Screening of cell populations

Details Screening of cell populations Screening of cell populations

1998-06-04

2001-08-21

Gitomer, Ralph (Department: 1623)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving viable micro-organism

C435S029000, C435S034000

Reexamination Certificate

active

06277588

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention generally relates to screening of large cell populations e.g. bacteria, yeast, fungi or mammalian cells, for the isolation of mutants expressing envisaged or a target activity like an enzymatic activity. More particularly, the invention relates to screening of expression libraries for the isolation of rare mutants expressing enzymatic activity catalysing non-biological chemical reactions or enzymes with altered specificity.
2. Description of Related Art
The isolation of novel biocatalysts has been based for many years on screening of cell cultures derived from natural and artificial environments affecting selective pressure, resulting in the generation of enzymes exhibiting envisaged special activity and stability (P. S. J. Cheetham, 1987. Screening for new biocatalysts. Enzyme and Microbial Technology, 9, 194-213). The isolation of useful enzymes, such as proteases, lipases and esterases, was carried out by a multi-step screening including enrichment for target microorganisms, screening for desired activity and secondary screening for the isolation of enzyme overproducers. These screenings were traditionally based on spreading a diluted sample of the cell population to be screened on solid agar, including appropriate growth medium, in petri dishes. Colonies expressing the envisaged activity were isolated on the basis of selection pressure affected by the medium composition employed or by adding substrate and following observable changes e.g. colour development or clear zones on turbid background. Some of these methods were automated into robotic screening (D. B. Steele and M. D. Stowers, 1991. Techniques for selection of industrially important microorganisms. Annual Reviews in Microbiology, 45, 89-106). More recently, the application of flow cytometry to mutant selection and industrial strain improvement was successfully employed in a number of cases. Single cells were encapsulated within small e.g.
30
&mgr; agar microbeads, allowed to proliferate into micro-colonies and the microbead population screened for enzymatic activities resulting in the production of fluorescence by flow cytometer (E. Sahar, R. Nirand R. Lamed, 1994. Flow cytometric analysis of entire microbial colonies. Cytometry, 15, 213-221.
However, the entrapment of cells and the subsequent propagation of these cells to form colonies within microbeads composed of polysaccharides is a complicated process. In addition, the screening and detection of desired mutants requires that a fluorescent or colored product accumulates within the microbead. Thus, special measures must be taken to retain the fluorescent/colored product. For example, the product must either be insoluble in the medium in which the reaction is carried out, or alternatively must be immobilized within the microbead to prevent its diffusion into the surrounding medium. Finally, the method of Sahar et al. requires very expensive instrumentation, e.g., a flow cytometer, and specially trained personnel to operate the instrument, both of which decrease the general applicability of the overall approach. The method of the invention overcomes these prior art disadvantages by immobilizing cells directly onto the surface of derivitized microbeads, which are subsequently immobilized as a monolayer on a solid surface which in a method that requires no special processing steps. The substrate used for the screening and detection of desired target cells or colonies is not subject to the solubility constraints of the method of Sahar et al., since the detection can be performed visually after the solvent is removed from the solid surface containing the immobilized microbeads. In addition, the detection method only requires simple and relatively inexpensive instrumentation that is present in, or readily available to any laboratory, e.g., a microscope, to identify the target cells/colonies.
A major objective in the field of applied biocatalysis is the identification or creation of enzymes capable of catalysing chemical, non-biological reactions. Although in some cases enzymes exhibiting activity on a broad spectrum of substrates were found capable of catalysing chemical reactions of similarly structured unnatural substrates (C. H. Who and G. M. Whitesides, 1994. Enzymes in organic synthesis. Pergamon Press), the systematic generation of new enzymes for this purpose remained a major challenge.
Recent developments in the field of genetic engineering and, in particular, options of introducing either predetermined or random changes into isolated genes by PCR created new tools for the generation of improved enzymes. A variety of techniques, including chemical mutagenesis of isolated DNA, gene amplification by error prone PCR and DNA shuffling, have been employed to generate large libraries of mutant genes, including a few rare mutants expressing the envisaged activity. Multiple rounds of selection and mutagenesis were successfully employed for the isolation of increasingly improved enzymes. Thus, a mutant of &bgr;-lactamase exhibiting a 32,000 fold increase of activity on cefotaxime (W. P. Stemmer, 1994. Rapid evolution of a protein in vitro by DNA shuffling. Nature, 370, 389-391) and the conversion of a galactosidase into fucosidase (J. H. Zhang, G. Dawes and W. P. Stemmer, 1997. Directed evolution of a fucosidase from a galactosidase by DNA shuffling and screening. Proceedings of National Academy of Sciences, USA. 94, 4504-4509) were demonstrated for DNA shuffling. Directed evolution of an esterase into a variant exhibiting higher stability in the presence of water miscible organic solvent (J. C. Moore and F. H. Arnold, 1996. Directed evolution of a para-nitrobenzyl esterase for aqueous-organic solvents. Nature Biotechnology, 14, 458-467) and significant increase of enantioselectivity of lipase (M. T. Reetz, A. Zonta, K. Schmossek, K. Liebeton and K. E. Jaeger 1997. Creation of enantioselective biocatalysts for organic chemistry by in vitro evolution. Angew. Chemie (International) 36, 2830-2832) were successfully demonstrated for error prone PCR.
Accumulating experience in these and related studies indicated that isolation of rare clones producing desired enzymes by plate screening is tedious and impractical when large populations e.g. 100,000 and more have to be individually assayed for envisaged activity. There is a need for an efficient rapid screening method of large libraries for envisaged, sometimes non-biological enzymatic activity, for the exploitation of the potential inherent in the combination of mutagenesis, screening and isolation (M. K. Winson and D. B. Kell, 1997. If you've got it, flaunt it—rapid screening for microbial biocatalysts. Trends in Biotechnology, 15, 120-122).
The present invention contributes to overcome the disadvantages of the prior art and also provides valuable new products heretofore not available.
SUMMARY OF THE INVENTION
The invention provides a methodology for the convenient separation of large cell populations into individual colonies, each derived from a single cell. These colonies are readily assayed in parallel for the desired activity and rapidly screened for the isolation of selected mutants expressing the envisaged activity.
The invention also provides a methodology for the screening of large cell populations for rare mutants expressing a desired product or enzymatic activity. In a preferred embodiment, the method is based on the separation of the cell population into single cells which are adsorbed individually onto the surface of derivitized microbeads. The beads are subsequently immobilized as a monolayer, consisting of single beads or small groups of beads, on a solid surface. The resulting monolayer is, if desired, equilibrated in a suitable growth medium, allowing the immobilized cells to grow and generating immobilized cell colonies, thereby intensifying the signal by that the colonies which have the target activity will be detected. When the enzyme is a highly active enzyme and/or when the assay is a very sensitive assay, it will not be necessar

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