Protein expression strains

Details Protein expression strains Protein expression strains

1999-12-15

2002-04-30

Ketter, James (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S483000, C435S484000, C435S254110, C435S254200, C435S254210

Reexamination Certificate

active

06379924

ABSTRACT:

FIELD OF THE INVENTION
This invention relates primarily to the development of fungal strains which express proteins at levels substantially higher than the parental strains.
BACKGROUND AND PRIOR ART
For some 20 years, desired foreign proteins have been produced in microorganisms. However, having introduced the necessary coding sequence and obtained expression, much still remains to be done in order to optimise the process for commercial production One area of interest concerns strain improvement, that is to say finding or making strains of the host microorganism which enable the protein to be made in higher yields or better purity, for example.
To increase the yield, once a good expression system (eg transcription promoter) has been devised, one might envisage trying to increase the copy number of the coding sequence (although this will have the desired effect only if DNA transcription was the limiting factor), or to increase the stability of the mnRNA or to decrease the degradation of the protein. Thus, as an example of the latter approach, yeast strains (eg pep4-3) which are deficient in certain proteases have been used for producing desired foreign proteins. In another approach, the number of 2 &mgr;m-based plasmids in the yeast
Saccharomyces cerevisiae
has been increased by introducing a FLP gene into the genome under the control of a regulated promoter, eg GAL. Upon switching to a growth medium containing galactose as the sole carbon source, plasmid copy number rises (11), but the plasmid copy number increase is uncontrolled since the GAL promoter is not repressed by REP1/REP2. This leads to reduced growth rate and thence clonal selection of cir
o
derivatives of the original cir
+
strain (11,20).
We have mutated yeast strains by application of mutagens in order to generate mutants randomly and thereby hopefully find mutant strains which produce heterologous proteins in better yield (16,21). We have now characterised such a randomly-produced mutant which maintained a higher number of copies of the plasmid expressing the desired protein and have found that the mutation occurred in one of the genes encoding ubiquitin-conjugating enzymes, namely UBC4. The UBC4-encoded enzyme (and the closely related UBC5-encoded) enzyme are involved in degrading aberrant and short lived proteins and there was no reason to have supposed that the deletion of either of them would have enabled an increased yield of a normal, desired, protein to have been obtained.
Several genes encoding ubiquitin conjugating enzymes (UBC) have been implicated in the bulk protein degradation and in the stress response of yeast. UBC1, UBC4 and UBC5 act together to mediate important functions for cell growth and cell viability (2,3). Yeast strains with a mutation in a single gene are viable and have similar growth rates to the parental strains, but ubc4/ubc5 double mutants have reduced growth rates and are sensitive to amino acid analogues, while a triple mutant is inviable, indicating that their activities overlap. The UBC4 and UBC5 genes are closely related and the two coding DNA sequences share 77% identical residues, while the predicted amino acid sequences of the two proteins show 92% identical residues (3). Because of the near identity of the Ubc4 and Ubc5 proteins (hereafter abbreviated to Ubc4p and Ubc5p) it has been suggested that UBC4 could complement for the loss of function of the ubc5 mutant and vice versa (3). This would explain why the dramatic reduction in growth rate was only observed in ubc4/ubc5 double mutants. Pulse chase experiments have indicated that Ubc4p and Ubc5p are responsible for the degradation of short-lived and abnormal proteins, but that the turnover of these proteins was only reduced in strains with the ubc4/ubc5 double mutation. It was not reduced in strains with single ubc4 or ubc5 mutations (3). This reference, therefore, suggested that the use of single ubc4 and ubc5 mutant fungal strains would not be beneficial.
Structurally, all known UBC genes encode a conserved domain (known as the UBC domain) of approximately 16kDa containing the conserved conjugating cysteine (1,22). Transfer of activated ubiquitin results in the covalent attachment of the C-terminus of ubiquitin via a thioester bond to the cysteine residue. UBC genes have been divided into different classes (reviewed in 22). Class I UBC genes are composed almost exclusively of the conserved UBC domain, class II and class III UBC genes have C-terminal or N-terminal extension, respectively, while class IV UBC genes have both C- and N-terminal extensions (22).
The fungal genome is composed of chromosomes, extrachromosomal copies of chromosomal genes, eg nucleosomes, and occasionally stable extrachromosomal elements. These extrachromosomal elements have developed a benignly parasitic relationship with their host, where they successfully balance the theft of cellular resource for the replication and segregation of the element, while not compromising the fitness of the host. General reviews of fligal extrachromosomal elements are covered by references 5 and 6, while the DNA plasmids of the yeasts Saccharomyces species are covered by references 7 and 8 and Kluyveromyces species are covered by reference 9.
The 2 &mgr;m plasmids of Saccharomyces species are extrachromosomal DNA species which have evolved mechanisms to ensure their long term autonomous survival without any associated phenotype. The 2 &mgr;m plasmid resides in the nucleus and is packaged into chromatin. The plasmid origin of replication acts as an autonomously replicating sequence, while other sequences ensure the maintenance of a controlled high copy number and allow the plasmid to partition uniformly into the daughter cells at mitosis. The plasmid is not required for normal mitotic growth and does not provide the host with any selective advantage since Saccharomyces species devoid of 2 &mgr;m plasmid, denoted as cir
o
, grow only slightly faster than their 2 &mgr;m plasmid containing, or cir
+
, parents.
The 2 &mgr;m plasmid is a double stranded circular plasmid of approximately 6,318 bp, comprising two unique regions of 2,774 and 2,346 bp separated by a pair of exact inverted repeats, each 599 bp long (10). In vivo the monomeric plasmid exists as an equal mixture of the two inversion isomers (A and B) that form following site specific recombination between the two inverted repeats. The 2 &mgr;m plasmid has four open reading frames known as FLP (also known as A), REP1 (also known as B), REP2 (also known as C) and RAF (also known as D). The plasmid also contains a region, located between RAF and the origin of replication, called STB or REP3, which is composed of a series of imperfect 62 bp repeat elements This element is required in cis, along with the trans acting elements, REP1 and REP2, to enable efficient partitioning of the plasmid between the mother and the daughter cell.
The 2 &mgr;m plasmid copy number is also indirectly under the control of chromosomal genes, since it is known that 2 &mgr;m plasmid copy number does vary between different
Saccharomyces cerevisiae
strains and because the chromosomal recessive mutation, known as nib1, results in clonal lethality due to uncontrolled amplification of 2 &mgr;m plasmid copy number (39). Yeast strains carrying the nib1 mutation resemble engineered yeast strains where FLP gene expression is galactose induced. The involvement of proteins of the fungal ATP-dependent ubiquitin protein degradation pathway in the regulation of fungal plasmid copy number is not described in the art. Nor is it disclosed that genes of the fungal ATP-dependent ubiquitin protein degradation pathway can be manipulated to control fungal plasmid copy number.
Although the 2 &mgr;m plasmid is a very common genetic component of
Saccharomyces cerevisiae,
other yeast strains are known to contain identifiable DNA plasmids, notably the pSR1 and pSB3 plasmids (6,251 bp and 6,615 bp) of
Zygosaccharomyces rouxii,
the pSB1 and pSB2 plasmids (6,550 bp and 5,415 bp) of
Zygosaccharomyces baijii,
the pSM1 plasmid (5,416) of
Zygosacchar

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