Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Fungi
Reexamination Certificate
2000-04-13
2003-06-03
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Fungi
C435S069100, C435S209000
Reexamination Certificate
active
06573086
ABSTRACT:
SUMMARY OF THE INVENTION
The subject invention pertains to a novel transformation system in the field of filamentous fungal hosts for expressing and secreting heterologous proteins or polypeptides. The invention also covers a process for producing large amounts of polypeptide in an economical manner. The system comprises a transformed or transfected fungal strain of the genus Chrysosporium, more particularly of
Chrysosporium lucknowense
and mutants or derivatives thereof It also covers transformants containing Chrysosporium coding sequences. Novel mutant Chrysosporium strains are disclosed as are novel enzymes derived therefrom. The subject invention further relates to novel enzymes derived from filamentous fungi, especially from strains of the genus Chrysosporium, and to coding sequences and expression-regulating sequences for these enzymes.
BACKGROUND TO THE INVENTION
A number of hosts for gene expression and methods of transformation have been disclosed in the prior art. Bacteria are often mentioned e.g.
Escherichia coli. E. coli
is however a micro-organism incapable of secretion of a number of proteins or polypeptides and as such is undesirable as host cell for production of protein or polypeptide at the industrial level. An additional disadvantage for
E. coli
, which is valid also for bacteria in general, is that prokaryotes cannot provide additional modifications required for numerous eukaryotic proteins or polypeptides to be produced in an active form. Glycosylation of proteins and proper folding of proteins are examples of processing required to ensure an active protein or polypeptide is produced. To ensure such processing one can sometimes use mammalian cells; however, the disadvantage of such cells is that they are often difficult to maintain and require expensive media. Such transformation systems are therefore not practical for production of proteins or polypeptides at the industrial level. They may be cost efficient for highly priced pharmaceutical compounds requiring relatively low amounts, but certainly not for industrial enzymes.
A number of fungal expression systems have been developed e.g.
Aspergillus niger, Aspergillus awamori, Aspergillus nidulans, Trichoderma reesei
. A number of others have been suggested but for various reasons have not found wide-spread acceptance or use. In general terms the ideal host must fulfil a large number of criteria:
The ideal host must be readily fermented using inexpensive medium.
The ideal host should use the medium efficiently.
The ideal host must produce the polypeptide or protein in high yield, i.e. must exhibit high protein
The ideal host should be capable of efficient secretion of the protein or polypeptide.
The ideal host must enable ease of isolation and purification of the desired protein or polypeptide.
The ideal host must process the desired protein or polypeptide such that it is produced in an active form not requiring additional activation or modification steps.
The ideal host should be readily transformed.
The ideal host should allow a wide range of expression regulatory elements to be used thus ensuring ease of application and versatility.
The ideal host should allow use of easily selectable markers that are cheap to use.
The ideal host should produce stable transformants.
The ideal host should allow cultivation under conditions not detrimental to the expressed protein or polypeptide e.g. low viscosity, low shear.
Fungal systems that have not yet found widespread use are described e.g. in U.S. Pat. No. 5,578,463 by Berka et al suggesting Neurospora, Podospora, Endothia, Mucor, Cochoibolus and Pyricularia together with Aspergillus and Trichoderma. However only illustrations of transformation and expression are provided for Aspergillus and Trichoderma and no details are provided for any of the other suggested hosts.
1 WO 96/02563 and U.S. Pat. Nos. 5,602,004, 5,604,129 and 5,695,985 to Novo Nordisk describe the drawbacks of Aspergillus and Trichoderma systems and suggests cultivation conditions for other fungi may be more suited to large scale protein production. The only examples provided for any transformed cultures are those of
Myceliophthora thermophila, Acremonium alabamense, Thielavia terrestris
and
Sporotrichum cellulophilum
strains. The Sporotrichum strain is reported to lyse and produce green pigment under fermentation conditions not leading to such results for the other strains. A non-sporulating mutant of
Thielavia terrestris
is described as being the organism of choice by virtue of its morphology. However it is also stated that the protoplasting efficiency of Thielavia and Acremonium (whereby the Acremonium strain used was the imperfect state of the Thielavia strain used) is low and that hygromycin was not useful as a selection marker. A large number of others are suggested as being potentially useful by virtue of their morphology but no transformation thereof is described. The suggested strains are Corynascus, Thermoascus, Chaetomium, Ctenomyces, Scytalidium and Talaromyces. The transformed hosts are mentioned as only producing low levels of the introduced Humicola xylanase with Thielavia producing the lowest amount; however, the information is ambiguous and could actually infer Thielavia was the best embodiment. The nomenclature of this reference is based on the ATCC names of Industrial Fungi of 1994. Thus it is apparent no high degree of heterologous expression was achieved and in fact no positive correlation could be derived between the postulated morphology and the degree of expression. If any correlation could be made, it was more likely to be negative. According to the 1996 ATCC fungal classification
Sporotrichum thermophilum
ATCC 20493 is a
Myceliophthora thermophila
strain. Currently the strain is still identified as
Myceliophthora thermophila
. The unpredicatability of the art is apparent from these recent disclosures.
Also Allison et al (
Curr. Genetics
21:225-229, 1992) described transformation of
Humicola grisea
var. thermoidea using the lithium acetate method and a Humicola enzyme-encoding sequence, but no report of expression of heterologous protein from such a strain has been provided.
In 1997 a patent issued to Hawaii Biotechnology Group for transformed Neurospora for expression of mammalian peptide such as chymosin. The transformation of auxotrophic Neurospora crassa occurred with spheroplasts. Endogenous transcriptional regulatory regions were introduced and cotransformation was carried out. Nothing is mentioned concerning other hosts and other transformation protocols. Nothing is apparent from the disclosure concerning the degree of expression. It is doubtful whether the degree of expression is high, as immunotechniques (which are useful for detecting small amounts of protein) are the only techniques used to illustrate the presence of the protein. No actual isolation of the protein is disclosed.
WO 97/26330 of Novo Nordisk suggests a method of obtaining mutants of filamentous fungal parent cells having an improved property for production of heterologous polypeptide. The method comprises first finding a specific altered morphology followed by assessing whether a transformant produces more heterologous polypeptide than the parent. The method is illustrated only for strains of Fusarium A3/5 and
Aspergillus oryzae
. The method is suggested to be applicable for Aspergillus, Trichoderma, Thielavia, Fusarium, Neurospora, Acremonium, Tolyplocadium, Humicola, Scytalidium, Myceliophthora or Mucor. As stated above the unpredictability in the art and also the unpredictability of the method of the cited application do not provide a generally applicable teaching with a reasonable expectation of success.
DETAILED DESCRIPTION OF THE INVENTION
We now describe an alternative fungal expression system with the simplicity of use of the above-mentioned Aspergillus and Trichoderma fulfilling the above requirements. The new system has not been taught or suggested in the prior art. The new system according to the invention provides the additional advantages that transformation rates a
Bousson Jean Christophe
Burlingame Richard Paul
Emalfrab Mark Aaron
Olson Philip Terry
Parriche Martine
Dyadic International, Inc.
Ketter James
Morgan & Finnegan , LLP
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