Protease A-deficient Saccharomyces cerevisiae

Details Protease A-deficient Saccharomyces cerevisiae Protease A-deficient Saccharomyces cerevisiae

1998-10-29

2001-04-03

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Fungi

C435S069100, C435S200000, C435S252300, C435S255200, C435S320100

Reexamination Certificate

active

06210954

ABSTRACT:

BACKGROUND OF THE INVENTION
Research efforts have been made to develop a recombinant yeast capable of expressing a heterologous protein, e.g., hepatitis B surface antigen (HBsAg), in the secretion pathway, thereby simplifying the purification of the yeast-derived recombinant protein.
The gene product of PEP4, protease A, regulates several yeast vacuolar hydrolases at the post-translational level. Woolford et al., Mol. Cell Biol. 6: 2500-2510, 1986. In earlier studies, HBsAg expressed in the secretion pathway was found to be toxic to various protease A-deficient strains of host yeast. See, e.g., Jones, Genetics 85: 23-33, 1977; Zubenco et al., Genetics 102: 679-690, 1982; and Achstetter et al., Yeast 1: 139-157, 1985. According to a more recent report, such toxicity could be progressively reduced in media containing lower concentrations of ammonium sulphate; the non-inhibitory transformants thus obtained were characterized by the phenotypes of enlarged cell and colony morphology, dimorphic transition to pseudohyphal-like and invasive growth in nitrogen-starved solid media, increase in HBsAg particle production, and enhancement of growth rate in liquid media. Chen et al., Curr Genet 27: 201-206, 1995.
SUMMARY OF THE INVENTION
The present invention features a stable protease A-deficient host strain of
Saccharomyces cerevisiae
, which undergoes a pseudohyphal-like growth mode when starved for a nitrogen source and is capable of expressing and secrets a heterologous protein (e.g., HBsAg or &agr;-amylase) when transformed with a secretion vector containing a DNA sequence which encodes the heterologous protein. A strain is protease A deficiency if no or only residual activity can be detected by the APE test (described below).
This strain, preferably, is further characterized by one or more of the following parameters: a cell volume of 150 &mgr;m
3
at 28° C., a generation time of 90 min at 28° C., a viability rate of 45% at 37° C., a cell density of 7×10
8
cells/ml at 37° C., a cell mass of 14 OD
600
units at 37° C., a colony diameter of 2 mm at 28° C., a viability rate of 65% at 28° C., a cell density of 4.5×10
8
cells/ml at 28° C., a cell mass of 9.2° D
600
units at 28° C., and a higher growth rate at 37° C. than at 28° C. and a higher growth at 28° C. than at 20° C. All of such parameters can be measured employing the methods, materials, and conditions described in an actual working example provided below. Note that the above-recited values of the parameters are minima and a strain which exhibits one or more higher values is within the scope of this invention.
Also contemplated within the scope of this invention is a transformed
Saccharomyces cerevisiae
obtained by transforming the strain described above with a secretion vector containing a DNA sequence which encodes a desirable heterologous protein such as HBsAg or &agr;-amylase.
The present invention also features a high-proliferative host strain of
Saccharomyces cerevisiae
obtained by a process which includes the following steps: (1) transforming cells of a protease A-deficient
Saccharomyces cerevisiae
parent strain, e.g., 20B12 (CCRC Accession No. 51837, Taiwan), with a secretion vector that expresses and secrets a heterologous protein which inhibits the growth of the transformed cells; (2) cultivating the transformed cells in a medium containing a reduced nitrogen source and selecting a non-inhibited mutant strain, the non-inhibited mutant strain having unstable phenotypes; (3) maintaining cells of the unstable non-inhibited mutant strain in the stationary phase for an extended period of time and selecting a stable non-inhibited mutant strain; and (4) growing cells of the stable non-inhibited mutant strain under conditions which favor the removal of the secretion vector to obtain a high-proliferative strain of
Saccharomyces cerevisiae
. NI-C (CCRC Accession No. 920007, Taiwan) is a high-proliferative strain obtained by the just-described method. A strain obtained by transforming such a high-proliferative strain with a secretion vector containing a DNA sequence which encodes a heterologous protein (e.g., HBsAg or &agr;-amylase) is also within the scope of this invention. An example of transformants thus prepared is pYAS/12S-transformed NI-C (CCRC Accession No. 940112, Taiwan).
Other features or advantages of the present invention will be apparent from the following detailed description and also from the appending claims.
DETAILED DESCRIPTION OF THE INVENTION
Studies of the non-inhibitory (NI) yeast HBsAg-expressing transformants described by Chen et al. (see Id.) show that their NI phenotypes (defined as: enlarged cell and colony morphology, increase in HBsAg particle production, and enhancement of growth rate) are not stable and degenerate after several subcultures in non-selective broth. A long-term cultivation of NI transformants in the stationary phase has led to an unexpected discovery that the survival cells display more stable NI phenotypes than their parent NI transformants, i.e., having NI phenotypes that remain unchanged following repeated subculturing.
Culturing a stable NI transformant thus obtained under conditions which favor the curing of the secretion vector enables one to acquire a stable host strain, NI-C. NI-C cells are capable of receiving a secretion vector and expressing a desirable heterologous protein encoded by a DNA sequence in that vector. Also, they undergo pseudohyphal-like growth after a long-term (e.g., two weeks) cultivation under nitrogen-starved conditions. An appropriate reporter gene, such as mouse &agr;-amylase, can be used as a positive selective marker for NI-C cells as this novel variant is able to proliferate on nitrogen-starved starch plates to form transformed colonies.
Without further elaboration, it is believed that one of ordinary skill in the art can, based on the above description, utilize the present invention to its fullest extent. The specific examples described below are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Further, all publications cited herein are incorporated by reference.


REFERENCES:
Bitter et al., “Expression of heterologous genes inSaccharomyces cerevisiaefrom vectors untilizing the glyceraldehyde-3-phosphate dehydrogenase gene promoter”, Gene 32:263-274 (1984).
Chen et al., “Abnormal growth induced by expression of HBsAg in the secretion pathway ofS. cerevisiae pep4 mutants”, Curr Genet 27:201-206 (1995).
Kim et al., “High-Efficiency, One-Step Starch Utilization by Transformed Saccharomyces Cells Which Secrete Both Yeast Glucoamylase and Mouse &agr;-Amylase”, Applied and Environmental Microbiology, Apr. 1988, p. 966-971.
Teichert et al., “Lysosomal (Vacuolar) Proteinases of Yeast Are Essential Catalysts for Protein Degradation, Differentiation, and Cell Survival”, The Journal of Biological Chemistry, 264(27):16037-16045 (Sep. 25, 1989).

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