Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Fungi
Reexamination Certificate
2001-01-05
2004-10-05
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Fungi
C435S440000, C435S471000, C435S483000, C435S243000, C435S255200, C435S259000, C435S320100, C536S023100, C536S023740, C536S024100
Reexamination Certificate
active
06800476
ABSTRACT:
The present invention relates to genetically modified yeasts in which the expression of genes responsible for cell wall synthesis are modulated.
The yeast cell wall is a dynamic organelle responsible for a number of cellular functions, the most important being physical and osmotic protection, selective permeability, cell-cell recognition and adhesion during mating and flocculation.
The cell wall of the yeast
Saccharomyces cerevisiae
is composed of three components, &bgr;-glucan (a glucose polymer), mannoproteins and chitin (an N-acetylglucosamine polymer). The &bgr;-glucan component of the cell wall consists of two polymers: a large, linear &bgr;-1,3-glucan and a smaller, highly branched &bgr;-1,6-glucan moiety whereas the mannoproteins are a complex of proteins modified by the attachment, via N- and O-glycosidic bonds, of mannose-containing carbohydrate chains of different length and structure.
The yeast cell wall is a very rigid, highly complex structure which determines the shape of the yeast cell and enables it to be protected from and adjusted to its ever-changing environment. A growing number of genes have been shown to participate in the biosynthesis and assembly of the major cell wall components, some of them as part of well-defined signal transduction pathways. For instance, PKC1 (the yeast homologue of protein kinase C) regulates the biosynthesis and assembly of major cell wall components by a PKC1-mediated signal transduction pathway. PKC1, in conjunction with Rho1p, regulates &bgr;1-3 glucan synthetase Null mutants of PKC1 can only grow in the presence of osmotic stabilisers. Loss of PKC1 function results in a cell-cycle-specific osmotic stability defect.
A further gene relevant to the cell wall is the recently identified SRB1 (also known as PSA1 or VIG9) yeast gene which is essential for growth and encodes GDP-mannose pyrophosphorylase, an enzyme responsible for the production of a major substrate for all kinds of mannosylation reactions, including the biosynthesis of cell wall mannoprotein. A SRB1/PSA1 null mutation is lethal whereas a decrease in SRB1/PSA1 function (by inhibiting the expression of SRB1/PSA1) leads to defects in bud growth, bud site selection, and cell separation, in addition to increases in cell permeability and cell lysis.
Another gene which regulates the cell wall is PDE2 (the gene encoding a high-affinity cAMP phosphodiesterase) which is part of the RAS/cAMP dependent pathway in yeast. We recently identified PDE2 as a multi-copy suppressor of srb1-1 (a mutation which depends on sorbitol for growth). Moreover, strains carrying a pde2 deletion share a number of phenotypes with srb1-1 mutants, including lysis upon osmotic shock.
The yeast cell wall influences the characteristics of the yeast cell in a number of ways and it is desirable to modulate the wall to confer desirable properties on yeast.
For instance, the yeast cell wall acts as a barrier which can obstruct the liberation of protein expressed within yeast cells. This effect is of particular significance to the biotechnology industry which uses yeasts for protein production and for the production of recombinant proteins in particular. For some proteins, it is possible to use the yeast secretion pathway to release the protein from the cell, thus obviating the need for mechanical or enzymic degradation of the cell wall. However, many proteins cannot be exported by the secretion pathway and are retained within the cell in a membrane-associated form. Some protein complexes, such as virus-like particles (VLPs) are also impossible to recover by the secretion route. It may be seen, therefore, that the rigid cell wall of yeast is a major barrier to the efficient operation of downstream processes leading to protein isolation and purification. In the food industry, yeast cell extracts are used as dietary supplements and flavourings, again requiring efficient methods of cell lysis that do not compromise the nutritional or organoleptic properties of the yeast cell extract.
The use of lysis mutants represent an alternative to mechanical/chemical disruption for the efficient recovery of yeast cell contents. Some attempts have been made to develop such mutants. For instance, WO 92/01798 concerns the use of a srb1-1 mutant. However this specification related to a DNA sequence deposited under the Budapest treaty which was first believed to code for the SRB1 gene but has since been shown to be the PDE2 gene. WO 96/02629 also relates to a lysis mutant and concerns the use of a mpk1/slt2 mutant which is stated to release intracellular proteins, including VLPs, following a temperature shift and osmotic shock. However, the application of both types of lytic mutants has significant drawbacks. The osmotic stabiliser required to enable the growth of a srb1-1 mutant is either expensive (sorbitol) or corrosive to the fermentor (NaCl) thus precluding the use of this mutant nil in large-scale processes. The temperature shift involved in the use of the mpk1/slt2 mutant requires considerable energy input and can also trigger the degradation or re-modelling of the proteins released from the lysed cells.
It is therefore an object of the present invention to provide yeast mutants which obviate or mitigate the abovementioned disadvantages.
According to a first aspect of the invention, there is provided a yeast cell containing the SRB1/PSA1 gene and the PKC1 gene or functional derivatives thereof each operatively linked to a heterologous inducible promoter.
We have found that yeast cells containing the SRB1/PSA1 gene and the PKC1 gene or functional derivatives thereof each operatively linked to an inducible promoter (e.g. the cells according to the first aspect of the invention) may be used in applications where the induction of cell lysis is desirable. For instance, induction of yeast cell lysis is useful for isolating protein expressed within a yeast cell which is not readily secreted into the medium in which the cells are growing. Thus according to a second aspect of the present invention, there is provided a method of regulating yeast cell lysis comprising:
(i) growing yeast cells containing the SRB1/PSA1 gene and the PKC1 gene or functional derivatives thereof each operatively linked to an inducible promoter in a growth medium which activates the inducible promoter such that SRB1/PSA1 and PKC1 are expressed from said cells; and
(ii) when lysis is required, growing the cells in a modified growth medium which represses SRB1/PSA1 and PKC1 expression such that cell lysis is induced.
The present invention is based upon our efforts to develop conditional lysis mutants that do not require special media in which to grow (e.g. sorbitol supplemented) or temperature shifts. We placed various genes that have been shown to contribute, in different ways, to cellular integrity of
S. cerevisiae
under the control of inducible promoters and examined A whether or not repression of the gene (by altering the composition of the media in which the yeast is grown such that the promoter is inactivated) modulates cell lysis. We found that repression of some of the genes tested, for instance PDE2, did not significantly influence lysis. These genes were therefore unsuitable candidates to be modulated to generate an improved lytic yeast strain.
We found that repression of the SRB1/PSA1 gene in yeast cells grown in normal media resulted in a reduction in cell growth, cells gradually losing their viability and integrity and the release of about 7% of total protein into the medium 24 hours after repression was induced. Furthermore we found that repression of PKC1 led to more extensive release of cellular protein into the medium (approximately 18% of total protein into the medium 24 hours after repression was induced) although cell growth was not significantly altered. These results were expected because, although it is known that yeast cells carrying the srb1-1 allele can grow in osmotically-buffered media, such mutanits lyse upon hypoosmotic shock. It is also known that cells bearing the pkc1 mutation can grow in osmotically-bufferred media (e
Oliver Stephen
Stateva Lubomira I.
Zhang Nianshu
Ketter James
Lambertson David A.
Nixon & Vanderhye PC
The Victoria University of Manchester
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