Super-secreting saccharomyces cerevisiae strains

Details Super-secreting saccharomyces cerevisiae strains Super-secreting saccharomyces cerevisiae strains

2000-06-16

2003-05-20

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Fungi

C435S183000, C435S200000, C435S252300, C435S243000, C435S320100

Reexamination Certificate

active

06566122

ABSTRACT:

BACKGROUND
Yeast species can be used as hosts for the production of heterologous proteins. As unicellular microorganisms, yeasts share the advantages of bacterial systems with regard to ease of manipulation and growth. Yet, unlike bacteria, yeast cells possess an eukaryotic subcellular organization that is capable of accurate posttranslational processing and modification of many mammalian proteins.
The yeast
Saccharomyces cerevisiae
has been used extensively for the production of many heterologous proteins, given that host-vector systems, genetic information, and recombinant DNA techniques are well established for this organism. In addition, industrial-scale production of heterologous proteins by yeast benefits from an established fermentation technology.
Protease A-deficient strains of
S. cerevisiae
are commonly used as host cells because of the associated decrease in protein hydrolysis; however, these strains exhibit a reduced proliferative capacity. A recently isolated protease A-deficient strain having enhanced ability to secrete heterologous protein showed a higher viability and mitotic capacity, as compared with the parental and wild-type strains. Chen et al.,
Appl Microbiol Biotechnol
51:185-192, 1999.
The PMR1 gene encodes a Ca
2+
-dependent ATPase in
S. cerevisiae
. According to earlier reports, pmr1 mutants exhibited a 5- to 50-fold increase in the abundance of secreted prochymosin, bovine growth hormone, or scuPA. Smith et al.,
Science
229:219-1224, 1985; Turner et al.,
Biotechnol Bioeng
37:869-875, 1991.
SUMMARY OF THE INVENTION
This invention relates to a super-secreting protease A-deficient strain of
Saccharoinyces cerevisiae
, which, when starved for a nitrogen source, undergoes a pseudohyphal-like growth mode, and, when transformed with a secretion vector containing a DNA sequence that encodes a mouse a-amylase (e.g., pMS12; see below for details), is capable of secreting the mouse a-amylase at 2,000 to 15,000 units/liter (e.g., 3,000 to 14,000, 6,000 to 13,000, or 9,000 to 12,000 units/liter). The strain of this invention further has one or more of the characteristics of reduced glycosylation of the mouse a-amylase, high stability of the vector, inability to grow at 37° C., and cell cycle-dependent secretion of the mouse a-amylase. It can be either a PMR1 -positive strain (e.g., NI-C-D4 ) or a pmr1-deficient strain (e.g., DP-1). Deposit of the strain NI-C-D4 was made on Mar. 13, 2000 and deposit of the strain DP-1 was made on Jun. 22, 2000, both with the Culture Collection Research Center, Hsinchu, Taiwan, where the deposits were given Accession Number CCRC 920020 and Accession Number CCRC 920021, respectively.
Set forth below is a process for preparing a strain of the present invention: (1) transforming cells of a protease A-deficient and PMR1 positive
S. cerevisiae
parent strain with a secretion vector that expresses and secrets a heterologous protein, e.g., hepatitis surface antigen HBsAg, 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 choosing a stable non-inhibited mutant strain; (4) growing cells of the stable non-inhibited mutant strain under conditions which favor the curing of the secretion vector; (5) crossing the vector-cured strain with a protease A-positive strain of
S. cerevisiae
(PMR1-positive or pmr1-deficient) to acquire heterozygotes; (6) allowing the heterozygotes to sporulate to produce haploid segregants; and (7) screening the segregants to select a strain of
S. cerevisiae
which has a higher secretion capability than the parent strain. Before crossing a PMR1-positive vector-cured strain with a protease A-positive strain, both strains may be transformed with a vector encoding a marker secretion protein. In any event, if the vector-cured strain is crossed with a protease A-positive and PMR1-positive strain, the PMR1 gene of the selected super-secreting strain can be disrupted to produce a strain with an even higher secretion capability.
For the sole purpose of describing a characteristic of the strains of this invention, the capability of each strain to secrete a heterologous protein is based on the amount of a mouse &agr;-amylase in the supernatant from a 4-day culture of a pMS12-transformed strain determined by the procedure described in the actual examples below. Thus, it will be understood that the above-described strains can be used to produce various valuable heterologous proteins, such as HbsAg or human GM-CSF (granulocytelmacrophage colony-stimulating factor. Similarly, mouse &agr;-amylase is also used to demonstrate reduced glycosylation of the secreted protein (i.e., as compared with wild-type strains) and cell cycle dependent secretion, and pMS12 is used to demonstrate high stability of the vector (i.e., >50% of cells exhibiting amylolytic activity at 96 h). Again see the actual examples below for the detailed procedures.


REFERENCES:
patent: 5312735 (1994-05-01), Fink et al.
patent: 5705616 (1998-01-01), Lehle et al.
patent: 5798226 (1998-08-01), Lehle et al.
patent: 6210954 (2001-04-01), Kuo et al.
Turner et al. 1991, Optimization of uro-kinase secretion from recombinant S.cerevisiae. Biotechnol. Bioeng. vol. 37:869-875.*
Chen et al., “A Variant ofSaccharomyces cerevisiae pep4Strain With Improved Oligotrophic Proliferation, Cell Survival and Heterologous Secretion of &agr;-amylase”, Appl Microbiol Biotechnol 51:185-192, 1999.
Sakai et al., Enhanced Secretion of Human Nerve Growth Factor FromSaccharomyces CerevisiaeUsing an Advanced &dgr;-Integration System, Bio/Technology 9:1382-1385, 1991.
Sakai et al., “Isolation and Characterization of Mutants Which Show an Oversecretion Phenotype inSaccharomyces Cerevisiae”, Genetics 119:499-506, 1988.
Smith et al., “Heterologous Protein Secretion from Yeast”, Science 229:1219-1224, 1985.
Suzuki et al., “Yeast Mutants with Enhanced Ability to Secrete Human Lysozyme: Isolation and Indentification of a Protease-deficient Mutant”, MGG 58-63, 1989.

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