U.S. patent application number 09/909574 was filed with the patent office on 2002-11-07 for production of polyhydroxyalkanoates from polyols.
Invention is credited to Sholl, Martha, Skraly, Frank A..
Application Number | 20020164729 09/909574 |
Document ID | / |
Family ID | 22821601 |
Filed Date | 2002-11-07 |
United States Patent
Application |
20020164729 |
Kind Code |
A1 |
Skraly, Frank A. ; et
al. |
November 7, 2002 |
Production of polyhydroxyalkanoates from polyols
Abstract
Recombinant processes are provided whereby additional genes are
introduced into E. coli which have been genetically engineered to
produce PHA so that the improved strains produce PHA homopolymers
and copolymers directly from diols. In preferred embodiments, PHAs
containing 4-hydroxybutyrate monomers are produced directly from
1,4-butanediol; PHAs containing 5-hydroxyvalerate are produced from
1,5-pentanediol; PHAs containing 6-hydroxyhexanoate (6HH) are
produced from 1,6-hexanediol; PHAs containing 3-hydroxypropionate
are produced from 1,3-propanediol; PHAs containing
2-hydroxypropionate (lactate) are produced from 1,2-propanediol
(propylene glycol); PHAs containing 2-hydroxyethanoate (glycolate)
are produced from 1,2-ethanediol (ethylene glycol). Genes encoding
these same enzyme activities can be introduced or their expression
amplified in wild type PHA producers to improve the production of
PHA homopolymers and copolymers directly from diol and other
alcohol feedstocks. The PHA polymers are readily recovered and
industrially useful as polymers or as starting materials for a
range of chemical intermediates.
Inventors: |
Skraly, Frank A.;
(Cambridge, MA) ; Sholl, Martha; (Haverhill,
MA) |
Correspondence
Address: |
PATREA L. PABST
HOLLAND & KNIGHT LLP
SUITE 2000, ONE ATLANTIC CENTER
1201 WEST PEACHTREE STREET, N.E.
ATLANTA
GA
30309-3400
US
|
Family ID: |
22821601 |
Appl. No.: |
09/909574 |
Filed: |
July 20, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60219995 |
Jul 21, 2000 |
|
|
|
Current U.S.
Class: |
435/135 ;
435/252.3; 435/252.33; 536/23.2 |
Current CPC
Class: |
C12N 15/52 20130101;
C12P 7/625 20130101 |
Class at
Publication: |
435/135 ;
435/252.3; 435/252.33; 536/23.2 |
International
Class: |
C12P 007/62; C07H
021/04; C12N 001/21 |
Claims
We claim:
1. A method for producing polyhydroxyalkanoates comprising
providing genetically engineered organisms which express enzymes
selected from the group consisting of diol oxidoreductase, aldehyde
dehydrogenase, acyl-CoA transferase, acyl-CoA synthetase,
.beta.-ketothiolase, acetoacetyl-CoA reductase, and PHA synthase,
providing diols which can be converted into hydroxyalkanoate
monomers by enzymes expressed by the organisms, and culturing the
organisms under conditions wherein the hydroxyalkanoate monomers
are polymerized to form polyhydroxyalkanoates.
2. The method of claim 1 wherein the diol is 1,6-hexanediol and the
hydroxyalkanoate monomer is 6-hydroxyhexanoate.
3. The method of claim 1 wherein the diol is 1,5-pentanediol and
the hydroxyalkanoate monomer is 5-hydroxyvalerate.
4. The method of claim 1 wherein the diol is 1,4-butanediol and the
hydroxyalkanoate is 4-hydroxybutyrate.
5. The method of claim 1 wherein the diol is 1,3-propanediol and
the hydroxyalkanoate monomer is 3-hydroxypropionate.
6. The method of claim 1 wherein the diol is 1,2-ethanediol and the
hydroxyalkanoate is 2-hydroxyethanoate.
7. The method of claim 1 wherein the diol is 1,2-propanediol and
the hydroxyalkanoate is 2-hydroxypropionate.
8. A genetically engineered organism for use in the method of claim
1 comprising an organism which expresses the aldH and dhaT
genes.
9. The organism of claim 8 wherein the organism is selected from
the group consisting of Escherichia coli, Ralstonia eutropha,
Klebsiella spp., Alcaligenes latus, Azotobacter spp., and Comamonas
spp.
10. A system for making polyhydroxyalkanoates comprising an
organism genetically engineered to express enzymes selected from
the group consisting of a diol oxidoreductase, aldehyde
dehydrogenase, acyl-CoA transferase, acyl-CoA synthetase,
.beta.-ketothiolase, acetoacetyl-CoA reductase, and PHA synthase,
wherein the organism can convert diols into hydroxyalkanoate
monomers which are polymerized to form polyhydroxyalkanoates.
11. A composition comprising a polyhydroxyalkanoate copolymer which
includes 2-hydroxypropionate or 2-hydroxyethanoate or both, and at
least one comonomer selected from the group consisting of
3-hydroxybutyrate, 4-hydroxybutyrate, 3-hydroxypropionate,
2-hydroxybutyrate, 4-hydroxyvalerate, 5-hydroxyvalerate,
6-hydroxyhexanoate, and 3-hydroxyhexanoate, having a weight-average
molecular weight (Mw) of at least 300,000.
12. The composition of example 11 where the comonomer is
3-hydroxybutyrate.
13. The composition of example 11 where the comonomer is
4-hydroxybutyrate.
14. The composition of example 11 where the comonomer is
3-hydroxypropionate.
15. The composition of example 11 where the comonomer is
2-hydroxybutyrate.
16. The composition of example 11 where the comonomer is
4-hydroxyvalerate.
17. The composition of example 11 where the comonomer is
5-hydroxyvalerate.
18. The composition of example 11 where the comonomer is
6-hydroxyhexanoate.
19. The composition of example 11 where the comonomer is
3-hydroxyhexanoate.
20. A method for improving a biological system for making
polyhydroxyalkanoates with an organism genetically engineered to
express enzymes selected from the group consisting of a diol
oxidoreductase, aldehyde dehydrogenase, acyl-CoA transferase,
acyl-CoA synthetase, .beta.-ketothiolase, acetoacetyl-CoA
reductase, and PHA synthase, wherein the organism can convert diols
into hydroxyalkanoate monomers which are polymerized to form
polyhydroxyalkanoates, the method comprising selecting for mutants
with increased enzyme activities by i) introducing mutations into a
specific host, and ii) screening pools of the mutants generated for
increased ability to synthesize PHA from a selected diol or
diols.
21. A DNA fragment encoding a diol oxidoreductase and an aldehyde
dehydrogenase, wherein the expressed enzymes can produce
hydroxyalkanoate monomer selected from the group consisting of
3-hydroxybutyrate, 4-hydroxybutyrate, 3-hydroxypropionate,
2-hydroxybutyrate, 4-hydroxyvalerate, 5-hydroxyvalerate,
6-hydroxyhexanoate, 3-hydroxyhexanoate, 2-hydroxypropionate, and
2-hydroxyethanoate from diol.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] Priority is claimed to U.S. Provisional Application Serial
No. 60/219,995 filed on Jul. 21, 2000.
BACKGROUND OF THE INVENTION
[0002] This invention is generally in the field of production of
polyhydroxyalkanoates (PHAs) by genetic engineering of
bacteria.
[0003] Synthesis of PHA polymers containing the monomer
4-hydroxybutyrate (4HB), such as
poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (PHB4HB) (Doi, 1995,
Macromol Symp. 98:585-99) or 4-hydroxyvalerate and
4-hydroxyhexanoate containing PHA polyesters have been described,
for example, in Valentin et al., 1992, Appl. Microbiol. Biotechnol.
36:507-14 and Valentin et al., 1994, Appl. Microbiol. Biotechnol.
40:710-16. Production of PHB4HB, for example, has been accomplished
by feeding glucose and 4HB or a substrate that is converted to
4-hydroxybutyrate to Ralstonia eutropha (Kunioka, et al., 1988,
Polym. Commun. 29:174; Doi, et al., 1990, Int. J. Biol. Macromol.
12:106; Nakamura, et al., 1992, Macromolecules 25:423), to
Alcaligenes latus (Hiramitsu, et al., 1993, Biotechnol. Lett.
15:461), to Pseudomonas acidovorans (Kimura, et al., 1992,
Biotechnol. Lett. 14:445), and to Comamonas acidovorans (Saito
& Doi, 1994, Int. J. Biol. Macromol. 16:18). Substrates that
are converted to 4HB include 1,4-butanediol, 1,6-hexanediol,
1,8-octanediol, 1,10-decanediol, 1,12-dodecanediol and
gamma-butyrolactone. The PHB4HB copolymers can be produced with a
range of monomer compositions which provide a range of polymer
properties. In particular, as the amount of 4HB increases above 15
wt. %, the melting temperature (T.sub.m) decreases below
130.degree. C. and the elongation to break increases above 400%
(Saito, et al., 1996, Polym. Int. 39:169).
[0004] It would be highly advantageous, however, to develop more
cost effective ways of producing PHAs containing 4HB by biological
systems. For economic production of PHA, several factors are
critical, including substrate costs, fermentation time, and
efficiency of downstream processing. A general characteristic of
the wild type PHA-producing bacteria is that their growth rate is
low, they are often difficult to break open and their amenity to
genetic engineering is limited. Therefore, it would be desirable to
develop transgenic organisms that provide improved economics of PHA
production.
[0005] The production of the copolymer PHB4HB in recombinant E.
coli has been described (e.g., PCT WO 00/011188 by Huisman et al.;
PCT WO 98/39453 by Hein et al.). A range of novel biologically
produced 4HB polymers produced in recombinant E. coli have been
described by Skraly and Peoples (e.g., PCT WO 99/61624). In these
studies only the Huisman reference demonstrated the incorporation
of small amounts of 4HB co-monomer from 1,4-butanediol. It would be
highly advantageous to develop genetically engineered systems
capable of the production of a range of 4HB copolymers and poly-4HB
homopolymer using 1,4-butanediol as the source of the 4HB
monomer.
[0006] It is therefore an object of the present invention to
provide improved recombinant systems and methods for the production
of PHAs, such as PHAs containing the 4HB monomer, using a variety
of simple sugars and alcohols as substrates.
SUMMARY OF THE INVENTION
[0007] Recombinant processes are provided whereby additional genes
are introduced into E. coli which have been genetically engineered
to produce PHA so that the improved strains produce PHA
homopolymers and copolymers directly from diols. In preferred
embodiments, PHAs containing 4-hydroxybutyrate (4HB) monomers are
produced directly from 1,4-butanediol; PHAs containing
5-hydroxyvalerate (5HV) are produced from 1,5-pentanediol; PHAs
containing 6-hydroxyhexanoate (6HH) are produced from
1,6-hexanediol; PHAs containing 3-hydroxypropionate (3HP) are
produced from 1,3-propanediol (also called propylene glycol); PHAs
containing 2-hydroxypropionate (2HP, lactate) are produced from
1,2-propanediol (propylene glycol); PHAs containing
2-hydroxyethanoate (2HE, glycolate) are produced from
1,2-ethanediol (ethylene glycol). Genes encoding these same enzyme
activities can be introduced or their expression amplified in wild
type PHA producers to improve the production of PHA homopolymers
and copolymers directly from diol and other alcohol feedstocks. The
PHA polymers are readily recovered and industrially useful as
polymers or as starting materials for a range of chemical
intermediates.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 illustrates the pathway from 1,4-butanediol to
4-hydroxybutyryl-CoA that is employed in one embodiment.
DETAILED DESCRIPTION OF THE INVENTION
[0009] Processes are provided whereby additional genes are
introduced into microorganisms which have been genetically
engineered to produce PHA so that the improved strains produce PHA
homopolymers and copolymers directly from simple alcohol and sugar
substrates. These processes are based on recombinant bacteria e.g.,
Escherichia coli as a production organism and PHA biosynthetic
genes from PHA-producing microbes such as Ralstonia eutropha or
Alcaligenes latus although many other sources of PHA genes are now
known (Madison & Huisman, 1999, Microbiol. & Molecular
Biology Reviews, 63:21-53). Recombinant E. coli has many advantages
over the wild type PHA producing organisms including ease of
genetic manipulation, complete availability of the genome sequence,
fast growth rate, flexibility of growth substrates and ready
lysis.
[0010] Organisms to be Engineered
[0011] In one embodiment, genes for the entire pathway illustrated
in FIG. 1 are introduced into the production organism. An organism
that does not naturally produce PHAs, such as Escherichia coli, may
be used. A number of recombinant E. coli PHB production systems
have been described (Madison & Huisman, 1999, Microbiology
& Molecular Biology Reviews, 63:21-53). The genes encoding a
diol oxidoreductase and aldehyde dehydrogenase are introduced into
this host. In the case of 1,4-butanediol, the diol oxidoreductase
converts the substrate to 4-hydroxybutyraldehyde, which is then
converted to 4-hydroxybutyrate by the aldehyde dehydrogenase. In
the case of 1,3-propanediol, the diol oxidoreductase converts the
substrate to 3-hydroxypropionaldehyde, which is then converted to
3-hydroxypropionate by the aldehyde dehydrogenase. Other diols may
be treated in an analogous way. In some instances incorporation
into PHA of a hydroxyacid that is two carbons shorter than the diol
feedstock may occur. This is due to endogenous catabolism
resembling that of the beta-oxidation pathway of fatty acid
catabolism. For example, 4HB units, or both 4HB and 6HH units, may
be produced in the polymer as a result of feeding 1,6-hexanediol.
Optionally an exogenous acyl-CoA transferase or acyl-CoA synthetase
may be included to facilitate activation of the hydroxyacid with
coenzyme A. The activated monomer may then be incorporated into PHA
by the action of an appropriate PHA synthase present in the
production host. The enzyme activities provide a system for the
synthesis in the production host of a polymer containing one or
more monomer types, depending upon the diol feedstocks.
[0012] It is often very useful to synthesize copolymers containing
monomers like those mentioned above and 3HB. In this case, the
production host will also contain the .beta.-ketothiolase and
acetoacetyl-CoA reductase genes, the products of which convert
acetyl-CoA to 3HB-CoA. Acetyl-CoA may be derived from the diol or
from another carbon source such as a sugar. Both 3HB-CoA and
hydroxyacyl-CoAs such as those mentioned above can be accepted by
various PHA synthases such as the one expressed in the recombinant
host, and therefore copolymers of PHB are synthesized by the
recombinant host. Whatever the desired PHA composition, the diol
can be fed to the cells either during growth or after a separate
growth phase, either alone or in combination with at least one
other feedstock, such as a sugar, and PHA is accumulated within the
cells.
[0013] In another embodiment, a recombinant organism that naturally
contains at least part of the pathway shown in FIG. 1 can be used.
In this embodiment one or more of the activities discussed above
(diol oxidoreductase, aldehyde dehydrogenase, acyl-CoA transferase
or acyl-CoA synthetase, .beta.-ketothiolase, PHA synthase, and
acetoacetyl-CoA reductase) can be derived from the endogenous
machinery of the host. For example, only diol oxidoreductase and
aldehyde dehydrogenase might be expressed in R. eutropha, a natural
PHA-producing organism, to augment its ability to convert
1,4-butanediol to 4HB, and the natural ability of the host may be
relied upon to accomplish the rest of the necessary metabolic
steps. Many natural PHA-producing organisms are well-known to those
skilled in the art (Braunegg et al. 1998, J. Biotechnology
65:127-61). If the host is not capable of PHA production, a PHA
synthase or an entire PHB biosynthetic pathway and optionally an
exogenous acyl-CoA transferase or acyl-CoA synthetase may be
introduced into this organism to enable PHA production. Techniques
for doing this are well known in the art (for example, Dennis et
al., 1998, J. Biotechnology 64:177-86). Here also, the diol can be
fed to the cells either during growth or after a separate growth
phase, either alone or in combination with at least one other
feedstock, such as a sugar, and PHA is accumulated within the
cells.
[0014] The implementation of the production of PHAs with diol
feedstocks is not limited to bacteria as described in the examples.
The same genes may be introduced into eukaryotic cells, including
but not restricted to, yeast cells and cultured plant cells.
[0015] Genes for Utilization of Substrates
[0016] Genes and techniques for developing recombinant PHA
producers suitable for use as described herein are generally known
to those skilled in the art (Madison & Huisman, 1999,
Microbiology and Molecular Biology Reviews, 63:21-53; PCT WO
99/14313). Because all of the genes necessary to implement the
production of PHAs from feedstocks such as diols and sugars have
been cloned and are available in genetically manipulatable form,
any combination of plasmid-borne and integrated genes may be used,
and the implementation of this pathway is therefore not restricted
to the schemes outlined herein. Many different implementations will
be apparent to those skilled in the art.
[0017] 1,3-Propanediol oxidoreductase (EC 1.1.1.202) is found in
several species of bacteria. Often it is induced under anaerobic
conditions in the presence of glycerol (Forage & Foster, 1982,
J. Bacteriol. 149:413-419). This enzyme catalyzes the reversible
formation of 3-hydroxypropionaldehyde and other hydroxyaldehydes
from the corresponding diol. Physiologically the enzyme is thought
to be primarily used in diol formation, when the aldehyde is needed
as an electron acceptor at the expense of NADH (Johnson & Lin,
J. Bacteriol. 169:2050-54). Organisms that contain 1,3-propanediol
oxidoreductase typically are able to convert glycerol to
1,3-propanediol, though similar activities are found in other
organisms. Bacterial species noted for the ability to convert
glycerol to 1,3-propanediol include Klebsiella pneumoniae
(Streekstra et al., 1987, Arch. Microbiol. 147:268-75), Klebsiella
oxytoca (Homann et al., 1990, Appl. Microbiol. Biotechnol.
33:121-26), Klebsiella planticola (Id.) and Citrobacter freundii
(Boenigk et al., 1993, Appl. Microbiol. Biotechnol. 38:453-57)
although many other examples are generally known.
[0018] Aldehyde dehydrogenases are extremely common in biological
systems. Probing the E. coli genome database for homology shows
that this organism alone contains at least seven putative enzymes
of this type. They are so numerous and varied that even attempts to
classify them all are complicated (e.g. Vasiliou et al., 1999,
Pharmacogenetics 9:421-34). A discussion of all of the types and
physiological roles of these enzymes is beyond the scope of this
discussion. The choice of an appropriate aldehyde dehydrogenase for
use in metabolic engineering should be done after evaluation of the
substrate specificity of several candidates. Enzyme assays such as
that described in Baldom & Aguilar (1987, J. Biol. Chem.
262:13991-6) are useful for such diagnoses.
[0019] Acyl-CoA transferases (EC 2.8.3.x) and acyl-CoA synthetases
(EC 6.2.1.x) both catalyze the formation of thioesters of organic
acids with coenzyme A. Acyl-CoA transferases, such as OrfZ (also
called HbcT) (Sohling and Gottschalk, 1996, J. Bacteriol
178:871-80) transfer the CoA moiety from a donor such as acetyl-CoA
to a free organic acid, such as a fatty acid. Acyl-CoA synthetases
such as AlkK (van Beilen et al., 1992, Mol. Microbiol. 6:3121-36)
ligate free organic acid and free coenzyme A, deriving the energy
for the reaction from ATP and leaving AMP and pyrophosphate as
byproducts.
[0020] Improvements in the Enzymes in the Pathway
[0021] It may be advantageous to improve the specific activity or
substrate specificity of the enzymes in the diol-to-PHA pathway
described herein. For example, a specific diol may not be converted
to PHA at an acceptable rate in a specific organism. Improvements
of this nature will generally involve mutagenesis and screening;
the DNA sequence(s) to be improved are subjected to one or more
rounds of mutagenesis followed by an assessment of improvements
made.
[0022] Mutagenesis can be implemented using any of a variety of
ways known to those skilled in the art (e.g., error-prone PCR or
cassette mutagenesis, passage through bacterial mutator strains,
treatment with chemical mutagens), such as those described by
Cadwell et al., 1992, PCR Methods and Applications 2:28-33;
Erickson et al., 1993, Appl. Environ. Microbiol. 59:3858-62; Hermes
et al., 1990, Proc. Natl. Acad. Sci. USA 87:696-700; Ho et al.,
1989, Gene 77:51-59; Kellog et al., 1981, Science 214:1133-35;
Reidhaar-Olson et al., 1988, Science 241:53-57; Stemmer, 1994,
Nature 370:389-91; and Stemmer, 1994, Proc. Natl. Acad. Sci. USA
91:10747-51.
[0023] Screening for an improved diol-to-PHA pathway involves
culturing a population of mutants generated as described above in
such a way that cells improved in some property relating to the
pathway can be selected readily. One embodiment is the selection
for improved growth on the diol of interest. An organism deficient
in uptake or utilization of a particular diol will not grow well
with that diol as the sole carbon source. A pool of mutants can be
inoculated into liquid medium or onto agar plates containing the
diol as sole carbon source, along with all other nutrients
necessary for growth of the organism in question, and cells able to
grow may be readily isolated. Another embodiment is the selection
of cells able to produce polymer when cultured in the presence of
the diol. If an organism is unable to convert the diol at a
significant rate to a monomer precursor that can subsequently be
polymerized, plating that organism on agar containing the diol as
the sole carbon source (other than carbon contained in any complex
supplements added, such as yeast extract) will yield cells with
little or no PHA content. Culturing a pool of mutants on such a
plate can identify strains that have gained the ability to convert
the diol to polymerizable intermediates. These cells will appear
more opaque and white than the non-PHA-producing cells.
Alternatively, another carbon source such as glucose may be added
if the cells to be screened cannot synthesize polymer from that
carbon source. Plates can also serve to eliminate strains that
cannot grow in the conditions it presents; for example, a cell that
has gained via mutagenesis the ability to produce PHA from diol,
but has lost an industrially important characteristic such as the
ability to grow on minimal glucose medium, will not grow on plates
containing diol and glucose, especially if it cannot grow on the
diol as sole carbon source. Only the cells that can produce PHA
from diol and can grow on minimal glucose in this case will appear
as opaque colonies. PHA can be visualized within cells, especially
on plates, by methods more sensitive than visual screening of
untreated colonies, such as by staining with Nile red (as in, e.g.,
Spiekermann et al, 1999, Arch Microbiol. 171:73-80.). Methods such
as those above may be repeated for several rounds to further
optimize the diol-to-PHA pathway. Methods of screening are
illustrated by, but not restricted to, the aspects of the
discussion above, and other useful screening procedures will be
apparent to those skilled in the art.
[0024] Regulation of Expression
[0025] In any of the aforementioned embodiments, it is possible to
control the composition of the polymer produced by controlling the
expression of the diol oxidoreductase and aldehyde dehydrogenase or
by controlling the availability of the diol. The higher the
activities of diol oxidoreductase and aldehyde dehydrogenase, the
more activated monomer will be derived as a result of their
activities, up to the point where another factor such as substrate
availability or an enzyme activity downstream of these becomes
limiting. Methods for modulation of gene expression (and thus
enzyme activity) in various organisms are well-known to those
skilled in the art. The rate of diol feed to the cultured cells can
be controlled by various techniques well-known to those skilled in
fermentation and cell culture.
[0026] In the case of some microorganisms, some or all of the genes
can be integrated into the host chromosome and some or all provided
on a plasmid. In some cases, compatible plasmid systems can be
used, for example, with several steps of the pathway encoded on one
plasmid and the other steps encoded by a second plasmid. A
combination of the two approaches may also be used.
[0027] Substrates
[0028] As discussed above, substrates that can be used to make PHAs
in the context of the systems described herein include alcohols,
preferably diols. Examples of suitable diols include
1,6-hexanediol, 1,5-pentanediol, 1,4-butanediol, 1,3-propanediol,
1,2-ethanediol, and 1,2-propanediol.
[0029] These diols are nontoxic to many microorganisms, in many
cases even at high concentrations. They can be superior feedstocks
for fermentation as compared to organic acids, which often become
toxic at low concentrations to many microorganisms. Many diols are
readily available and relatively inexpensive. For example,
1,4-butanediol had a global demand of about 1 billion pounds in
1995 and is very widely used for synthetic polymer production
(Morgan, Chemistry & Industry, Mar. 3, 1997, pp. 166-8).
[0030] The present invention will be further understood by
reference to the following non-limiting examples.
EXAMPLE 1
Enzymatic Assay of Escherichia coli AldH
[0031] On the basis of its homology with other aldehyde
dehydrogenases, the aldH gene was cloned by PCR from the E. coli
genome. Plasmid pMS33 contains aldH under the control of the trc
promoter. E. coli DH5.alpha. was transformed with pMS33 or pFS14,
as a negative control. Plasmid pFS14 contains the Clostridium
kluyveri 4hbD (4HB dehydrogenase) gene, as described in Sohling and
Gottschalk (1996, J. Bacteriol. 178:871-80).
[0032] DH5.alpha./pMS33 and DH5.alpha./pFS14 were grown at
37.degree. C. with shaking in Luria-Bertani (LB; Difco; Detroit,
Mich.) broth to an optical density (600 mn) of 0.5 and subsequently
induced with 1 mM isopropyl-.beta.-D-thiogalactopyranoside (IPTG).
The incubation continued for 3 hours, after which the cells were
removed from the medium by centrifugation (2000 g, 10 min.), washed
in 0.1 M Tris (pH 8.0), centrifuged again, and resuspended in a
volume of 0.1 M Tris (pH 8.0) roughly equal to the size of the cell
pellet. Each sample was sonicated (XL sonicator, Heat
Systems-Ultrasonics, Inc., Farmingdale, N.Y.) with a microtip in
3-mL aliquots on ice for 2 min. each at a 70% cycle with a
one-second interval. The lysate was spun in a microcentrifuge at
14,000 g for 10 min. and the supernatant was collected and
designated crude cell extract.
[0033] The enzyme assays were conducted in a total volume of 1 mL
containing 100 mM sodium glycine (pH 9.5), 1 mM
3-hydroxypropionaldehyde (3HPA), 1 mM NAD.sup.+ or NADP.sup.+, 6 mM
dithiothreitol (DTT), and a volume of crude cell extract containing
20-100 .mu.g total protein. A baseline was established prior to
adding 3HPA, which started the reaction. The activity given by the
DH5.alpha./pFS14 extract was 0.00 U/mg when NAD.sup.+ was used and
0.03 U/mg when NADP.sup.+ was used. The activity given by the
DH5.alpha./pMS33 extract was 1.89 U/mg when NAD.sup.+ was used and
0.32 U/mg when NADP.sup.+ was used. Thus cells expressing the E.
coli AldH protein gain the ability to convert 3HPA to
3-hydroxypropionic acid with either NAD.sup.+ or NADP.sup.+ as
cofactor.
[0034] Construction of pFS14
[0035] The 4hbD gene was cloned by PCR using the plasmid pCK3
(Sohling & Gottschalk, 1996, J. Bacteriol. 178:871-80) as a
template. The following oligonucleotide primers were used:
1 5'-CTCTGAATTCAAGGAGGAAAAAATATGAAGTTATTAAAATTGGC-3' (4hbD 5'
EcoRI) 5'-TTTCTCTGAGCTCGGGATATTTAATGATTGTAGG-3' (4hbD 3' SacI)
[0036] The resulting PCR product was digested with EcoRI and SacI
and ligated to plasmid pTrcN that had been digested with the same
enzymes. pTrcN is a derivative of pTrc99a (Pharmacia; Uppsala,
Sweden); the modification that distinguishes pTrcN is the removal
of the NcoI restriction site by digestion with NcoI, treatment with
T4 DNA polymerase, and self-ligation.
[0037] Construction of pMS33
[0038] On the basis of its homology with other aldehyde
dehydrogenases, the aldH gene was cloned by PCR from the E. coli
genome using the following oligonucleotide primers:
2 5'-GGTGGTACCTTAAGAGGAGGTTTTTATGAATTTTCATCACCTGGCTT-3' (aldH 5'
Acc65I) 5'-GGTGCGGCCGCTCAGGCCTCCAGGCTTATCCA-- 3' (aldH 3' NotI)
[0039] The resulting PCR product was digested with Acc65I and NotI
and ligated to pSE380 (Invitrogen; Carlsbad, Calif.) that had been
digested with the same enzymes to form pMS33.
EXAMPLE 2
Growth of E. coli with 1,4-Butanediol as Sole Carbon Source
[0040] E. coli strain LS5218 (obtained from the Yale E. coli
Genetic Stock Center, New Haven, Conn., as strain CGSC 6966) was
transformed with either of two plasmids, pFS76 or pFS77. pFS76
contains the 4HB dehydrogenase (gbd) gene from Ralstonia eutropha,
as described in Valentin et al. (1995, Eur. J. Biochem. 227:43-60).
Plasmid pFS77 contains the gbd gene as well as the E. coli aldehyde
dehydrogenase (aldH) gene and the Klebsiella pneumoniae
1,3-propanediol oxidoreductase (dhaT) gene, arranged in a single
operon. Both plasmids contain the trc promoter for transcription of
the genes.
[0041] LS5218/pFS76 and LS5218/pFS77 were streaked onto
minimal-medium plates containing 5 g/L of either 4HB
(4-hydroxybutyrate, as the sodium salt) or 1,4-butanediol. The
plate medium also contained, per liter: 15 g agar; 1 mmol
MgSO.sub.4; 10 mg thiamine; 25.5 mmol Na.sub.2HPO.sub.4; 33.3 mmol
K.sub.2HPO.sub.4; 27.2 mmol KH.sub.2PO.sub.4; 2.78 mg
FeSO.sub.4.7H.sub.2O; 1.98 mg MnCl.sub.2.4H.sub.2O; 2.81 mg
CoSO.sub.4.7H.sub.2O;0.17 mg CuCl.sub.2.2H.sub.2O; 1.67 mg
CaCl.sub.2.2H.sub.2O; 0.29 mg ZnSO.sub.4.7H.sub.2O; 100 .mu.g
ampicillin; and 0.1 mmol IPTG. The plates were incubated overnight
at 37.degree. C. Both strains grew on the 4HB plate, but only
LS5218/pFS77 grew on the 1,4-butanediol plate. Therefore, it was
shown that the pathway consisting of the gbd, aldH, and dhaT genes
is sufficient for growth of E. coli LS5218 with 1,4-butanediol as
the sole carbon source.
[0042] Construction of pFS76
[0043] The gbd gene was amplified by PCR from the genome of R.
eutropha H16 (obtained from the American Type Culture Collection,
Rockville, Md., as strain ATCC 17699) using the following
oligonucleotide primers:
3 5'-CCTGAATTCAGGAGGTTTTTATGGCGTTTA TCTACTATCTGACCCAC-3' (gbd 5'
EcoRI) 5'-CCTGAGCTCCTACCTGCAAGTGCTCGCCGCTC-3' (gbd 3' SacI)
[0044] The resulting PCR product was digested with EcoRI and SacI
and ligated to pSE380 (Invitrogen; Carlsbad, Calif.) that had been
digested with the same enzymes to form pFS76.
[0045] Construction of pFS77
[0046] The aldH-dhaT region was removed from pMS59 by digestion
with NheI and HindIII. Plasmid pFS76 was digested with SpeI and
HindIII. NheI and SpeI form compatible sticky ends. The aldH-dhaT
fragment from pMS59 and the large fragment of pFS76 were ligated
together to give pFS77, containing the gbd, aldH, and dhaT genes,
all under control of the trc promoter.
EXAMPLE 3
Production of Poly(4HB) From 1,4-Butanediol
[0047] Escherichia coli strain LS5218 (CGSC 6966) was transformed
with either of two plasmids, pFS30 or pMS60. pFS30 contains the
Ralstonia eutropha PHA synthase (phaC) gene and the Clostridium
kluyveri 4HB-CoA transferase (hbcT) gene, both under control of the
trc promoter. pMS60 contains the aldH and dhaT genes along with the
two genes in pFS30, all under control of the trc promoter. The
objective of the experiment was to determine whether the addition
of the aldH and dhaT genes would be beneficial to the conversion of
1,4-butanediol to 4HB in the PHA polymer.
[0048] Each strain was grown in LB broth supplemented with 100
.mu.g/mL ampicillin overnight at 37.degree. C. with shaking at 250
rpm. The cells were then removed from the medium by centrifugation
(2000 g, 10 min.) and resuspended in 100 mL of a medium containing,
per liter: 2.5 g LB powder; 50 mmol potassium phosphate, pH 7.0; 2
g glucose; 5 g 1,4-butanediol; 100 .mu.g ampicillin; and 0.1 mmol
IPTG. These incubations were at 30.degree. C. with shaking at 250
rpm for 25 hours. The cells from one-quarter of the volume of the
flask were centrifuged as above, washed with water, centrifuged
again, and lyophilized. About 20 mg of lyophilized cell mass from
each flask was subjected to simultaneous extraction and butanolysis
at 110.degree. C. for 3 hours in 2 mL of a mixture containing (by
volume) 90% 1-butanol and 10% concentrated hydrochloric acid, with
2 mg/mL benzoic acid added as an internal standard. The
water-soluble components of the resulting mixture were removed by
extraction with 3 mL water. The organic phase (1 .mu.L at a split
ratio of 1:50 at an overall flow rate of 2 mL/min) was analyzed on
an SPB-1 fused silica capillary GC column (30 m; 0.32 mm ID; 0.25
.mu.m film; Supelco; Bellefonte, Pa.) with the following
temperature profile: 80.degree. C., 2 min; 10.degree. C. per min.
to 250.degree. C.; 250.degree. C., 2 min. The standard used to test
for the presence of 4-hydroxybutyrate units in the polymer was
gamma-butyrolactone (Aldrich Chemical Co.; Milwaukee, Wis.).
[0049] Strain LS5218/pFS30 reached an optical density (600 nm) of
3.9 and had accumulated poly-4HB to 3.3% of the dry cell weight,
while strain LS5218/pMS60 reached an optical density (600 nm) of
6.5 and had accumulated poly-4HB to 12.3% of the dry cell weight.
Thus expression of the aldH and dhaT genes is sufficient to
increase the ability of E. coli LS5218 to synthesize poly-4HB from
1,4-butanediol.
[0050] Construction of pFS16
[0051] The plasmid pFS16 was constructed by ligating the
Clostridium kluyveri orfZ (also called hbcT) PCR product to pTrcN.
The orfZ gene was amplified by PCR from plasmid pCK3 (Sohling and
Gottschalk, 1996, J. Bacteriol 178:871-80) using the following
oligonucleotide primers:
4 5'-TCCCCTAGGATTCAGGAGGTTTTTATGGAGTGGGAA GAGATATATAAAG-3' (orfZ 5'
AvrII) 5'-CCTTAAGTCGACAAATTCTAAAATCTCTTTTTAAATTC-3' (orfZ 3'
SalI)
[0052] The resulting PCR product was digested with AvrII and SalI
and ligated to pTrcN that had been digested with XbaI (which is
compatible with AvrII) and SalI to form pFS16.
[0053] Construction of pFS30
[0054] The plasmid pFS30 was derived from pFS16 by adding the
Ralstonia eutropha PHA synthase (phaC) gene. The plasmid pAeT414
was digested with XmaI and StuI so that the R. eutropha promoter
and the structural phaC gene were present on one fragment. pFS16
was cut with BamHI, treated with T4 DNA polymerase to create blunt
ends, then digested with XmaI. The two DNA fragments thus obtained
were ligated together to form pFS30.
[0055] Construction of pMS59
[0056] The aldH gene was removed from pMS33 by digestion with SpeI
and BglII. Plasmid pTC42 (Skraly et al., 1998, Appl. Environ.
Microbiol. 64:98-105), which contains the Klebsiella pneumoniae
dhaT gene under the control of the trc promoter, was digested with
NheI and BglII. SpeI and NheI form compatible sticky ends. The
aldH-containing fragment of pMS33 and the large fragment of pTC42
were ligated together to form pMS59.
[0057] Construction of pMS60
[0058] The aldH-dhaT region was isolated from pMS59 by digestion
with SpeI, followed by treatment with the Klenow fragment of DNA
polymerase I and subsequent digestion with MfeI. This fragment had
one blunt end and one sticky end compatible with EcoRI-generated
sticky ends. pFS30 was digested with XmaI, followed by treatment
with the Klenow fragment of DNA polymerase I and subsequent
digestion with EcoRI. The large fragment of pFS30 and the
aldH-dhaT-containing fragment of pMS59 were ligated together to
form pMS60.
EXAMPLE 4
Synthesis of Poly(3HB-co-4HB) from Glucose and 1,4-Butanediol
[0059] E. coli strain MBX1493 is a poly(3HB-co-4HB) producing
strain with the C. kluyveri orjZ (also called hbcT) gene (Sohling
& Gottschalk, 1996, J. Bacteriol. 178:871-80) integrated into
its chromosome. It was derived from strain MBX1335, a PHB-producing
strain with the phaA, phaB, and phaC genes integrated into its
chromosome. MBX1335 was itself derived from MBX820 (see PCT WO
00/011188 by Metabolix) by bacteriophage P1 transduction of the
phaA, phaB, and phaC genes into strain LS5218.
[0060] Strain MBX1493 was transformed with four plasmids in
separate procedures: pTrcN, pTC42, pMS33, and pMS59. These plasmids
contain, under control of the trc promoter, the following genes,
respectively: none, dhaT only, aldH only, both aldH and dhaT. Each
of these strains was grown in 3 mL LB supplemented with 100
.mu.g/mL ampicillin at 37.degree. C. with shaking overnight. A
volume of 1 mL of each of these cultures was used as an inoculum
into 50 mL of a medium containing, per liter: 1 mmol MgSO.sub.4; 10
mg thiamine; 25.5 mmol Na.sub.2HPO.sub.4; 33.3 mmol
K.sub.2HPO.sub.4; 27.2 mmol KH.sub.2PO.sub.4; 2.78 mg
FeSO.sub.4.7H.sub.2O; 1.98 mg MnCl.sub.2.4H.sub.2O; 2.81 mg
CoSO.sub.4.7H.sub.2O; 0.17 mg CuCl.sub.2.2H.sub.2O; 1.67 mg
CaCl.sub.2.2H.sub.2O; 0.29 mg ZnSO.sub.4.7H.sub.2O; 10 g glucose; 5
g 4-hydroxybutyrate or 10 g 1,4-butanediol; 100 .mu.g ampicillin;
25 .mu.g chloramphenicol; and 0.01 mmol IPTG. These cultures were
incubated at 30.degree. C. with shaking at 250 rpm for 88 hours.
The cells were removed from this medium by centrifugation (2000 g,
10 min.), and they were lyophilized and analyzed for PHA content
and composition by GC. Table 1 shows the composition of the
polymers made by these strains and the final optical densities (600
nm) of the cultures.
[0061] As shown in Table 1, all strains produce a copolymer with a
significant percentage of 4HB when fed 4HB. However, when fed
1,4-butanediol, only the pMS59-containing cells, that is, the cells
expressing both aldH and dhaT, achieved a significant level of 4HB
incorporation into the polymer. Thus the aldH-dhaT pathway was
shown to enable the conversion of 1,4-butanediol to 4HB and not to
interfere significantly with cell health or the subsequent
incorporation of 4HB into a PHA.
5TABLE 1 Conversion of 1,4-butanediol to 4HB PHA 4HB Substrate
Plasmid OD (600 nm) % dcw.sup.a % of polymer.sup.b 4HB pTrcN 19.2
53.0 36.6 4HB pTC42 18.8 62.7 16.0 4HB pMS33 14.8 49.8 21.4 4HB
pMS59 22.8 43.5 32.2 1,4-BD pTrcN 16.0 41.0 1.1 1,4-BD pTC42 10.4
38.7 0.7 1,4-BD pMS33 10.8 40.6 2.9 1,4-BD pMS59 10.4 34.6 25.3
.sup.aPercent of total dry cell weight. .sup.bPercent of total
polymer weight.
EXAMPLE 5
Production of Poly(3HP) from 1,3-Propanediol
[0062] Escherichia coli strain LS5218 (CGSC 6966) was transformed
with either of two plasmids, pFS30 or pMS60. The objective of the
experiment was to determine whether the addition of the aldH and
dhaT genes would be beneficial to the conversion of 1,3-propanediol
to 3HP.
[0063] Each strain was grown in LB broth supplemented with 100
.mu.g/mL ampicillin overnight at 37.degree. C. with shaking at 250
rpm. The cells were then removed from the medium by centrifugation
(2000 g, 10 min.) and resuspended in 50 mL of a medium containing,
per liter: 2.5 g LB powder; 50 mmol potassium phosphate, pH 7.0; 5
g glucose; 0 or 10 g 1,3-propanediol; 100 .mu.g ampicillin; and 0.1
mmol IPTG. These incubations were at 30.degree. C. with shaking at
250 rpm for 25 hours. The cells were removed from the medium by
centrifugation as described above, washed with water, centrifuged
again, lyophilized, and analyzed for PHA content and composition by
GC. The standard used to test for the presence of
3-hydroxypropionate units in the polymer was beta-propiolactone
(Aldrich Chemical Co.; Milwaukee, Wis.).
[0064] In the flasks without added 1,3-propanediol, no PHP
formation was detected; strains LS5218/pFS30 and LS5218/pMS60
reached optical densities (600 nm) of 4.6 and 8.2, respectively. In
the flasks with added 1,3-propanediol, strain LS5218/pFS30 reached
an optical density (600 nm) of 5.2 and did not accumulate poly-3HP
to a detectable level, while strain LS5218/pMS60 reached an optical
density (600 nm) of 6.6 and had accumulated poly-3HP to 5.0% of the
dry cell weight. Thus expression of the aldH and dhaT genes is
sufficient to increase the ability of E. coli LS5218 to synthesize
poly-3HP from 1,3-propanediol.
EXAMPLE 6
Synthesis of Poly(3HB-co-3HP) from Glucose and 1,3-Propanediol
[0065] Strain MBX1493 was transformed with four plasmids in
separate procedures: pTrcN, pTC42, pMS33, and pMS59. Each of these
strains was grown in 100 mL LB supplemented with 100 .mu.g/mL
ampicillin at 37.degree. C. with shaking overnight. The cells were
decanted from each flask, and the residual liquid was retained. To
each flask was then added 80 mL of a medium containing, per liter:
6.25 g LB powder; 1 mmol MgSO.sub.4; 10 mg thiamine; 25.5 mmol
Na.sub.2HPO.sub.4; 33.3 mmol K.sub.2HPO.sub.4; 27.2 mmol
KH.sub.2PO.sub.4; 2.78 mg FeSO.sub.4.7H.sub.2O; 1.98 mg
MnCl.sub.2.4H.sub.2O; 2.81 mg CoSO.sub.4.7H.sub.2O; 0.17 mg
CuCl.sub.2.2H.sub.2O; 1.67 mg CaCl.sub.2.2H.sub.2O; 0.29 mg
ZnSO.sub.4.7H.sub.2O; 10 g glucose; 100 .mu.g ampicillin; 25 .mu.g
chloramphenicol; and 0.01 mmol IPTG. These cultures were incubated
at 37.degree. C. with shaking at 250 rpm for 7 hours. To each flask
was then added 20 mL of the same medium given above, except that in
this medium LB was increased to 12.5 g/L, glucose was increased to
100 g/L, IPTG was increased to 0.25 mM, and 1,3-propanediol was
added at 50 g/L. Thus the final concentrations added at this stage
were 2.5 g/L LB, 20 g/L glucose, 10 g/L 1,3-propanediol, and 0.05
mM IPTG. These flasks were incubated at 30.degree. C. for 24 hours
with shaking at 250 rpm. The cells were then removed from this
medium by centrifugation (2000 g, 10 min.), and they were
lyophilized and analyzed for PHA content and composition by GC.
Table 2 shows the composition of the polymers made by these strains
and the final optical densities (600 nm) of the cultures.
[0066] In the absence of 1,3-propanediol, each strain synthesized
only PHB. When fed 1,3-propanediol, only the pMS59-containing
cells, that is, the cells expressing both aldH and dhaT, achieved a
significant level of 3HP incorporation into the polymer. The cells
containing pMS33, or aldH alone, do accomplish 3HP incorporation,
but to only a small extent. Thus the aldH-dhaT pathway was shown to
enable the conversion of 1,3-propanediol to 3HP. The cells
containing the dhaT gene (pTC42 and pMS59) made less total polymer
when 1,3-propanediol was present, and this is most likely due to
the toxicity of 3-hydroxypropionaldehyde. Increasing the ratio of
aldH to dhaT expression and/or reducing 1,3-propanediol
concentration should subdue this phenomenon.
6TABLE 2 Incorporation of 3HP into PHA by MBX1493 Containing
Various Plasmids 1,3-Propanediol PHA 3HP g/L Plasmid % dcw.sup.a %
of polymer.sup.b 0 pTrcN 35.2 0.0 0 pTC42 46.0 0.0 0 pMS33 31.2 0.0
0 pMS59 37.7 0.0 10 pTrcN 36.6 0.0 10 pTC42 23.9 0.0 10 pMS33 39.6
0.3 10 pMS59 20.0 3.8 .sup.aPercent of total dry cell weight.
.sup.bPercent of total polymer weight.
EXAMPLE 7
Use of Acyl-CoA Synthetase for Poly(4HB) Synthesis from
1,4-Butanediol
[0067] Strain MBX1668, which has the aldH and dhaT genes integrated
into its chromosome as an operon along with the tetracycline
resistance marker from Tn10, was transformed with either of two
plasmids: pFS73 or pMS92. The plasmid pFS73 is the same as pFS30
described in previous examples except that the ampicillin
resistance marker has been replaced with the kanamycin resistance
marker from pACYC177 (GenBank Accession No. X06402). The plasmid
pMS92 is derived from pFS73, the orfZ gene having been replaced
with the alkK gene from Pseudomonas oleovorans (van Beilen et al.,
1992, Mol. Microbiol. 6:3121-36). Each of these strains was grown
in 3 mL LB supplemented with 50 .mu.g/mL kanamycin and 10 .mu.g/mL
tetracycline at 37.degree. C. with shaking overnight. One
milliliter of each culture was added as an inoculum to a 200-mL
square bottle. Each bottle held 50 mL of a medium containing, per
liter: 0.1 g casamino acids; 5 mmol MgSO.sub.4; 10 mg thiamine;
25.5 mmol Na.sub.2HPO.sub.4; 33.3 mmol K.sub.2HPO.sub.4; 27.2 mmol
KH.sub.2PO.sub.4; 2.78 mg FeSO.sub.4.7H.sub.2O; 1.98 mg
MnCl.sub.2.4H.sub.2O; 2.81 mg CoSO.sub.4.7H.sub.2O; 0.17 mg
CuCl.sub.2.2H.sub.2O; 1.67 mg CaCl.sub.2.2H.sub.2O; 0.29 mg
ZnSO.sub.4.7H.sub.2O; 10 g glucose; 10 g 1,4-butanediol; 50 .mu.g
kanamycin; and 10 .mu.g tetracycline. These cultures were incubated
at 30.degree. C. with shaking at 250 rpm for 48 hours. The cells
were then removed from this medium by centrifugation (2000 g, 10
min.), and they were lyophilized and analyzed for PHA content and
composition by GC. Strain MBX1668 harboring pFS73 contained 5.8%
poly(4HB) by dry weight, while MBX1668 harboring pMS92 contained
27.7% poly(4HB) by dry weight. Thus the alkK gene is an acceptable,
and in this case better, substitute for the orfZ gene in the
synthesis of PHAs from diols.
[0068] Modifications and variations of the methods and materials
described herein will be obvious to those skilled in the art and
are intended to come within the scope of the following claims.
Sequence CWU 1
1
8 1 44 DNA Artificial sequence 4hbD 5' EcoRI primer 1 ctctgaattc
aaggaggaaa aaatatgaag ttattaaaat tggc 44 2 34 DNA Artificial
sequence 4hbD 3' SacI primer 2 tttctctgag ctcgggatat ttaatgattg
tagg 34 3 47 DNA E. coli misc_feature aldH 5' Acc65I primer 3
ggtggtacct taagaggagg tttttatgaa ttttcatcac ctggctt 47 4 32 DNA E.
coli misc_feature aldH 3' NotI 4 ggtgcggccg ctcaggcctc caggcttatc
ca 32 5 47 DNA Ralstonia eutropha misc_feature gbd 5' EcoRI primer
5 cctgaattca ggaggttttt atggcgttta tctactatct gacccac 47 6 32 DNA
Ralstonia eutropha misc_feature gbd 3' SacI primer 6 cctgagctcc
tacctgcaag tgctcgccgc tc 32 7 49 DNA Clostridium kluyveri
misc_feature orfZ 5' AvrII primer 7 tcccctagga ttcaggaggt
ttttatggag tgggaagaga tatataaag 49 8 38 DNA Clostridium kluyveri
orfZ misc_feature orfZ 3' SalI primer 8 ccttaagtcg acaaattcta
aaatctcttt ttaaattc 38
* * * * *