Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1998-10-09
2000-02-22
Ketter, James
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 691, 4353201, 4352572, 536 234, 536 241, C12Q 168
Patent
active
060279001
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
The importance of algae as a resource for new products in both the present and the future cannot be overemphasized (Cannell, 1990, "Algal Biotechnology," Applied Biochemistry and Biotechnolgy, 26, 85-106). Algae have the potential to be used in high density photobioreactors (Lee et al., 1994, "High density algal photobioreactors using light emitting diodes". Biotech. Bioengineering, 44, 1161-1167; Chaumont, 1990, "Biotechnology of algal biomass production. A review of systems for outdoor mass culture," J Appl. Phycology 5, 593-604), bioreactors for sewage (Sawayama et al., 1994, "Continuous cultures of hydrocarbon-rich microalga Botryococcus braunii in secondarily treated sewage," Appl. Micro. Biotech., 41, 729-731; Lincoln 1993, Bulletin De L'institut Oceangraphique (Monaco), 12, 109-115) and waste water treatments, elimination of heavy metals from contaminated water (Wilkinson, 1989, "Mercury accumulation and volatilization in immobilized algal cell systems," Biotech. Letters, 11, 861-864), the production of .beta.-carotene (Yamaoka, 1994, Seibutsu-Kogaku Kaishi, 72, 111-114) and pharmaceutical compounds (Cannell, 1990), as nutritional supplements for both humans and animals (Becker, 1993, "Development of Spirulina research in a developing country: India". Bulletin De L'institut Oceanographique (Monaco), 12, 141-155) and for the production of other compounds of nutritional value. None of these areas has been adequately explored and exploited at this time. The full potential of algae can only be fully developed with an increase in our ability to genetically tailor algae for specific purposes. Genetic engineering can potentially be used to improve the productivity of algae. Unfortunately, until recently most algae were refractory to any type of genetic manipulation, this especially true for the eukaryotic algae. The molecular biological manipulation of algal systems has thus seriously lagged behind other systems. Many of the techniques that have been developed for the introduction of DNA into bacterial, yeast, insect and animal cells have not been adapted to algal systems.
Algal synthesis of valuable molecules on a large scale requires precise methods to enhance the quantity and quality of the product being made, either by changing what is being produced or producing more of a desirable substance. The only way this can be accomplished, in an economical manner, is by molecular biological manipulation. Previous academic investigations used a small number model algal species that have little possible use in a production mode (i.e., Chlamydomonas reinhartii and Synechococcus sp. PCC 7942), their choice dictated by special characteristics that have little to do with commercial utility.
Thus, there is a need for new methods and tools for use in the genetic engineering of eukaryotic algae in order to manufacture products.
SUMMARY OF THE INVENTION
It is an object of the invention to provide a genetic fusion for selection or expression in algae.
It is another object of the invention to provide DNA vectors and cells comprising the genetic fusions.
It is yet another object of the invention to provide methods of transforming eukaryotic algae.
These and other objects of the invention are provided by one or more of the following embodiments. In one embodiment of the invention, a genetic fusion is provided. The genetic fusion consists of a promoter of a photosynthetic algal light harvesting protein 5' and fused to a coding sequence of one or more additional proteins.
In another embodiment of the invention a method of transforming eukaryotic algae is provided. The method comprises the steps of:
introducing DNA to eukaryotic algae, wherein the DNA contains a zeocin resistance determinant; and
selecting for eukaryotic algae transformants by culturing in a medium comprising between about 10 and about 35 g/L salt and a concentration of zeocin or phleomycin which is inhibitory to untransformed eukaryotic algae.
The present inventors have discovered that eukaryotic algae can be transformed with D
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Allnutt F. C. Thomas
Apt Kirk R.
Grossman Arthur R.
Kyle David J.
Carnegie Institution of Washington
Ketter James
Martek Biosciences Corporation
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