Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-08-04
2003-04-01
Yucel, Remy (Department: 1636)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S069100, C435S071100, C435S483000
Reexamination Certificate
active
06541622
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates specifically to the isolation of a yeast gene regulatory sequence (promoter), which is native to
Schwanniomyces castellii
and can regulate gene expression in a heterologous yeast host using starch as the sole carbon source. More specifically, the starch can be used as an inducing agent for the expression of native or foreign genes, which are fused to the isolated yeast promoter. The transformed host cells bearing the promoter-gene fusion can grow in culture medium containing various carbon sources, and the gene expression is induced by starch addition as a gene expression inducing-agent. The heterologous host is preferably bacteria, yeast, mold, plant cell, plant tissue, and whole plant.
DESCRIPTION OF THE RELATED ART
Natural yeast strains have been identified that can use starch as a primary growth substrate via complete or partial enzymatic hydrolysis. These yeast strains include but are not limited to
Saccharomycopsis fibuligera, Schwanniomyces castellii,
and
Saccharomyces diastaticus,
which can produce and secrete both alpha-amylase and glucoamylase to, liquefy and hydrolyze starch into glucose. A fusion yeast cell strain of
Saccharomyces diastaticus
and
Saccharomyces cerevisiae
could degrade 60% of starch present in culture media within two days. In addition, other natural Saccharomyces species can ferment starch and dextrin to ethanol, as well as improve ethanol production from starch and higher sugars.
The ability to genetically modify yeast strains has greatly advanced both protein expression engineering and metabolic engineering for the past two decades. The use of yeast for producing transgenic prokaryotic and eukaryotic heterologous proteins has the added advantage that yeast and filamentous mold are microbial eukaryotes, and they are more closely related to animal cells. Hence, yeasts possess the molecular genetic manipulation and growth characteristics of prokaryotic organisms, together with the subcellular machinery for performing eukaryotic post-translational protein modification. For example,
Pichia pastoris
is able to synthesize functional recombinant protein and its glycosylation abilities are very similar to those of animal cells, though the glycosylation in another yeast strain,
Saccharomyces cerevisiae,
is different from that of an animal. In addition, the metabolic pathway of a regular ethanol producing yeast strain can be genetically altered to accumulate large amounts of lactic acid and to increase xylose utilization rate. However, only a few yeast systems (transformation vector and promoters) are available for protein engineering and metabolic engineering, which include
Saccharomyces cerevisiae, Pichia pastoris,
among others.
Starch utilizing yeast strain,
Schwanniomyces castellii
or
Schwanniomyces occidentalis,
is one of the most important microorganisms, since it can directly use starch as its growth medium. Due to the low level of glycosylation and the ability of protein secretion,
Schwanniomyces castellii
is a promising host for heterologous protein expression. However, the molecular study of
Schwanniomyces sp.
is very limited. Only about 21 genes have been cloned, and very few promoter sequences have been cloned and characterized in their full length from
Schwanniomyces sp.
The ability to genetically manipulate
Schwanniomyces sp.
depends on the successfulness in developing the transformation methods and gene expression systems. To effectively direct the transcription or expression of an interested gene, strong gene regulating elements or promoters are required. These promoters can be isolated from the upstream sequences of strongly expressed gene clones.
Alpha-amylase, a 56-kDa protein, is one of the highly expressed clones in
Schwanniomyces castellii,
and different carbohydrates such as starch regulate its expression. The expression level of alpha-amylase can be increased about 100 times when the glucose is depleted in the culture medium. The gene regulatory element (promoter) of the alpha-amylase gene would be a useful genetic element to be used for the regulation of foreign gene expression. However, the full alpha-amylase promoter of
Schwanniomyces castellii
has never been sequenced and characterized. To genetically manipulate
Schwanniomyces sp,
either for the purpose of metabolic pathway modification, conferring necessary traits such as chemical production, or producing biocatalyst of interest, high levels of mRNA expression are always desirable. Therefore, there is a need to isolate strong promoter sequences of
Schwanniomyces sp.
and characterize its function.
The following references disclose technical information useful in this art:
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88: 313-316.
Hongpattarakere T, H-Kittikun A. 1995. Optimization of single-cell-protein production from cassava starch using
Schwanniomyces castellii J. Microbiol. Biotechnol.
11:607-609.
Lemmel S A, Heimsch R C, Korus R A. 1980. Kinetics of growth and amylase production of Saccharomycopsis fibuligera on potato processing.
Appl. Environ. Microbiol
39:387-393.
Kim K, Park C S, Mattoon J R. 1988. High-efficiency one-step starch utilization by transformed Saccharomyces cells which secrete both yeast glucoamylase and mouse alpha amylase.
Appl. Environ. Microbiol
54:966-971.
Laluce C, Bertolini M C, Emandes J R, Martini A V, Martini A. 1988. New amylolytic yeast strains for starch and dextrin fermentation.
Appl. Environ. Microbiol
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Saccharomyces cerevisiae World J. Microbiol. Biotechnol
9:338-341.
Ryu Y W, Ko S H, Byun S Y, Kim C. 1994. Direct alcohol fermentation of starch by a derepressed mutant of
Schwanniomyces castellii Biotechnol. Lett
16:107-112.
Sreekrishna K, Nelles L, Potenz R, Cruze J, Mazzaferro P, Fish W, Fuke M, Holden K, Phelps D, Wood P, Parker K. 1989. High-level expression, purification, and characterization of recombinant human tumor necrosis factor synthesized in the methylotrophic yeast
Pichia pastoris Biochemistry
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Annual Green Chemistry and Engineering Conference, Washington, D.C. Pp. 77-78.
Wang T T, Lee C F, Lee B H. 1999. The molecular biology of Schwanniomyces occidentalis Klocker. Critical Review in Biotechnol. 19(2):113-143.
Jefferson R A, Kavanagh T A, Bevan M W. 1987. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.
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Dohmen R J, Strasser A W M, Martens F B, Dahlems U M, Hollenberg C P. 1990. Cloning of the
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glucoamylase gene (GAM1) and its expression in
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Strasser A, Martens F B, Dohlmen J, Hollenberg C P. 1992. Amylolytic enzymes producing microorganisms, constructed by recombinant DNA technology and their use for fermentation processes. U.S. Pat. No. 5,100,794.
Claros M G, Abarca D, Femandez-Lobato M, Jimenez A. 1993. Molecular structure of the SWA2 gene encoding an AMY1-related alph-amylase from
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Piper P. 1996. Isolation of yeast DNA, Methods in Molecular Biology, 53:103-107
SUMMARY OF THE INVENTION
The present invention provides the promoter clone discovery and isolation of alpha-amylase gene of a starch util
Anderson Daniel B.
Gao Johnway
Hooker Brian S.
Skeen Rodney S.
Battelle (Memorial Institute)
Loeb Bronwen M.
Wells St. John P.S.
Yucel Remy
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