Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification
Reexamination Certificate
2000-01-24
2001-10-30
Whisenant, Ethan (Department: 1655)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
C435S006120, C536S023100, C536S024300
Reexamination Certificate
active
06309883
ABSTRACT:
COPYRIGHT NOTIFICATION
Pursuant to 37 C.F.R. 1.71(e), Applicants note that a portion of this disclosure contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
BACKGROUND OF THE INVENTION
Metabolic engineering is the manipulation of intermediary metabolism through the use of both classical genetics and genetic engineering techniques. Cellular engineering is generally a more inclusive term referring to the modification of cellular properties. Cameron et al. (
Applied Biochem. Biotech
. 38:105-140 (1953)) provide a summary of equivalent terms to describe this type of engineering, including “metabolic engineering”, which is most often used in the context of industrial microbiology and bioprocess engineering, “in vitro evolution” or “directed evolution”, most often used in the context of environmental microbiology, “molecular breeding”, most often used by Japanese researchers, “cellular engineering”, which is used to describe modifications of bacteria, animal, and plant cells, “rational strain development”, and “metabolic pathway evolution”. In this application, the terms “metabolic engineering” and “cellular engineering” are used preferentially for clarity; the term “evolved” genes is used as discussed below.
Metabolic engineering can be divided into two basic categories: modification of genes endogenous to the host organism to alter metabolite flux and introduction of foreign genes into an organism. Such introduction can create new metabolic pathways leading to modified cell properties including but not limited to synthesis of known compounds not normally made by the host cell, production of novel compounds (e.g. polymers, antibiotics, etc.) and the ability to utilize new nutrient sources. Specific applications of metabolic engineering can include the production of specialty and novel chemicals, including antibiotics, extension of the range of substrates used for growth and product formation, the production of new catabolic activities in an organism for Ada toxic chemical degradation, and modification of cell properties such as resistance to salt and other environmental factors.
Bailey (
Science
252:1668-1674 (1991)) describes the application of metabolic engineering to the recruitment of heterologous genes for the improvement of a strain, with the caveat that such introduction can result in new compounds that may subsequently undergo further reactions, or that expression of a heterologous protein can result in proteolysis, improper folding, improper modification, or unsuitable intracellular location of the protein, or lack of access to required substrates. Bailey recommends careful configuration of a desired genetic change with minimal perturbation of the host.
Liao (
Curr. Opin. Biotech
. 4:211-216 (1993)) reviews mathematical modelling and analysis of metabolic pathways, pointing out that in many cases the kinetic parameters of enzymes are unavailable or inaccurate.
Stephanopoulos et al. (
Trends. Biotechnol
. 11:392-396 (1993)) describe attempts to improve productivity of cellular systems or effect radical alteration of the flux through primary metabolic pathways as having difficulty in that control architectures at key branch points have evolved to resist flux changes. They conclude that identification and characterization of these metabolic nodes is a prerequisite to rational metabolic engineering. Similarly, Stephanopoulos (
Curr. Opin. Biotech
. 5:196-200 (1994)) concludes that rather than modifying the “rate limiting step” in metabolic engineering, it is necessary to systematically elucidate the control architecture of bioreaction networks.
The present invention is generally directed to the evolution of new metabolic pathways and the enhancement of bioprocessing through a process herein termed recursive sequence recombination. Recursive sequence recombination entails performing iterative cycles of recombination and screening or selection to “evolve” individual genes, whole plasmids or viruses, multigene clusters, or even whole genomes (Stemmer,
Bio/Technoloqy
13:549-553 (1995)). Such techniques do not require the extensive analysis and computation required by conventional methods for metabolic engineering. Recursive sequence recombination allows the recombination of large numbers of mutations in a minimum number of selection cycles, in contrast to traditional, pairwise recombination events.
Thus, because metabolic and cellular engineering can pose the particular problem of the interaction of many gene products and regulatory mechanisms, recursive sequence recombination (RSR) techniques provide particular advantages in that they provide recombination between mutations in any or all of these, thereby providing a very fast way of exploring the manner in which different combinations of mutations can affect a desired result, whether that result is increased yield of a metabolite, altered catalytic activity or substrate specificity of an enzyme or an entire metabolic pathway, or altered response of a cell to its environment.
SUMMARY OF THE INVENTION
One aspect of the invention is a method of evolving a biocatalytic activity of a cell, comprising:
(a) recombining at least a first and second DNA segment from at least one gene conferring ability to catalyze a reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes;
(b) screening at least one recombinant gene from the library that confers enhanced ability to catalyze the reaction of interest by the cell relative to a wildtype form of the gene;
(c) recombining at least a segment from at least one recombinant gene with a further DNA segment from at least one gene, the same or different from the first and second segments, to produce a further library of recombinant genes;
(d) screening at least one further recombinant gene from the further library of recombinant genes that confers enhanced ability to catalyze the reaction of interest in the cell relative to a previous recombinant gene;
(e) repeating (c) and (d), as necessary, until the further recombinant gene confers a desired level of enhanced ability to catalyze the reaction of interest by the cell.
Another aspect of the invention is a method of evolving a gene to confer ability to catalyze a reaction of interest, the method comprising:
(1) recombining at least first and second DNA segments from at least one gene conferring ability to catalyze a reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes;
(2) screening at least one recombinant gene from the library that confers enhanced ability to catalyze a reaction of interest relative to a wildtype form of the gene;
(3) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from the first and second segments, to produce a further library of recombinant genes;
(4) screening at least one further recombinant gene from the further library of recombinant genes that confers enhanced ability to catalyze a reaction of interest relative to a previous recombinant gene;
(5) repeating (3) and (4), as necessary, until the further recombinant gene confers a desired level of enhanced ability to catalyze a reaction of interest.
A further aspect of the invention is a method of generating a new biocatalytic activity in a cell, comprising:
(1) recombining at least first and second DNA segments from at least one gene conferring ability to catalyze a first reaction related to a second reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes;
(2) screening at least one recombinant gene from the library that confers a new ability to catalyze the second reaction of interest;
(3) recombin
Minshull Jeremy
Stemmer Willem P. C.
Fujita Sharon
Law Offices of Jonathan Alan Quine
Maxygen Inc.
Quine Jonathan Alan
Whisenant Ethan
LandOfFree
Methods and compositions for cellular and metabolic engineering does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Methods and compositions for cellular and metabolic engineering, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methods and compositions for cellular and metabolic engineering will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2571072