transformés peuvent être utilisés pour la production de quantités plus importantes d'une protéine pour l'isolement, la caractérisation et l'essai d'application et peut convenir à la production commerciale de ladite protéine.
PCT/US2000/010199 1999-10-06 2000-04-13 Criblage a rendement eleve de banques d'adn exprimees, dans des champignons filamenteux WO2001025468A1 (fr)

Priority Applications (19)

Application Number Priority Date Filing Date Title
AU43527/00A AU4352700A (en) 1999-10-06 2000-04-13 High-throughput screening of expressed dna libraries in filamentous fungi
JP2001576942A JP5138855B2 (ja) 2000-04-13 2001-04-13 糸状菌において発現されるｄｎａライブラリーのハイスループットスクリーニング
AT01927056T ATE433486T1 (de) 2000-04-13 2001-04-13 Durchmusterung von bibliotheken exprimierter dna in filamentösen pilzen mit hoher durchsatzrate
US09/834,434 US7122330B2 (en) 2000-04-13 2001-04-13 High-throughput screening of expressed DNA libraries in filamentous fungi
MXPA01012905A MXPA01012905A (es) 2000-04-13 2001-04-13 Clasificacion de alto rendimiento de bibliotecas de adn expresado en hongo filamentos.
BR0105795-2A BR0105795A (pt) 2000-04-13 2001-04-13 Triagem de alta produção de bibliotecas de dna expressadas em fungos filamentosos
DE60138947T DE60138947D1 (de) 2000-04-13 2001-04-13 Durchmusterung von bibliotheken exprimierter dna in filamentösen pilzen mit hoher durchsatzrate
EA200200035A EA006873B1 (ru) 2000-04-13 2001-04-13 Трансформированные грибы-гифомицеты, способ их получения и способы экспрессии и получения белков при их использовании
CN01801513.1A CN1380905A (zh) 2000-04-13 2001-04-13 丝状真菌中dna表达文库的高通量筛选
IL14693501A IL146935A0 (en) 2000-04-13 2001-04-13 High-throughput screening of expressed dna libraries in filamentous fungi
ES01927056T ES2328011T3 (es) 2000-04-13 2001-04-13 Selecion de alto rendimiento de bibliotecas de adn expresadas en hongos filamentosos.
CA002376552A CA2376552A1 (fr) 2000-04-13 2001-04-13 Criblage a debit eleve de bibliotheques d'adn exprimees dans des champignons filamenteux
AU53544/01A AU5354401A (en) 2000-04-13 2001-04-13 High-throughput screening of expressed DNA libraries in filamentous fungi
DK01927056T DK1272669T3 (da) 2000-04-13 2001-04-13 High-throughput screening af udtrykte DNA-biblioteker i trådsvampe
EP01927056A EP1272669B1 (fr) 2000-04-13 2001-04-13 Criblage a debit eleve de bibliotheques d'adn exprimees dans des champignons filamenteux
CN2010106218825A CN102174551A (zh) 2000-04-13 2001-04-13 丝状真菌中dna表达文库的高通量筛选
PCT/US2001/012335 WO2001079558A1 (fr) 2000-04-13 2001-04-13 Criblage a debit eleve de bibliotheques d'adn exprimees dans des champignons filamenteux
KR1020017016040A KR20020026456A (ko) 2000-04-13 2001-04-13 사상균에서 발현된 ｄｎａ 라이브러리의 고산출량 스크리닝
US11/490,761 US7794962B2 (en) 2000-04-13 2006-07-21 High-throughput screening of expressed DNA libraries in filamentous fungi

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PCT/NL1999/000618 WO2000020555A2 (fr) 1998-10-06 1999-10-06 Systeme de transformation dans le domaine des hotes fongiques filamenteux
NLPCT/NL99/00618 1999-10-06

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WO2003000941A3 (fr) * 2001-06-26 2004-03-25 Novozymes As Polypeptides presentant une activite de cellobiohydrolase i et polynucleotides codant pour ceux-ci
WO2009033071A2 (fr) 2007-09-07 2009-03-12 Dyadic International, Inc. Enzymes fongiques inédites
EP2102366A2 (fr) * 2006-12-10 2009-09-23 Dyadic International, Inc. Expression et criblage a haut debit de bibliotheques d'adn exprime complexes dans les champignons filamenteux
EP2385104A1 (fr) * 2003-04-01 2011-11-09 Danisco US Inc. CBH1 de Scytalidium thermophilium variant
WO2011161004A1 (fr) * 2010-06-22 2011-12-29 Dsm Ip Assets B.V. Procédé de fermentation à bulles d'air
US8268585B2 (en) 1998-10-06 2012-09-18 Dyadic International (Usa), Inc. Transformation system in the field of filamentous fungal hosts
US8673618B2 (en) 1996-10-10 2014-03-18 Dyadic International (Usa), Inc. Construction of highly efficient cellulase compositions for enzymatic hydrolysis of cellulose
US8999696B2 (en) 2007-05-10 2015-04-07 Novozymes, Inc. Compositions and methods for enhancing the degradation or conversion of cellulose-containing material
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CN116135979A (zh) * 2021-11-16 2023-05-19 中国科学院天津工业生物技术研究所 一种基于流式细胞术的免涂板操作的菌株工程改造的方法及应用

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US5811381A (en) * 1996-10-10 1998-09-22 Mark A. Emalfarb Cellulase compositions and methods of use

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US8916363B2 (en) 1996-10-10 2014-12-23 Dyadic International (Usa), Inc. Construction of Highly efficient cellulase compositions for enzymatic hydrolysis of cellulose
US8673618B2 (en) 1996-10-10 2014-03-18 Dyadic International (Usa), Inc. Construction of highly efficient cellulase compositions for enzymatic hydrolysis of cellulose
US7906309B2 (en) * 1998-10-06 2011-03-15 Dyadic International (Usa), Inc. Expression-regulating sequences and expression products in the field of filamentous fungi
US8268585B2 (en) 1998-10-06 2012-09-18 Dyadic International (Usa), Inc. Transformation system in the field of filamentous fungal hosts
EP1276876B1 (fr) * 2000-04-13 2007-04-04 Mark Aaron Emalfarb Sequences de regulation de l'expression des champignons filamenteux chrysosporium
EP1276876A2 (fr) * 2000-04-13 2003-01-22 Mark Aaron Emalfarb Nouvelles sequences de regulation de l'expression et produits d'expression dans le domaine des champignons filamenteux
EP1854888A3 (fr) * 2000-04-13 2009-10-07 Dyadic International (USA), Inc. Séquences de régulation de l'expression et produits de l'expression dans le domaine des champignons filamenteux
US8507238B2 (en) 2001-06-26 2013-08-13 Novozymes A/S Polypeptides having cellobiohydrolase 1 activity and polynucleotides encoding same
US9187739B2 (en) 2001-06-26 2015-11-17 Novozymes A/S Polypeptides having cellobiohydrolase I activity and polynucleotides encoding same
US9447399B2 (en) 2001-06-26 2016-09-20 Novozymes A/S Polypeptides having cellobiohydrolase I activity and polynucleotides encoding same
US9309504B2 (en) 2001-06-26 2016-04-12 Novozymes A/S Polypeptides having cellobiohydrolase I activity and polynucleotides encoding same
US7785853B2 (en) 2001-06-26 2010-08-31 Novozymes A/S Polypeptides having cellobiohydrolase I activity and polynucleotides encoding same
US8993299B2 (en) 2001-06-26 2015-03-31 Novozymes A/S Polypeptides having cellobiohydrolase I activity and polynucleotides encoding same
US8986969B2 (en) 2001-06-26 2015-03-24 Novozymes A/S Polypeptides having cellobiohydrolase I activity and polynucleotides encoding same
US8338156B2 (en) 2001-06-26 2012-12-25 Novozymes A/S Polypeptides having cellobiohydrolase I activity and polynucleotides encoding same
WO2003000941A3 (fr) * 2001-06-26 2004-03-25 Novozymes As Polypeptides presentant une activite de cellobiohydrolase i et polynucleotides codant pour ceux-ci
US8603793B2 (en) 2001-06-26 2013-12-10 Novozymes A/S Polypeptides having cellobiohydrolase 1 activity and polynucleotides encoding same
US8603794B2 (en) 2001-06-26 2013-12-10 Novozymes A/S Polypeptides having cellobiohydrolase 1 activity and polynucleotides encoding same
EP2385104A1 (fr) * 2003-04-01 2011-11-09 Danisco US Inc. CBH1 de Scytalidium thermophilium variant
EP2102366A4 (fr) * 2006-12-10 2010-01-27 Dyadic International Inc Expression et criblage a haut debit de bibliotheques d'adn exprime complexes dans les champignons filamenteux
US20120030839A1 (en) * 2006-12-10 2012-02-02 Dyadic International, Inc. Expression and High-Throughput Screening of Complex Expressed DNA Libraries in Filamentous Fungi
US8680252B2 (en) * 2006-12-10 2014-03-25 Dyadic International (Usa), Inc. Expression and high-throughput screening of complex expressed DNA libraries in filamentous fungi
EP2505651A3 (fr) * 2006-12-10 2013-01-09 Dyadic International, Inc. Isolat de champignon avec activité protéase réduite
EP2505651A2 (fr) 2006-12-10 2012-10-03 Dyadic International, Inc. Isolat de champignon avec activité protéase réduite
EP2102366A2 (fr) * 2006-12-10 2009-09-23 Dyadic International, Inc. Expression et criblage a haut debit de bibliotheques d'adn exprime complexes dans les champignons filamenteux
US8999696B2 (en) 2007-05-10 2015-04-07 Novozymes, Inc. Compositions and methods for enhancing the degradation or conversion of cellulose-containing material
US9631213B2 (en) 2007-05-10 2017-04-25 Novozymes, Inc. Compositions and methods for enhancing the degradation or conversion of cellulose-containing material
US9914946B2 (en) 2007-05-10 2018-03-13 Novozymes, Inc. Polypeptides having xylanase activity and polynucleotides encoding same
WO2009033071A2 (fr) 2007-09-07 2009-03-12 Dyadic International, Inc. Enzymes fongiques inédites
US8551751B2 (en) 2007-09-07 2013-10-08 Dyadic International, Inc. BX11 enzymes having xylosidase activity
WO2011161004A1 (fr) * 2010-06-22 2011-12-29 Dsm Ip Assets B.V. Procédé de fermentation à bulles d'air
CN116135979A (zh) * 2021-11-16 2023-05-19 中国科学院天津工业生物技术研究所 一种基于流式细胞术的免涂板操作的菌株工程改造的方法及应用
CN115399202A (zh) * 2022-08-10 2022-11-29 吐鲁番市农业技术推广中心 一种西瓜种植期间的病虫害防治方法

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