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
2002-04-05
2004-03-02
McKelvey, Terry (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...
C435S243000, C435S254200, C435S255400, C435S255500, C435S255600, C435S320100, C435S325000, C435S419000, C536S024100
Reexamination Certificate
active
06699691
ABSTRACT:
FIELD OF THE INVENTION
The current invention is generally directed toward isolated polynucleotides comprising regulatory nucleotide sequences, vectors and expression cassettes containing such regulatory sequences, and host cells comprising the vectors and/or expression cassettes. In particular, the invention relates to 5′ upstream regulatory nucleotide sequences within the promoter region of the alcohol oxidase 1 gene, vectors and expression cassettes containing such regulatory sequences, and host cells comprising the vectors and/or expression cassettes.
BACKGROUND OF THE INVENTION
Methylotrophic yeast are capable of utilizing methanol as their sole energy source. The methylotrophic yeast comprise two groups, the asporagenous consisting of the Candida and Torulopsis species, and the ascomycetous consisting of the Hansenula and Pichia species. These yeast possess a conserved methanol utilization pathway. In the first step of this pathway, the enzyme alcohol oxidase catalyzes the oxidation of methanol to formaldehyde. Concomitantly, this reaction also generates hydrogen peroxide, a substance that is highly toxic to most cellular organelles. In order to avoid hydrogen peroxide toxicity, methanol metabolism is highly compartmentalized in specialized organelles, called peroxisomes, which sequester this toxic byproduct from the rest of the cell. Alcohol oxidase has a low affinity for its substrate, oxygen. The partitioning of alcohol oxidase in the peroxisome, therefore, serves the additional role of concentrating the enzyme and substrate, thereby compensating, at least in part, for its low oxygen affinity.
The regulation of methanol metabolism in yeast generally occurs at the level of transcription, and includes control of the synthesis and activation of corresponding enzymes, as well as their degradation. Synthesis of methanol metabolizing enzymes is induced by methanol, formaldehyde and formate, and repressed by both glucose and ethanol. As stated above, a key enzyme in methanol oxidation is alcohol oxidase, which is also controlled both positively and negatively at the transcription level. Several genes encoding alcohol oxidase from
Pichia pastoris
(AOX1 and AOX2),
Candida boidinii
S2 (AOD1), and methanol oxidase from
Hansenula polymorpha
(MOX1) are well characterized. Methanol induction causes the rapid de novo synthesis of alcohol oxidase, which is accompanied by a corresponding increase in alcohol oxidase mRNA. For example, in methanol grown
P. pastoris
cells, AOX rapidly accumulates up to 30% of the total soluble protein.
Yeasts have been extensively employed for the production of heterologous proteins and offer several advantages over prokaryotic expression systems. For example, some eukaryotic proteins that are produced in prokaryotic cells are either unstable or lack biological activity. Accordingly, in these cases, yeasts offer advantages over their prokaryotic counterparts which include an intracellular environment that is more conducive for correct folding of eukaryotic proteins. Additionally yeasts, unlike prokaryotic hosts, have the ability to glycosylate proteins, which is important for both the stability and biological activity of the protein.
Saccharomyces cerevisiae
was the first, and remains the most commonly employed eukaryotic expression system because its genome and physiology have been extensively characterized. It is not always the optimal expression system, however, for the large-scale production of heterologous proteins because of plasmid loss during scale-up, hyperglycosylation, and low protein yields. Recently, methylotrophic yeast expression systems have been developed and offer several advantages over their counterpart,
S. cerevisiae
. For example, methylotrophic yeast expression systems achieve very high cell densities in a simple defined medium, have strong inducible promoters, and the availability of methods, host strains, and expression vectors that facilitate genetic manipulations.
In particular, the methylotrophic yeast,
P. pastoris
, has been extensively utilized for the production of heterologous proteins at the industrial scale.
P. pastoris
is a particularly suitable expression system for the production of proteins at the industrial scale because of the presence of a unique promoter, AOX1, used to drive gene expression. The AOX1 promoter, as stated above, is derived from the methanol regulated alcohol oxidase I gene and is one of the most efficient and tightly regulated promoters known. For example, as delineated above, in methanol grown
P. pastoris
cells, AOX rapidly accumulates up to 30% of the total soluble protein.
In addition to the AOX1 gene,
P. pastoris
also harbors a second functional AOX gene, AOX2. AOX2 encodes a protein that shares approximately 97% homology, and has the same specific activity as its AOX1 counterpart. However, the 5′ upstream regulatory regions of AOX1 and AOX2 do not have significant regions of homology, despite the fact that both genes are repressed during growth on glucose, derepressed during carbon limitation, and induced by growth on methanol as the sole carbon source. In addition, AOX2 induction is responsible for only 15% of total alcohol oxidase activity in methanol-grown cell cultures. Due to the limited accumulation of AOX2 relative to AOX1, the AOX1 promoter is more advantageous to employ in an expression system for large-scale protein production.
Although high-level expression of heterologous proteins has been achieved employing the AOX1 promoter from
P. pastoris
, very little is known about the molecular mechanisms involved in methanol induction, either in
P. pastoris
or methylotrophic yeasts in general. The promoter region for AOX1 has been identified (SEQ ID NO: 1), however, regulatory sequences within this region have not been extensively characterized. See Stroman et al., U.S. Pat. No. 4,855,231. In addition, the promoter region for the AOX2 gene has also been identified (SEQ ID NO: 2), and consists of three cis-acting regulatory elements that have been characterized (one positive and the other two negative). See Cregg., U.S. Pat. No. 5,032,516. The positive cis-acting element, the AOX2 upstream activator site, is required for a response to transcriptional induction by methanol. The two negative cis-acting elements are responsible for repression of the AOX2 promoter. However, as delineated above, the 5′ upstream regulatory regions of AOX1 and AOX2 do not have significant regions of homology and thus, the extensive characterization of AOX2 regulation provides little insight into the mechanism by which the AOX1 promoter is regulated.
In yet a further attempt to characterize regulatory regions of the AOX1 promoter, its sequence was compared to the 5′ upstream regulatory regions of an alcohol oxidase gene, ZZA (SEQ ID NO: 3), isolated from an uncharacterized
P. pastoris
strain. See Kumagai et al., U.S. Pat. No. 5,641, 661. This strain also contains two copies of the AOX genes. The deduced amino acid sequence of ZZA revealed that 14 of the first 16 amino acids are identical to that of the AOX1 and AOX2 genes. However, similar to the 5′ regulatory regions of AOX1 and AOX2, the 5′ upstream regulatory regions of ZZA and AOX1 share only 66% homology. The two promoters, thus, may be regulated by completely distinct mechanisms.
Highly conserved nucleotide sequences, (TTGNNNGCTTCCAANNNTGGT) (SEQ ID NO: 4) and CCNCTTTTTG (SEQ ID NO: 5) have been found in the 5′ flanking regions of alcohol oxidase, methanol oxidase, and dihydroxyacetone synthase genes in
P. pastoris, H. polymorpha
and
C. boidini
S2. See Kumagai et al., U.S. Pat. No. 5,641,661. It was postulated that these conserved regions were involved in binding methanol-specific trans-acting factors. However, in a study characterizing the AOX2 promoter, these sequences were not involved in transcriptional regulation of the AOX2 gene since deletion of these regions did not impact regulation of the AOX2 promoter (Ohi, et al. (1994) Mol.Gen Genet 243:489-99). Thus, although these regions may be
Benson Andrew K.
Inan Mehmet
Meagher Michael M.
McKelvey Terry
Scully Scott Murphy & Presser
The Board of Regents of the University of Nebraska
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