Methods for generating and screening novel metabolic pathways

Details Methods for generating and screening novel metabolic pathways Methods for generating and screening novel metabolic pathways

1999-03-05

2001-06-05

Brusca, John S. (Department: 1631)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

C435S091410, C435S091520, C435S320100, C435S463000, C435S468000, C435S477000, C536S023500, C536S023700

Reexamination Certificate

active

06242211

ABSTRACT:

TABLE OF CONTENTS
1. Field of the Invention
2. Background of the Invention
2.1. Sources of Drug Leads
2.2. Expression Libraries
3. Summary of the Invention
3.1. Definitions
4. Description of the Figures
5. Detailed Description of the Invention
5.1. Preparation of Combinatorial Gene Expression Libraries
5.1.1. Donor Organisms
5.1.2. Preparation of High Quality Nucleic Acids from Donor Organisms
5.1.3. Host Organisms and Vectors
5.1.4. Combinatorial Natural Pathway Expression Libraries
5.1.5. Combinatorial Chimeric Pathway Expression Libraries
5
.
1
.
6
. Biased Combinatorial Expression Libraries
5.1.7. Recombined Combinatorial Expression Libraries
5.2. Screening Combinatorial Expression Libraries
5.2.1. Reporter Constructs
5.2.2. Physiological Probes and Reporter Precursors
5.2.3. Pre-screening and Screening of the Library
5.3. Protocols for the Preparation of High Quality Nucleic Acids from Donor Organisms
5.3.1. Guanidinium Isothiocyanate Nucleic Acid Isolation
5.3.2. Isolation of Poly(A)-containing RNA
5.3.3. Enrichment of Non-ribosomal Sequences from Total RNA
5.3.4. Fill-in Reaction Using the Klenow Fragment
5.3.5. Protocols for Preparation of Subtracted DNA Probes for Pre-screening
5.3.6. Purification of Nucleic Acids from Soil or Other Mixed Environmental Samples
5.3.7. Repair of DNA
5.4. Protocols for Prokaryotic Expression Libraries
5.4.1. Bacterial Species, Strains, and Culture Conditions
5.4.2. Preparation of Donor Genomic DNA
5.4.3. Generation of Prokaryotic Promoter Fragments
5.4.4. Preparation of Gene Cassettes for Combinatorial Chimeric Pathway Expression Libraries
5.4.5. Preparation of Solid Support
5.4.6. Assembly of a Combinatorial Chimeric Pathway Expression Library
5.4.7. Assembly of a Combinatorial Natural Pathway Expression Library
5.4.8. Pre-screening of Expression Libraries
5.4.9. Metabolic Testing of Marine Gram(−)/
E. Coli
Library by Plate Replication
5.4.10. Metabolic Testing of Marine Gram(−)/
E. Coli
Library by Macrodroplet Encapsulation
5.4.11. Metabolic Testing of Marine Gram(−)/
E. Coli
Libraries by Microdroplet Encapsulation
5.4.12. Metabolic Testing of
Actinomycetes/streptomyces Lividans
Library by Plate Replication
5.4.13. Metabolic Testing of
Actinomycetes/streptomyces Lividans
Library by Macrodroplet Encapsulation
5.4.14. Pre-screening of Clones by Co-encapsulation with Indicator Cells
5.5. Protocols for Eukaryotic Expression Libraries
5.5.1. Removal of Satellite Genomic DNA by Density Gradient Centrifugation
5.5.2. Generation of Eukaryotic Promoters and Terminator Fragments
5.5.3. Preparation of DNA Inserts
5.5.4. Ligation of Insert DNA to Promoters and Terminators
5.5.5. Serial Ligations of Gene Cassettes to Form Concatemers
5.5.6. Circularization and Transformation of Vector Containing Concatemer Constructs
5.5.7. Preparation and Ligation of Prepared Vector for Expression in Yeast
5.5.8. Plant Expression Libraries
6. Example: Construction and Screening of Combinatorial Gene Expression Library
6.1. Materials and Methods
6.1.1. Media Preparation
6.2. Pre-screening of
Actinomycetes/streptomyces Lividans
Combinatorial Natural Pathway Expression Library by Plate Replication and Macrodroplet Encapsulation
6.3. Pre-screening of Actinomycetes/
E. Coli
Combinatorial Chimeric Pathway Expression Library by Macrodroplet Encapsulation
6.4. Pre-screening of Fungal/
schizosaccharomyces Pombe
Combinatorial Chimeric Pathway Expression Libraries by Macrodroplet Encapsulation
6.5. Pre-screening of Marine Gram(−)/
E. Coli
Library by Plate Replication
6.6. Pre-screening of Marine Gram(−)/
E. Coli
Library by Macrodroplet Encapsulation
7. Example: Construction And Screening of
Actinomycetes/Streptomyces Lividans
Combinatorial Gene Expression Library
7.1. Materials and Methods
7.2. Results
1. FIELD OF THE INVENTION
The present invention relates to a novel approach to drug discovery. More particularly, the invention relates to a system for preserving the genomes of organisms that are good or promising sources of drugs; for randomly combining genetic materials from one or more species of organisms to generate novel metabolic pathways; and for pre-screening or screening such genetically engineered cells for the generation of novel biochemical pathways and the production of novel classes of compounds. The novel or reconstituted metabolic pathways can have utility in commercial production of the compounds.
2. BACKGROUND OF THE INVENTION
2.1. Sources of Drug Leads
The basic challenges in drug discovery are to identify a lead compound with the desirable activity, and to optimize the lead compound to meet the criteria required to proceed with further drug development. One common approach to drug discovery involves presenting macromolecules implicated in causing a disease (disease targets) in bioassays in which potential drug candidates are tested for therapeutic activity. Such molecules could be receptors, enzymes or transcription factors.
Another approach involves presenting whole cells or organisms that are representative of the causative agent of the disease. Such agents include bacteria and tumor cell lines.
Traditionally, there are two sources of potential drug candidates, collections of natural products and synthetic chemicals. Identification of lead compounds has been achieved by random screening of such collections which encompass as broad a range of structural types as possible. The recent development of synthetic combinatorial chemical libraries will further increase the number and variety of compounds available for screening. However, the diversity in any synthetic chemical library is limited to human imagination and skills of synthesis.
Random screening of natural products from sources such as terrestrial bacteria, fungi, invertebrates and plants has resulted in the discovery of many important drugs (Franco et al. 1991, Critical Rev Biotechnol 11:193-276; Goodfellow et al. 1989, in “Microbial Products: New Approaches”, Cambridge University Press, pp. 343-383; Berdy 1974, Adv Appl Microbiol 18:309-406; Suffness et al. 1988, in Biomedical Importance of Marine Organisms, D. G. Fautin, California Academy of Sciences, pages 151-157). More than 10,000 of these natural products are biologically active and at least 100 of these are currently in use as antibiotics, agrochemicals and anti-cancer agents. The success of this approach of drug discovery depends heavily on how many compounds enter a screening program. Typically, pharmaceutical companies screen compound collections containing hundreds of thousands of natural and synthetic compounds. However, the ratio of novel to previously-discovered compounds has diminished with time. In screens for anti-cancer agents, for example, most of the microbial species which are biologically active may yield compounds that are already characterized. Partly, this is due to the difficulties of consistently and adequately finding, reproducing and supplying novel natural product samples. Since biological diversity is largely due to underlying molecular diversity, there is insufficient biological diversity in the organisms currently selected for random screening, which reduces the probability that novel compounds will be isolated.
Novel bioactivity has consistently been found in various natural sources. See for example, Cragg et al., 1994. (in “Enthnobotany and the search for new drugs” Wiley, Chichester. p178-196). Few of these sources have been explored systematically and thoroughly for novel drug leads. For example, it has been estimated that only 5000 plant species have been studied exhaustively for possible medical use. This is a minor fraction of the estimated total of 250,000-3,000,000 species, most of which grow in the tropics (Abelson 1990, Science 247:513). Moreover, out of the estimated millions of species of marine microorganisms, only a small number have been characterized. Indeed, there is tremendous biodiversity that remains untapped as sources of lead compounds.
Terrestrial microorganisms, fungi, invertebrates and plants have historically been used as sources of natural pro

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