Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-03-27
2002-10-15
Yucel, Remy (Department: 1636)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S069100, C435S320100
Reexamination Certificate
active
06465635
ABSTRACT:
FIELD OF THE INVENTION
The present invention is a promoter sequence of 3-phosphogycerate kinase gene 1 of lactic acid-producing fungus
Rhizopus oryzae
and a method of expressing a gene of interest in fungal species.
BACKGROUND OF THE INVENTION
The genus of Rhizopus is versatile in the production of biocatalysts such as glucoamylase and lipase and chemicals including L-(+)-lactic acid, fumaric acid, and ethanol. Rhizopus is the member of the order Mucorales, which is within the class Zygomycetes of the division Amastigomycota.
Rhizopus oryzae
(ATCC 9363) is the best lactic acid producer found in the Rhizopus genus, while
Rhizopus delemar
and
Rhizopus niveus
can produce significant amount of extracellular lipase. In addition,
R. oryzae
can also secrete large amount of glucoamylase in the solid culture for starch hydrolysis. Therefore,
R. oryzae
could be potentially a host for upgrading lactic acid production as well as foreign protein production. However, in the current literature, there is very limited information available on gene clones as well as gene regulatory elements (promoters) for
R. oryzae.
Less than nine gene clone and partial gene sequences are reported for
R. oryzae,
which include glucoamylase, ribosomal genes, and aspartic proteinase genes.
The ability to genetically manipulate filamentous fungi largely depends on the successfulness to develop the transformation methods and gene expression systems. Transformation methods have been developed for filamentous fungi, in particular,
Aspergillus nidulans
and
Neurospora crassa,
including others such as
Aspergillus niger, Aspergillus oryzae, Penicillium nalgiovense.
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. Phosphoglycerate kinase gene is one of the highly expressed genes found in yeast and filamentous fungi. This gene encodes some of the most abundant mRNA in the yeast cells, accounting for up to 5% of the total cellular protein expression. After the discovery and characterization of
Saccharomyces cerevisiae
gene, other phosphoglycerate kinase genes were also isolated from various fungal species such as
Penicillium chrysogenum
and
Rhizopus niveus
using
S. cerevisiae
phosphoglycerate kinase gene as homologous gene probe. However, only a few of phosphoglycerate kinase gene promoters were isolated and characterized, which were from
S. cerevisiae, Trichoderma reesei,
and
R. niveus,
among others.
To genetically manipulate
R. oryzae,
either for the purpose of metabolic pathway modification, conferring necessary traits such as acid tolerance and upgrading of lactic acid 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
R. oryzae
and design/develop expression vectors, harboring the isolated phosphoglycerate kinase gene promoter.
SUMMARY OF THE INVENTION
The present invention provides the promoter clone discovery of phosphoglycerate kinase gene 1 of a lactic acid-producing filamentous fungal strain,
Rhizopus oryzae.
The isolated promoter can constitutively regulate gene expression under various carbohydrate conditions. In addition, the present invention also provides a design of an integration vector for the transformation of a foreign gene in
Rhizopus oryzae.
REFERENCES:
patent: 3-247286 (1991-11-01), None
CR Soccol et al. “Production of L-Lactic Acid by Rhizopus Species”, p. 433-435, 1994.
MJ Haas et al. “Cloning, Expression and Characterization of a cDNA Encoding a Lipase FromRhizopus Delemar”, p. 107-113, 1991.
P Yin et al. “Enhanced Production of L(+)-Lactic Acid From Corn Starch in a Culture ofRhizopus OryzaeUsing an Air-Lift Bioreactor”, p. 249-253. 1997.
P Van Solingen et al. “Sequence of thePenicillium ChrysogenumPhosphoglycerate Kinase Gene”, p. 11823, 1988.
RA Hitzeman et al. “The Primary Structure of theSaccharomyces CerevisiaeGene for 3-Phosphoglycerate Kinase”, p. 7791-7808. 1982.
N Takaya et al. “Analysis of the 3-Phosphoglycerate Kinase 2 Promoter inRhizopus Niveus”, p. 121-125. 1995.
S Vanhanen et al. “Promoter Structure and Expression of the 3-Phosphoglycerate Kinase-Encoding Gene (pgk ) ofTrichoderma Reesei”, p. 129-133. 1991.
MJ Haas et al. “Lipases of theGenera Rhizopusand Rhizomucor: Versatile Catalysts in Nature and the Laboratory”, p. 549-588. 1994.
N Takaya et al. “Cloning and Characterization of Two 3-Phosphoglycerate Kinase Genes ofRhizopus Niveusand Heterologous Gene Expression Using Their Promoters”, p. 524-530. 1994.
DJ Ballance. “Transformation Systems for Filamentous Fungi and an Overview of Fungal Gene Structure”, p. 1-29. 1991.
MG Richey et al. “Transformation of Filamentous Fungi with Plasmid DNA by Electroporation”, p. 844-847. 1989.
M. Kapoor. “Gene Transfer by Electroporation of Filamentous Fungi”, p. 279-289. 1996.
MJ Holland et al. “isolation and Identification of Yeast Messenger Ribonuclide Acids Coding for Enolase, Glyceraldehyde-3-Phosphate Dehydrogenase, and Phosphoglycerate Kinase”, p. 4900-4907. 1978.
J Mellor et al. “Efficient Synthesis of Enzymatically Active Calf Chymosin inSaccharomyces Cerevisiae”, p. 1-14. 1983.
Gao Johnway
Skeen Rodney S.
Battelle (Memorial Institute)
Loeb Bronwen M.
Wells St. John P.S.
Yucel Remy
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