Metabolic selection methods

Details Metabolic selection methods Metabolic selection methods

1998-10-14

2002-04-09

McGarry, Sean (Department: 1635)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S252100, C435S252300, C435S325000, C435S375000

Reexamination Certificate

active

06368793

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to methods for screening for enzymatic pathways, and the isolation of the genes and proteins that make up these pathways.
BACKGROUND OF THE INVENTION
The following description of the background of the invention is provided to aid in understanding the invention, but is not admitted to be, or to describe, prior art to the invention.
Biological synthesis of compounds is frequently more cost effective and more productive than chemical synthesis, which can have low yields, require expensive and toxic reagents, and require lengthy purifications. In contrast, biological synthesis using known pathways can be rapid, with high yields. However, the identification of new biological pathways for syntheses of interest is difficult and time consuming.
Currently, the biochemical screening of isolates is a major means by which people find new pathways for the production of chemicals, antibacterials, and other anti-infectives. However, screening is inherently several orders of magnitude slower than selection and requires that the organism be cultured in the laboratory. Since at least 99% of the microbes in the environment do not grow on laboratory media, less than 1% can be tested using a biochemical screen. Thus, biological pathways in 99% of organisms will never be found by classical biochemical screening technologies.
SUMMARY OF THE INVENTION
The metabolic selection strategy of this invention is designed to find an enzymatic pathway for the conversion of any source compound to any target compound. Conservatively, this technique allows at least a million-fold increase in the discovery rate over classical biochemical screening approaches, and allows testing of the 99% of the environmental microbes that are currently unable to be cultured in the laboratory.
A biocatalytic or metabolic pathway consists of a series of protein catalysts (enzymes) which catalyze the conversion of a starting material to the final product. A general process to identify the metabolic pathway from a source compound to a target compound involves the creation/identification of an easily genetically-manipulatable organism containing an inducible signal, which is activated when a target compound is metabolized. This is followed by the screening of nucleic acid in this organism to identify genes which metabolize the source compound to the target compound.
An example of a selection strategy which can be used to identify the metabolic pathway from a source compound to a target compound is diagrammed in FIG.
11
. As a first step, microbial isolates are selected that are capable of metabolizing a target compound “T”, but not a source compound “S”, to an essential factor. Essential factors can include elements like carbon, sulfur, phosphorous, and nitrogen, or other essential nutrients, e.g. some amino acids, fatty acids, and carbohydrates. In a second step, the pathway responsible for the catabolism of compound “T” is identified and made conditional. That is, the gene(s) for the pathway is cloned and placed under control of an inducible promoter such that growth on the target compound is turned “ON” only when the inducer is present. This engineered strain is referred to as the “tester strain”. The third part of the strategy is the transfer of foreign DNA from environmental sources into the tester strain, followed by selection for growth on the source compound “S” in the presence of inducer. Such positive clones either are capable of metabolizing compound “S” in the absence of inducer, in which case utilization of “S” does not require prior conversion to compound “T” (
FIG. 11
; pathway I), or alternatively metabolize compound “S” only when “T” catabolism is “ON”, suggesting that utilization of “S” proceeds via compound “T” to intermediary metabolism (
FIG. 11
; pathway II). These latter clones are further analyzed and the biocatalysts for the conversion of “S” to “T” are characterized. A specific embodiment of the metabolic selection strategy is shown in
FIG. 12
, where “S” is 2-keto-L-gulonate (2-KLG), and “T” is ascorbic acid (AsA) which can be metabolized to carbon and energy.
Thus, in a first aspect, the invention features a method of screening for one or more nucleic acid sequences which express a product or products that convert a source compound into a target compound. The method comprises contacting a cell with one or more test nucleic acid sequences, where the cell expresses one or more genes encoding one or more proteins which, in the presence of the target compound, provide a detectable signal. The detectable signal indicates the presence of the desired nucleic acid sequence or sequences.
The term “screening” as used herein refers to methods for identifying a nucleic acid sequence of interest. Preferably, the method permits the identification of a nucleic acid sequence of interest among one or more sequences, more preferably among hundreds (100, 200, . . . 900), most preferably among thousands (1,000, 2,000, . . . etc.) or more. The sequences to be screened can be isolated from one or more organisms. Preferably, the sequences are isolated from hundreds of organisms, more preferably from thousands or more organisms. The term “screening” may include both classical screening, whereby expression of the nucleic acid results in a phenotype that can be identified (for example by having a colony with the nucleic acid of interest change color, fluoresce, or luminesce), and may also include classical selection, where typically the phenotype to be identified is growth on selective media. By “selective” is meant media on which the host strain will not grow or grows poorly, but that strains with the nucleic acid of interest will grow in a manner which can be readily distinguished from host strain growth by methods well-known in the art.
The term “nucleic acid” as used herein refers to either deoxyribonucleic acid or ribonucleic acid that may be isolated, enriched, or purified from natural sources or synthesized recombinantly. These methods are well-known in the art and specific examples are also given herein. Preferably, a “nucleic acid” to be identified in the screening method comprises a nucleic acid encoding a metabolic pathway that is not normally found in the cell. Thus, preferably, the pathway has not simply been inactivated through a mutation and the relevant genes are now being identified through complementation. Rather the nucleic acid being identified does not normally exist in the cell in which it is being screened for. Typically, the screening is cross strains, more typically, cross-species, and even more preferably, cross-genera or with further remoteness.
By “isolated, purified, or enriched” in reference to nucleic acid is meant a polymer of 6 (preferably 21, more preferably 39, most preferably 75) or more nucleotides conjugated to each other, including DNA and RNA that is isolated from a natural source or that is synthesized. In certain embodiments of the invention, longer nucleic acids are preferred, for example those of 300, 600, 900 or more nucleotides and/or those having at least 50%, 60%, 75%, 90%, 95% or 99% identity to the sequence shown in SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:19.
The isolated nucleic acid of the present invention is unique in the sense that it is not found in a pure or separated state in nature. Use of the term “isolated” indicates that a naturally occurring sequence has been removed from its normal cellular (i.e., chromosomal) environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only nucleotide chain present, but that it is essentially free (about 90-95% pure at least) of non-nucleotide material naturally associated with it, and thus is distinguished from isolated chromosomes.
By the use of the term “enriched” in reference to nucleic acid is meant that the specific DNA or RNA sequence constitutes a significantly higher fraction (2-5 fold)

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