Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-09-08
2001-02-20
Schwartzman, Robert A. (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...
C435S254210
Reexamination Certificate
active
06190883
ABSTRACT:
FIELD OF THIS INVENTION
The present invention is related to a method for the production and secretion of heterologous proteins or polypeptides in Crabtree negative Saccharomyces yeast species, and DNA-sequences, vectors and transformed cell lines for use in such method.
BACKGROUND OF THIS INVENTION
Saccharomyces cerevisiae
is an attractive organism for heterologous protein production and many suitable expression systems have been developed to produce high level of proteins with this organism. This yeast is also the best studied eukaryotic organism and many molecular tools have been developed. However,
S. cerevisiae
has some limitations in its commercial application, due to its relatively poor secretion efficiency of proteins, the need to use fed-batch fermentation techniques to attain high cell densities and hence improve the protein synthesis, and secretion of O- and N-glycosylated proteins which are often hyperglycosylated. Although,
S.cerevisiae
is regarded as a very proteolysis weak host organism, a further problem encountered in production of some heterologous proteins in
S. cerevisiae
is low yield, presumably due to proteolytic processing both in intracellular compartments and at the plasma membrane cf. Gabrielsen et al. Gene, 90:255-262, 1990, (secretion of human parathyroid hormone by
S. cerevisiae
), Rokkones et al., J. Biotechnol. 33:293-306 (secretion of human parathyroid hormone by
S. cerevisiae
), and Bitter et al. Proc. Natl. Acad. Sci. USA, 81:5330-5334, 1984 (secretion of &bgr;-endorphine by
S. cerevisiae
).
According to Waldron and Lacroute, J. Bacteriol., 122:855-865,1975, the protein synthesis rate in yeast is dependent on the specific growth rate. They demonstrated that the net rate of protein synthesis in yeast lowered with decreasing specific growth rate. This was due to the decrease in average ribosomal efficiency i.e. the rate of protein synthesis per ribosome.
In batch culture with glucose as the carbon and energy source, the yeast
S. cerevisiae
will mainly ferment glucose to ethanol. Under anaerobic conditions, this is the only mode of energy production. In the presence of oxygen respiration occurs. However, alcoholic fermentation may s et in even under aerobic conditions if the glucose concentration surpasses a critical threshold value (Verduyn et al, J. Microbiol. Methods, 2:15-25, 1984; and vanDijken and Scheffers, FEMS Microbiol. Review, 32:199-244, 1986). This affects the biomass yield drastically (Reiger et al, J. Gen. Microbiol., 129:653-661, 1983; and von Meyenburg, Arch. Microbiol., 66:289-303, 1969). At the level of pyruvate, respiration competes with alcoholic fermentation via the mitochondrial pyruvate dehydrogenase complex and the cytosolic pyruvate decarboxylase. Acetaldehyde formed by the activity of pyruvate decarboxylase can after oxidation to acetic acid enter the tricarboxylic acid (TCA) cycle via acetyl CoA. Alternatively, acetaldehyde may be reduced to ethanol instead of being oxidized to carbon dioxide.
S. cerevisiae
can secrete all its fermentive metabolites acetate, pyruvate, ethanol, glycerol and succinate in glucose limited aerobic batch fermentation.
The overflow metabolism and repression of respiration in yeast strains resulting in redirection of substrate towards fermentative metabolism resulting in formation of ethanol and other primary products of fermentative activity, is referred to as the “Crabtree effect”. This effect is remarkable in the species
Saccharomyces cerevisiae.
However, so far it has not been clear what the situation is with other species belonging to the genus Saccharomyces. Presently the genus Saccharomyces consists of more than ten species (Piskur, J. et al Int. J. System. Bacteriol. 48:1015-1024, 1998).
In literature, most of the yeast species employed for heterologous protein expression like
Kluveromyces lactis, Pichia pastoris, Hansenuela polymorpha, Schwanniomyces occidentalis
and
Yarrowia lipolytica,
are not respiration-limited yeasts (Kreger-van Rij, Classification of yeasts, in Yeast vol 1:5-66, 1987 and Heslot et. al., J. Bacteriol., 104:473-491, 1970). These strains differ from
S. cerevisiae
in being Crabtree negative yeast strains, thus having the advantage of utilizing the substrate more efficiently for protein and biomass synthesis. However, from a molecular biology aspect these yeast strains are not very well characterized and they are not as easy to manipulate as
S. cerevisiae.
Moreover, the secretion properties of these yeast strains are not characterized as well as for
S. cerevisiae.
The present invention is based on the surprising recognition by the inventors hereof that at least one Saccharomyces species, namely
Saccharomyces kluyveri
is Crabtree negative or is only effected in a very small degree by glucose surplus during aerobic batch fermentation.
S. kluyveri
is a distant relative of
S. cerevisiae
and showed higher biomass yield on glucose than
S. cerevisiae
in batch fermentation confirming its higher respiratory capacity over
S. cerevisiae.
This is a desirable feature for the protein biosynthesis.
FIG. 1
a,
1
b,
2
a
and
2
b
clearly demonstrate that the
Saccharomyces kluyveri
strains are “Crabtree negative”.
SUMMARY OF THE INVENTION
The present invention is related to an industrial Saccharomyces yeast fermentation method for production of a heterologous product encoded by a plasmid or DNA contained in said strain which method utilizes the substrate more efficiently and without or with reduced fermentative metabolism resulting in formation of ethanol and other unwanted primary products of fermentative activity whereby high yields of the heterologous product are obtained.
The invention is related to a method for producing a heterologous product comprising (a) cultivation under industrial conditions a Saccharomyces yeast strain which comprises a plasmid or DNA encoding the protein wherein the strain utilizes the substrate more efficiently and has less or no fermentative metabolism and (b) recovering the protein.
More specifically the present invent ion is related to an industrial fermentation method comprising culturing a Crabtree negative Saccharomyces yeast species in a suitable culture medium said Crabtree negative yeast species comprising DNA coding for the desired product operably linked to transcriptional and translational control sequences and other sequences necessary for expression in yeast whereupon the expressed product is isolated from either the cells or the culture medium.
In a preferred embodiment of the present invention the Crabtree negative Saccharomyces species is
Saccharomyces kluyveri.
The transcriptional and translational control sequences may preferably be derived from
Saccharomyces cerevisiae
genes, from
Saccharomyces kluyveri
genes or from genes from both species.
By “Crabtree negative yeast species” in this context is meant that the yeast strain produces no or a substantially lowered amount of ethanol than
S. cerevisiae
under aerobic condition irrespective of the mode of cultivation (growth under sugar limitation or growth with excess sugar).
By “a substantially lowered amount of ethanol produced” is meant that less than 10 mg ethanol is formed per g glucose taken up by the cells when
S. kluyveri
GRY1175 and GRY1183 are cultured in the medium given by Verduyn et al. in 1990 or in optimized medium (see Example 1). Preferably the amount of ethanol produced should be less than 5 mg per g glucose, more preferably the amount should be less than 2.5 mg per g glucose and even more preferred it should be zero.
With “excess sugar” is meant up to 40 g/liter of glucose present in Verduyn medium or present in the optimized medium (see Example 1).
By “reduced fermentative metabolism” or “less or no fermentative metabolism” is meant that the amount of ethanol produced per g glucose during fermentation is at least 25% reduced as compared to that observed in
S. cerevisiae
under same substrate concentration strain which is about 0.3 g ethanol/g glucose. The reduction will typically be at least 50%. It is preferred that t
Egel-Mitani Michi
Nielsen Jens
Piskur Jure
Srivastava Aradhana
Davis Katharine F.
Gregg, Esq. Valeta A.
Novo Nordisk A S
Schwartzman Robert A.
Zelson, Esq. Steve T.
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