Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1997-11-13
2001-07-17
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S252300, C536S023100
Reexamination Certificate
active
06261842
ABSTRACT:
BACKGROUND OF THE INVENTION
Until recently, it was assumed that cultivation of microorganisms from the environment resulted in the isolation of a good proportion of the microorganisms present. Phylogenetic analysis of rRNA sequences obtained from direct sampling of environments has shown that this is not the case. Giovannoni et al. (1990)
Nature
345:60-63; Pace et al. (1996)
ASM News
62:463-470; Stahl et al. (1985)
Appl. Environ. Microbiol
. 49:1379-1384; Suzuki et al. (1997)
Appl. Environ. Microbiol
. 63:983-989; Ward et al. (1990)
Nature
345:63-65. It is now apparent that the microorganisms that can be cultured from any environment using standard techniques probably represent the minority of the total species present in that environment, indicating that a vastly greater diversity of prokaryotes exists than suggested by culturing methods. Pace et al, supra; Stahl (1993)
ASM News
59:609-613. The idea that perhaps the vast majority of bacteria in an environment are currently nonculturable has revolutionized thinking in microbiology, and has stimulated new approaches to the study of microbes. Woese et al. (1990) PNAS 87:4576-4579.
For instance, it is estimated that the number of species currently culturable from soil represents 1% or less of the total population. Griffiths et al. (1996)
Microbial Ecol
. 31:269-280; Torsvik et al. (1996)
J. Ind. Microbiol
. 17:170-178. DNA-DNA reassociation measurements have been used to determine total genetic diversity in one soil sample. The data indicated that greater than 4000 species might be present. Torsvik et al. (1990)
Appl. Environ. Microbiol
. 56:782-787. This represented at least 200 times more diversity than was observed by examining culturable bacteria from the same sample. Another study based on methods that did not involve culturing suggested 13,000 species in 100 g soil. Torsvik et al. (1994) p.39-48, In
Beyond the Biomass
, K. Ritz, J. Dighton and K. E. Giller (eds.), John Wiley and Sons, Chichester. By estimating the total number of cells at 5×10
11
per gram of soil, this suggested an average of 5×10
7
cells per species assuming even species distribution. Thus even rare species might have fairly large population sizes in the soil. A recent analysis in our labs indicated that novel phyla of Bacteria and Archaea are present in soil. Bintrim et al. (1997) PNAS 94:277-282; Bintrim et al., in press. Of 144 cloned Bacterial 16S rRNA gene sequences examined, 45 had the closest affiliation to members of the phylum Proteobacteria, but of these clones, only 6 had close affiliation to known genera (Pseudomonas, Hafnia, Azospirillum). The clones were distributed across the entire Domain, and none were identical to any previously known sequence. Moreover, these studies revealed entirely new lineages of microbes in soil, both from the Domain Archaea and the Domain Bacteria. This indicates the enormous diversity of noncultured microorganisms from soil.
SUMMARY OF THE INVENTION
The present invention, in one aspect, provides methods and reagents for identifying genes from microbial organisms, the gene products of which are involved in biochemical transformation reactions that produce, for example, small organic molecules by de novo synthesis, or that chemically modify molecules ectopically provided in the microbe's environment. In general, the method provides host cells which have been engineered to express the opening reading frames of genomic DNA sub-cloned from a heterologous microorganism. The subject method detects changes in the phenotype of the host cell which are dependent on expression of open reading frames from the genomic DNA, e.g., which may be marked by altered biosynthetic capabilities.
Another aspect of the invention provides methods and reagents for identifying biosynthetic products, preferably other than those produced by ribosomal synthesis, which are generated by recapitulation of a heterologous microbial biosynthesis pathway in a host cell, or generated by a chimeric metabolic pathway involving both heterologous and endogenous gene products in the host cell. As above, the assay generally detects biochemical transformation reactions that produce, for example, small organic molecules by de novo synthesis, or that chemically modify molecules ectopically provided in the host microbe's environment. In general, the method provides host cells which have been engineered to express the opening reading frames of genomic DNA sub-cloned from a heterologous microorganism. Likewise, this embodiment of the subject method can be disposed to detect changes in the phenotype of the host cell which are dependent on the formation of a natural product, or the transformation of an ectopically added agent.
Thus, for example, there is provided a method for identifying a product of a biosynthetic pathway, comprising
i) providing host cells containing a replicable vector including genomic DNA isolated from a source of uncultivated microrganisms, which host cells are provided under conditions wherein expression of open reading frame sequence(s) of the genomic DNA occurs; and
ii) detecting a compound produced by the host cells, e.g., relative to host cells lacking the genomic DNA.
In another embodiment, the present invention provides a method for cloning genes of a biosynthetic pathway, comprising
i) providing host cells containing a replicable vector including genomic DNA isolated from a source of uncultivated microrganisms, which host cells are provided under conditions wherein expression of open reading frame sequence(s) of the genomic DNA occurs; and
ii) detecting the presence or absence of a biosynthetic pathway which is dependent on expression of at least one of the opening reading frames by the host microorganisms.
There is also provided a method for cloning genes of a biosynthetic pathway, comprising
i) cloning, into a replicable vector, genomic DNA from a source of uncultivated microrganisms;
ii) expressing open reading frame sequence(s) of the genomic DNA in a host microorganism harboring the vector; and
iii) detecting the presence or absence of a biosynthetic pathway which is dependent on expression of at least two of the opening reading frames by the host microorganism.
That method can also be used to identify a product of such a biosynthetic pathway produced in the host microorganism. In preferred embodiments, the biosynthetic pathway produces or transmutes a non-polymeric and/or non-proteinaceous compound. In certain preferred embodiments, the biosynthetic pathway produces a product having a molecular weight less than 7500 amu, more preferably less than 5000 amu, and even more preferably less than 2000 amu.
Yet another aspect provides a method for detecting a non-proteinaceous compound produced by a microorganism, comprising
i) sub-cloning, into a replicable vector, genomic DNA from one or more uncultivated microrganisms;
ii) expressing open reading frame sequence(s) of the genomic DNA in a host microorganism harboring the vector;
iii) detecting ectopic production of a non-proteinaceous compound by the host microorganism.
In other embodiments, there is provided a method for cloning two or more genes encoding gene products functioning in a biological pathway of a microorganism, comprising
i) sub-cloning, into a replicable vector, genomic DNA from one or more uncultivated microrganisms;
ii) expressing at least two open reading frame (ORF) sequences of the cloned genomic DNA in a cultivable host microorganism transfected with the vector;
iii) identifying ORF sequences which confer a phenotypic change on the host microorganism, which phenotypic change is dependent on the expression of at least two ORF sequences of the cloned genomic DNA.
In still other embodiments, there is provided a method for cloning genes encoding gene products functioning in the chemical transformation of a non-proteinaceous compound by a microorganism, comprising
i) sub-cloning, into a replicable vector, genomic DNA from an uncultivated microrganism;
ii) expressing open reading frame (ORF) sequence(s) of the cloned genomic DNA in
Goodman Robert M.
Handelsman Jo
Rondon Michelle R.
Clauss Isabelle M.
Foley Hoag & Eliot
Wisconsin Alumni Research Foundation
Yucel Remy
LandOfFree
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