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  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • 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
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
• • 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
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi

Definitions

• the invention relates to novel means of transformation of fungi and to their use for production of heterologous proteins.
• the means involve genetically engineered fungi belonging to the taxonomic group Aspergillus sojae. Suggestions have been provided in the past to use Aspergillus sojae as a host strain for transformation. However to date no data are provided on successful transformation and/or expression of heterologous proteins. In addition it has been found, that so far certain proteins, such as phytase which were difficult to express in large amounts, due to several reasons including proteolytic degradation in expression hosts other than Aspergillus sojae, can surprisingly be expressed in Aspergillus sojae.
• the subject of the invention further covers a process for obtaining improved Aspergillus sojae strains for expression purposes, characterized by, on the one hand, a decreased proteolytic activity, and, on the other hand improved fermentation characteristics related to the morphology of the fungi.
• WO97/04108 for example describes the isolation of a protease encoding nucleic acid sequence, specifically a leucine aminopeptidase encoding sequence, and the transformation of a variety of host organisms, i.a. Aspergillus sojae, with a leucine aminopeptidase encoding sequence.
• Aspergillus sojae a variety of host organisms
• no illustration of this particular transformation actually having been carried out is provided.
• vector p3SR2 with the amdS marker has frequently been described in the literature as being useful for transforming various strains, for example Aspergillus oryzae (in EP 0.238.023), Trichoderma reesei (in EP 0.244.234) and Aspergillus niger (EMBO Journal 4, pages 475-479). Consequently, the analogous use for transforming Aspergillus in general is put forward in WO97/04108 on the basis of these previous publications.
• the suggested transformation protocol is, however, unsuccessful with Aspergillus sojae.
• the selection criteria described in the prior art are insufficient to ensure practical selection of desirable transformants when using the vector p3SR2.
• auxotrophic pyrG-based system has many favourable characteristics.
• Experiments were carried out to obtain A. sojae pyrG mutant strains, using a standard procedure based on direct selection for resistance to fluoro-orotic acid (FOA) on plates containing uridine to support growth of the mutant strain (Van Hartingsveldt et al. in Mol. Gen. Genet. (1987) 206, pages 71-75).
• FOA fluoro-orotic acid
• use of the analogous method on Aspergillus sojae strains did not lead to pyrG mutants.
• the usual method did lead to fluoro-orotic acid resistant strains but all the strains were able to grow without uridine.
• the required argB mutant can be obtained through random mutagenesis followed by screening of tens of thousands of colonies.
• the situation for pyrG is better in that the mutant is itself selectable.
• no mutant is required as the presence of amdS works as dominant selectable marker.
• protease profile of this strain is incompatible for application as a production host. So even though a transformation protocol has been suggested in the prior art for this particular Aspergillus sojae strain it could not possibly lead to a high level of expression of heterologous protein even if the protocol for transformation was successful.
• the subject invention is directed at Aspergillus sojae strains and the application thereof for production of recombinant proteins and polypeptides. Firstly, a description of Aspergillus sojae strains is provided.
• the fungal taxonomy is a complex issue.
• the Aspergillus genus comprises Aspergillus sojae in the Flavi/Tamarii section (see Table 1).
• A. sojae is clearly shown to be distinct from A. oryzae which is located in the same section (see Table 2).
• strains belonging to Aspergillus sojae can be distinguished from taxonomically closely related Aspergillus oryzae and also closely related Aspergillus parasiticus strains in a number of manners recognised in the art.
• Aspergillus oryzae further differs from Aspergillus sojae upon comparison of the alpA sequence of these strains.
• Aspergillus sojae comprises an Xmnl restriction site at a specific location in the alpA gene. The corresponding location in the alpA gene of several Aspergillus oryzae strains does not possess such a restriction site.
• Aspergillus sojae as used throughout the patent application is meant to imply a strain that preferably fulfils all the requirements described in the cited references in combination with the presence of the Xmnl restriction site in the alpA gene.
• Specific homologous primers for both the Aspergillus oryzae and Aspergillus sojae sequences are also provided.
• the invention in one aspect covers a recombinant Aspergillus sojae comprising an introduced acetamidase S (amdS) gene as a selectable marker.
• an A. sojae is selectable on a medium comprising a substrate for the introduced amdS protein as sole source of nitrogen, said medium further comprising a carbon substrate and said medium being free of endogenous amdS inducing substrate.
• a suitable medium comprises acrylamide as substrate for the introduced amdS as sole source of nitrogen.
• a suitable medium at least further comprises minimum substrates required for growth of Aspergillus sojae.
• a suitable category of A. sojae according to the invention is formed by A. sojae that are not selectable on acetamide comprising medium.
• An A. sojae according to the invention is suitably an A. sojae selectable on a medium free of glucose, i.e. a medium wherein the carbon source is not glucose.
• Such a medium can be a medium having sorbitol as carbon source. Best results in the case of sorbitol are achieved when sorbitol is the sole carbon source.
• An Aspergillus sojae according to the invention may comprise a further introduced nucleic acid sequence, said further introduced sequence preferably encoding a protein or polypeptide.
• the further introduced sequence may be adapted for optimised codon usage to the host strain codon usage or may have the original codons from the host from which it has been derived.
• the introduced sequence is in principle any sequence the skilled person wishes to express.
• the introduced sequence can suitably be heterologous, i.e. foreign to the Aspergillus sojae into which it is introduced. It can also be native but introduced in the form of one or more additional copies.
• One of the subjects of the invention is aimed at expressing phytase or proteins having phytase activity.
• Numerous sequences are known to the skilled person concerning sequence data of phytases. We refer to and incorporate by reference the contents of EP 684.313, EP 897.010, WO 99/49022, EP 911.416 and EP 897.985. These documents describe various natural and modified phytase sequences. They also describe a consensus sequence. A suitable embodiment is formed by phytase sequences from Peniophora being either the natural sequences or modified versions thereof.
• the new system is more flexible than prior systems and thus heterologous sequences, including heterologous sequences encoding phytase or proteins having phytase activity that were difficult to express in the prior art fungal systems can be expressed in the novel system according to the invention.
• An Aspergillus sojae according to the invention as defined in any of the embodiments defined above comprising an introduced amdS gene as selectable marker may suitably have no active endogenous amdS gene.
• the Aspergillus sojae according to such an embodiment may by way of example have an endogenous amdS gene comprising an endogenous amdS inactivating mutation. Any type of inactivating mutation known or conceivable to the skilled person may have occurred. A suitable example of such inactivating mutation may be a deletion or disruption. The mutation may inactivate the gene or the gene product. The skilled person will realise that numerous options are available to achieve this and that they can readily be achieved.
• the invention is also directed at a recombinant Aspergillus sojae free of an active endogenous amdS gene and further comprising an introduced amdS gene as selectable marker.
• the recombinant Aspergillus sojae according to the invention is selectable on a medium comprising a substrate for the amdS as sole source of nitrogen, said medium further comprising a carbon substrate.
• a suitable medium at least further comprises minimum substrates required for growth of A. sojae.
• the endogenous amdS gene can for example have been inactivated. This inactivation can be any type of inactivation known or conceivable to a person skilled in the art that still leaves the A. sojae viable.
• the endogenous amdS gene can comprise an inactivating mutation in the form of a substitution, deletion or insertion of the gene or part thereof, or by virtue of a mutation affecting expression of the gene such as to render it inactive.
• the complete endogenous amdS gene can also be absent.
• An Aspergillus sojae in any of the described embodiments according to the invention may be an A. sojae into an amdS gene has been introduced. This can be achieved e.g. by transformation or transfection. The resulting Aspergillus sojae according to the invention must then subsequently have been separated from non transformed or transfected A. sojae. Any of the embodiments described above as such or in combination are covered by the invention.
• the invention not only covers Aspergillus sojae as such, but also covers a method of introducing a nucleic acid sequence into A. sojae. The method comprises subjecting Aspergillus sojae to introduction of a nucleic acid sequence in a manner known per se for introduction of a nucleic acid sequence into a fungus.
• Such a manner can e.g. be transformation or transfection of the A. sojae.
• the method comprises the introduction of the amdS gene as the nucleic acid sequence followed by selection of the resulting transformed or transfected A. sojae on a medium free of endogenous amdS inducing substrate, said medium further comprising a substrate for the introduced amdS as sole source of nitrogen and said medium further comprising a carbon substrate, said medium enabling the desired A. sojae comprising introduced amdS gene to grow whilst eliminating growth of A. sojae devoid of a functional amdS gene.
• a suitable embodiment of such a method involves applying a medium comprising a substrate for amdS other than acetamide.
• such a medium comprises acrylamide as substrate for the introduced amdS as sole source of nitrogen.
• a medium for the method according to the invention comprises a carbon source other than glucose.
• a medium for use in a method according to the invention comprises sorbitol as carbon source, preferably as sole carbon source.
• a suitable medium at least further comprises minimum substrates required for growth of A. sojae.
• a method according to the invention as defined above in any of the embodiments comprises introduction of an additional nucleic acid sequence besides the amdS gene.
• the additional nucleic acid sequence for example encodes a protein or polypeptide, such as a phytase or proteins having phytase activity.
• the sequence does not necessarily have to be a non Aspergillus sojae sequence, but can also include A. sojae derived sequences It is however intended to indicate that the sequence that is introduced is absent in the non- transformed strain or else is present in a lower copy number than in the A. sojae according to the invention.
• the subject invention also covers any Aspergillus sojae obtained by the method described above.
• the method is directed at introducing a sequence capable of realising the presence of sufficient active amdS to function as selectable marker as opposed to the A. sojae into which the sequence is introduced which cannot for some reason or another produce sufficient active amdS to enable growth on a substrate for amdS as sole source of nitrogen.
• a method of selecting transformed or transfected A. sojae also falls within the scope of the invention
• the method comprises subjecting sojae (with no active endogenous amdS gene as defined according to any of the embodiments described) to a method of transformation or transfection of the A. sojae in a manner known per se for transformation or transfection of fungi with a nucleic acid sequence.
• the method comprises the introduction of an amdS gene as the nucleic acid sequence, followed by selection of the resulting transformed or transfected A. sojae, said selection occurring on a medium comprising a substrate for the introduced amdS as sole source of nitrogen, said medium further comprising a carbon substrate, said medium enabling the desired A. sojae to grow whilst eliminating growth of non transformed or transfected A.
• a suitable medium at least further " comprises minimum substrates required for growth of A. sojae.
• the invention is also directed at a method for producing recombinant Aspergillus sojae. This method comprises introducing a desired nucleic acid sequence e.g. by transformation or transfection in a manner known per se into an A. sojae, said desired nucleic acid sequence being flanked by sections of the endogenous amdS gene of a length and homology sufficient to ensure recombination. The introduction is followed by selection of the recombinant A. sojae having the desired nucleic acid sequence.
• the selection occurs for a selectable marker comprised in or transformed in cotransformation with the desired nucleic acid sequence, said selectable marker being absent in the A. sojae prior to introduction of the desired nucleic acid sequence.
• the flanking sequences may also be sequences corresponding to the endogenous amdS gene sufficient to ensure recombination. The skilled person can readily assess which sequences will suffice on the basis of hybridisation knowledge and the sequence data of the endogenous amdS gene. The recombination event eliminates the endogenous amdS activity in both cases.
• the selectable marker can quite suitably be pyrG, with, however, uracil instead of uridin in the selection medium.
• a further embodiment of the invention comprises Aspergillus sojae exhibiting growth on medium comprising uracil and fluoro-orotic acid, said A. sojae further not exhibiting growth on medium comprising uridine and fluoro-orotic acid.
• the A. sojae exhibits uracil auxotrophy, is unable to utilize uridine, is pyrG negative and exhibits resistance to fluoro-orotic acid.
• the uracil auxotrophy and the fluoro-orotic acid resistance are relievable upon complementation with an active introduced pyrG gene.
• Such an A. sojae according to the invention can be free of active endogenous pyrG genes.
• sojae according to the invention may comprise an endogenous pyrG gene with a mutation inactivating it.
• the mutation can be any mutation known or conceivable to a person skilled in the art, said mutation inactivating a pyrG gene or the expression product thereof.
• Such a mutation can by way of example be in the form of an insert of a nucleic acid sequence in the gene, a substitution of a part of the encoding sequence of the gene, a deletion of a part of the encoding sequence of the gene or a deletion of the whole encoding sequence of the gene.
• the mutation can also occur in the regulating part of the gene.
• said Aspergillus sojae can have a nucleic acid sequence for the mutated pyrG gene different to that of the wild type A. sojae pyrG gene.
• a further embodiment comprises pyrG negative A. sojae according to the invention as described in any of the above embodiments which further comprise any of the characteristics described for any of the amdS variant A. sojae according to the invention as such or in combination.
• a method of selecting transformed or transfected Aspergillus sojae also falls within the scope of the invention. The method comprises subjecting A.
• sojae of the pyrG negative type according to any of the embodiments of the invention as described above to a method of transformation or transfection with a nucleic acid sequence, said method comprising introducing an active pyrG gene into the pyrG negative A. sojae in a manner known per se for transformation or transfection.
• the introduction step is then followed by selection of the resulting transformed or transfected A. sojae on a medium free of uracil and fluoro- orotic acid, said medium at least further comprising minimum substrates required for growth of A. sojae, said medium enabling the desired A. sojae to grow whilst eliminating growth of non-transformed or -transfected A.
• the active pyrG gene that is introduced is flanked by identical nucleic acid sequence fragments, and the pyrG positive A. sojae resulting from the introduction of the pyrG gene and the flanking sequences is selected on a medium free of uracil and fluoro-orotic acid. Subsequently the pyrG positive A. sojae is cultivated on medium comprising uracil and fluoro-orotic acid, thereby eliminating the pyrG gene that had been introduced and thus resulting in a pyrG negative A.
• flanking sequences and the pyrG gene are further flanked by sequences that direct integration of the pyrG gene and the flanking sequences into a specific location, due to the fact that the integration directing sequences are homologous to a specific sequence of the A. sojae to be transformed. This enables knock out (if desired) of the gene associated with the specific sequence.
• the process of knock-out mutant creation as such is well known to the person skilled in the art.
• any of the embodiments of the selection method just described may further comprise the step wherein the Aspergillus sojae is transformed or transfected with a further heterologous nucleic acid sequence.
• the further heterologous nucleic acid sequence preferably encodes a protein or polypeptide and the same remarks are valid here as made elsewhere in this description for the nature of such further nucleic acid sequences for the other embodiments of Aspergillus sojae and fungi in general according to the invention.
• the further sequence can be introduced with the active pyrG gene either on the same vector or by cotransformation with the active pyrG gene that is introduced. The method of selecting transformed or transfected A.
• sojae as described may also be carried out in combination with the method for introducing a nucleic acid comprising introduction of a heterologous amdS gene in any of the embodiments according to the invention disclosed therefore above.
• the invention covers any recombinant A. sojae obtained by the method of selecting transformed or transfected A. sojae according to the invention.
• the invention is also directed at a method for producing recombinant Aspergillus sojae, said method comprising transformation or transfection in a manner known per se of a pyrG positive Aspergillus sojae with a nucleic acid sequence comprising the sequence to be introduced flanked by sections of the pyrG gene or corresponding sequences of a length and homology sufficient to ensure recombination eliminating the pyrG gene and introducing the desired sequence, followed by selection of the recombinant Aspergillus sojae with the desired sequence by selecting for the A sojae with a pyrG negative phenotype. Determination of the corresponding sequences lies within the reach of the skilled person by virtue of their knowledge of hybridisation processes with nucleic acid sequences and their knowledge of required sequence data of the pyrG genes.
• any Aspergillus sojae strain obtained by either the amdS and/or pyrG introduction method according to the invention is a novel strain falling within the scope of the invention as is any subsequent use of such a novel strain.
• Such a novel strain can comprise nucleic acid sequences that do not occur in the original corresponding Aspergillus sojae strain or even do not occur in Aspergillus sojae, Aspergilli or fungi.
• the sequences can be of mammalian origin or derived from any animal, plant or microbe.
• Nucleic acid sequences can also be expressed that are naturally present in the Aspergillus sojae strain but that are present in a lower copy number in the corresponding non-transformed A sojae.
• the production of homologous proteins is also covered by the invention when pyrG and/or amdS Aspergillus sojae strains according to the invention are involved.
• a preferred embodiment is that wherein the particular protein or polypeptide to be produced is absent in the corresponding non-treated A. sojae and/or is present in a lower copy number in the corresponding non-treated A. sojae, i.e. the A. sojae prior to introduction of the nucleic acid sequence.
• a process of production comprises cultivating the fungus under suitable conditions for expression of the desired sequence to occur.
• the process of production optionally includes the step of isolation of the resulting polypeptide or protein in a manner known per se for protein or polypeptide production by fungi.
• the protein or polypeptide will be secreted into the culture medium.
• a preferred protein or polypeptide is a protein or polypeptide susceptible to degradation upon expression by Aspergillus niger or Aspergillus awamori.
• a number of such proteins and polypeptides have already been disclosed in the prior art and a large number remain yet to be determined. Such determination is however a matter of routine for the skilled person.
• Another preferred embodiment of the protein or polypeptide to be expressed is one whereby the protein or polypeptide differs from an Aspergillus sojae protease and amylase.
• a preferred embodiment involves a non Aspergillus sojae protein or polypeptide.
• a particularly interesting embodiment comprises a combination of the two processes for introducing nucleic acid sequences according to the invention as described above.
• the advantage thereof lies in the fact that the frequency of transformation obtained with the pyrG marker is clearly much higher than that of the amdS marker.
• secondary screening of the pyrG+ strains for the best growth on acrylamide selective plates allows the identification of those recombinant Aspergillus sojae showing the highest copy number and thus most likely the highest level of gene expression.
• homologous and heterologous expression regulating sequences can be used by Aspergillus sojae i.e. natively occurring sequences of the strain itself or sequences foreign to the strain can be used.
• the transformants according to the invention can comprise any such regulatory sequences.
• the selection of the suitable regulatory region is a matter of choice that lies well within the range of the standard capabilities of the skilled person and will depend on the particular application.
• the regulating sequences can be constitutive or inducible.
• the regulating sequences can be fungal or non-fungal. A broad range are exemplified in the examples.
• a large number of expression regulating sequences are regularly used in the art for other systems, in particular fungal systems such as Aspergilli, and can routinely be applied without undue burden in the Aspergilli according to the invention.
• any vector may be used that is suitable for introducing nucleic acid sequences into fungal host cells. Numerous examples are available in the art.
• vectors that have been found suitable for transformation, transfection or expression in Aspergilli such as Aspergillus niger, Aspergillus awamori and Aspergillus oryzae can suitably be applied.
• the subject invention describes efficient protein production for recombinant Aspergillus sojae.
• Such efficient production is disclosed in those strains having a protease profile superior to ATCC42251 or at least as good as any of ATCC9362, ATCC 11906 and ATCC20387.
• the subject description thus reveals that some known strains of A. sojae are well suited already as such for production of proteins, polypeptides and metabolites. These Aspergillus sojae strains exhibit a lower proteolytic activity than the reference strain A. sojae ATCC42251.
• the two known strains ATCC 11906 and ATCC20387 are well suited. So preferred A. sojae strains for production of proteins, polypeptides and metabolites will be those expressing equal to or less proteolytic activity than the two preferred strains.
• Strain ATCC 11906 is the best embodiment of the deposited ATCC A. sojae strains according to the prior art. Suitable proteins or polypeptides will be produced. Now that the subject invention has enabled introduction of nucleic acid sequences, such can serve to provide any protein or polypeptide of choice using an A. sojae as expression host.
• the subject invention offers an improvement over existing expression systems. A number of existing protein production systems exhibit expression problems due to proteolysis. In particular the new system is better than the currently frequently applied expression systems Aspergillus niger and Aspergillus awamori.
• the subject invention now renders it possible to provide a recombinant Aspergillus sojae comprising a introduced nucleic acid sequence encoding a protein or a polypeptide for expression, said protein or polypeptide being susceptible to degradation upon expression by A. niger or A. awamori.
• the invention also provides a recombinant A. sojae comprising an introduced nucleic acid sequence encoding a protein or polypeptide for expression, said protein or polypeptide being other than A. sojae protease and amylase.
• a preferred embodiment is that wherein the introduced nucleic acid sequence encodes a non-A. sojae protein or polypeptide.
• Such recombinant A. sojae strains also fall within the scope of the invention.
• Aspergillus sojae strains that have been modified in order to enhance their suitability as expression hosts is currently provided. These modifications can be reduced proteolytic activity as induced by any means. Specifically, the use of UV random mutagenesis is illustrated. Also specific mutation of one or more protease genes is illustrated. The means by which mutations can be introduced are common knowledge to the skilled person, and numerous alternative embodiments are thus readily available to arrive at the desired mutants. A suitable embodiment is formed by mutants in which alkaline proteolytic activity has been reduced. In particular elimination of activity of specifically the major 35 kDa alkaline protease is illustrated as ensuring increased expression of proteins and polypeptides.
• novel strains exhibiting reduced proteolytic activity specifically reduced alkaline proteolytic activity.
• Such strains are obtainable using any specific mutation route known or conceivable to the skilled person.
• a preferred embodiment of such expression hosts exhibiting reduced proteolytic activity as described above further comprises a selectable marker. Quite suitably the selectable marker will be amdS, pyrG or a combination thereof.
• the invention in particular covers a method of producing protease deficient mutants of A. sojae by knocking out the 35 kDa alkaline protease gene.
• This can potentially be achieved on the basis of the sequence data provided for this gene.
• a method using recombination with a pyrG selection marker linked to two flanking regions eliciting cross over of the 35 kDa alkaline protease gene, whereby the resulting strain has the pyrG selection marker and misses the 35 kDa alkaline protease gene is an elegant one.
• the pyrG selection marker can be eliminated, thus providing a 35 kDa alkaline protease negative Aspergillus sojae mutant that can be used for expression purposes of any desired sequence to be introduced therein.
• sequence to be introduced can have been incorporated in the previous steps already either on the same vector as the pyrG marker or in a cotransformation event.
• the method can be carried out analogously where a different protease gene than the 35 kDa alkaline protease gene is to be knocked out.
• the analogous measures to be taken are obvious to the skilled person on the basis of the illustration provided herein in combination with knowledge of other protease sequences.
• amdS selectable marker can be used in accordance with the invention as described elsewhere in this description.
• Mutant fungi exhibiting improved fermentation characteristics are also provided as an additional aspect of the subject invention.
• the invention is directed at a fungus comprising a mutation inhibiting the activity of proprotein convertase or an equivalent protein.
• proprotein convertases are known in the art.
• Figure 1 providing sequence data of a number of such proteins.
• a fungus according to the invention is suitably selected from Agaricus, Aspergillus, Trichoderma, Rhizopus, Mucor, Phanerochaete, Trametes, Penicillium, Cephalosporium, Neurospora,
• fungi are Aspergillus niger, Aspergillus foetidus, Aspergillus sojae, Aspergillus awamori, Aspergillus oryzae, Trichoderma reesei,
• Penicillium chrysosporum Cephalosporium acremonium, Neurospora crassa,
• a preferred embodiment covers the mutant when it is an Aspergillus sojae, most particular preference is extended to Aspergillus sojae as defined above according to the invention, i.e. comprising heterologous nucleic acid sequences, e.g. in combination with the selectable markers amdS and/or pyrG.
• the functionally equivalent protein may suitably have a nucleic acid sequence capable of hybridising under stringent conditions to a nucleic acid sequence according to SEQ ID Nos. 3 to 9. Stringent hybridisation conditions can readily be determined by the skilled person.
• a suitable example of stringent hybridisation conditions are hybridisation at 50°C and preferably at 56°C and final washes at 3xSCC.
• PE4, PCL1 and PCL2 are specifically mentioned as examples of suitable oligonucleotide mixtures corresponding to the coding strand (i.e. SEQ ID Nos. 10, 11 and 12).
• PCL2-rev, PCL3 and PCL4 are mentioned (i.e. SEQ ID Nos. 13, 14, 15 and 16, respectively).
• Use of these primers in amplification procedures common in the art will provide equivalent sequences and such use and the resulting newly found sequences and application thereof in the manner analogous to that described in the subject description fall within the scope of the invention.
• sequences for which the oligonucleotides were made are well conserved as could be determined from comparison of the various amino acid sequences for the proteins provided (see Figure 1). Any other nucleic acid sequences exhibiting the same or higher degree of identity, similarity or homology with the sequences provided in the subject patent application for the proteins or relevant active parts thereof are covered by the invention as is the use thereof as primers or probes to find other proprotein convertase or equivalent protein encoding sequences and/or for subsequently introducing mutations in such protein encoding sequences.
• the equivalent protein or polypeptide will exhibit the activity of a proprotein convertase as the one having an amino acid sequence according to SEQ ID Nos. 3 to 9.
• the mutant fungus can comprise a substitution, insertion or deletion in the encoding sequence of the proprotein convertase or equivalent protein.
• the mutant fungus can suitably comprise a mutation in the regulation of the expression of the gene encoding proprotein convertase or equivalent protein.
• a mutant fungus according to the invention in a suitable embodiment exhibits reduced viscosity vis a vis the corresponding non mutated fungus under equivalent cultivation conditions.
• a mutant fungus according to any of the above embodiments exhibiting increased expression of a desired introduced nucleic acid sequence encoding a protein or polypeptide is included within the scope of the invention, said fungus exhibiting increased production of a protein or polypeptide under equivalent conditions vis a vis the corresponding wild type fungus.
• the activity site for the A. sojae proprotein convertase has been ascertained to be comprised within the amino acid sequences inferred by SEQ ID Nos. 3 and 4.
• a process for producing a phytase or protein having phytase activity or any other protein or polypeptide, preferably a recombinant phytase or any other heterologous protein or any other polypeptide, said process comprising cultivating a mutant fungus according to any of the embodiments described above falls within the scope of the invention.
• a process for obtaining the resulting protein or polypeptide either from the cell as such or after secretion thereof from the cell is also included.
• any of the described novel strains for transformation of any nucleic acid sequence encoding a phytase or protein having phytase activity or any other protein or polypeptide thereto and any subsequent expression of any nucleic acid sequence introduced therein and also optionally any following processing and/or secretion and/or isolation is covered by the invention.
• Any phytase or phytase-like or any other heterologous protein or polypeptide encoding sequence can suitably be used. This can be of fungal or non-fungal origin.
• a preferred embodiment is formed by acid labile protein or polypeptide encoding sequences.
• the protein encoding sequence encodes non protease-like proteins.
• the examples show a phytase sequence and a number of heterologous sequences suitable for use in transformation and also for expression in Aspergillus sojae hosts. Further examples of suitable proteins to be expressed are obvious to a person skilled in the art.
• Genomic DNA of A. sojae was isolated from protoplasts obtained from ATCC 11906 using a previously described protocol (Punt, van den Hondel, 1992). After isolation, DNA was extracted from the protoplasts using the protocol described by Kolar et al., 1988. Subsequently the DNA was partially digested with Mbol to result in DNA fragments of an average size of 30-50 kb.
• Vector pAOpyrGcosarpl which was used for the construction of the gene library, was constructed by ligation of a 3 kb BamRl-Hindlll fragment from pANsCosl (Osiewacs,
• This cosmid vector carries the A. oryzae pyrG selection marker and is self-replicating in filamentous fungi.
• Mbol digested genomic DNA was ligated to if ⁇ mHI-digested pAOpyrGcosarpl, and the ligation mixture was packaged into phage particles using the Stratagene Supercosl vector kit. In total 30,000 individual clones were obtained 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. AMDS TRANSFORMATION METHOD AND TRANSFORMANTS.
• vector p3SR2 (carrying the amdS marker) was used in combination with pAOpyrGcosARPl.
• This latter vector is a derivative of the autonomously replicating Aspergillus vector Arpl, which in all Aspergillus species tested so far, resulted in highly increased numbers of (instable) transformants when used as a cotransforming vector. For nearly all strains sufficient protoplasts (about 10E6-10E7 per transformation) were obtained.
• Analysis of appropriate AmdS selection conditions for the various A. sojae strains revealed vigorous growth of most strains on the commonly used selective acetamide medium. Clearly, the acetamide selection conditions proposed for A.
• sojae amdS transformants as reported in WO97/04108, were not appropriate for the selection of A. sojae transformants.
• Figures 2a, b and c show the background growth observed for selected strains on the selection medium described in WO97/04108 and the improved acrylamide selection medium described in Table 3. Further transformation experiments with the three selected A. sojae strains revealed that protoplasting efficiencies for ATCC 11906 and ATCC20387 were better using the NaCl-method. Successful protoplasting was obtained using various commercially available protoplasting enzyme preparations such as NOVOZYM, Caylase, Glucanex, etc. Based on the NaCl transformation protocol the three selected A. sojae strains were transformed with amdS selection vector p3SR2 or derivatives thereof. Using the modified acrylamide selection plates a number of vigorously growing transformants were obtained, while no growth was observed in a control transformation without DNA.
• Another approach to circumvent background growth of non-transformed mycelium is the elimination of the activity of the wild type A. sojae amdS gene. This can be achieved for example by disruption of the A. sojae amdS gene.
• specific DNA fragments carrying ATCC 11906 amdS sequences were PCR-amplified using primers derived from published A. oryzae amdS sequences (Gomi et al; 1991, Gene 108, 91-98).
• Previous experiments had shown that cloning by stringent hybridisation would be unsuccessful due to a low level of sequence conservation between A. nidulans and A. sojae amdS sequences.
• Transformation of A. sojae pyrG mutants with this vector results in a similar number of PyrG+ transformants as with the vector pAB4-l.
• subsequent plating of spores of selected pAB4-l and pAB4-Irep transtormants to FOA selection plates resulted in many more FOA resistant/uracil requiring colonies for the pAB4-lrep transformant.
• Southern analysis of these FOA resistant/uracil requiring clones showed that in most of the pAB4-lrep strains the A. niger pyrG marker gene had been deleted leaving only the small 0.7 kb repeat region at the locus of integration, while in the pAB4-l strains the A. niger gene was still present and had presumably acquired a mutation resulting in the pyrG-negative phenotype.
• sojae culture fluid To analyse protease activity of the culture fluids of the various strains, a milk clearing assay was performed. In addition medium samples were incubated with different proteins (e.g. bovine serum albumin (BSA)), and degradation of these proteins was followed in time in order to assess the suitability of the tested strains as expression hosts for a range of products. BSA was chosen as in our previous experiments with A. niger. This protein was shown to be very susceptible to proteases. A. terreus phytase was chosen as example of another proteolytically instable protein. Degradation of milk proteins as shown by the formation of a milk clearing zone at the periphery of growing colonies is a generally accepted criterion for protease activity.
• BSA bovine serum albumin
• Detection of BSA was carried out by Coomassie staining of SDS-PAGE gels.
• Western analysis using specific antibodies was carried out.
• Table 4 clear differences of degradation in A. niger culture fluid are evident when this is compared with that in A. sojae culture fluid.
• A. niger culture fluid pH 3-4
• rapid degradation of BSA occurs.
• A. sojae culture fluids from richer media degradation of BSA occurs, albeit less than in A. niger culture fluid.
• A. sojae culture fluids pH 7-8
• rapid degradation of A. terreus phytase occurs, with the exception of ATCC9362, ATCC11906 and ATCC20387 culture fluids.
• strains with the lowest phytase degradation also show low BSA degradation under the conditions tested.
• the two A. oryzae strains ATCC20235 and ATCC46250 show much higher proteolytic activity than most A. sojae strains.
• protease assays resulted in the identification of three low protease A. sojae strains, namely ATCC9362, ATCC 11906 and ATCC20387.
• A. sojae can cleary be used as expression host for a range of proteins and provides a series of advantages over prior art transformation and expression systems.
• Aspergillus sojae means by which additional strains could be created with enhanced characteristics for expression were considered. Two different approaches which can be used as such or in combination were developed to provide novel improved strains for expression of proteins.
• protease deficient mutants On the one hand the possibility of developing protease deficient mutants was investigated and the impact of such on levels of expression was assessed. On the other hand strains with amended morphology were developed with a view to improve fermentation characteristics. To achieve this a hitherto non-disclosed or suggested route was followed which is applicable not only to Aspergillus sojae but to Aspergilli and in fact to fungi in general. Development of protease deficient mutants
• alpA alkaline protease
• ATCCl 1906 was disrupted using a disruption vector carrying the re-usable pyrG selection marker as described in this description.
• An ATCCl 1906 cosmid library (in a PyrG cosmid) was constructed. From 10.000 independent cosmid clones initially 4 were found to hybridize under homologous conditions with an A. sojae alp A fragment obtained by PCR with primers MBL1784 and MBL1785. Rescreening of the 4 clones revealed only strong hybridisation with one clone. A 4 kb EcoRI and a 2.5 kb Hindlll fragment from this clone, together expected to carry the complete gene, were subcloned and characterised by restriction enzyme digestion and sequence analysis. Based on these subclones a new gene-replacement vector was constructed as described in Figure 6.
• low viscosity mutants can be isolated by various ways of screening.
• WO96/02653 and WO97/26330 describe non defined mutants exhibiting low viscosity.
• WO96/02653 and WO97/26330 describe non defined mutants exhibiting low viscosity.
• a proprotein processing mutant from this organism had an unexpected aberrant growth phenotype (hyper-branching) while no detrimental effect on the productivity of proteins was observed.
• Controlled fermentation experiments with this strain revealed that increased biomass concentrations were obtained at considerably lower viscosity values. The observed characteristics were not only present in A. sojae but other fungi as well, e.g. in A. niger.
• proprotein convertase encoding gene from A. niger
• PCR was used. Based on the comparison of various proprotein convertase genes from various yeast species and higher eukaryotes ( Figure 1) different PCR primers were designed (SEQ ID Nos. 10, 13 and 18-23) which are degenerated, respectively, 2048, 49152, 4, 2, 2, 512, 2048, and 4608 times. From the amplification using primers PE4 and PE6, two individual clones were obtained of which the encoded protein sequence did show significant homology to the S. cerevisiae KEX2 sequence (SEQ ID No. 24). These clones were used for further experiments.
• A. niger gene was designated pclA (from proprotein convertase-/ike). 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 stringent hybridisation conditions (50°C; washes at 6xSSC), no additional hybridisation signals were evident.
• a first screening of an EMBL3 genomic library of A. niger N401 van Hartingsveldt et al, 1987 did not result in any positively hybridising plaques although about 10-20 genome equivalents were screened.
• sojae pclA gene a replacement vector was generated using the EcoRV-site in the A. sojae pclA gene to clone the re-usable pyrG marker as a Smal fragment inside ( Figure 11).
• the resulting vector was partially digested with Clal to obtain the delta-pel fragment of 10.5 kb (see Figure 11). This fragment was isolated to be used for transformation of A. sojae pyrG strains.
• the gene replacement vector was used to generate pel A mutants in ATCCl 1906 and ATCCl 1906 derivatives.
• the resulting strains were used for the expression of homologous and heterologous proteins. Controlled fermentation experiments with some of the resulting transformants revealed improved fermentation characteristics, in particular a lower viscosity/biomass ratio of the culture.
• oligonucleotide mixtures were used in PCR with chromosomal DNA from Trichoderma reesei QM9414, Fusarium venenatum ATCC20334, Penicillium chrysogenum P2, Trametes versicolor, Rhizopus oryzae ATCC200076, and Agaricus bisporus HORST.
• PCR amplifications (30 cycles; 1 min 94°C; 1 min 40°C; 2 min 68°C) with one or more of the combinations of coding and non-coding strand oligonucleotides resulted in specific PCR products. Table 6 gives the results of the various amplification reactions.
• Viscosity ranges have been determined for fermentations using the specified fungal organism using the above procedure (Table 7).
• Biomass is determined by the following procedure: Pre weigh 5.5 cm filter paper (Whatman 54) in an aluminium weighing dish. Filter 25.0 ml whole broth through the 5.5 cm paper on a Buchner funnel, wash the filter cake with 25.0 ml deionised water, place the washed cake and filter in a weighing pan and dry overnight at 60°C. Finish drying at 100°C for 1 hour, then place in desiccator to cool. Measure the weight of dried material. Total biomass (g/1) is equal to the difference between the initial and final weights multiplied by 40.
• Protein levels were determined using the BioRad Assay Procedure. The data presented above represent values determined 48 hours into the fermentation process until fermentation end; all values of Aspergilli and Trichoderma are for commercially relevant fungal organisms and reflect actual commercial data.
• a fungal strain such as A. sojae IfvA and A. sojae pclA has the advantage that the low viscosity permits the use of lower power input and/or shear the in the fermentation to meet oxygen demands for those cases where shear stress on the product may be detrimental to productivity due to physical damage of the product molecule.
• the lower biomass production at high protein production indicates a more efficient organism in the conversion of fermentation media to product.
• A. sojae mutants provides better biomass and viscosity data whilst also delivering at least as much protein, and in fact a lot more protein than the two commercially used systems which obviously are better than for typically deposited Aspergillus or Trichoderma reesei strains in general public collections.
• sojae IjvA would allow development of fermentation conditions with higher multiples of increase in biomass, if increasing biomass results in increased productivity, for the desired product before reaching limiting fermentation conditions.
• the present high levels of biomass and viscosity produced by the T. longibrachiatum and A. niger organisms restrict the increase of biomass as the present levels of biomass and viscosity are near limiting practical fermentation conditions.
• the bipA promoter results in about 30% of the gpdA activity, which corresponds to expression data obtained in A. niger.
• the glaA promoter which is very active in A. niger results in less than 1% of the gpdA activity in A. sojae.
• A. sojae genes namely alp A (alkaline protease; inducible), amyA (amylase; inducible) and gpdA (glyceraldehyde-3-phosphate dehydrogenase; constitutive) was attempted using primers based on sequences available from A. oryzae (SEQ ID Nos. 26 to 31).
• Figures 13 a, b and c give the sequences and the position in published A. oryzae sequences of the various PCR primers used for this approach. Genomic template DNA from A. sojae ATCC 11906 was used for PCR amplification.
• the complete sequence of the cloned genes was determined. As shown in Figure 14 the A. sojae ATCCl 1906 gpdA promoter region has a very high homology with other gpdA promoter sequences and the alpA promoter was virtually identical to the A. oryzae alpA promoter (SEQ ID Nos. 32 and 33). Expression vectors carrying expression cassettes comprising these A. sojae promoters show significant levels of gene expression.
• heterologous proteins were tested which were known to be susceptible to acidic proteolysis and thus could not be expressed efficiently in other well known expression systems. Also proteins that are already efficiently expressed in alternative systems were tested in order to assess by way of comparison the levels of expression achieved with Aspergillus sojae vis a vis other known expression systems such as Aspergillus niger.
• DNA fragments carrying various fungal phytases (Wyss et al. (1999) Appl. Environ. Microbiol. 65, 359-366) were ligated as 5' Ncol or BspHl sites introduced at the ATG codon - 3' blunt-ended fragments downstream of the A.nidulans gpdA promoter in pA ⁇ 52-l ⁇ otI.
• the resulting vectors were used in cotransformation experiments of A. sojae using the amdS and or pyrG selection marker.
• Phytase producing transformants were screened using the described phytase plate-assay.
• AmdS+ clones were screened for phytase production using the phytase plate-assay. Further phytase expression vectors were generated using the GLA fusion approach (e.g. Broekhuijsen et al. (1993) J. Biotech. 31, 135-145). To this end phytase gene fragments, encoding the mature A. fumigatus phytase protein were fused, using convenient restriction sites and fusion PCR, to the 3 '-end of the glucoamylase carrier gene in vector pAN56-l (Genbank accession number Z32700).
• Vector pGLA6S ( Figure 15) is derived from pGLA6 (Punt et al. (1991) J. Biotech. 17, 19-334) by introducing a 5 kb EcoRI fragment carrying the A. nidulans amdS gene as selection marker into the unique EcoRI site of pGLA6.
• Vector pGLA6S ( Figure 15) carrying the amdS selection marker and the glucoamlyase gene under control of the A. nidulans gpdA promoter was introduced into A. sojae ATCCl 1906pyrG using cotransformation with vector pAB4.1.
• Starch plate-assays demonstrated the production of increased levels of amylolytic activity in these transformants. From the resulting transformants those showing proficient growth on acrylamide medium were analysed for glucoamylase production. On a Coomassie Briljant Blue-stained SDS PAGE gel from the culture supernatant of some of these transformants a single dominant protein band corresponding to glucoamylase was observed . Western analysis using a monoclonal antibody against glucoamylase (Verdoes et al. (1993) Transgenic Research 2, 84-92) was used to confirm the identity of this protein band. Interleukin-6 production
• interleukin-6 which is an example of a highly sensitive protein for proteolytic degradation, was shown to be virtually impossible in A. niger without the use of the gla-fusion strategy and protease deficient strains. Even with all these improvements the yields of IL-6 were only a few mg per litre culture fluid.
• A. sojae Another type of acid labile protein we have attempted to produce in A. sojae is GFP from the jelly fish Aequoria victoria. This protein is not only proteolytically sensitive but furthermore it loses its activity at acid pH.
• Vectors carrying GFP or GLA-GFP fusion genes were introduced into A. sojae by cotransformation using either the pyrG or amdS selection marker. Expression resulted in brightly fluorescent A. sojae transformants for both vector types.
• Figure 1 This figure provides a comparison of amino acid sequences of KEX2-like processing proteases from X. laevis (XENPC2 and XNFURIN), S. cerevisiae (SCKEX2), K. lactis (KLKEX1), C. albicans (CAKEX2), S pombe (SPKRP) and Y. lipolytica (YLKEX2).
• the primers which encode for the amino acid sequences with the highest overall identity (indicated with lightblue boxes), are indicated: MBL793, MBL1208, MBL 794, MBL1158, PE6, PCL1, PCL2(rev), PE6, PCL3, MBL789, PCL4 and MBL1219. Regions of overall identity (4 out of 7 entries) are indicated with purple boxes. Gaps are indicated with . ; no sequence data are indicated with ⁇ ; asteriks indicate the stop codon of the protein.
• Figure 2 This consists of 2a, b and c
• Figure 2a provides the background growth of the A. sojae strain described in patent WO97/04108 after 5 days of incubation at 33°C.
• the top picture reveals growth on non selection medium.
• the bottom left picture shows selection medium according to WO97/04108 and the bottom right picture shows the results using improved medium (acrylamide) according to the invention.
• Figure 2b provides the background growth of the A. sojae strain ATCCl 1906 after 5 days of incubation at 33°C.
• the top picture reveals growth on non selection medium.
• the bottom left picture shows selection medium according to WO97/04108 and the bottom right picture shows the results using improved medium (acrylamide) according to the invention.
• Figure 2c provides the background growth of the A.
• the top picture reveals growth on non selection medium.
• the bottom left picture shows selection medium according to WO97/04108 and the bottom right picture shows the results using improved medium (acrylamide) according to the invention.
• Figure 3 (a and b): This figure provides a comparison of A. sojae ATCCl 1906 and A. oryzae amdS sequences from both ends. A and B indicate the two ends. The cloned 1.6 kb A. sojae sequence was used. Underlined bold bases differ between species/strains. Intron I sequences are indicated in small letters.
• Figure 4 (a and b): This figure illustrates the construction of a pyrG disruption vector via pAO4-13 and pAO4-13deltaCla.
• Figure 5 This figure illustrates the construction of pAB4-lrep going from pAB4-l via isolation ofXhol fragment and Hindlll fragment followed by cloning into pMTL24.
• Figure 6 The construction of the alpA gene replacement vector is disclosed in this figure.
• a 4.4 kb EcoRI-StwI fragment from pASl-1 with the ATCCl 1906 genomic fragment, the 2.6 kb Smal-Ncol fragment from pAB4-lrep and the 4.4 kb NcoI-EcoRI fragment from pASl-2A are ligated in a 3 way ligation thus providing pASl-deltaalp.
• Figure 7 This figure provides the restriction map of the D ⁇ A fragment carrying the A. niger pclA gene.
• Figure 8 This figure provides the structure (functional organisation) of the A. niger pclA encoded protein. It shows pre, pro, activity and P domains from left to right.
• the light coloured triangles indicate KR sites.
• the dark coloured triangles indicate glycosylation sites.
• the vertically striped light box is an S/P/T rich region.
• the dark weavepatterned box at the right end is a D/ ⁇ rich region.
• Figure 9 This figure illustrates growth phenotype of an A. niger pclA mutant strain.
• Figure 10 This figure provides a D ⁇ A sequence comparison between the A. sojae and A. niger pclA genes.
• a vertical bar indicates identity; : indicates 5; • indicates 1. 72.139%) similarity and 12.013% identity were found.
• FIG. 11 The construction of the pclA gene replacement vector is disclosed in this figure.
• a 7.6 kb Clal fragment which is a ATCCl 1906 genomic fragment, was cloned into pMTL23p.
• the 2.6 kb Smal fragment from pAB4-lrep was cloned into the EcoRV-site, thus providing pAS2-delta pel.
• Figure 12 This figure shows the amino acid sequence comparison of the various PclA homolous from S. cerevisiae (Sckex2), K. lactis (Klkexl), A. sojae (Aspcla), A. niger (A. niger), P. chrysogenum (Penpcll), A. bisporus (Agarmbll29), T. reesei (Trichpcll), R. oryzae (Rhizpcll), F. venenatum (Fuspcll), S. pombe (Spkrp), C. albicans (Cakex2) and Y. lipolytica (Ylkex2).
• Regions of overall identity (8 out of 12 entries) are indicated with yellow boxes. Gaps are indicated with .. ; no sequence data are indicated with ⁇ .
• Figure 13 Sequence data are provided in figure 13a for the A. oryzae alpA promoter sequences (Q11755). The primer position for PCR cloning is indicated. In figure 13b the sequence data are provided for the A. oryzae amyA promoter sequences also including primer positions (A02532).
• Figure 13c provides the ATCC42149 A. oryzae derived gpdA promoter sequences (EP0.436.858 al) also including primer positions.
• Figure 14 This figure provides a comparison between various gpdA promoter sequences of Aspergillus: From top to bottom, A sojae ATCCl 1906, A. oryzae, A. niger and A. nidulans. Asterisks indicate the putative intron present in the 5' untranslated region of the promoters. Arrowhats indicate the CT rich regions. Bold underlined letters indicate the differences between the A. oryzae and A. sojae sequences.
• Figure 15 This figure shows a map of the vector pGLA6S of 12700bp.
• MBL1784 5'-CGGAATTCGAGCGCAACTACAAGATCAA-3'
• GGTGCCATTC ATGACGATAA CTGTAACTTT GACGGTTACA CCAACAGTAT 1151 CTACAGCATC ACGGTGGGTG CCATTGATCG GGAGGGTAAC CATCCTCCGT 1201 ATTCGGAATC CTGCTCGGCG CAACTGGTGG TTGCCTACAG CAGCGGCGCC 1251 AGTGATGCAA TTCATACCAC GGACGTCGGC ACAGACAAGT GCTCGACTAC 1301 CCATGGTGGA ACTTCGGCGG CCGGCCCGCT CGCTGCGGGA ACCGTGGCGCGC
• SEQ ID No. 10 coding strand it ⁇ mHI-site is underlined PE4 5*- CG CGGATC CACT/C) GGX ACX (C/A)GX TG(T/C) GCX GG -3' degenerated 2048 times
• PE6 5'- CGC GGA TCC XCC (A/G)TT XCC X(C/G)(A/G) XGC
• MBL 1208 Clal is underlined 5'- CGG ATC GAXT/C) GGX ACX (C/A)GX TGTT/C) GCX GG -3' degenerated 2048 times
• GGGCCATGGT CAATGGTATC CAAAATGGTC GAGGTGGAAA AGGCTCGGTT TTTGTCTGCG 300
• Ala Ala Gin Lys Arg lie Ala Ser Glu Leu Gly lie Ala Asp Pro
• Flavi/Tamarii A.orvzae (amylase, protease)
• Circumdati A. ochraceus tox (xulanase A. alliaceus tox
• Candidi A. candidus (lipase, glucosidase)
• BSA, phytase and buffer were added after the mediumsample was taken from the culture. This sample was incubated at 30°C and after certain timepoints the sample was analysed for the degradation of BSA and phytase.

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