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
2001-07-24
2003-07-22
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, C435S254110, C435S254200, C435S320100, C435S325000, C435S410000, C536S024100
Reexamination Certificate
active
06596513
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of molecular biology and more particularly to a novel DNA sequence possessing regulated transcriptional promoter activity in two directions, to expression vectors containing this sequence, and to their use for the production of recombinant proteins from either homologous or heterologous sources. The invention also relates to the recombinant cells containing this DNA sequence.
BACKGROUND OF THE INVENTION
Model bacterial species, such as
Escherichia coli
, have been used in important molecular biological processes such as the amplification of desirable nucleic acid sequences and the production of heterologous gene products. Unfortunately the ability of bacteria to make functional products from eukaryotic genes is often limited due to alternate prokaryotic codon biases, improper protein folding, and divergent pathways of secondary modifications such as protein glycosylation. For this reason many researchers have turned their attention to yeast species, some of which have been found to be suitable hosts for the expression of proteins of eukaryotic origin.
Yeasts are more easily cultured than cells of many higher eukaryotes and lend themselves to genetic manipulations commonly performed with prokaryotes yet have the capacity for complex post-translational modifications including glycosylation events, proteolytic maturation, and disulphide bond formation (Eckart and Bussineau 1996). Traditionally
Saccharomyces cereviseae
has been used for this production but in recent years focus has shifted to other yeast species, including the dairy yeast
Kluyveromyces lactis
, which has become a model system of studies on the molecular physiology of “non-conventional” yeasts (Breunig et al 2000, Flores et al 2000). One of the difficulties with
S. cereviseae
as a production system is its preference for fermentation under aerobic conditions. This is known as the Crabtree effect. The Pasteur effect is the opposite: the repression of fermentation in aerobic conditions (Breunig et al 2000).
Crabtree-positive yeasts need to be cultured under sugar limited conditions to prevent the decarboxylation of pyruvate to ethanol. Such conditions typically result in low rates of growth and protein synthesis (Breunig et al 2000). If
S. cereviseae
is not grown under sugar-limited conditions, each molecule of pyruvate that is reduced to ethanol is not available to the TCA cycle again resulting in reduced biomass yields and ATP production (Schaffrath and Breunig 2000).
Kluyveromyces lactis
, like many yeast species, differs from
S. cereviseae
in that it prefers respiration as opposed to fermentation under aerobic conditions. Under aerobic conditions
K. lactis
oxidizes pyruvate to CO
2
through the TCA cycle and therefore produces more ATP and reducing equivalents per molecule of pyruvate (Flores et al 2000). Besides relief from the Crabtree effect
K. lactis
possesses a number of other attributes that make it an attractive host for the expression of heterologous proteins. As a naturally occurring dairy yeast,
K. lactis
is generally regarded as a safe organism and has been considered a food grade organism by the US FDA. It has excellent fermentative characteristics and can be cultured at high cell densities (>100 g dry cell weight/l) making it amenable to large-scale industrial applications.
Genetic tools have been developed to make use of Kluyveromyces as a host system for the production of recombinant proteins. Several heterologous gene expression systems have been successfully developed for
K. lactis
making this organism an attractive alternative for heterologous gene expression in industrial applications.
Kluyveromyces lactis
is capable of using many of the vectors, promoters and marker genes already used in
S. cereviseae
systems however these have not been optimized for
K. lactis
. With the exception of natural promoters for the 3-phospoglycerate kinase (U.S. Pat. No. 5,646,012), RP28 ribosomal protein (U.S. Pat. No. 5,627,049), alcohol dehydrogenase (U.S. Pat. No. 5,624,046), transaldolase (U.S. Pat. No. 5,616,474) and pyruvate decarboxylase (U.S. Pat. No. 5,631,143) genes no other endogenous promoters have been developed for expression in
K. lactis
. The rate at which an introduced gene will be transcribed depends not only on the nature of the gene but also on the promoter associated with the gene. The selection of the appropriate promoter is therefore crucial for efficient heterologous protein expression. Certain applications favor the use of strong constitutive promoters to drive gene expression while other applications require promoters that can be externally regulated to give a phased expression. Regulated expression is particularly useful for the expression of products that may be toxic to the host organism.
Fermentation conditions can be controlled such that the toxin is not produced while host cell biomass is increased. This is commonly achieved by the use of glucose as a major carbon source due to its ability to suppress the expression of genes necessary for the utilization of alternate carbon sources (Dong and Dickson 1997). Regulated expression can be achieved by utilizing promoters derived from one of these alternate pathways. Many of the proteins involved in this type of carbon catabolite repression have been identified in
S. cereviseae
and most are believed to have functional analogues in
K lactis.
The Saccharomyces ScSNF1p kinase, which has been conserved across many eukaryotic taxa from yeasts, plants and mammals (Dong and Dickson 1997, Carlson 1998), is part of the signal cascade that directs a coordinated response to changing glucose concentrations. One of the targets of ScSNF1p is Mig1p, a DNA-binding transcriptional repressor of many glucose-repressed genes (Lutfiyya and Johnston 1996, Hu et al 1999, Breunig et al 2000). Mig1p binds to conserved GC-elements present in the promoters of glucose-repressed genes and disruption of Mig1p gene function results in dramatic de-repression of expression (Wang et al 1997, Zaragosa et al 2000).
Kluyveromyces lactis
possess a Mig1p homologue (KlMig1p) responsible for the repression of the glucose-repressed lactose-galactose regulon (Dong and Dickson 1997, Hu et al 2000). De-repression is not complete in the absence of KlMig1p suggesting the presence of an additional KlMig1p independent pathway and regulation by other repressors (Dong and Dickson 1997). Glucose repressed genes in alternative sugar metabolism also involve transcriptional activator proteins. In Saccharomyces, Gal4p is a transcriptional activator of many glucose-repressed genes and is turn repressed by Gal80p. This secondary repression is relieved by raised intracellular concentrations of non-glucose sugars (Hu et al 2000). The
K. lactis
homologue KlGal4p (also called Lac9p) escapes inhibition by KlGal80p and is thought to activate its own expression, thereby increasing the concentration of the activator. The concentration of KlGal4p (Lac9p) limits expression of many glucose repressed regulons. In addition to KlMig1p and KlGal4p(Lac9p) there are other transcription factors, many of which are yet to be identified, that add specificity to each of the regulated alternate pathways.
The selection of an appropriate promoter is crucial for efficient heterologous protein expression. Although relatively rare, bi-directional promoters are generally believed to regulate related gene products that are produced in stoichiometric quantities (Levine et al 1992, Menne et al 1994). An artificial construct utilizing the bi-directional expression of the reporter genes gusA and lacZ, driven by the promoter of a-aminoadipyl-cysteinyl-valine [ACV] synthetase/isopenicillin N-synthetase from
Acremonium chrysogenum
, has shown that the pcbC promoter (isopenicillin N-synthetases) was at least 5 times stronger than pcbAB (ACV synthetase) promoter (Menne et al 1994).
References Cited
Breunig K D, Bolotin-Fukuhara M, Bianchi M M, Bourgarel D, Falcaone C, Ferrero I, Frontali L, Goffrini P, Krijger J J, Mazzoni C,
Hintz William
Williams Diane P.
Klarquist & Sparkman, LLP
McKelvey Terry
University of Victoria Innovation and Development Corporation
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