U.S. patent application number 09/188777 was filed with the patent office on 2002-02-28 for methods and compositions for cellular and metabolic engineering.
Invention is credited to MINSHULL, JEREMY, STEMMER, WILLEM P. C..
Application Number | 20020025517 09/188777 |
Document ID | / |
Family ID | 27417292 |
Filed Date | 2002-02-28 |
United States Patent
Application |
20020025517 |
Kind Code |
A1 |
MINSHULL, JEREMY ; et
al. |
February 28, 2002 |
METHODS AND COMPOSITIONS FOR CELLULAR AND METABOLIC ENGINEERING
Abstract
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. Such techniques do not require the extensive analysis and
computation required by conventional methods for metabolic
engineering.
Inventors: |
MINSHULL, JEREMY; (SAN
FRANCISCO, CA) ; STEMMER, WILLEM P. C.; (LOS GATOS,
CA) |
Correspondence
Address: |
LAW OFFICE OF JONATHAN ALAN QUINE
P.O. BOX 458
ALAMEDA
CA
94501
|
Family ID: |
27417292 |
Appl. No.: |
09/188777 |
Filed: |
November 9, 1998 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09188777 |
Nov 9, 1998 |
|
|
|
08650400 |
May 20, 1996 |
|
|
|
Current U.S.
Class: |
435/6.18 ;
435/6.1; 435/91.2 |
Current CPC
Class: |
C12N 15/1027 20130101;
C12N 15/1093 20130101; C12N 9/86 20130101; C12N 15/64 20130101;
C12Y 302/01023 20130101; C12Q 1/6811 20130101; C07K 14/43595
20130101; C12N 15/52 20130101; C07K 16/00 20130101; C12N 15/1037
20130101; B07C 2501/009 20130101; C12N 9/2471 20130101; C07K
2317/622 20130101; C12N 15/1058 20130101; C07K 2317/565 20130101;
C07K 14/545 20130101; C40B 40/02 20130101 |
Class at
Publication: |
435/6 ;
435/91.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Claims
What is claimed is:
1. 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 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; (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 by
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.
2. The method of claim 1, wherein the reaction of interest is the
ability to utilize a substrate as a nutrient source.
3. The method of claim 1, wherein the reaction of interest is the
ability to catabolize a compound.
4. The method of claim 1, wherein the reaction of interest is the
ability to detoxify a compound.
5. The method of claim 1, wherein the reaction of interest is the
ability to synthesize a compound of interest.
6. The method of claim 4, wherein the compound is an
antibiotic.
7. The method of claim 4, wherein the compound is an amino
acid.
8. The method of claim 4, wherein the compound is a polymer.
9. The method of claim 4, wherein the compound is a carotenoid.
10. The method of claim 4, wherein the compound is vitamin C.
11. The method of claim 4, wherein the compound is indigo.
12. The method of claim 1, wherein at least one recombining step is
performed in vitro, and the resulting library of recombinants is
introduced into the cell whose biocatalytic activity is to be
enhanced generating a library of cells containing different
recombinants.
13. The method of claim 12, wherein the in vitro recombining step
comprises: cleaving the first and second segments into fragments;
mixing and denaturing the fragments; and incubating the denatured
fragments with a polymerase under conditions which result in
annealing of the denatured fragments and formation of the library
of recombinant genes.
14. The method of claim 1, wherein at least one recombining step is
performed in vivo.
15. The method of claim 1, wherein the recombining step is
performed in the cell whose biocatalytic activity is to be
enhanced.
16. The method of claim 1, wherein at least one DNA segment
comprises a cluster of genes collectively conferring ability to
catalyze a reaction of interest.
17. 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.
18. 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) recombining at least
a segment from 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 the second reaction of interest in the
cell 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 the second reaction
of interest in the cell.
19. A modified form of a cell, wherein the modification comprises a
metabolic pathway evolved by recursive sequence recombination.
20. A method of optimizing expression of a gene product, the method
comprising: (1) recombining at least first and second DNA segments
from at least one gene conferring ability to produce the gene
product, 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 optimized expression of the gene product 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 optimized ability
to produce the gene product relative to a previous recombinant
gene; (5) repeating (3) and (4), as necessary, until the further
recombinant gene confers a desired level of optimized ability to
express the gene product.
21. The method of claim 20, wherein the at least one gene encodes
the gene product.
22. The method of claim 20, wherein the at least one gene is a
vector comprising a gene encoding the gene product.
23. The method of claim 20, wherein at least one recombining step
is performed in vivo.
24. The method of claim 23, wherein the recombining step is
performed in a host cell wherein the gene product is expressed.
25. The method of claim 20, wherein the at least one gene is a host
cell gene and wherein the host cell gene does not encode the gene
product.
26. The method of claim 20, wherein optimization results in
increased expression of the gene product.
27. A method of evolving a biosensor for a compound A of interest,
the method comprising: (1) recombining at least first and second
DNA segments from at least one gene conferring ability to detect a
related compound B, 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 optimized ability to detect compound A 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 optimized ability
to detect compound A relative to a previous recombinant gene; (5)
repeating (3) and (4), as necessary, until the further recombinant
gene confers Et desired level of optimized ability to detect
compound A.
28. The method of claim 27, wherein optimization results in
increased amplitude of response by the biosensor.
29. The method of claim 27, wherein compound A and compound B are
different.
30. The method of claim 27, wherein compound A and compound B are
identical.
Description
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 08/198,431, filed Feb. 17, 1994, Ser. No.
PCT/US95/02126, filed, Feb. 17, 1995, Ser. No. 08/537,874, filed
Oct. 30, 1995, Ser. No. 08/621,859, filed Mar. 25, 1996, Ser. No.
08/621,430, filed Mar. 25, 1996, and Ser. No. 08/425,684, filed
Apr. 18, 1995, the specifications of which are herein incorporated
by reference in their entirety for all purposes.
BACKGROUND OF THE INVENTION
[0002] 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
(1993)) 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.
[0003] 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 toxic
chemical degradation, and modification of cell properties such as
resistance to salt and other environmental factors.
[0004] 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.
[0005] 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.
[0006] 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.
[0007] 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/Technology 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.
[0008] 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
[0009] One aspect of the invention is a method of evolving a
biocatalytic activity of a cell, comprising:
[0010] (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;
[0011] (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;
[0012] (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;
[0013] (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;
[0014] (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.
[0015] Another aspect of the invention is a method of evolving a
gene to confer ability to catalyze a reaction of interest, the
method comprising:
[0016] (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;
[0017] (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;
[0018] (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;
[0019] (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;
[0020] (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.
[0021] A further aspect of the invention is a method of generating
a new biocatalytic activity in a cell, comprising:
[0022] (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;
[0023] (2) screening at least one recombinant gene from the library
that confers a new ability to catalyze the second reaction of
interest;
[0024] (3) recombining at least a segment from 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;
[0025] (4) screening at least one further recombinant gene from the
further library of recombinant genes that confers enhanced ability
to catalyze the second reaction of interest in the cell relative to
a previous recombinant gene;
[0026] (5) repeating (3) and (4), as necessary, until the further
recombinant gene confers a desired level of enhanced ability to
catalyze the second reaction of interest in the cell.
[0027] Another aspect of the invention is a modified form of a
cell, wherein the modification comprises a metabolic pathway
evolved by recursive sequence recombination.
[0028] A further aspect of the invention is a method of optimizing
expression of a gene product, the method comprising:
[0029] (1) recombining at least first and second DNA segments from
at least one gene conferring ability to produce the gene product,
the segments differing from each other in at least two nucleotides,
to produce a library of recombinant genes;
[0030] (2) screening at least one recombinant gene from the library
that confers optimized expression of the gene product relative to a
wildtype form of the gene;
[0031] (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;
[0032] (4) screening at least one further recombinant gene from the
further library of recombinant genes that confers optimized ability
to produce the gene product relative to a previous recombinant
gene;
[0033] (5) repeating (3) and (4), as necessary, until the further
recombinant gene confers a desired level of optimized ability to
express the gene product.
[0034] A further aspect of the invention is a method of evolving a
biosensor for a compound A of interest, the method comprising:
[0035] (1) recombining at least first and second DNA segments from
at least one gene conferring ability to detect a related compound
B, the segments differing from each other in at least two
nucleotides, to produce a library of recombinant genes;
[0036] (2) screening at least one recombinant gene from the library
that confers optimized ability to detect compound A relative to a
wildtype form of the gene;
[0037] (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;
[0038] (4) screening at least one further recombinant gene from the
further library of recombinant genes that confers optimized ability
to detect compound A relative to a previous recombinant gene;
[0039] (5) repeating (3) and (4), as necessary, until the further
recombinant gene confers a desired level of optimized ability to
detect compound A.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1 is a drawing depicting a scheme for in vitro
recursive sequence, recombination.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0041] The invention provides a number of strategies for evolving
metabolic and bioprocessing pathways through the technique of
recursive sequence recombination. One strategy entails evolving
genes that confer the ability to use a particular substrate of
interest as a nutrient source in one species to confer either more
efficient use of that substrate in that species, or comparable or
more efficient use of that substrate in a second species. Another
strategy entails evolving genes that confer the ability to detoxify
a compound of interest in one or more species of organisms. Another
strategy entails evolving new metabolic pathways by evolving an
enzyme or metabolic pathway for biosynthesis or degradation of a
compound A related to a compound B for the ability to biosynthesize
or degrade compound B, either in the host of origin or a new host.
A further strategy entails evolving a gene or metabolic pathway for
more efficient or optimized expression of a particular metabolite
or gene product. A further strategy entails evolving a host/vector
system for expression of a desired heterologous product. These
strategies may involve using all the genes in a multi-step pathway,
one or several genes, genes from different organisms, or one or
more fragments of a gene.
[0042] The strategies generally entail evolution of gene(s) or
segment(s) thereof to allow retention of function in a heterologous
cell or improvement of function in a homologous or heterologous
cell. Evolution is effected generally by a process termed recursive
sequence recombination. Recursive sequence recombination can be
achieved in many different formats and permutations of formats, as
described in further detail below. These formats share some common
principles. Recursive sequence recombination entails successive
cycles of recombination to generate molecular diversity, i.e., the
creation of a family of nucleic acid molecules showing substantial
sequence identity to each other but differing in the presence of
mutations. Each recombination cycle is followed by at least one
cycle of screening or selection for molecules having a desired
characteristic. The molecule(s) selected in one round form the
starting materials for generating diversity in the next round. In
any given cycle, recombination can occur in viva or in vitro.
Furthermore, diversity resulting from recombination can be
augmented in any cycle by applying prior methods of mutagenesis
(e.g., error-prone PCR or cassette mutagenesis, passage through
bacterial mutator strains, treatment with chemical mutagens) to
either the substrates for or products of recombination.
[0043] I. Formats for Recursive Sequence Recombination
[0044] Some formats and examples for recursive sequence
recombination, sometimes referred to as DNA shuffling or molecular
breeding, have been described by the present inventors and
co-workers in co-pending applications, U.S. patent application Ser.
No. 08/621,430, filed Mar. 25, 1996; Ser. No. PCT/US95/02126, filed
Feb. 17, 1995; Ser. No. 08/621,859, filed Mar. 25, 1996; Ser. No.
08/198,431, filed Feb. 17, 1994; Stemmer, Science 270:1510 (1995);
Stemmer et al., Gene 164:49-53 (1995); Stemmer, Bio/Technology
13:549-553 (1995); Stemmer, Proc. Natl. Acad. Sci. U.S.A.
91:10747-10751 (1994); Stemmer, Nature 370:389-391 (1994); Crameri
et al. Nature Medicine 2(1):1-3 (1996); Crameri et al. Nature
Biotechnology 14:315-319 (1996), each of which is incorporated by
reference in its entirety for all purposes.
[0045] (1) In Vitro Formats
[0046] One format for recursive sequence recombination in vitro is
illustrated in FIG. 1. The initial substrates for recombination are
a pool of related sequences. The X's in FIG. 1, panel A, show where
the sequences diverge. The sequences can be DNA or RNA and can be
of various lengths depending on the size of the gene or DNA
fragment to be recombined or reassembled. Preferably the sequences
are from 50 bp to 100 kb.
[0047] The pool of related substrates can be fragmented, usually at
random, into fragments of from about 5 bp to 5 kb or more, as shown
in FIG. 1, panel B. Preferably the size of the random fragments is
from about 10 bp to 1000 bp, more preferably the size of the DNA
fragments is from about 20 bp to 500 bp. The substrates can be
digested by a number of different methods, such as DNAseI or RNAse
digestion, random shearing or restriction enzyme digestion. The
concentration of nucleic acid fragments of a particular length or
sequence is often less than 0.1% or 1% by weight of the total
nucleic acid. The number of different specific nucleic acid
fragments in the mixture is usually at least about 100, 500 or
1000.
[0048] The mixed population of nucleic acid fragments are denatured
by heating to about 80.degree. C. to 100.degree. C., more
preferably from 90.degree. C. to 96.degree. C., to form
single-stranded nucleic acid fragments and then reannealed.
Single-stranded nucleic acid fragments having regions of sequence
identity with other single-stranded nucleic acid fragments can then
be reannealed by cooling to 20.degree. C. to 75.degree. C., and
preferably from 40.degree. C. to 65.degree. C. Renaturation can be
accelerated by the addition of polyethylene glycol ("PEG") or salt.
The salt concentration is preferably from 0 mM to 600 mM, more
preferably the salt concentration is from 10 mM to 100 mM. The salt
may be such salts as (NH.sub.4).sub.2SO.sub.4, KCl, or NaCl. The
concentration of PEG is preferably from 0% to 20%, more preferably
from 5% to 10%. The fragments that reanneal can be from different
substrates as shown in FIG. 1, panel C.
[0049] The annealed nucleic acid fragments are incubated in the
presence of a nucleic acid polymerase, such as Taq or Klenow, and
dNTP's (i.e. DATP, dCTP, dGTP and dTTP). If regions of sequence
identity are large, Iaq or other high-temperature polymerase can be
used with an annealing temperature of between 45-65.degree. C. If
the areas of identity are small, Klenow or other low-temperature
polymerases can be used with an annealing temperature of between
20-30.degree. C. The polymerase can be added to the random nucleic
acid fragments prior to annealing, simultaneously with annealing or
after annealing.
[0050] The cycle of denaturation, renaturation and incubation of
random nucleic acid fragments in the presence of polymerase is
sometimes referred to as "shuffling" of the nucleic acid in vitro.
This cycle is repeated for a desired number of times. Preferably
the cycle is repeated from 2 to 100 times, more preferably the
sequence is repeated from 10 to 40 times. The resulting nucleic
acids are a family of double-stranded polynucleotides of from about
50 bp to about 100 kb, preferably from 500 bp to 50 kb, as shown in
FIG. 1, panel D. The population represents variants of the starting
substrates showing substantial sequence identity thereto but also
diverging at several positions. The population has many more
members than the starting substrates. The population of fragments
resulting from recombination is preferably first amplified by PCR,
then cloned into an appropriate vector and the ligation mixture
used to transform host cells.
[0051] In a variation of in vitro shuffling, subsequences of
recombination substrates can be generated by amplifying the
full-length sequences under conditions which produce a substantial
fraction, typically at least 20 percent or more, of incompletely
extended amplification products. The amplification products,
including the incompletely extended amplification products are
denatured and subjected to at least one additional cycle of
reannealing and amplification. This variation, wherein at least one
cycle of reannealing and amplification provides a substantial
fraction of incompletely extended products, is termed "stuttering."
In the subsequent amplification round, the incompletely extended
products anneal to and prime extension on different
sequence-related template species.
[0052] In a further variation, at least one cycle of amplification
can be conducted using a collection of overlapping single-stranded
DNA fragments of related sequence, and different lengths. Each
fragment can hybridize to and prime polynucleotide chain extension
of a second fragment from the collection, thus forming
sequence-recombined polynucleotides. In a further variation,
single-stranded DNA fragments of variable length can be generated
from a single primer by Vent DNA polymerase on a first DNA
template. The single stranded DNA fragments are used as primers for
a second, Kunkel-type template, consisting of a uracil-containing
circular single-stranded DNA. This results in multiple
substitutions of the first template into the second (see Levichkin
et al. Mol. Biology 29:572-577 (1995)).
[0053] Gene clusters such as those involved in polyketide synthesis
(or indeed any multi-enzyme pathways catalyzing analogous metabolic
reactions) can be recombined by recursive sequence recombination
even if they lack DNA sequence homology. Homology can be introduced
using synthetic oligonucleotides as PCR primers. In addition to the
specific sequences for the gene being amplified, all of the primers
used to amplify one type of enzyme (for example the acyl carrier
protein in polyketide synthesis) are synthesized to contain an
additional sequence of 20-40 bases 5' to the gene (sequence A) and
a different 20-40 base sequence 3' to the gene (sequence B). The
adjacent gene (in this case the keto-synthase) is amplified using a
5' primer which contains the complementary strand of sequence B
(sequence B'), and a 3' primer containing a different 20-40 base
sequence (C). Similarly, primers for the next adjacent gene
(keto-reductases) contain sequences C' (complementary to C) and D.
If 5 different polyketide gene clusters are being shuffled, all
five acyl carrier proteins are flanked by sequences A and B
following their PCR amplification. In this way, small regions of
homology are introduced, making the gene clusters into
site-specific recombination cassettes. Subsequent to the initial
amplification of individual genes, the amplified genes can then be
mixed and subjected to primerless PCR. Sequence B at the 3' end of
all of the five acyl carrier protein genes can anneal with and
prime DNA synthesis from sequence B' at the 5' end of all five keto
reductase genes. In this way all possible combinations of genes
within the cluster can be obtained. Oligonucleotides allow such
recombinants to be obtained in the absence of sufficient sequence
homology for recursive sequence recombination described above. Only
homology of function is required to produce functional gene
clusters.
[0054] This method is also useful for exploring permutations of any
other multi-subunit enzymes. An example of such enzymes composed of
multiple polypeptides that have shown novel functions when the
subunits are combined in novel ways are dioxygenases. Directed
recombination between the four protein subunits of biphenyl and
toluene dioxygenases produced functional dioxygenases with
increased activity against trichloroethylene (Furukawa et. al. J.
Bacteriol. 176: 2121-2123 (1994)). This combination of subunits
from the two dioxygenases could also have been produced by
cassette-shuffling of the dioxygenases as described above, followed
by selection for degradation of trichloroethylene.
[0055] In some polyketide synthases, the separate functions of the
acyl carrier protein, keto-synthase, keto-reductase, etc. reside in
a single polypeptide. In these cases domains within the single
polypeptide may be shuffled, even if sufficient homology does not
exist naturally, by introducing regions of homology as described
above for entire genes. In this case, it may not be possible to
introduce additional flanking sequences to the domains, due to the
constraint of maintaining a continuous open reading frame. Instead,
groups of oligonucleotides are synthesized that are homologous to
the 3' end of the first domain encoded by one of the genes to be
shuffled, and the 5' ends of the second domains encoded by all of
the other genes to be shuffled together. This is repeated with all
domains, thus providing sequences that allow recombination between
protein domains while maintaining their order.
[0056] The cassette-based recombination method can be combined with
recursive sequence recombination by including gene fragments
(generated by DNase, physical shearing, DNA stuttering, etc.) for
one or more of the genes. Thus, in addition to different
combinations of entire genes within a cluster (e.g., for polyketide
synthesis), individual genes can be shuffled at the same time
(e.g., all acyl carrier protein genes can also be provided as
fragmented DNA), allowing a more thorough search of sequence
space.
[0057] (2) In Vivo Formats
(a) Plasmid-plasmid Recombination
[0058] The initial substrates for recombination are a collection of
polynucleotides comprising variant forms of a gene. The variant
forms usually show substantial sequence identity to each other
sufficient to allow homologous recombination between substrates.
The diversity between the polynucleotides can be natural (e.g.,
allelic or species variants), induced (e.g., error-prone PCR or
error-prone recursive sequence recombination), or the result of in
vitro recombination. Diversity can also result from resynthesizing
genes encoding natural proteins with alternative codon usage. There
should be at least sufficient diversity between substrates that
recombination can generate more diverse products than there are
starting materials. There must be at least two substrates differing
in at least two positions. However, commonly a library of
substrates of 10.sup.3-10.sup.8 members is employed. The degree of
diversity depends on the length of the substrate being recombined
and the extent of the functional change to be evolved. Diversity at
between 0.1-25% of positions is typical. The diverse substrates are
incorporated into plasmids. The plasmids are often standard cloning
vectors, e.g., bacterial multicopy plasmids. However, in some
methods to be described below, the plasmids include mobilization
(MOB) functions. The substrates can be incorporated into the same
or different plasmids. Often at least two different types of
plasmid having different types of selectable markers are used to
allow selection for cells containing at least two types of vector.
Also, where different types of plasmid are employed, the different
plasmids can come from two distinct incompatibility groups to allow
stable co-existence of two different plasmids within the cell.
Nevertheless, plasmids from the same incompatibility group can
still co-exist within the same cell for sufficient time to allow
homologous recombination to occur.
[0059] Plasmids containing diverse substrates are initially
introduced into cells by any method (e.g., chemical transformation,
natural competence, electroporation, biolistics, packaging into
phage or viral systems). Often, the plasmids are present at or near
saturating concentration (with respect to maximum transfection
capacity) to increase the probability of more than one plasmid
entering the same cell. The plasmids containing the various
substrates can be transfected simultaneously or in multiple rounds.
For example, in the latter approach cells can be transfected with a
first aliquot of plasmid, transfectants selected and propagated,
and then infected with a second aliquot of plasmid.
[0060] Having introduced the plasmids into cells, recombination
between substrates to generate recombinant genes occurs within
cells containing multiple different plasmids merely by propagating
the cells. However, cells that receive only one plasmid are unable
to participate in recombination and the potential contribution of
substrates on such plasmids to evolution is not fully exploited
(although these plasmids may contribute to some extent if they are
progagated in mutator cells). The rate of evolution can be
increased by allowing all substrates to participate in
recombination. Such can be achieved by subjecting transfected cells
to electroporation. The conditions for electroporation are the same
as those conventionally used for introducing exogenous DNA into
cells (e.g., 1,000-2,500 volts, 400 .mu.F and a 1-2 mM gap). Under
these conditions, plasmids are exchanged between cells allowing all
substrates to participate in recombination. In addition the
products of recombination can undergo further rounds of
recombination with each other or with the original substrate. The
rate of evolution can also be increased by use of conjugative
transfer. To exploit conjugative transfer, substrates can be cloned
into plasmids having MOB genes, and tra genes are also provided in
cis or in trans to the MOB genes. The effect of conjugative
transfer is very similar to electroporation in that it allows
plasmids to move between cells and allows recombination between any
substrate and the products of previous recombination to occur,
merely by propagating the culture. The rate of evolution can also
be increased by fusing cells to induce exchange of plasmids or
chromosomes. Fusion can be induced by chemical agents, such as PEG,
or viral proteins, such as influenza virus hemagglutinin, HSV-1 gB
and gD. The rate of evolution can also be increased by use of
mutator host cells (e.g., Mut L, S, D, T, H in bacteria and Ataxia
telangiectasia human cell lines).
[0061] The time for which cells are propagated and recombination is
allowed to occur, of course, varies with the cell type but is
generally not critical, because even a small degree of
recombination can substantially increase diversity relative to the
starting materials. Cells bearing plasmids containing recombined
genes are subject to screening or selection for a desired function.
For example, if the substrate being evolved contains a drug
resistance gene, one would select for drug resistance. Cells
surviving screening or selection can be subjected to one or more
rounds of screening/selection followed by recombination or can be
subjected directly to an additional round of recombination.
[0062] The next round of recombination can be achieved by several
different formats independently of the previous round. For example,
a further round of recombination can be effected simply by resuming
the electroporation or conjugation-mediated intercellular transfer
of plasmids described above. Alternatively, a fresh substrate or
substrates, the same or different from previous substrates, can be
transfected into cells surviving selection/screening. optionally,
the new substrates are included in plasmid vectors bearing a
different selective marker and/or from a different incompatibility
group than the original plasmids. As a further alternative, cells
surviving selection/screening can be subdivided into two
subpopulations, and plasmid DNA from one subpopulation transfected
into the other, where the substrates from the plasmids from the two
subpopulations undergo a further round of recombination. In either
of the latter two options, the rate of evolution can be increased
by employing DNA extraction, electroporation, conjugation or
mutator cells, as described above. In a still further variation,
DNA from cells surviving screening/selection can be extracted and
subjected to in vitro recursive sequence recombination.
[0063] After the second round of recombination, a second round of
screening/selection is performed, preferably under conditions of
increased stringency. If desired, further rounds of recombination
and selection/screening can be performed using the same strategy as
for the second round. With successive rounds of recombination and
selection/screening, the surviving recombined substrates evolve
toward acquisition of a desired phenotype. Typically, in this and
other methods of recursive recombination, the final product of
recombination that has acquired the desired phenotype differs from
starting substrates at 0.1%-25% of positions and has evolved at a
rate orders of magnitude in excess (e.g., by at least 10-fold,
100-fold, 1000-fold, or 10,000 fold) of the rate of naturally
acquired mutation of about 1 mutation per 10.sup.-9 positions per
generation (see Anderson et al. Proc. Natl. Acad. Sci. U.S.A.
93:906-907 (1996)). The final products may be transferred to
another host more desirable for utilization of the "shuffled" DNA.
This is particularly advantageous in situations where the more
desirable host is less efficient as a host for the many cycles of
mutation/recombination due to the lack of molecular biology or
genetic tools available for other organisms such as E. coli.
(b) Virus-plasmid Recombination
[0064] The strategy used for plasmid-plasmid recombination can also
be used for virus-plasmid recombination; usually, phage-plasmid
recombination. However, some additional comments particular to the
use of viruses are appropriate. The initial substrates for
recombination are cloned into both plasmid and viral vectors. It is
usually not critical which substrate(s) are inserted into the viral
vector and which into the plasmid, although usually the viral
vector should contain different substrate(s) from the plasmid. As
before, the plasmid (and the virus) typically contains a selective
marker. The plasmid and viral vectors can both be introduced into
cells by transfection as described above. However, a more efficient
procedure is to transfect the cells with plasmid, select
transfectants and infect the transfectants with virus. Because the
efficiency of infection of many viruses approaches 100% of cells,
most cells transfected and infected by this route contain both a
plasmid and virus bearing different substrates.
[0065] Homologous recombination occurs between plasmid and virus
generating both recombined plasmids and recombined virus. For some
viruses, such as filamentous phage, in which intracellular DNA
exists in both double-stranded and single-stranded forms, both can
participate in recombination. Provided that the virus is not one
that rapidly kills cells, recombination can be augmented by use of
electroporation or conjugation to transfer plasmids between cells.
Recombination can also be augmented for some types of virus by
allowing the progeny virus from one cell to reinfect other cells.
For some types of virus, virus infected-cells show resistance to
superinfection. However, such resistance can be overcome by
infecting at high multiplicity and/or using mutant strains of the
virus in which resistance to superinfection is reduced.
[0066] The result of infecting plasmid-containing cells with virus
depends on the nature of the virus. Some viruses, such as
filamentous phage, stably exist with a plasmid in the cell and also
extrude progeny phage from the cell. Other viruses, such as lambda
having a cosmid genome, stably exist in a cell like plasmids
without producing progeny virions. Other viruses, such as the
T-phage and lytic lambda, undergo recombination with the plasmid
but ultimately kill the host cell and destroy plasmid DNA. For
viruses that infect cells without killing the host, cells
containing recombinant plasmids and virus can be screened/selected
using the same approach as for plasmid-plasmid recombination.
Progeny virus extruded by cells surviving selection/screening can
also be collected and used as substrates in subsequent rounds of
recombination. For viruses that kill their host cells, recombinant
genes resulting from recombination reside only in the progeny
virus. If the screening or selective assay requires expression of
recombinant genes in a cell, the recombinant genes should be
transferred from the progeny virus to another vector, e.g., a
plasmid vector, and retransfected into cells before
selection/screening is performed.
[0067] For filamentous phage, the products of recombination are
present in both cells surviving recombination and in phage extruded
from these cells. The dual source of recombinant products provides
some additional options relative to the plasmid-plasmid
recombination. For example, DNA can be isolated from phage
particles for use in a round of in vitro recombination.
Alternatively, the progeny phage can be used to transfect or infect
cells surviving a previous round of screening/selection, or fresh
cells transfected with fresh substrates for recombination.
(c) Virus-virus Recombination
[0068] The principles described for plasmid-plasmid and
plasmid-viral recombination can be applied to virus-virus
recombination with a few modifications. The initial substrates for
recombination are cloned into a viral vector. Usually, the same
vector is used for all substrates. Preferably, the virus is one
that, naturally or as a result of mutation, does not kill cells.
After insertion, some viral genomes can be packaged in vitro or
using a packaging cell line. The packaged viruses are used to
infect cells at high multiplicity such that there is a high
probability that a cell will receive multiple viruses bearing
different substrates.
[0069] After the initial round of infection, subsequent steps
depend on the nature of infection as discussed in the previous
section. For example, if the viruses have phagemid genomes such as
lambda cosmids or M13, F1 or Fd phagemids, the phagemids behave as
plasmids within the cell and undergo recombination simply by
propagating the cells. Recombination is particularly efficient
between single-stranded forms of intracellular DNA. Recombination
can be augmented by electroporation of cells.
[0070] Following selection/screening, cosmids containing
recombinant genes can be recovered from surviving cells, e.g., by
heat induction of a cos.sup.- lysogenic host cell, or extraction of
DNA by standard procedures, followed by repackaging cosmid DNA in
vitro.
[0071] If the viruses are filamentous phage, recombination of
replicating form DNA occurs by propagating the culture of infected
cells. Selection/screening identifies colonies of cells containing
viral vectors having recombinant genes with improved properties,
together with phage extruded from such cells. Subsequent options
are essentially the same as for plasmid-viral recombination.
(d) Chromosome Recombination
[0072] This format can be used to especially evolve chromosomal
substrates. The format is particularly useful in situations in
which many chromosomal genes contribute to a phenotype or one does
not know the exact location of the chromosomal gene(s) to be
evolved. The initial substrates for recombination are cloned into a
plasmid vector. If the chromosomal gene(s) to be evolved are known,
the substrates constitute a family of sequences showing a high
degree of sequence identity but some divergence from the
chromosomal gene. If the chromosomal genes to be evolved have not
been located, the initial substrates usually constitute a library
of DNA segments of which only a small number show sequence identity
to the gene or gene(s) to be evolved. Divergence between
plasmid-borne substrate and the chromosomal gene(s) can be induced
by mutagenesis or by obtaining the plasmid-borne substrates from a
different species than that of the cells bearing the
chromosome.
[0073] The plasmids bearing substrates for recombination are
transfected into cells having chromosomal gene(s) to be evolved.
Evolution can occur simply by propagating the culture, and can be
accelerated by transferring plasmids between cells by conjugation
or electroporation. Evolution can be further accelerated by use of
mutator host cells or by seeding a culture of nonmutator host cells
being evolved with mutator host cells and inducing intercellular
transfer of plasmids by electroporation or conjugation. Preferably,
mutator host cells used for seeding contain a negative selectable
marker to facilitate isolation of a pure culture of the nonmutator
cells being evolved. Selection/screening identifies cells bearing
chromosomes and/or plasmids that have evolved toward acquisition of
a desired function.
[0074] Subsequent rounds of recombination and selection/screening
proceed in similar fashion to those described for plasmid-plasmid
recombination. For example, further recombination can be effected
by propagating cells surviving recombination in combination with
electroporation or conjugative transfer of plasmids. Alternatively,
plasmids bearing additional substrates for recombination can be
introduced into the surviving cells. Preferably, such plasmids are
from a different incompatibility group and bear a different
selective marker than the original plasmids to allow selection for
cells containing at least two different plasmids. As a further
alternative, plasmid and/or chromosomal DNA can be isolated from a
subpopulation of surviving cells and transfected into a second
subpopulation. Chromosomal DNA can be cloned into a plasmid vector
before transfection.
(e) Virus-chromosome Recombination
[0075] As in the other methods described above, the virus is
usually one that does not kill the cells, and is often a phage or
phagemid. The procedure is substantially the same as for
plasmid-chromosome recombination. Substrates for recombination are
cloned into the vector. Vectors including the substrates can then
be transfected into cells or in vitro packaged and introduced into
cells by infection. Viral genomes recombine with host chromosomes
merely by propagating a culture. Evolution can be accelerated by
allowing intercellular transfer of viral genomes by
electroporation, or reinfection of cells by progeny virions.
Screening/selection identifies cells having chromosomes and/or
viral genomes that have evolved toward acquisition of a desired
function.
[0076] There are several options for subsequent rounds of
recombination. For example, viral genomes can be transferred
between cells surviving selection/recombination by electroporation.
Alternatively, viruses extruded from cells surviving
selection/screening can be pooled and used to superinfect the cells
at high multiplicity. Alternatively, fresh substrates for
recombination can be introduced into the cells, either on plasmid
or viral vectors.
[0077] II. Recursive Sequence Recombination Techniques for
Metabolic and Cellular Engineering
[0078] A. Starting Materials
[0079] Thus, a general method for recursive sequence recombination
for the embodiments herein is to begin with a gene encoding an
enzyme or enzyme subunit and to evolve that gene either for ability
to act on a new substrate, or for enhanced catalytic properties
with an old substrate, either alone or in combination with other
genes in a multistep pathway. The term "gene" is used herein
broadly to refer to any segment or sequence of DNA associated with
a biological function. Genes can be obtained from a variety of
sources, including cloning from a source of interest or
synthesizing from known or predicted sequence information, and may
include sequences designed to have desired parameters. The ability
to use a new substrate can be assayed in some instances by the
ability to grow on a substrate as a nutrient source. In other
circumstances such ability can be assayed by decreased toxicity of
a substrate for a host cell, hence allowing the host to grow in the
presence of that substrate. Biosynthesis of new compounds, such as
antibiotics, can be assayed similarly by growth of an indicator
organism in the presence of the host expressing the evolved genes.
For example, when an indicator organism used in an overlay of the
host expressing the evolved gene(s), wherein the indicator organism
is sensitive or expected to be sensitive to the desired antibiotic,
growth of the indicator organism would be inhibited in a zone
around the host cell or colony expressing the evolved gene(s).
[0080] Another method of identifying new compounds is the use of
standard analytical techniques such as mass spectroscopy, nuclear
magnetic resonance, high performance liquid chromatography, etc.
Recombinant microorganisms can be pooled and extracts or media
supernatants assayed from these pools. Any positive pool can then
be subdivided and the procedure repeated until the single positive
is identified ("sib-selection").
[0081] In some instances, the starting material for recursive
sequence recombination is a discrete gene, cluster of genes, or
family of genes known or thought to be associated with metabolism
of a particular class of substrates.
[0082] One of the advantages of the instant invention is that
structural information is not required to estimate which parts of a
sequence should be mutated to produce a functional hybrid
enzyme.
[0083] In some embodiments of the invention, an initial screening
of enzyme activities in a particular assay can be useful in
identifying candidate enzymes as starting materials. For example,
high throughput screening can be used to screen enzymes for
dioxygenase-type activities using aromatic acids as substrates.
Dioxygenases typically transform indole-2-carboxylate and
indole-3-carboxylate to colored products, including indigo (Eaton
et. al. J. Bacteriol. 177:6983-6988 (1995)). DNA encoding enzymes
that give some activity in the initial assay can then be recombined
by the recursive techniques of the invention and rescreened. The
use of such initial screening for candidate enzymes against a
desired target molecule or analog of the target molecule can be
especially useful to generate enzymes that catalyze reactions of
interest such as catabolism of man-made pollutants.
[0084] The starting material can also be a segment of such a gene
or cluster that is recombined in isolation of its surrounding DNA,
but is relinked to its surrounding DNA before screening/selection
of recombination products. In other instances, the starting
material for recombination is a larger segment of DNA that includes
a coding sequence or other locus associated with metabolism of a
particular substrate at an unknown location. For example, the
starting material can be a chromosome, episome, YAC, cosmid, or
phage P1 clone. In still other instances, the starting material is
the whole genome of an organism that is known to have desirable
metabolic properties, but for which no information localizing the
genes associated with these characteristics is available.
[0085] In general any type of cells can be used as a recipient of
evolved genes. Cells of particular interest include many bacterial
cell types, both gram-negative and gram-positive, such as
Rhodococcus, Streptomycetes, Actinomycetes, Corynebacteria,
Penicillium, Bacillus, Escherichia coli, Pseudomonas, Salmonella,
and Erwinia. Cells of interest also include eukaryotic cells,
particularly mammalian cells (e.g., mouse, hamster, primate,
human), both cell lines and primary cultures. Such cells include
stem cells, including embryonic stem cells, zygotes, fibroblasts,
lymphocytes, Chinese hamster ovary (CHO), mouse fibroblasts
(NIH3T3), kidney, liver, muscle, and skin cells. Other eukaryotic
cells of interest include plant cells, such as maize, rice, wheat,
cotton, soybean, sugarcane, tobacco, and arabidopsis; fish, algae,
fungi (Penicillium, Fusarium, Aspergillus, Podospora, Neurospora),
insects, yeasts (Picchia and Saccharomyces).
[0086] The choice of host will depend on a number of factors,
depending on the intended use of the engineered host, including
pathogenicity, substrate range, environmental hardiness, presence
of key intermediates, ease of genetic manipulation, and likelihood
of promiscuous transfer of genetic information to other organisms.
Particularly advantageous hosts are E. coli, lactobacilli,
Streptomycetes, Actinomycetes and filamentous fungi.
[0087] The breeding procedure starts with at least two substrates,
which generally show substantial sequence identity to each other
(i.e., at least about 50%, 70%, 80% or 90% sequence identity) but
differ from each other at certain positions. The difference can be
any type of mutation, for example, substitutions, insertions and
deletions. Often, different segments differ from each other in
perhaps 5-20 positions. For recombination to generate increased
diversity relative to the starting materials, the starting
materials must differ from each other in at least two nucleotide
positions. That is, if there are only two substrates, there should
be at least two divergent positions. If there are three substrates,
for example, one substrate can differ from the second as a single
position, and the second can differ from the third at a different
single position. The starting DNA segments can be natural variants
of each other, for example, allelic or species variants. The
segments can also be from nonallelic genes showing some degree of
structural and usually functional relatedness (e.g., different
genes within a superfamily such as the immunoglobulin superfamily).
The starting DNA segments can also be induced variants of each
other. For example, one DNA segment can be produced by error-prone
PCR replication of the other, or by substitution of a mutagenic
cassette. Induced mutants can also be prepared by propagating one
(or both) of the segments in a mutagenic strain. In these
situations, strictly speaking, the second DNA segment is not a
single segment but a large family of related segments. The
different segments forming the starting materials are often the
same length or substantially the same length. However, this need
not be the case; for example; one segment can be a subsequence of
another. The segments can be present as part of larger molecules,
such as vectors, or can be in isolated form.
[0088] The starting DNA segments are recombined by any of the
recursive sequence recombination formats described above to
generate a diverse library of recombinant DNA segments. Such a
library can vary widely in size from having fewer than 10 to more
than 10.sup.5, 10.sup.7, or 10.sup.9 members. In general, the
starting segments and the recombinant libraries generated include
full-length coding sequences and any essential regulatory
sequences, such as a promoter and polyadenylation sequence,
required for expression. However, if this is not the case, the
recombinant DNA segments in the library can be inserted into a
common vector providing the missing sequences before performing
screening/selection.
[0089] If the recursive sequence recombination format employed is
an in vivo format, the library of recombinant DNA segments
generated already exists in a cell, which is usually the cell type
in which expression of the enzyme with altered substrate
specificity is desired. If recursive sequence recombination is
performed in vitro, the recombinant library is preferably
introduced into the desired cell type before screening/selection.
The members of the recombinant library can be linked to an episome
or virus before introduction or can be introduced directly. In some
embodiments of the invention, the library is amplified in a first
host, and is then recovered from that host and introduced to a
second host more amenable to expression, selection, or screening,
or any other desirable parameter. The manner in which the library
is introduced into the cell type depends on the DNA-uptake
characteristics of the cell type, e.g., having viral receptors,
being capable of conjugation, or being naturally competent. If the
cell type is insusceptible to natural and chemical-induced
competence, but susceptible to electroporation, one would usually
employ electroporation. If the cell type is insusceptible to
electroporation as well, one can employ biolistics. The biolistic
PDS-1000 Gene Gun (Biorad, Hercules, Calif.) uses helium pressure
to accelerate DNA-coated gold or tungsten microcarriers toward
target cells. The process is applicable to a wide range of tissues,
including plants, bacteria, fungi, algae, intact animal tissues,
tissue culture cells, and animal embryos. One can employ electronic
pulse delivery, which is essentially a mild electroporation format
for live tissues in animals and patients. Zhao, Advanced Drug
Delivery Reviews 17:257-262 (1995). Novel methods for making cells
competent are described in co-pending application U.S. patent
application Ser. No. 08/621,430, filed Mar. 25, 1996. After
introduction of the library of recombinant DNA genes, the cells are
optionally propagated to allow expression of genes to occur.
[0090] B. Selection and Screening
[0091] Screening is, in general, a two-step process in which one
first determines which cells do and do not express a screening
marker and then physically separates the cells having the desired
property. Selection is a form of screening in which identification
and physical separation are achieved simultaneously, for example,
by expression of a selectable marker, which, in some genetic
circumstances, allows cells expressing the marker to survive while
other cells die (or vice versa). Screening markers include, for
example, luciferase, .beta.-galactosidase, and green fluorescent
protein. Screening can also be done by observing such aspects of
growth as colony size, halo formation, etc. Additionally, screening
for production of a desired compound, such as a therapeutic drug or
"designer chemical" can be accomplished by observing binding of
cell products to a receptor or ligand, such as on a solid support
or on a column. Such screening can additionally be accomplished by
binding to antibodies, as in an ELISA. In some instances the
screening process is preferably automated so as to allow screening
of suitable numbers of colonies or cells. Some examples of
automated screening devices include fluorescence activated cell
sorting, especially in conjunction with cells immobilized in
agarose (see Powell et. al. Bio/Technology 8:333-337 (1990); Weaver
et. al. Methods 2:234-247 (1991)), automated ELISA assays, etc.
Selectable markers can include, for example, drug, toxin
resistance, or nutrient synthesis genes. Selection is also done by
such techniques as growth on a toxic substrate to select for hosts
having the ability to detoxify a substrate, growth on a new
nutrient source to select for hosts having the ability to utilize
that nutrient source, competitive growth in culture based on
ability to utilize a nutrient source, etc.
[0092] In particular, uncloned but differentially expressed
proteins (e.g., those induced in response to new compounds, such as
biodegradable pollutants in the medium) can be screened by
differential display (Appleyard et al. Mol. Gen. Gent. 247:338-342
(1995)). Hopwood (Phil Trans R. Soc. Lond B 324:549-562) provides a
review of screens for antibiotic production. Omura (Microbio. Rev.
50:259-279 (1986) and Nisbet (Ann Rep. Med. Chem. 21:149-157
(1986)) disclose screens for antimicrobial agents, including
supersensitive bacteria, detection of .beta.-lactamase and
D,D-carboxypeptidase inhibition, .beta.-lactamase induction,
chromogenic substrates and monoclonal antibody screens. Antibiotic
targets can also be used as screening targets in high throughput
screening. Antifungals are typically screened by inhibition of
fungal growth. Pharmacological agents can be identified as enzyme
inhibitors using plates containing the enzyme and a chromogenic
substrate, or by automated receptor assays. Hydrolytic enzymes
(e.g., proteases, amylases) can be screened by including the
substrate in an agar plate and scoring for a hydrolytic clear zone
or by using a calorimetric indicator (Steele et al. Ann. Rev.
Microbiol. 45:89-106 (1991)). This can be coupled with the use of
stains to detect the effects of enzyme action (such as congo red to
detect the extent of degradation of celluloses and hemicelluloses).
Tagged substrates can also be used. For example, lipases and
esterases can be screened using different lengths of fatty acids
linked to umbelliferyl. The action of lipases or esterases removes
this tag from the fatty acid, resulting in a quenching of
umbelliferyl fluorescence. These enzymes can be screened in
microtiter plates by a robotic device.
[0093] Fluorescence activated cell sorting (FACS) methods are also
a powerful tool for selection/screening. In some instances a
fluorescent molecule is made within a cell (e.g., green fluorescent
protein). The cells producing the protein can simply be sorted by
FACS. Gel microdrop technology allows screening of cells
encapsulated in agarose microdrops (Weaver et al. Methods 2:234-247
(1991)). In this technique products secreted by the cell (such as
antibodies or antigens) are immobilized with the cell that
generated them. Sorting and collection of the drops containing the
desired product thus also collects the cells that made the product,
and provides a ready source for the cloning of the genes encoding
the desired functions. Desired products can be detected by
incubating the encapsulated cells with fluorescent antibodies
(Powell et al. Bio/Technology 8:333-337 (1990)). FACS sorting can
also be used by this technique to assay resistance to toxic
compounds and antibiotics by selecting droplets that contain
multiple cells (i.e., the product of continued division in the
presence of a cytotoxic compound; Goguen et al. Nature 363:189-190
(1995)). This method can select for any enzyme that can change the
fluorescence of a substrate that can be immobilized in the agarose
droplet.
[0094] In some embodiments of the invention, screening can be
accomplished by assaying reactivity with a reporter molecule
reactive with a desired feature of, for example, a gene product.
Thus, specific functionalities such as antigenic domains can be
screened with antibodies specific for those determinants.
[0095] In other embodiments of the invention, screening is
preferably done with a cell-cell indicator assay. In this assay
format, separate library cells (Cell A, the cell being assayed) and
reporter cells (Cell B, the assay cell) are used. Only one
component of the system, the library cells, is allowed to evolve.
The screening is generally carried out in a two-dimensional
immobilized format, such as on plates. The products of the
metabolic pathways encoded by these genes (in this case, usually
secondary metabolites such as antibiotics, polyketides,
carotenoids, etc.) diffuse out of the library cell to the reporter
cell. The product of the library cell may affect the reporter cell
in one of a number of ways.
[0096] The assay system (indicator cell) can have a simple readout
(e.g., green fluorescent protein, luciferase, .beta.-galactosidase)
which is induced by the library cell product but which does not
affect the library cell. In these examples the desired product can
be detected by calorimetric changes in the reporter cells adjacent
to the library cell.
[0097] In other embodiments, indicator cells can in turn produce
something that modifies the growth rate of the library cells via a
feedback mechanism. Growth rate feedback can detect and accumulate
very small differences. For example, if the library and reporter
cells are competing for nutrients, library cells producing
compounds to inhibit the growth of the reporter cells will have
more available nutrients, and thus will have more opportunity for
growth. This is a useful screen for antibiotics or a library of
polyketide synthesis gene clusters where each of the library cells
is expressing and exporting a different polyketide gene
product.
[0098] Another variation of this theme is that the reporter cell
for an antibiotic selection can itself secrete a toxin or
antibiotic that inhibits growth of the library cell. Production by
the library cell of an antibiotic that is able to suppress growth
of the reporter cell will thus allow uninhibited growth of the
library cell.
[0099] Conversely, if the library is being screened for production
of a compound that stimulates the growth of the reporter cell (for
example, in improving chemical syntheses, the library cell may
supply nutrients such as amino acids to an auxotrophic reporter, or
growth factors to a growth-factor-dependent reporter. The reporter
cell in turn should produce a compound that stimulates the growth
of the library cell. Interleukins, growth factors, and nutrients
are possibilities.
[0100] Further possibilities include competition based on ability
to kill surrounding cells, positive feedback loops in which the
desired product made by the evolved cell stimulates the indicator
cell to produce a positive growth factor for cell A, thus
indirectly selecting for increased product formation.
[0101] In some embodiments of the invention it can be advantageous
to use a different organism (or genetic background) for screening
than the one that will be used in the final product. For example,
markers can be added to DNA constructs used for recursive sequence
recombination to make the microorganism dependent on the constructs
during the improvement process, even though those markers may be
undesirable in the final recombinant microorganism.
[0102] Likewise, in some embodiments it is advantageous to use a
different substrate for screening an evolved enzyme than the one
that will be used in the final product. For example, Evnin et al.
(Proc. Natl. Acad. Sci. U.S.A. 87:6659-6663 (1990)) selected
trypsin variants with altered substrate specificity by requiring
that variant trypsin generate an essential amino acid for an
arginine auxotroph by cleaving arginine .beta.-naphthylamide. This
is thus a selection for arginine-specific trypsin, with the growth
rate of the host being proportional to that of the enzyme
activity.
[0103] The pool of cells surviving screening and/or selection is
enriched for recombinant genes conferring the desired phenotype
(e.g. altered substrate specificity, altered biosynthetic ability,
etc.). Further enrichment can be obtained, if desired, by
performing a second round of screening and/or selection without
generating additional diversity.
[0104] The recombinant gene or pool of such genes surviving one
round of screening/selection forms one or more of the substrates
for a second round of recombination. Again, recombination can be
performed in vivo or in vitro by any of the recursive sequence
recombination formats described above. If recursive sequence
recombination is performed in vitro, the recombinant gene or genes
to form the substrate for recombination should be extracted from
the cells in which screening/selection was performed. Optionally, a
subsequence of such gene or genes can be excised for more targeted
subsequent recombination. If the recombinant gene(s) are contained
within episomes, their isolation presents no difficulties. If the
recombinant genes are chromosomally integrated, they can be
isolated by amplification primed from known sequences flanking the
regions in which recombination has occurred. Alternatively, whole
genomic DNA can be isolated, optionally amplified, and used as the
substrate for recombination. Small samples of genomic DNA can be
amplified by whole genome amplification with degenerate primers
(Barrett et al. Nucleic Acids Research 23:3488-3492 (1995)). These
primers result in a large amount of random 3' ends, which can
undergo homologous recombination when reintroduced into cells.
[0105] If the second round of recombination is to be performed in
vivo, as is often the case, it can be performed in the cell
surviving screening/selection, or the recombinant genes can be
transferred to another cell type (e.g., a cell type having a high
frequency of mutation and/or recombination). In this situation,
recombination can be effected by introducing additional DNA
segment(s) into cells bearing the recombinant genes. In other
methods, the cells can be induced to exchange genetic information
with each other by, for example, electroporation. In some methods,
the second round of recombination is performed by dividing a pool
of. cells surviving screening/selection in the first round into two
subpopulations. DNA from one subpopulation is isolated and
transfected into the other population, where the recombinant
gene(s) from the two subpopulations recombine to form a further
library of recombinant genes. In these methods, it is not necessary
to isolate particular genes from the first subpopulation or to take
steps to avoid random shearing of DNA during extraction. Rather,
the whole genome of DNA sheared or otherwise cleaved into
manageable sized fragments is transfected into the second
subpopulation. This approach is particularly useful when several
genes are being evolved simultaneously and/or the location and
identity of such genes within chromosome are not known.
[0106] The second round of recombination is sometimes performed
exclusively among the recombinant molecules surviving selection.
However, in other embodiments, additional substrates can be
introduced. The additional substrates can be of the same form as
the substrates used in the first round of recombination, i.e.,
additional natural or induced mutants of the gene or cluster of
genes, forming the substrates for the first round. Alternatively,
the additional substrate(s) in the second round of recombination
can be exactly the same as the substrate(s) in the first round of
replication.
[0107] After the second round of recombination, recombinant genes
conferring the desired phenotype are again selected. The selection
process proceeds essentially as before. If a suicide vector bearing
a selective marker was used in the first round of selection, the
same vector can be used again. Again, a cell or pool of cells
surviving selection is selected. If a pool of cells, the cells can
be subject to further enrichment.
[0108] III. Recursive Sequence Recombination of Genes For
Bioremediation
[0109] Modern industry generates many pollutants for which the
environment can no longer be considered an infinite sink. Naturally
occurring microorganisms are able to metabolize thousands of
organic compounds, including many not found in nature (e.g
xenobiotics). Bioremediation, the deliberate use of microorganisms
for the biodegradation of man-made wastes, is an emerging
technology that offers cost and practicality advantages over
traditional methods of disposal. The success of bioremediation
depends on the availability of organisms that are able to detoxify
or mineralize pollutants. Microorganisms capable of degrading
specific pollutants can be generated by genetic engineering and
recursive sequence recombination.
[0110] Although bioremediation is an aspect of pollution control, a
more useful approach in the long term is one of prevention before
industrial waste is pumped into the environment. Exposure of
industrial waste streams to recursive sequence
recombination-generated microorganisms capable of degrading the
pollutants they contain would result in detoxification of
mineralization of these pollutants before the waste stream enters
the environment. Issues of releasing recombinant organisms can be
avoided by containing them within bioreactors fitted to the
industrial effluent pipes. This approach would also allow the
microbial mixture used to be adjusted to best degrade the
particular wastes being produced. Finally, this method would avoid
the problems of adapting to the outside world and dealing with
competition that face many laboratory microorganisms.
[0111] In the wild, microorganisms have evolved new catabolic
activities enabling them to exploit pollutants as nutrient sources
for which there is no competition. However, pollutants that are
present at low concentrations in the environment may not provide a
sufficient advantage to stimulate the evolution of catabolic
enzymes. For a review of such naturally occurring evolution of
biodegradative pathways and the manipulation of some of
microorganisms by classical techniques, see Ramos et al.,
Bio/Technology 12:1349-1355 (1994).
[0112] Generation of new catabolic enzymes or pathways for
bioremediation has thus relied upon deliberate transfer of specific
genes between organisms (Wackett et al., supra), forced matings
between bacteria with specific catabolic capabilities (Brenner et
al. Biodegradation 5:359-377 (1994)), or prolonged selection in a
chemostat. Some researchers have attempted to facilitate evolution
via naturally occurring genetic mechanisms in their chemostat
selections by including microorganisms with a variety of catabolic
pathways (Kellogg et. al. Science 214:1133-1135 (1981); Chakrabarty
American Society of Micro. Biol. News 62:130-137 (1996)). For a
review of efforts in this area, see Cameron et al. Applied Biochem.
Biotech. 38:105-140 (1993).
[0113] Current efforts in improving organisms for bioremediation
take a labor-intensive approach in which many parameters are
optimized independently, including transcription efficiency from
native and heterologous promoters, regulatory circuits and
translational efficiency as well as improvement of protein
stability and activity (Timmis et al. Ann. Rev. Microbiol.
48:525-527 (1994)).
[0114] A recursive sequence recombination approach overcomes a
number of limitations in the bioremediation capabilities of
naturally occurring microorganisms. Both enzyme activity and
specificity can be altered, simultaneously or sequentially, by the
methods of the invention. For example, catabolic enzymes can be
evolved to increase the rate at which they act on a substrate.
Although knowledge of a rate-limiting step in a metabolic pathway
is not required to practice the invention, rate-limiting proteins
in pathways can be evolved to have increased expression and/or
activity, the requirement for inducing substances can be
eliminated, and enzymes can be evolved that catalyze novel
reactions.
[0115] Some examples of chemical targets for bioremediation include
but are not limited to benzene, xylene, and toluene, camphor,
naphthalene, halogenated hydrocarbons, polychlorinated biphenyls
(PCBs), trichlorethylene, pesticides such as pentachlorophenyls
(PCPs), and herbicides such as atrazine.
[0116] A. Aromatic Hydrocarbons
[0117] Preferably, when an enzyme is "evolved" to have a new
catalytic function, that function is expressed, either
constitutively or in response to the new substrate. Recursive
sequence recombination subjects both structural and regulatory
elements (including the structure of regulatory proteins) of a
protein to recombinogenic mutagenesis simultaneously. Selection of
mutants that are efficiently able to use the new substrate as a
nutrient source will be sufficient to ensure that both the enzyme
and its regulation are optimized, without detailed analysis of
either protein structure or operon regulation.
[0118] Examples of aromatic hydrocarbons include but are not
limited to benzene, xylene, toluene, biphenyl, and polycyclic
aromatic hydrocarbons such as pyrene and naphthalene. These
compounds are metabolized via catechol intermediates. Degradation
of catechol by Pseudomonas putida requires induction of the
catabolic operon by cis, cis-muconate which acts on the CatR
regulatory protein. The binding site for the CatR protein is
G-N.sub.11-A, while the optimal sequence for the LysR class of
activators (of which CatR is a member) is T-N.sub.11-A. Mutation of
the G to a T in the CatR binding site enhances the expression of
catechol metabolizing genes (Chakrabarty, American Society of
Microbiology News 62:130-137 (1996)). This demonstrates that the
control of existing catabolic pathways is not optimized for the
metabolism of specific xenobiotics. It is also an example of a type
of mutant that would be expected from recursive sequence
recombination of the operon followed by selection of bacteria that
are better able to degrade the target compound.
[0119] As an example of starting materials, dioxygenases are
required for many pathways in which aromatic compounds are
catabolized. Even small differences in dioxygenase sequence can
lead to significant differences in substrate specificity (Furukawa
et al. J. Bact. 175:5224-5232 (1993); Erickson et al. App. Environ.
Micro. 59:3858-3862 (1993)). A hybrid enzyme made using sequences
derived from two "parental" enzymes may possess catalytic
activities that are intermediate between the parents (Erickson,
ibid.), or may actually be better than either parent for a specific
reaction (Furukawa et al. J. Bact. 176:2121-2123 (1994)). In one of
these cases site directed mutagenesis was used to generate a single
polypeptide with hybrid sequence (Erickson, ibid.); in the other, a
four subunit enzyme was produced by expressing two subunits from
each of two different dioxygenases (Furukawa, ibid.). Thus,
sequences from one or more genes encoding dioxygenases can be used
in the recursive sequence recombination techniques of the instant
invention, to generate enzymes with new specificities. In addition,
other features of the catabolic pathway can also be evolved using
these techniques, simultaneously or sequentially, to optimize the
metabolic pathway for an activity of interest.
[0120] B. Halogenated Hydrocarbons
[0121] Large quantities of halogenated hydrocarbons are produced
annually for uses as solvents and biocides. These include, in the
United States alone, over 5 million tons of both 1,2-dichloroethane
and vinyl chloride used in PVC production in the U.S. alone. The
compounds are largely not biodegradable by processes in single
organisms, although in principle haloaromatic catabolic pathways
can be constructed by combining genes from different
microorganisms. Enzymes can be manipulated to change their
substrate specificities. Recursive sequence recombination offers
the possibility of tailoring enzyme specificity to new substrates
without needing detailed structural analysis of the enzymes.
[0122] As an example of possible starting materials for the methods
of the instant invention, Wackett et al. (Nature 368:627-629
(1994)) recently demonstrated that through classical techniques a
recombinant Pseudomonas strain in which seven genes encoding two
multi-component oxygenases are combined, generated a single host
that can metabolize polyhalogenated compounds by sequential
reductive and oxidative techniques to yield non-toxic products.
These and/or related materials can be subjected to the techniques
discussed above so as to evolve and optimize a biodegradative
pathway in a single organism.
[0123] Trichloroethylene is a significant groundwater contaminant.
It is degraded by microorganisms in a cometabolic way (i.e., no
energy or nutrients are derived). The enzyme must be induced by a
different compound (e.g., Pseudomonas cepacia uses
toluene-4-monoxygenase, which requires induction by toluene, to
destroy trichloroethylene). Furthermore, the degradation pathway
involves formation of highly reactive epoxides that can inactivate
the enzyme (Timmis et al. Ann. Rev. Microbiol. 48:525-557 (1994)).
The recursive sequence recombination techniques of the invention
could be used to mutate the enzyme and its regulatory region such
that it is produced constitutively, and is less susceptible to
epoxide inactivation. In some embodiments of the invention,
selection of hosts constitutively producing the enzyme and less
susceptible to the epoxides can be accomplished by demanding growth
in the presence of increasing concentrations of trichloroethylene
in the absence of inducing substances.
[0124] C. Polychlorinated Biphenyls (PCBs) and Polycyclic Aromatic
Hydrocarbons (PAHs)
[0125] PCBs and PAHs are families of structurally related compounds
that are major pollutants at many Superfund sites. Bacteria
transformed with plasmids encoding enzymes with broader substrate
specificity have been used commercially. In nature, no known
pathways have been generated in a single host that degrade the
larger PAHs or more heavily chlorinated PCBs. Indeed, often the
collaboration of anaerobic and aerobic bacteria are required for
complete metabolism.
[0126] Thus, likely sources for starting material for recursive
sequence recombination include identified genes encoding
PAH-degrading catabolic pathways on large (20-100 KB) plasmids
(Sanseverino et al. Applied Environ. Micro. 59:1931-1937 (1993);
Simon et al. Gene 127:31-37 (1993); Zylstra et al. Annals of the NY
Acad. Sci. 721:386-398 (1994)); while biphenyl and PCB-metabolizing
enzymes are encoded by chromosomal gene clusters, and in a number
of cases have been cloned onto plasmids (Hayase et al. J.
Bacteriol. 172:1160-1164 (1990); Furukawa et al. Gene 98:21-28
(1992); Hofer et al. Gene 144:9-16 (1994)). The materials can be
subjected to the techniques discussed above so as to evolve a
biodegradative pathway in a single organism.
[0127] Substrate specificity in the PCB pathway largely results
from enzymes involved in initial dioxygenation reactions, and can
be significantly altered by mutations in those enzymes (Erickson et
al. Applied Environ. Micro. 59:3858-38662 (1993); Furukawa et al.
J. Bact. 175:5224-5232 (1993). Mineralization of PAHs and PCBs
requires that the downstream pathway is able to metabolize the
products of the initial reaction (Brenner et al. Biodegradation
5:359-377 (1994)). In this case, recursive sequence recombination
of the entire pathway with selection for bacteria able to use the
PCB or PAH as the sole carbon source will allow production of novel
PCB and PAH degrading bacteria.
[0128] D. Herbicides
[0129] A general method for evolving genes for the catabolism of
insoluble herbicides is exemplified as follows for atrazine.
Atrazine
[2-chloro-4-(ethylamino)-6-(isopropylamino)-1,3,5-triazine] is a
moderately persistent herbicide which is frequently detected in
ground and surface water at concentrations exceeding the 3 ppb
health advisory level set by the EPA. Atrazine can be slowly
metabolized by a Pseudomonas species (Mandelbaum et al. Appl.
Environ. Micro. 61:1451-1457 (1995)). The enzymes catalyzing the
first two steps in atrazine metabolism by Pseudomonas are encoded
by genes AtzA and AtzB (de Souza et al. Appl. Environ. Micro.
61:3373-3378 (1995)). These genes have been cloned in a 6.8 kb
fragment into pUC18 (AtzAB-pUC). E. coli carrying this plasmid
converts atrazine to much more soluble metabolites. It is thus
possible to screen for enzyme activity by growing bacteria on
plates containing atrazine. The herbicide forms an opaque
precipitate in the plates, but cells containing AtzAB-pU18 secrete
atrazine degrading enzymes, leading to a clear halo around those
cells or colonies. Typically, the size of the halo and the rate of
its formation can be used to assess the level of activity so that
picking colonies with the largest halos allows selection of the
more active or highly produced atrazine degrading enzymes. Thus,
the plasmids carrying these genes can be subjected to the recursive
sequence recombination formats described above to optimize the
catabolism of atrazine in E. coli or another host of choice,
including Pseudomonas. After each round of recombination, screening
of host colonies expressing the evolved genes can be done on agar
plates containing atrazine to observe halo formation. This is a
generally applicable method for screening enzymes that metabolize
insoluble compounds to those that are soluble (e.g.,
polycyclicaromatic hydrocarbons). Additionally, catabolism of
atrazine can provide a source of nitrogen for the cell; if no other
nitrogen is available, cell growth will be limited by the rate at
which the cells can catabolize nitrogen. Cells able to utilize
atrazine as a nitrogen source can thus be selected from a
background of non-utilizers or poor-utilizers.
[0130] E. Heavy Metal Detoxification
[0131] Bacteria are used commercially to detoxify arsenate waste
generated by the mining of arsenopyrite gold ores. As well as
mining effluent, industrial waste water is often contaminated with
heavy metals (e.g., those used in the manufacture of electronic
components and plastics). Thus, simply to be able to perform other
bioremedial functions, microorganisms must be resistant to the
levels of heavy metals present, including mercury, arsenate,
chromate, cadmium, silver, etc.
[0132] A strong selective pressure is the ability to metabolize a
toxic compound to one less toxic. Heavy metals are toxic largely by
virtue of their ability to denature proteins (Ford et al.
Bioextraction and Biodeterioration of Metals, p. 1-23).
Detoxification of heavy metal contamination can be effected in a
number of ways including changing the solubility or bioavailability
of the metal, changing its redox state (e.g. toxic mercuric
chloride is detoxified by reduction to the much more volatile
elemental mercury) and even by bioaccumulation of the metal by
immobilized bacteria or plants. The accumulation of metals to a
sufficiently high concentration allows metal to be recycled;
smelting burns off the organic part of the organism, leaving behind
reusable accumulated metal. Resistances to a number of heavy metals
(arsenate, cadmium, cobalt, chromium, copper, mercury, nickel,
lead, silver, and zinc) are plasmid encoded in a number of species
including Staphylococcus and Pseudomonas (Silver et al. Environ.
Health Perspect. 102:107-113 (1994); Ji et al. J. Ind. Micro.
14:61-75 (1995)). These genes also confer heavy metal resistance on
other species as well (e.g., E. coli). The recursive sequence
recombination techniques of the instant invention (RSR) can be used
to increase microbial heavy metal tolerances, as well as to
increase the extent to which cells will accumulate heavy metals.
For example, the ability of E. coli to detoxify arsenate can be
improved at least 100-fold by RSR (see co-pending application Ser.
No. 08/621,859, filed Mar. 25, 1996).
[0133] Cyanide is very efficiently used to extract gold from rock
containing as little as 0.2 oz per ton. This cyanide can be
microbially neutralized and used as a nitrogen source by fungi or
bacteria such as Pseudomonas fluorescens. A problem with microbial
cyanide degradation is the presence of toxic heavy metals in the
leachate. RSR can be used to increase the resistance of bioremedial
microorganisms to toxic heavy metals, so that they will be able to
survive the levels present in many industrial and Superfund sites.
This will allow them to biodegrade organic pollutants including but
not limited to aromatic hydrocarbons, halogenated hydrocarbons, and
biocides.
[0134] F. Microbial Mining
[0135] "Bioleaching" is the process by which microbes convert
insoluble metal deposits (usually metal sulfides or oxides) into
soluble metal sulfates. Bioleaching is commercially important in
the mining of arsenopyrite, but has additional potential in the
detoxification and recovery of metals and acids from waste dumps.
Naturally occurring bacteria capable of bioleaching are reviewed by
Rawlings and Silver (Bio/Technology 13:773-778 (1995)). These
bacteria are typically divided into groups by their preferred
temperatures for growth. The more important mesophiles are
Thiobacillus and Leptospirillum species. Moderate thermophiles
include Sulfobacillus species. Extreme thermophiles include
Sulfolobus species. Many of these organisms are difficult to grow
in commercial industrial settings, making their catabolic abilities
attractive candidates for transfer to and optimization in other
organisms such as Pseudomonas, Rhodococcus, T. ferrooxidans or E.
coli. Genetic systems are available for at least one strain of T.
ferrooxidans, allowing the manipulation of its genetic material on
plasmids.
[0136] The recursive sequence recombination methods described above
can be used to optimize the catalytic abilities in native hosts or
heterologous hosts for evolved bioleaching genes or pathways, such
as the ability to convert metals from insoluble to soluble salts.
In addition, leach rates of particular ores can be improved as a
result of, for example, increased resistance to toxic compounds in
the ore concentrate, increased specificity for certain substrates,
ability to use different substrates as nutrient sources, and so
on.
[0137] G. Oil Desulfurization
[0138] The presence of sulfur in fossil fuels has been correlated
with corrosion of pipelines, pumping, and refining equipment, and
with the premature breakdown of combustion engines. Sulfur also
poisons many catalysts used in the refining of fossil fuels. The
atmospheric emission of sulfur combustion products is known as acid
rain.
[0139] Microbial desulfurization is an appealing bioremediation
application. Several bacteria have been reported that are capable
of catabolizing dibenzothiophene (DBT), which is the representative
compound of the class of sulfur compounds found in fossil fuels.
U.S. Pat. No. 5,356,801 discloses the cloning of a DNA molecule
from Rhodococcus rhodochrous capable of biocatalyzing the
desulfurization of oil. Denome et al. (Gene 175:6890-6901 (1995))
disclose the cloning of a 9.8 kb DNA fragment from Pseudomonas
encoding the upper naphthalene catabolizing pathway which also
degrades dibenzothiophene. Other genes have been identified that
perform similar functions (disclosed in U.S. Pat. No.
5,356,801).
[0140] The activity of these enzymes is currently too low to be
commercially viable, but the pathway could be increased in
efficiency using the recursive sequence recombination techniques of
the invention. The desired property of the genes of interest is
their ability to desulfurize dibenzothiophene. In some embodiments
of the invention, selection is preferably accomplished by coupling
this pathway to one providing a nutrient to the bacteria. Thus, for
example, desulfurization of dibenzothiophene results in formation
of hydroxybiphenyl. This is a substrate for the
biphenyl-catabolizing pathway which provides carbon and energy.
Selection would thus be done by "shuffling" the dibenzothiophene
genes and transforming them into a host containing the
biphenyl-catabolizing pathway. Increased dibenzothiophene
desulfurization will result in increased nutrient availability and
increased growth rate. Once the genes have been evolved they are
easily separated from the biphenyl degrading genes. The latter are
undesirable in the final product since the object is to desulfurize
without decreasing the energy content of the oil.
[0141] H. Organo-nitro Compounds
[0142] Organo-nitro compounds are used as explosives, dyes, drugs,
polymers and antimicrobial agents. Biodegradation of these
compounds occurs usually by way of reduction of the nitrate group,
catalyzed by nitroreductases, a family of broadly-specific enzymes.
Partial reduction of organo-nitro compounds often results in the
formation of a compound more toxic than the original (Hassan et al.
1979 Arch Bioch Biop. 196:385-395). Recursive sequence
recombination of nitroreductases can produce enzymes that are more
specific, and able to more completely reduce (and thus detoxify)
their target compounds (examples of which include but are not
limited to nitrotoluenes and nitrobenzenes). Nitro-reductases can
be isolated from bacteria isolated from explosive-contaminated
soils, such as Morganella morganii and Enterobacter cloacae (Bryant
et. al., 1991. J. Biol Chem. 266:4126-4130). A preferred selection
method is to look for increased resistance to the organo-nitro
compound of interest, since that will indicate that the enzyme is
also able to reduce any toxic partial reduction products of the
original compound.
[0143] IV. Use of Alternative Substrates for Chemical Synthesis
[0144] Metabolic engineering can be used to alter microorganisms
that produce industrially useful chemicals, so that they will grow
using alternate and more abundant sources of nutrients, including
human-produced industrial wastes. This typically involves providing
both a transport system to get the alternative substrate into the
engineered cells and catabolic enzymes from the natural host
organisms to the engineered cells. In some instances, enzymes can
be secreted into the medium by engineered cells to degrade the
alternate substrate into a form that can more readily be taken up
by the engineered cells; in other instances, a batch of engineered
cells can be grown on one preferred substrate, then lysed to
liberate hydrolytic enzymes for the alternate substrate into the
medium, while a second inoculum of the same engineered host or a
second host is added to utilize the hydrolyzate.
[0145] The starting materials for recursive sequence recombination
will typically be genes for utilization of a substrate or its
transport. Examples of nutrient sources of interest include but are
not limited to lactose, whey, galactose, mannitol, xylan,
cellobiose, cellulose and sucrose, thus allowing cheaper production
of compounds including but not limited to ethanol, tryptophan,
rhamnolipid surfactants, xanthan gum, and polyhydroxylalkanoate.
For a review of such substrates as desired target substances, see
Cameron et al. (Appl. Biochem. Biotechnol. 38:105-140 (1993)).
[0146] The recursive sequence recombination methods described above
can be used to optimize the ability of native hosts or heterologous
hosts to utilize a substrate of interest, to evolve more efficient
transport systems, to increase or alter specificity for certain
substrates, and so on.
[0147] V. Biosynthesis
[0148] Metabolic engineering can be used to alter organisms to
optimize the production of practically any metabolic intermediate,
including antibiotics, vitamins, amino acids such as phenylalanine
and aromatic amino acids, ethanol, butanol, polymers such as
xanthan gum and bacterial cellulose, peptides, and lipids. When
such compounds are already produced by a host, the recursive
sequence recombination techniques described above can be used to
optimize production of the desired metabolic intermediate,
including such features as increasing enzyme substrate specificity
and turnover number, altering metabolic fluxes to reduce the
concentrations of toxic substrates or intermediates, increasing
resistance of the host to such toxic compounds, eliminating,
reducing or altering the need for inducers of gene
expression/activity, increasing the production of enzymes necessary
for metabolism, etc.
[0149] Enzymes can also be evolved for improved activity in
solvents other than water. This is useful because intermediates in
chemical syntheses are often protected by blocking groups which
dramatically affect the solubility of the compound in aqueous
solvents. Many compounds can be produced by a combination of pure
chemical and enzymically catalyzed reactions. Performing enzymic
reactions on almost insoluble substrates is clearly very
inefficient, so the availability of enzymes that are active in
other solvents will be of great use. One example of such a scheme
is the evolution of a para-nitrobenzyl esterase to remove
protecting groups from an intermediate in loracarbef synthesis
(Moore, J. C. and Arnold, F. H. Nature Biotechnology 14:458-467
(1996)). In this case alternating rounds of error-prone PCR and
colony screening for production of a fluorescent reporter from a
substrate analogue were used to generate a mutant esterase that was
16-fold more active than the parent molecule in 30%
dimethylformamide. No individual mutation was found to contribute
more than a 2-fold increase in activity, but it was the combination
of a number of mutations which led to the overall increase.
Structural analysis of the mutant protein showed that the amino
acid changes were distributed throughout the length of the protein
in a manner that could not have been rationally predicted.
Sequential rounds of error-prone PCR have the problem that after
each round all but one mutant is discarded, with a concomitant loss
of information contained in all the other beneficial mutations.
Recursive sequence recombination avoids this problem, and would
thus be ideally suited to evolving enzymes for catalysis in other
solvents, as well as in conditions where salt concentrations or pH
were different from the original enzyme optimas.
[0150] In addition, the yield of almost any metabolic pathway can
be increased, whether consisting entirely of genes endogenous to
the host organisms or all or partly heterologous genes.
Optimization of the expression levels of the enzymes in a pathway
is more complex than simply maximizing expression. In some cases
regulation, rather than constitutive expression of an enzyme may be
advantageous for cell growth and therefore for product yield, as
seen for production of phenylalanine (Backman et al. Ann. NY Acad.
Sci. 589:16-24 (1990)) and 2-keto-L-gluconic acid (Anderson et al.
U.S. Pat. No. 5,032,514). In addition, it is often advantageous for
industrial purposes to express proteins in organisms other than
their original hosts. New host strains may be preferable for a
variety of reasons, including ease of cloning and transformation,
pathogenicity, ability to survive in particular environments and a
knowledge of the physiology and genetics of the organisms. However,
proteins expressed in heterologous organisms often show markedly
reduced activity for a variety of reasons including inability to
fold properly in the new host (Sarthy et al. Appl. Environ. Micro.
53:1996-2000 (1987)). Such difficulties can indeed be overcome by
the recursive sequence recombination strategies of the instant
invention.
[0151] A. Antibiotics
[0152] The range of natural small molecule antibiotics includes but
is not limited to peptides, peptidolactones, thiopeptides,
beta-lactams, glycopeptides, lantibiotics, microcins,
polyketide-derived antibiotics (anthracyclins, tetracyclins,
macrolides, avermectins, polyethers and ansamycins),
chloramphenicol, aminoglycosides, aminocyclitols, polyoxins,
agrocins and isoprenoids.
[0153] There are at least three ways in which recursive sequence
recombination techniques of the instant invention can be used to
facilitate novel drug synthesis, or to improve biosynthesis of
existing antibiotics.
[0154] First, antibiotic synthesis enzymes can be "evolved"
together with transport systems that allow entry of compounds used
as antibiotic precursors to improve uptake and incorporation of
function-altering artificial side chain precursors. For example,
penicillin V is produced by feeding Penicillium the artificial side
chain precursor phenoxyacetic acid, and LY146032 by feeding
Streptomyces roseosporus decanoic acid (Hopwood, Phil. Trans. R.
Soc. Lond. B 324:549-562 (1989)). Poor precursor uptake and poor
incorporation by the synthesizing enzyme often lead to inefficient
formation of the desired product. Recursive sequence recombination
of these two systems can increase the yield of desired product.
[0155] Furthermore, a combinatorial approach can be taken in which
an enzyme is shuffled for novel catalytic activity/substrate
recognition (perhaps by including randomizing oligonucleotides in
key positions such as the active site). A number of different
substrates (for example, analogues of side chains that are normally
incorporated into the antibiotic) can then be tested in combination
with all the different enzymes and tested for biological activity.
In this embodiment, plates are made containing different potential
antibiotic precursors (such as the side chain analogues). The
microorganisms containing the shuffled library (the library strain)
are replicated onto those plates, together with a competing,
antibiotic sensitive, microorganism (the indicator strain). Library
cells that are able to incorporate the new side chain to produce an
effective antibiotic will thus be able to compete with the
indicator strain, and will be selected for.
[0156] Second, the expression of heterologous genes transferred
from one antibiotic synthesizing organism to another can be
optimized. The newly introduced enzyme(s) act on secondary
metabolites in the host cell, transforming them into new compounds
with novel properties. Using traditional methods, introduction of
foreign genes into antibiotic synthesizing hosts has already
resulted in the production of novel hybrid antibiotics. Examples
include mederrhodin, dihydrogranatirhodin, 6-deoxyerythromycin A,
isovalerylspiramycin and other hybrid macrolides (Cameron et. al.
Appl. Biochem. Biotechnol. 38:105-140 (1993)). The recursive
sequence recombination techniques of the instant invention can be
used to optimize expression of the foreign genes, to stabilize the
enzyme in the new host cell, and to increase the activity of the
introduced enzyme against its new substrates in the new host cell.
In some embodiments of the invention, the host genome may also be
so optimized.
[0157] Third, the substrate specificity of an enzyme involved in
secondary metabolism can be altered so that it will act on and
modify a new compound or so that its activity is changed and it
acts at a different subset of positions of its normal substrate.
Recursive sequence recombination can be used to alter the substrate
specificities of enzymes. Furthermore, in addition to recursive
sequence recombination of individual enzymes being a strategy to
generate novel antibiotics, recursive sequence recombination of
entire pathways, by altering enzyme ratios, will alter metabolite
fluxes and may result, not only in increased antibiotic synthesis,
but also in the synthesis of different antibiotics. This can be
deduced from the observation that expression of different genes
from the same cluster in a foreign host leads to different products
being formed (see p. 80 in Hutchinson et. al., (1991) Ann NY Acad
Sci, 646:78-93). Recursive sequence recombination of the introduced
gene clusters may result in a variety of expression levels of
different proteins within the cluster (because it produces
different combinations of, in this case regulatory, mutations).
This in turn may lead to a variety of different end products. Thus,
"evolution" of an existing antibiotic synthesizing pathway could be
used to generate novel antibiotics either by modifying the rates or
substrate specificities of enzymes in that pathway.
[0158] Additionally, antibiotics can also be produced in vitro by
the action of a purified enzyme on a precursor. For example
isopenicillin N synthase catalyses the cyclization of many
analogues of its normal substrate
(d-(L-a-aminoadipyl)-L-cysteinyl-D-valine) (Hutchinson, Med. Res.
Rev. 8:557-567 (1988)). Many of these products are active as
antibiotics. A wide variety of substrate analogues can be tested
for incorporation by secondary metabolite synthesizing enzymes
without concern for the initial efficiency of the reaction.
Recursive sequence recombination can be used subsequently to
increase the rate of reaction with a promising new substrate.
[0159] Thus, organisms already producing a desired antibiotic can
be evolved with the recursive sequence recombination techniques
described above to maximize production of that antibiotic.
Additionally, new antibiotics can be evolved by manipulation of
genetic material from the host by the recursive sequence
recombination techniques described above. Genes for antibiotic
production can be transferred to a preferred host after cycles of
recursive sequence recombination or can be evolved in the preferred
host as described above. Antibiotic genes are generally clustered
and are often positively regulated, making them especially
attractive candidates for the recursive sequence recombination
techniques of the instant invention. Additionally, some genes of
related pathways show cross-hybridization, making them preferred
candidates for the generation of new pathways for new antibiotics
by the recursive sequence recombination techniques of the
invention. Furthermore, increases in secondary metabolite
production including enhancement of substrate fluxes (by increasing
the rate of a rate limiting enzyme, deregulation of the pathway by
suppression of negative control elements or over expression of
activators and the relief of feedback controls by mutation of the
regulated enzyme to a feedback-insensitive deregulated protein) can
be achieved by recursive sequence recombination without exhaustive
analysis of the regulatory mechanisms governing expression of the
relevant gene clusters.
[0160] The host chosen for expression of evolved genes is
preferably resistant to the antibiotic produced, although in some
instances production methods can be designed so as to sacrifice
host cells when the amount of antibiotic produced is commercially
significant yet lethal to the host. Similarly, bioreactors can be
designed so that the growth medium is continually replenished,
thereby "drawing off" antibiotic produced and sparing the lives of
the producing cells. Preferably, the mechanism of resistance is not
the degradation of the antibiotic produced.
[0161] Numerous screening methods for increased antibiotic
expression are known in the art, as discussed above, including
screening for organisms that are more resistant to the antibiotic
that they produce. This may result from linkage between expression
of the antibiotic synthesis and antibiotic resistance genes
(Chater, Bio/Technology 8:115-121 (1990)). Another screening method
is to fuse a reporter gene (e.g. xylE from the Pseudomonas TOL
plasmid) to the antibiotic production genes. Antibiotic synthesis
gene expression can then be measured by looking for expression of
the reporter (e.g. xylE encodes a catechol dioxygenase which
produces yellow muconic semialdehyde when colonies are sprayed with
catechol (Zukowski et al. Proc. Natl. Acad. Sci. U.S.A.
80:1101-1105 (1983)).
[0162] The wide variety of cloned antibiotic genes provides a
wealth of starting materials for the recursive sequence
recombination techniques of the instant invention. For example,
genes have been cloned from Streptomyces cattleya which direct
cephamycin C synthesis in the non-antibiotic producer Streptomyces
lividans (Chen et al. Bio/Technology 6:1222-1224 (1988)). Clustered
genes for penicillin biosynthesis
(.delta.-(L-.alpha.-aminoadipyl)-L-cysteinyl-D-valine synthetase;
isopenicillin N synthetase and acyl coenzyme A:6-aminopenicillanic
acid acyltransferase) have been cloned from Penicillium
chrysogenum. Transfer of these genes into Neurospora crassa and
Aspergillus niger result in the synthesis of active penicillin V
(Smith et al. Bio/Technology 8:39-41 (1990)). For a review of
cloned genes involved in Cephalosporin C, Penicillins G and V and
Cephamycin C biosynthesis, see Piepersberg, Crit. Rev. Biotechnol.
14:251-285 (1994). For a review of cloned clusters of
antibiotic-producing genes, see Chater Bio/Technoloqv 8:115-121
(1990). Other examples of antibiotic synthesis genes transferred to
industrial producing strains, or over expression of genes, include
tylosin, cephamycin C, cephalosporin C, LL-E33288 complex (an
antitumor and antibacterial agent), doxorubicin, spiramycin and
other macrolide antibiotics, reviewed in Cameron et al. Appl.
Biochem. Biotechnol. 38:105-140 (1993).
[0163] B. Biosynthesis to Replace Chemical Synthesis of
Antibiotics
[0164] Some antibiotics are currently made by chemical
modifications of biologically produced starting compounds. Complete
biosynthesis of the desired molecules may currently be impractical
because of the lack of an enzyme with the required enzymatic
activity and substrate specificity. For example,
7-aminodeacetooxycephalosporanic acid (7-ADCA) is a precursor for
semi-synthetically produced cephalosporins. 7-ADCA is made by a
chemical ring expansion from penicillin V followed by enzymatic
deacylation of the phenoxyacetal group. Cephalosporin V could in
principle be produced biologically from penicillin V using
penicillin N expandase, but penicillin V is not used as a substrate
by any known expandase. The recursive sequence recombination
techniques of the invention can be used to alter the enzyme so that
it will use penicillin V as a substrate. Similarly, penicillin
transacylase could be so modified to accept cephalosporins or
cephamycins as substrates.
[0165] In yet another example, penicillin amidase expressed in E.
coli is a key enzyme in the production of penicillin G derivatives.
The enzyme is generated from a precursor peptide and tends to
accumulate as insoluble aggregates in the periplasm unless
non-metabolizable sugars are present in the medium (Scherrer et al.
Appl. Microbiol. Biotechnol. 42:85-91 (1994)). Evolution of this
enzyme through the methods of the instant invention could be used
to generate an enzyme that folds better, leading to a higher level
of active enzyme expression.
[0166] In yet another example, Penicillin G acylase covalently
linked to agarose is used in the synthesis of penicillin G
derivatives. The enzyme can be stabilized for increased activity,
longevity and/or thermal stability by chemical modification
(Fernandez-Lafuente et. al. Enzyme Microb. Technol. 14:489-495
(1992). Increased thermal stability is an especially attractive
application of the recursive sequence recombination techniques of
the instant invention, which can obviate the need for the chemical
modification of such enzymes. Selection for thermostability can be
performed in vivo in E. coli or in thermophiles at higher
temperatures. In general, thermostability is a good first step in
enhancing general stabilization of enzymes. Random mutagenesis and
selection can also be used to adapt enzymes to function in
non-aqueous solvents (Arnold Curr Oin Biotechnol, 4:450-455 (1993);
Chen et. al. Proc. Natl. Acad. Sci. U.S.A., 90:5618-5622 (1993)).
Recursive sequence recombination represents a more powerful (since
recombinogenic) method of generating mutant enzymes that are stable
and active in non-aqueous environments. Additional screening can be
done on the basis of enzyme stability in solvents.
[0167] C. Polyketides
[0168] Polyketides include antibiotics such as tetracycline and
erythromycin, anti-cancer agents such as daunomycin,
immunosuppressants such as FK506 and rapamycin and veterinary
products such as monesin and avermectin. Polyketide syntheses
(PKS's) are multifunctional enzymes that control the chain length,
choice of chain-building units and reductive cycle that generates
the huge variation in naturally occurring polyketides. Polyketides
are built up by sequential transfers of "extender units" (fatty
acyl CoA groups) onto the appropriate starter unit (examples are
acetate, coumarate, propionate and malonamide). The PKS's determine
the number of condensation reactions and the type of extender
groups added and may also fold and cyclize the polyketide
precursor. PKS's reduce specific .beta.-keto groups and may
dehydrate the resultant .beta.-hydroxyls to form double bonds.
Modifications of the nature or number of building blocks used,
positions at which .beta.-keto groups are reduced, the extent of
reduction and different positions of possible cyclizations, result
in formation of different final products. Polyketide research is
currently focused on modification and inhibitor studies, site
directed mutagenesis and 3-D structure elucidation to lay the
groundwork for rational changes in enzymes that will lead to new
polyketide products.
[0169] Recently, McDaniel et al. (Science 262:1546-1550 (1995))
have developed a Streptomyces host-vector system for efficient
construction and expression of recombinant PKSs. Hutchinson
(Bio/Technology 12:375-308 (1994)) reviewed targeted mutation of
specific biosynthetic genes and suggested that microbial isolates
can be screened by DNA hybridization for genes associated with
known pharmacologically active agents so as to provide new
metabolites and large amounts of old ones. In particular, that
review focuses on polyketide synthase and pathways to
aminoglycoside and oligopeptide antibiotics.
[0170] The recursive sequence recombination techniques of the
instant invention can be used to generate modified enzymes that
produce novel polyketides without such detailed analytical effort.
The availability of the PKS genes on plasmids and the existence of
E. coli-Streptomyces shuttle vectors (Wehmeier Gene 165:149-150
(1995)) makes the process of recursive sequence recombination
especially attractive by the techniques described above. Techniques
for selection of antibiotic producing organisms can be used as
described above; additionally, in some embodiments screening for a
particular desired polyketide activity or compound is
preferable.
[0171] D. Isoprenoids
[0172] Isoprenoids result from cyclization of farnesyl
pyrophosphate by sesquiterpene synthases. The diversity of
isoprenoids is generated not by the backbone, but by control of
cyclization. Cloned examples of isoprenoid synthesis genes include
trichodiene synthase from Fusarium sprorotrichioides, pentalene
synthase from Streptomyces, aristolochene synthase from Penicillium
roquefortii, and epi-aristolochene syrithase from N. tabacum (Cane,
D. E. (1995). Isoprenoid antibiotics, pages 633-655, in "Genetics
and Biochemistry of Antibiotic Production" edited by Vining, L. C.
& Stuttard, C., published by Butterworth-Heinemann). Recursive
sequence recombination of sesquiterpene synthases will be of use
both in allowing expression of these enzymes in heterologous hosts
(such as plants and industrial microbial strains) and in alteration
of enzymes to change the cyclized product made. A large number of
isoprenoids are active as antiviral, antibacterial, antifungal,
herbicidal, insecticidal or cytostatic agents. Antibacterial and
antifungal isoprenoids could thus be preferably screened for using
the indicator cell type system described above, with the producing
cell competing with bacteria or fungi for nutrients. Antiviral
isoprenoids could be screened for preferably by their ability to
confer resistance to viral attack on the producing cell.
[0173] E. Bioactive Peptide Derivatives
[0174] Examples of bioactive non-ribosomally synthesized peptides
include the antibiotics cyclosporin, pepstatin, actinomycin,
gramicidin, depsipeptides, vancomycin, etc. These peptide
derivatives are synthesized by complex enzymes rather than
ribosomes. Again, increasing the yield of such non-ribosomally
synthesized peptide antibiotics has thus far been done by genetic
identification of biosynthetic "bottlenecks" and over expression of
specific enzymes (See, for example, p. 133-135 in "Genetics and
Biochemistry of Antibiotic Production" edited by Vining, L. C.
& Stuttard, C., published by Butterworth-Heinemann). Recursive
sequence recombination of the enzyme clusters can be used to
improve the yields of existing bioactive non-ribosomally made
peptides in both natural and heterologous hosts. Like polyketide
synthases, peptide synthases are modular and multifunctional
enzymes catalyzing condensation reactions between activated
building blocks (in this case amino acids) followed by
modifications of those building blocks (see Kleinkauf, H. and von
Dohren, H. Eur. J. Biochem. 236:335-351 (1996)). Thus, as for
polyketide synthases, recursive sequence recombination can also be
used to alter peptide synthases: modifying the specificity of the
amino acid recognized by each binding site on the enzyme and
altering the activity or substrate specificities of sites that
modify these amino acids to produce novel compounds with antibiotic
activity.
[0175] Other peptide antibiotics are made ribosomally and then
post-translationally modified. Examples of this type of antibiotics
are lantibiotics (produced by gram positive bacteria such
Staphylococcus, Streptomyces, Bacillus, and Actinoplanes) and
microcins (produced by Enterobacteriaceae). Modifications of the
original peptide include (in lantibiotics) dehydration of serine
and threonine, condensation of dehydroamino acids with cysteine, or
simple N- and C-terminal blocking (microcins). For ribosomally made
antibiotics both the peptide-encoding sequence and the modifying
enzymes may have their expression levels modified by recursive
sequence recombination. Again, this will lead to both increased
levels of antibiotic synthesis, and by modulation of the levels of
the modifying enzymes (and the sequence of the ribosomally
synthesized peptide itself) novel antibiotics.
[0176] Screening can be done as for other antibiotics as described
above, including competition with a sensitive (or even initially
insensitive) microbial species. Use of competing bacteria that have
resistances to the antibiotic being produced will select strongly
either for greatly elevated levels of that antibiotic (so that it
swamps out the resistance mechanism) or for novel derivatives of
that antibiotic that are not neutralized by the resistance
mechanism.
[0177] F. Polymers
[0178] Several examples of metabolic engineering to produce
biopolymers have been reported, including the production of the
biodegradable plastic polyhydroxybutarate (PHB), and the
polysaccharide xanthan gum. For a review, see Cameron et al.
Applied Biochem. Biotech. 38:105-140 (1993). Genes for these
pathways have been cloned, making them excellent candidates for the
recursive sequence recombination techniques described above.
Expression of such evolved genes in a commercially viable host such
as E. coli is an especially attractive application of this
technology.
[0179] Examples of starting materials for recursive sequence
recombination include but are not limited to genes from bacteria
such as Alcallgenes, Zoogloea, Rhizobium, Bacillus, and Azobacter,
which produce polyhydroxyalkanoates (PHAS) such as
polyhyroxybutyrate (PHB) intracellularly as energy reserve
materials in response to stress. Genes from Alcaligenes eutrophus
that encode enzymes catalyzing the conversion of acetoacetyl CoA to
PHB have been transferred both to E. coli and to the plant
Arabidopsis thaliana (Poirier et al. Science 256:520-523 (1992)).
Two of these genes (phbB and phbc, encoding acetoacetyl-CoA
reductase and PHB synthase respectively) allow production of PHB in
Arabidopsis. The plants producing the plastic are stunted, probably
because of adverse interactions between the new metabolic pathway
and the plants' original metabolism (i.e., depletion of substrate
from the mevalonate pathway). Improved production of PHB in plants
has been attempted by localization of the pathway enzymes to
organelles such as plastids. Other strategies such as regulation of
tissue specificity, expression timing and cellular localization
have been suggested to solve the deleterious effects of PHB
expression in plants. The recursive sequence recombination
techniques of the invention can be used to modify such heterologous
genes as well as specific cloned interacting pathways (e.g.,
mevalonate), and to optimize PHB synthesis in industrial microbial
strains, for example to remove the requirement for stresses (such
as nitrogen limitation) in growth conditions.
[0180] Additionally, other microbial polyesters are made by
different bacteria in which additional monomers are incorporated
into the polymer (Peoples et al. in Novel Biodegradable Microbial
Polymers, E A Dawes, ed., pp191-202 (1990)). Recursive sequence
recombination of these genes or pathways singly or in combination
into a heterologous host will allow the production of a variety of
polymers with differing properties, including variation of the
monomer subunit ratios in the polymer. Another polymer whose
synthesis may be manipulated by recursive sequence recombination is
cellulose. The genes for cellulose biosynthesis have been cloned
from Agrobacterium tumefaciens (Matthysse, A. G. et. al. J.
Bacterial. 177:1069-1075 (1995)). Recursive sequence recombination
of this biosynthetic pathway could be used either to increase
synthesis of cellulose, or to produce mutants in which alternative
sugars are incorporated into the polymer.
[0181] G. Carotenoids
[0182] Carotenoids are a family of over 600 terpenoids produced in
the general isoprenoid biosynthetic pathway by bacteria, fungi and
plants (for a review, see Armstrong, J. Bact. 176:4795-4802
(1994)). These pigments protect organisms against photooxidative
damage as well as functioning as anti-tumor agents, free
radical-scavenging anti-oxidants, and enhancers of the immune
response. Additionally, they are used commercially in pigmentation
of cultured fish and shellfish. Examples of carotenoids include but
are not limited to myxobacton, spheroidene, spheroidenone, lutein,
astaxanthin, violaxanthin, 4-ketorulene, myxoxanthrophyll,
echinenone, lycopene, zeaxanthin and its mono- and di-glucosides,
.alpha.-, .beta.-, .gamma.- and .delta.-carotene,
.beta.-cryptoxanthin monoglucoside and neoxanthin.
[0183] Carotenoid synthesis is catalyzed by relatively small
numbers of clustered genes: 11 different genes within 12 kb of DNA
from Myxococcus xanthus (Botella et al. Eur. J. Biochem.
233:238-248 (1995)) and 8 genes within 9 kb of DNA from Rhodobacter
sphaeroides (Lang et. al. J. Bact. 177:2064-2073 (1995)). In some
microorganisms, such as Thermus thermophilus, these genes are
plasmid-borne (Tabata et al. FEBS Letts 341:251-255 (1994)). These
features make carotenoid synthetic pathways especially attractive
candidates for recursive sequence recombination.
[0184] Transfer of some carotenoid genes into heterologous
organisms results in expression. For example, genes from Erwina
uredovora and Haematococcus pluvialis. will function together in E.
coli (Kajiwara et al. Plant Mol. Biol. 29:343-352 (1995)). E.
herbicola genes will function in R. sphaeroides (Hunter et al. J.
Bact. 176:3692-3697 (1994)). However, some other genes do not; for
example, R. capsulatus genes do not direct carotenoid synthesis in
E. coli (Marrs, J. Bact. 146:1003-1012 (1981)).
[0185] In an embodiment of the invention, the recursive sequence
recombination techniques of the invention can be used to generate
variants in the regulatory and/or structural elements of genes in
the carotenoid synthesis pathway, allowing increased expression in
heterologous hosts. Indeed, traditional techniques have been used
to increase carotenoid production by increasing expression of a
rate limiting enzyme in Thermus thermophilus (Hoshino et al. Appl.
Environ. Micro. 59:3150-3153 (1993)). Furthermore, mutation of
regulatory genes can cause constitutive expression of carotenoid
synthesis in actinomycetes, where carotenoid photoinducibility is
otherwise unstable and lost at a relatively high frequency in some
species (Kato et al. Mol. Gen. Genet. 247:387-390 (1995)). These
are both mutations that can be obtained by recursive sequence
recombination.
[0186] The recursive sequence recombination techniques of the
invention as described above can be used to evolve one or more
carotenoid synthesis genes in a desired host without the need for
analysis of regulatory mechanisms. Since carotenoids are colored, a
calorimetric assay in microtiter plates, or even on growth media
plates, can be used for screening for increased production.
[0187] In addition to increasing expression of carotenoids,
carotenogenic biosynthetic pathways have the potential to produce a
wide diversity of carotenoids, as the enzymes involved appear to be
specific for the type of reaction they will catalyze, but not for
the substrate that they modify. For example, two enzymes from the
marine bacterium Agrobacterium aurantiacum (CrtW and CrtZ)
synthesize six different ketocarotenoids from .beta.-carotene
(Misawa et al. J. Bact. 177:6576-6584 (1995)). This relaxed
substrate specificity means that a diversity of substrates can be
transformed into an even greater diversity of products.
Introduction of foreign carotenoid genes into a cell can lead to
novel and functional carotenoid-protein complexes, for example in
photosynthetic complexes (Hunter et al. J. Bact. 176:3692-3697
(1994)). Thus, the deliberate recombination of enzymes through the
recursive sequence recombination techniques of the invention is
likely to generate novel compounds. Screening for such compounds
can be accomplished, for example, by the cell competition/survival
techniques discussed above and by a calorimetric assay for
pigmented compounds.
[0188] Another method of identifying new compounds is to use
standard analytical techniques such as mass spectroscopy, nuclear
magnetic resonance, high performance liquid chromatography, etc.
Recombinant microorganisms can be pooled and extracts or media
supernatants assayed from these pools. Any positive pool can then
be subdivided and the procedure repeated until the single positive
is identified ("sib-selection").
[0189] H. Indigo Biosynthesis
[0190] Many dyes, i.e. agents for imparting color, are specialty
chemicals with significant markets. As an example, indigo is
currently produced chemically. However, nine genes have been
combined in E. coli to allow the synthesis of indigo from glucose
via the tryptophan/indole pathway (Murdock et al. Bio/Technology
11:381-386 (1993)). A number of manipulations were performed to
optimize indigo synthesis: cloning of nine genes, modification of
the fermentation medium and directed changes in two operons to
increase reaction rates and catalytic activities of several
enzymes. Nevertheless, bacterially produced indigo is not currently
an economic proposition. The recursive sequence recombination
techniques of the instant invention could be used to optimize
indigo synthesizing enzyme expression levels and catalytic
activities, leading to increased indigo production, thereby making
the process commercially viable and reducing the environmental
impact of indigo manufacture. Screening for increased indigo
production can be done by calorimetric assays of cultures in
microtiter plates.
[0191] I. Amino Acids
[0192] Amino acids of particular commercial importance include but
are not limited to phenylalanine, monosodium glutamate, glycine,
lysine, threonine, tryptophan and methionine. Backman et al. (Ann.
NY Acad. Sci. 589:16-24 (1990)) disclosed the enhanced production
of phenylalanine in E. coli via a systematic and downstream
strategy covering organism selection, optimization of biosynthetic
capacity, and development of fermentation and recovery
processes.
[0193] As described in Simpson et al. (Biochem Soc Trans,
23:381-387 (1995)), current work in the field of amino acid
production is focused on understanding the regulation of these
pathways in great molecular detail. The recursive sequence
recombination techniques of the instant invention would obviate the
need for this analysis to obtain bacterial strains with higher
secreted amino acid yields. Amino acid production could be
optimized for expression using recursive sequence recombination of
the amino acid synthesis and secretion genes as well as enzymes at
the regulatory phosphoenolpyruvate branchpoint, from such organisms
as Serratia marcescens, Bacillus, and the
Corynebacterium-Brevibacterium group. In some embodiments of the
invention, screening for enhanced production is preferably done in
microtiter wells, using chemical tests well known in the art that
are specific for the desired amino acid. Screening/selection for
amino acid synthesis can also be done by using auxotrophic reporter
cells that are themselves unable to synthesize the amino acid in
question. If these reporter cells also produce a compound that
stimulates the growth of the amino acid producer (this could be a
growth factor, or even a different amino acid), then library cells
that produce more amino acid will in turn receive more growth
stimulant and will therefore grow more rapidly.
[0194] J. Vitamin C Synthesis
[0195] L-Ascorbic acid (vitamin C) is a commercially important
vitamin with a world production of over 35,000 tons in 1984. Most
vitamin C is currently manufactured chemically by the Reichstein
process, although recently bacteria have been engineered that are
able to transform glucose to 2,5-keto-gluconic acid, and that
product to 2-keto-L-idonic acid, the precursor to L-ascorbic acid
(Boudrant, Enzyme Microb. Technol. 12:322-329 (1990)).
[0196] The efficiencies of these enzymatic steps in bacteria are
currently low. Using the recursive sequence recombination
techniques of the instant invention, the genes can be genetically
engineered to create one or more operons followed by expression
optimization of such a hybrid L-ascorbic acid synthetic pathway to
result in commercially viable microbial vitamin C biosynthesis. In
some embodiments, screening for enhanced L-ascorbic acid production
is preferably done in microtiter plates, using assays well known in
the art.
[0197] VI. Modification of Cell Properties.
[0198] Although not strictly examples of manipulation of
intermediary metabolism, recursive sequence recombination
techniques can be used to improve or alter other aspects of cell
properties, from growth rate to ability to secrete certain desired
compounds to ability to tolerate increased temperature or other
environmental stresses. Some examples of traits engineered by
traditional methods include expression of heterologous proteins in
bacteria, yeast, and other eukaryotic cells, antibiotic resistance,
and phage resistance. Any of these traits is advantageously evolved
by the recursive sequence recombination techniques of the instant
invention. Examples include replacement of one nutrient uptake
system (e.g. ammonia in Methylophilus methylotrophus) with another
that is more energy efficient; expression of haemoglobin to improve
growth under conditions of limiting oxygen; redirection of toxic
metabolic end products to less toxic compounds; expression of genes
conferring tolerance to salt, drought and toxic compounds and
resistance to pathogens, antibiotics and bacteriophage, reviewed in
Cameron et. al. Appl Biochem Biotechnol, 38:105-140 (1993).
[0199] The heterologous genes encoding these functions all have the
potential for further optimization in their new hosts by existing
recursive sequence recombination technology. Since these functions
increase cell growth rates under the desired growth conditions,
optimization of the genes by evolution simply involves recombining
the DNA recursively and selecting the recombinants that grow faster
with limiting oxygen, higher toxic compound concentration, or
whatever is the appropriate growth condition for the parameter
being improved.
[0200] Since these functions increase cell growth rates under the
desired growth conditions, optimization of the genes by "evolution"
can simply involve "shuffling" the DNA and selecting the
recombinants that grow faster with limiting oxygen, higher toxic
compound concentration or whatever restrictive condition is being
overcome.
[0201] Cultured mammalian cells also require essential amino acids
to be present in the growth medium. This requirement could also be
circumvented by expression of heterologous metabolic pathways that
synthesize these amino acids (Rees et al. Biotechnology 8:629-633
(1990). Recursive sequence recombination would provide a mechanism
for optimizing the expression of these genes in mammalian cells.
Once again, a preferred selection would be for cells that can grow
in the absence of added amino acids.
[0202] Yet another candidate for improvement through the techniques
of the invention is symbiotic nitrogen fixation. Genes involved in
nodulation (nod, ndv), nitrogen reduction (nif, fix), host range
determination (nod, hsp), bacteriocin production (tfx), surface
polysaccharide synthesis (exo) and energy utilization (dct, hup)
which have been identified (Paau, Biotech. Adv. 9:173-184
(1991)).
[0203] The main function of recursive sequence recombination in
this case is in improving the survival of strains that are already
known to be better nitrogen fixers. These strains tend to be less
good at competing with strains already present in the environment,
even though they are better at nitrogen fixation. Targets for
recursive sequence recombination such as nodulation and host range
determination genes can be modified and selected for by their
ability to grow on the new host. Similarly any bacteriocin or
energy utilization genes that will improve the competitiveness of
the strain will also result in greater growth rates. Selection can
simply be performed by subjecting the target genes to recursive
sequence recombination and forcing the inoculant to compete with
wild type nitrogen fixing bacteria. The better the nitrogen fixing
bacteria grow in the new host, the more copies of their recombined
genes will be present for the next round of recombination. This
growth rate differentiating selection is described above in
detail.
[0204] VI. Biodetectors/Biosensors
[0205] Bioluminescence or fluorescence genes can be used as
reporters by fusing them to specific regulatory genes (Cameron et.
al. Appl Biochem Biotechnol, 38:105-140 (1993)). A specific example
is one in which the luciferase genes luxCDABE of Vibrio fischeri
were fused to the regulatory region of the isopropylbenzene
catabolism operon from Pseudomonas putida RE204. Transformation of
this fusion construct into E. coli resulted in a strain which
produced light in response to a variety of hydrophobic compound
such as substituted benzenes, chlorinated solvents and naphthalene
(Selifonova et. al., Appl Environ Microbiol 62:778-783 (1996)).
This type of construct is useful for the detection of pollutant
levels, and has the added benefit of only measuring those
pollutants that are bioavailable (and therefore potentially toxic).
Other signal molecules such as jellyfish green fluorescent protein
could also be fused to genetic regulatory regions that respond to
chemicals in the environment. This should allow a variety of
molecules to be detected by their ability to induce expression of a
protein or proteins which result in light, fluorescence or some
other easily detected signal.
[0206] Recursive sequence recombination can be used in several ways
to modify this type of biodetection system. It can be used to
increase the amplitude of the response, for example by increasing
the fluorescence of the green fluorescent protein. Recursive
sequence recombination could also be used to increase induced
expression levels or catalytic activities of other
signal-generating systems, for example of the luciferase genes.
[0207] Recursive sequence recombination can also be used to alter
the specificity of biosensors. The regulatory region, and
transcriptional activators that interact with this region and with
the chemicals that induce transcription can also be shuffled. This
should generate regulatory systems in which transcription is
activated by analogues of the normal inducer, so that biodetectors
for different chemicals can be developed. In this case, selection
would be for constructs that are activated by the (new) specific
chemical to be detected. Screening could be done simply with
fluorescence (or light) activated cell sorting, since the desired
improvement is in light production.
[0208] In addition to detection of environmental pollutants,
biosensors can be developed that will respond to any chemical for
which there are receptors, or for which receptors can be evolved by
recursive sequence recombination, such as hormones, growth factors,
metals and drugs. These receptors may be intracellular and direct
activators of transcription, or they may be membrane bound
receptors that activate transcription of the signal indirectly, for
example by a phosphorylation cascade. They may also not act on
transcription at all, but may produce a signal by some
post-transcriptional modification of a component of the signal
generating pathway. These receptors may also be generated by fusing
domains responsible for binding different ligands with different
signaling domains. Again, recursive sequence recombination can be
used to increase the amplitude of the signal generated to optimize
expression and functioning of chimeric receptors, and to alter the
specificity of the chemicals detected by the receptor.
[0209] The following examples are offered by way of illustration,
not by way of limitation.
EXAMPLES
[0210] I. Alteration of Enzyme Activity and Specificity.
[0211] In this example, recursive sequence recombination techniques
of the instant invention were used to expand the range of
substrates efficiently hydrolyzed by E. coli .beta.-galactosidase.
The goal was to evolve wild type E. coli .beta.-galactosidase into
a fucosidase. The enzyme showed very weak activity with both
.rho.-nitrophenyl-.beta.-D-fucopyranoside and
o-nitrophenyl-.beta.-D-fucopyranoside (estimated respectively as
80- and 160-fold less efficient than for
.rho.-nitrophenyl-.beta.-D-galactopy- ranoside).
[0212] To increase the activity of E. coli .beta.-galactosidase
against these fucopyranoside derivatives, a lacZ gene (a 3.8 kb
Hind III-BamHI fragment from plasmid pCH110, Pharmacia) encoding E.
coli .beta.-galactosidase was subcloned into plasmid pl8SFI-BLA-SFI
(Stemmer, Nature, 370:389-391 (1994)). The resulting plasmid,
p18-lacZ, was used for recursive sequence recombination and mutant
screening.
[0213] Purified plasmid p18-lacZ (4-5 .mu.g) was used directly for
DNase I fragmentation. Fragments with sizes between 50 and 200 bp
were purified from a 2% agarose gel and used for reassembly PCR
(Stemmer, Nature 370:389-391 (1994)). Assembly reactions used Tth
polymerase (Perkin Elmer) in the manufacturer's supplied buffer.
The PCR program for assembly was as follows: 94.degree. C., 2 min.,
then 40 cycles of 94.degree. C. for 30 sec.; 55.degree. C. for 3
sec.; 72.degree. C. for 1 min. +5 sec. per cycle; then finally
72.degree. C. for S min.
[0214] This reaction was diluted 100-fold into a standard PCR
reaction using the 40 mer primers p50F
5'-AGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCC- -3' and pR34
5'-CTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCAT-3'. This resulted in
amplification of both the desired DNA band (about 4 kb in size) as
well as two smaller sized products (about 600bp and 100 bp bands).
The PCR products were digested with BamHI and Hind III and the
correct size product was cloned into BamHI-HindIII digested
p18-lacZ. The resulting plasmid containing a pool of recombined
lacZ mutants was plated out on LB plates supplemented with
kanamycin and 5-bromo-4-chloro-3-indolyl-.beta.-- D-fucopyranoside
(X-fuco). Plates were incubated at 37.degree. C. for 20 hours and
screened for colonies with slight blue tint, indicating hydrolysis
of the X-fuco. Plasmid DNA was prepared from positive colonies and
the procedure was repeated. Thus, six rounds of recursive sequence
recombination produced a ten-fold increase in X-fuco hydrolysis
activity.
[0215] II. Evolution of an Entire Metabolic Pathway
[0216] As an example of evolution of an entire metabolic pathway,
the recursive sequence recombination techniques of the invention
were used to modify a plasmid encoding resistance to mercury salts.
This plasmid, as disclosed by Wang et al. (J. Bact. 171:83-92
(1989)) contains at least 8 genes within 13.5 kb of Bacillus DNA
inserted in the cloning vector pUC9. The recursive sequence
recombination protocol used for this plasmid was as follows.
[0217] Plasmid DNA (at 130 .mu.g/ml) was digested with 0.09 U/ml
DNAse in 50 mM Tris-Cl, pH 7.4, 10 mM MnCl.sub.2, for 10 minutes at
25.degree. C. DNA fragments were not size-selected, but were
purified by phenol extraction and ethanol precipitation. The
assembly reaction was performed using Tth polymerase (Perkin Elmer)
using the manufacturer's supplied buffer, supplemented with the
following: 7.5% polyethylene glycol, 8000 MW; 35 mM
tetramethylammonium chloride; and 4 U/ml Pwo(Boehringer Mannheim),
Pfu(Stratagene), Vent (New England Biolabs), Deep Vent (New England
Biolabs), Tfl (Promega) or Tli (Promega) thermostable DNA
polymerases. DNA fragments were used at around 10 .mu.g/ml.
[0218] The PCR program for assembly was as follows: 94.degree. C.
for 20 sec., then 40 cycles of 940C for 15 sec., 40.degree. C. for
30 sec., 72.degree. C. for 30 sec. +2 sec./cycle, and finally
72.degree. C. for 10 min.
[0219] The recombinant plasmid was then amplified in three
fragments by using primers flanking the three relatively evenly
spaced AlwNI restriction sites contained in the plasmid. The
sequences of these primers were:
1 1) 5'-CAGGACTTATCGCCACTGGCAGC-3' 2) 5'-CTCGCTCTGCTAATCCTGTTACC-3'
3) 5'- GCATATTATGAGCGTTTAGGCTTAATTCC-3' 4)
5'-CGGTATCCTTTTTCCGTACGTTC-3' 5) 5'- GTTGAAGAGGTGAAGAAAGTTCTCC-3'
6) 5'-GTTCGTCGATTTCCACGCTT- GGC-3'.
[0220] Three fragments were amplified using primers 1+4 (6 kb
fragment), 2+5 (4 kb fragment) and 3+6 (6 kb fragment). These were
then digested with AlwNI, gel purified and ligated together. As
AlwNI is a non-palindromic cutter, the plasmid could only
reassemble in the correct (original) order. The resultant plasmids
were transformed into E. coli strain DHlOB (Gibco BRL) and selected
on nutrient agar containing ampicillin 50 .mu.g/ml and increasing
concentrations of mercuric chloride (100 .mu.M to 1000 .mu.M) or
phenylmercuric acetate (50 .mu.M to 400 .mu.M) Thus, in 2 rounds of
recursive sequence recombination the tolerance of E. coli to these
compounds increased by a factor of 10 (from about 100 to about
1,000 .mu.M).
[0221] III. Recursive Sequence Recombination of a Family of Related
Enzymes
[0222] In this example nucleotide sequences were recombined between
four homologous .beta.-lactamases from C. freundii, E. cloacae, K.
pneumonia, and Y. enterocolitica. The four genes were synthesized
from oligonucleotides as described in Stemmer, et al. Gene
164:49-53 (1995). Briefly, the entire coding sequences of the genes
were synthesized as overlapping 50-mer oligonucleotides on a
commercial oligonucleotide synthesizer. The oligonucleotides were
then assembled into full length genes by a standard recursive
sequence recombination reaction, followed by amplification using
primers common to all four genes. oligonucleotides were designed to
give optimal E. coli codon usage in the synthetic genes with the
goal of increasing the homology to increase the frequency of
recombination, and the same 5' and 3' terminal sequences. After
assembly of the genes and selection for active clones, which is
optional, they were DNase treated to produce fragments from 50 to
200 bp in length. The fragments were dissolved at 100 .mu.g/ml in
15 .mu.l of Klenow (DNA polymerase I large fragment) buffer (New
England Biolabs) and subjected to manual PCR as follows: 15 cycles
of 95.degree. C. for 1 min.; freeze on dry ice and ethanol; warm to
25.degree. C. and add 2 Al of Klenow (1 U/.mu.l) in Klenow buffer;
incubate for 2 min at 25.degree. C.
[0223] A 5 .mu.l aliquot of the manual PCR reaction was then
diluted 6-fold into a standard Taq reaction mix (without
oligonucleotide primers) and assembled using a standard PCR program
consisting of 30 cycles of 940C for 30 sec., 40.degree. C. for 30
sec., and 72.degree. C. for 30 sec.
[0224] A 4, 8 or 16 .mu.l aliquot of this second PCR reaction was
then diluted into a standard Taq reaction mix containing
oligonucleotide primers that prime on sequences contained in all
four .beta.-lactamase genes 5'-AGGGCCTCGTGATACGCCTATT-3' and
5'-ACGAAAACTCACGTTAAGGGATT-3'. Full-length product was amplified
using a standard PCR program consisting of 25 cycles of 94.degree.
C. for 30 sec., 45.degree. C. for 30 sec., 72.degree. C. for 45
sec.
[0225] This procedure produced hybrid .beta.-lactamase genes whose
activities can be tested against antibiotics including but not
limited to ampicillin, carbenicillin, cefotaxime, cefoxitine,
cloxacillin, ceftazidime, cephaloridine and moxalactam, to
determine the specificities of the hybrid enzymes so created.
Moxalactam was chosen as the test antibiotic for hybrid genes. The
best of the original .beta.-lactamase genes used in this study
conferred resistance to 0.125 .mu.g/ml of moxalactam. After the
first round of recursive sequence recombination hybrid genes were
isolated that conferred resistance to 0.5 .mu.g/ml moxalactam,
yielding a 4-fold increase.
[0226] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
[0227] All references cited herein are expressly incorporated in
their entirety for all purposes.
* * * * *