High throughput screening for novel enzymes

Abstract

Disclosed is a process for identifying clones having a specified activity of interest, which process comprises (i) generating one or more expression libraries derived from nuclei acid directly isolated from the environment; and (ii) screening said libraries utilizing a fluorescence activated cell sorter to identify said clones. More particularly, this is a process for identifying clones having a specified activity of interest by (i) generating one or more expression libraries derived from nucleic acid directly or indirectly isolated from the environment; (ii) exposing said libraries to a particular substrate or substrates of interest; and (iii) screening said exposed libraries utilizing a fluorescence activated cell sorter to identify clones which react with the substrate or substrates. Also provided is a process for identifying clones having a specified activity of interest by (i) generating one or more expression libraries derived from nucleic acid directly or indirectly isolated from the environment; and (ii) screening said exposed libraries utilizing an assay requiring co-encapsulation, a binding event or the covalent modification of a target, and a fluorescence activated cell sorter to identify positive clones.

Description

[0001] This application is a divisional of U.S. patent application Ser. No. 09 / 636,778, filed Aug. 11, 2000, which is a continuation of U.S. patent application Ser. No. 09 / 098,206, filed Jun. 16, 1998, continuation-in-part of U.S. application Ser. No. 08 / 876,276, filed Jun. 16, 1997, now abandoned.FIELD OF THE INVENTION[0002] The present invention relates generally to the identification of new bioactive molecules and particularly to methods for recovering such molecules by co-encapsulation and fluorescence activated cell sorting (FACS).BACKGROUND OF THE INVENTION[0003] There is a critical need in the chemical industry for efficient catalysts for the practical synthesis of optically pure materials; enzymes can provide the optimal solution. All classes of molecules and compounds that are utilized in both established and emerging chemical, pharmaceutical, textile, food and feed, detergent markets must meet stringent economical and environmental standards. The synthesis of polymers, pha...

AI Technical Summary

Benefits of technology

0039] The invention further provides a method of screening for an agent that modulates the activity of a target protein or other cell component (e.g., nucleic acid), wherein the target and a selectable marker are expressed by

Problems solved by technology

The synthesis of polymers, pharmaceuticals, natural products and agrochemicals is often hampered by expensive processes which produce harmful byproducts and which suffer from low enantioselectivity (Faber, 1995; Tonkovich and Gerber, U.S. Dept of Energy study, 1995).

These are often extremely difficult to duplicate chemically, especially in single-step reactions.

A current limitation to more widespread industrial use is primarily due to the relatively small number of commercially available enzymes.

The use of enzymes for technological applications also may require performance under demanding industrial conditions.

However, these drugs were developed using classical medicinal chemistry and without a knowledge of the molecular mechanism of action.

When attempting to identify genes encoding bioactivities of interest from complex environmental expression libraries, the rate limiting steps in discovery occur at the both DNA cloning level and at the screening level.

Furthermore there are no reports of recovering DNA encoding bioactivities screened by expression screening in E. coli using a FACS machine.

A limited number of papers describing various applications of flow cytometry in the field of microbiology and sorting of fluorescence activated microorganisms have, however, been published (Davey and Kell, 1996).

Such diffusion would not only lead to an underestimation of .beta.-galactosidase activity in highly active cells but could also lead to an overestimation of enzyme activity in inactive cells or those with low activity, as they may take up the leaked fluorescent compound, thus reducing the apparent heterogeneity of the population.

Using a chromogenic substrate, such as ONPG (o-nitrophenyl-(-D-galactopyranosid- e), one can measure expression of .beta.-galactosidase in cell cultures; but it is not possible to monitor expression in individual cells and to analyze the heterogeneity of expression in cell populations.

On this basis, one could only use fluorogenic reagents which could penetrate the cell and which are thus potentially cytotoxic.

FACS systems have typically been based on eukaryotic separations and have not been refined to accurately sort single E. coli cells; the low forward and sideward scatter of small particles like E. coli, reduces the ability of accurate sorting; enzyme substrates typically used in automated screening approaches, such as umbelifferyl based substrates, diffuse out of E. Coli at rates which interfere with quantitation.

Further, recovery of very small amounts of DNA from sorted organisms can be problematic.

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