Novel Enzymes Which Dehydrate Glycerol

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AU 40179/01 B2
(12) PATENT (11) Application No. AU 200140179 B2
(19) AUSTRALIAN PATENT OFFICE (10) Patent No. 780433
(54) Title
Novel enzymes which dehydrate glycerol
(51) 7  International Patent Classification(s)
C12P 007/04 C08G 063/87
C08G 063/78 G01N 033/554
C08G 063/82 G01N 033/569
(21) Application No: 200140179 (22) Application Date: 2000.09.22
(87) WIPO No: W001/21825
Priority Data
(31) Number (32) Date (33) Country
09/405692 1999.09.24 US
(43) Publication Date: 2001.04.24
(43) Publication Journal Date 2001.06.21
(44) Accepted Journal Date 2005.03.24
(71) Applicant(s)
Genencor International, Inc.; University of Georgia Research Foundation, Inc.
(72) Inventor(s)
Markus Seyfried; Juergen Wiegel; Gregory Whited
(74) Agent/Attorney
PHILLIPS ORMONDE and FITZPATRICK,367 Collins Street,MELBOURNE VIC
3000
(56) Related Art
HARTLEY BS PAYTON MA (1983) BIOCHEM.SOC.SYMP.VOL 48,133-46
VIELLE ET AL (1996) BIOTECHNOL. ANN. REV. (1996) 2 
WO 1996/036796

(12) ITRAINLAPPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
(19) World Intellectual Property Organization
International Bureau
(43) International Publication Date
29 March 2001 (29.03.2001)
(10) International Publication Numbher
WO1/21825 A3PCT
(SI) International patent classification": C12P 7/04.
GOIN 33/554, 33/569, C08G 63/78, 63/82, 63/87
(21) International Application Number. PCT/USOO/26042
(22) International Filing Date:
22 September 2000 (22.09.2000)
Filing Language: English
(26) Publication Language: English
(74) Agents: FRIIEBEL, Thomas, E. et al.; Pennie Edmonds
LLP, 1155 Avenue of the Americas, New York, NY 10036
(US).
(8I) Designated States (national): AE, AG, AL, AM, AT, AU,
AZ, BA. BB, BG. BR, BY. BZ, CA, CH, CN, CR, CU, CZ,
DE, DK. DM, DZ. EE, ES, Fl, GB, GD, GE, GH, GM, HR.
HU, ID, IL, IN, IS, JP. ICE, KG, KP, KR, KZ7, LC, LK, LR.
LS, LT, LU, LV, MA, MD, MG, MKC, MN, MW, MX, Mt,
NO, NZ, PL. PT, RO, RU, SD. SE, SG, SI, SK, SL. TJ, TM,
TR, iT, TZ, UA, UG, Ut, VN, YU, ZA, ZW.
(814) Designated States (regional): ARIPO patent (GH. GM,
ICE, LS, MW, MZ, SD, SL, SZ, 'FL, UG, ZW), Eurasian
patent (AM, AZ, BY, KG, KZ, MD, RU, TJ. TM), Eurupean
patent (AT, BE, CH, CY. DE, DK, ES, Fl, FR, GB, GR. IE.
IT. LU, MC, NL, PT, SE), QAPI patent (BF. BJ, CF CG,
Cl, CM, GA, GN, GW, ML, MR. NE, SN. TD, TG).
Published:
with international search report
(88) Date of publication of the international search report:
18 October 2001
For two-letter codes and other abbreviations, refer to the "Guid-
ance Notes on Codes andAbbreviations appearing at the begin-
ning of each regular issue of the PCT' Gazette.
Priouity Data:
09/405,692 24 September 1999 (24.09.1999) US
(71) Applicants: GENENCOR INERNATIONAL, INC.
[US/US]; 925 Page Mill Road, Palo Alto, CA 94304
UNIVERSITY OF GEORGIA RESEARCH
FOUNDATION, INC. [US/USI; Boyd Graduate Studies
Research Center, Athens, GA 30602 (US).
(72) Inventors: SEYFRIED, Markus; 8403 16th Street, Silver
Springs, MD 20910 WIEGEL, Juergen; 215 Biol.
Sciences. Athens, GA 30602 WHMITED, Gregory;,
304 South Road, Belmont, CA 94002 (US).
Title: NOVEL ENZYMES WHICH DEHYDRATE GLYCEROL
S(57) Abstract: The present invention relates to improved methods and reagents fur the production of 1,3-propanediol. In particular,
the present invention provides novel thermophilie organisms and thermostable enzymes capable of catalyzing the fermentation of
glycerol to I .3-propanediol. The present invention also relates to methods of isolating such thermopbilic organisms, methods of
Scloning polynucleotides that encode such enzymes, polynucleotides encoding such enzymes, and methods of' using sucb enzymes
and organisms for the production of I ,3-propanediol.

WO 01/21825 PCTIUS00/26042
Novel Enzymes Which Dehydrate
Glycerol
1 FIELD OF THE INVENTION
The present invention relates to improved methods and reagents for the
production of 1,3-propanediol. In particular, the present invention provides novel
thermophilic organisms and thermostable enzymes capable of catalyzing the
fermentation of glycerol to 1,3-propanediol. The present invention also relates to
methods of isolating such thermophilic organisms, methods of cloning polynucleotides
that encode such enzymes, polynucleotides encoding such enzymes, and methods of
using such enzymes and organisms for the production of 1,3-propanediol.
2 BACKGROUND
1,3-Propanediol is a monomer used in the production of polyester fibers and
the manufacture of polyurethanes and cyclic compounds.
A variety of synthetic routes to 1,3-propanediol are known. For example,
1,3-propanediol can be synthesized: by the conversion of ethylene oxide over a
catalyst in the presence of phosphine, water, carbon monoxide, hydrogen and an acid;
by the catalytic solution phase hydration of acrolein, followed by reduction; or (3)
by reacting a hydrocarbon glycerol) in the presence of carbon monoxide and
hydrogen over catalysts having atoms from group VIII of the periodic table. However,
traditional chemical synthesis methods are expensive and generate waste streams
containing environmental pollutants, and are thus far from ideal. It would be desirable

WO 01/21825 PCT/US00/26042
to develop alternate methods and reagents for the production of 1,3-propanediol that
are less expensive and more environmentally friendly.
An alternate approach is to use enzymes, either in vivo in a
microorganism) or in vitro, to catalyze the fermentation of glycerol to 1,3-propanediol.
See, WO 98/21339, WO 98/21341, and U.S. Patents Nos. 5,821,092, 5,254,467,
5,633,362 and 5,686,276. Bacterial strains able to produce 1,3-propanediol from
glycerol have been found, for example, in the groups Citrobacter, Clostridium,
Enterobacter, Ilyobacter, Klebsiella, Lactobacillus, and Pelobacter. These bacteria
convert glycerol to 1,3-propanediol by means of a two step, enzyme catalyzed reaction.
In the first step, a dehydratase catalyzes the conversion of glycerol to 3-hydroxy-
propionaldehyde (3-HP) and water (Equation In the second step, 3-HP is reduced
to 1,3-propanediol by a NAD*-linked oxidoreductase (Equation 2).
Glycerol 3-HP HO (Equation
1)
3-HP NADH H' 1,3-Propanediol NAD' (Equation
2)
The 1,3-propanediol is not metabolized further and, as a result, can accumulate in the
media to a high concentration. The overall reaction results in the oxidation of reduced
b-nicotinamide adenine dinucleotide (NADH) to nicotinamide adenine dinucleotide
(NAD').
The bioconversion ofglycerol to 1,3-propanediol is generally performed under
anaerobic conditions using glycerol as the sole carbon source and in the absence of
other exogenous reducing equivalent acceptors. In some bacterial strains, certain
strains of Citrobacter, Clostridium, and Klebsiella, a parallel pathway for glycerol
metabolism operates which first involves oxidation of glycerol to dihydroxyacetone
(DHA) by a NAD'- (or NADP4 linked glycerol dehydrogenase (Equation The
DHA, following phosphorylation to dihydroxyacetone phosphate (DHAP) by a DHA
kinase (Equation becomes available for biosynthesis and for supporting ATP
generation via, for example, glycolysis.

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Glycerol NAD' DHA NADH H' (Equation
3)
DHA ATP DHAP ADP (Equation
4)
In contrast to the 1,3-propanediol pathway, this pathway may provide carbon and
energy to the cell and produces rather than consumes NADH.
In Klebsiellapneumoniae and Citrobacterfreundii, the genes encoding the
functionally linked activities of glycerol dehydratase (dhaBCE), 1,3-propanediol
oxidoreductase (dhaT), glycerol dehydrogenase (dhaD), and dihydroxyacetone kinase
(dhaK) are found in the dha regulon. The dha regulons from Citrobacter and
Klebsiella have been expressed in Escherichia coli and have been shown to convert
glycerol to 1,3-propanediol (Tong et al., Appl. Environ. Microbiol. 57:3541-46 (1991);
Seyfried et al., J. of Bact. 178:5793-96 (1996); Tobimatsu et al., J. Biol. Chem.
271:22352-22357 (1996)).
In view of the potential advantages inherent in the use of biological methods
and reagents to produce 1,3-propanediol, their exists a need for the development and
identification of novel microorganisms and enzymes capable of converting glycerol
and other carbon substrates to 1,3-propanediol having superior characteristics. The
present invention satisfies that need by providing superior microorganisms and
enzymes, along with methods of identifying other superior microorganisms and
enzymes.
3 SUMMARY OF THE INVENTION
The present invention relates to improved methods and reagents for the
production of 1,3-propanediol. In particular, the present invention provides novel
thermophilic organisms and thermostable enzymes capable of catalyzing the
fermentation of glycerol to 1,3-propanediol. The present invention also relates to
methods of isolating such thermophilic organisms, methods of cloning polynucleotides
that encode such enzymes, polynucleotides encoding such enzymes, and methods of
using such enzymes and organisms for the production of 1,3-propanediol.
In one aspect, the invention provides a method of converting glycerol to 1,3-
propanediol in a thermophilic organism, the method comprising: providing a

WO 01/21825 PCT/US00/26042
thermophilic organism that ferments glycerol to 1,3-propanediol; and culturing the
thermophilic organism under conditions such that 1,3-propanediol is produced. In a
preferred embodiment, the method further comprises the step of collecting 1,3-
propanediol produced by the thermophilic organism. In another preferred
embodiment, the thermophilic organism is Caloramator viterbiensis, wherein a
thermophilic organism derived from the organism deposited as ATCC designation
PTA-584 is particularly preferred.
The invention further provides a method of producing 1,3-propanediol from
glycerol, the method comprising: incubating glycerol with a thermostable dehydratase
enzyme, thereby converting the glycerol to 3-hydroxypropionaldehyde; and reducing
the 3-hydroxypropionaldehyde to 1,3-propanediol. In a preferred embodiment, the
reduction of the 3-hydroxypropionaldehyde to 1,3-propanediol is catalyzed by a
thermostable 1,3-propanediol oxidoreductase. In another preferred embodiment, the
method further comprises the step of collecting 1,3-propanediol. In yet another
preferred embodiment, thermostable dehydratase enzyme is derived from a
thermophilic organism such as Caloramator viterbiensis, wherein a thermophilic
organism derived from the organism deposited as ATCC designation PTA-584 is
particularly preferred.
Still another aspect of the invention provides an isolated thermostable glycerol
fermentation enzyme that is derived from C. viterbiensis, wherein a thermostable
glycerol fermentation enzyme derived from the organism deposited as ATCC
designation PTA-584 is particularly preferred. In particular preferred embodiments,
the thermostable glycerol fermentation enzyme is a dehydratase, such as glycerol
dehydratase, or a NAD'-linked oxidoreductase, such as 1,3-propanediol
oxidoreductase. The invention also provides an isolated thermostable glycerol
fermentation enzyme that is homologous to a thermostable glycerol fermentation
enzyme derived from C. viterbiensis.
Also provided by the invention is an isolated culture or cell ofC. viterbiensis.
In a non-limiting embodiment, the genome of the culture or cell is at least 
identical to the genome of the organisms deposited as ATCC designation PTA-584,
preferably 90% identical to the genome of the organisms deposited as ATCC
designation PTA-584, more preferably 95% identical to the genome of the organisms

WO 01/21825 PCT/US00/26042
deposited as ATCC designation PTA-584, and most preferably at least 99% identical
to the genome of the organisms deposited as ATCC designation PTA-584. In another
non-limiting embodiment, the 16S rDNA sequence of the culture or cell is at least 
identical to the 16S rDNA sequence of the organisms deposited as ATCC designation
PTA-584, and preferably at least 98% identical to the 16S rDNA sequence of the
organisms deposited as ATCC designation PTA-584.
In another aspect, the present invention provides a method of cloning a
polynucleotide sequence that encodes a thermostable glycerol fermentation enzyme,
the method comprising: hybridizing polynucleotide probes homologous to a portion of
a known glycerol fermentation enzyme gene to a polynucleotide molecule from an
environmental sample suspected of containing a thermophilic organism; and isolating
a polynucleotide sequence that binds to at least one polynucleotide probe. In a non-
limiting embodiment, the method uses a polymerase chain reaction to amplify the
polynucleotide sequence that binds to the polynucleotide probes. In a preferred
embodiment, the thermostable glycerol fermentation enzyme is derived from a
thermophilic organism identified as fermenting glycerol to 1,3-propanediol, wherein
C. viterbiensis is particularly preferred. In another preferred embodiment, the
polynucleotide probes are homologous to a portion of a known dhaB gene, wherein
probes homologous to the dhaB gene from Klebsiella are particularly preferred.
The invention further provides a method of cloning a polynucleotide sequence
that encodes a thermostable glycerol fermentation enzyme, the method comprising:
transforming a target organism that cannot grow anaerobically on glycerol with DNA
from a thermophilic organism; and identifying those transformed target organisms that
contain the polynucleotide sequence that encodes an enzyme that ferments glycerol to
1,3-propanediol by their anaerobic growth on glycerol. In a non-limiting embodiment,
the thermostable glycerol fermentation enzyme is derived from a thermophilic
organism identified as fermenting glycerol to 1,3-propanediol, such as C. viterbiensis,
wherein a thermophilic organism derived from the organism deposited as ATCC
designation PTA-584 is particularly preferred.
Still another aspect of the invention provides a method of isolating a
thermophilic organism that catalyzes the fermentation of glycerol to 1,3-propanediol,
the method comprising: incubating a sample containing thermophilic organisms in

media containing glycerol as the primary carbon source; and isolating at least
one thermophilic organism that ferments glycerol into 1,3-propanediol. In non-
limiting embodiments, the sample is incubated at a temperature in the range of
about 400C to about 1000C and/or under anaerobic conditions. In another non-
limiting embodiment, the sample is obtained from a natural source having a
temperature of between about ambient to about 1000C, and more preferably
from about 500 to about 1000C. In a preferred embodiment, the method further
comprises the step of detecting production of 1,3-propanediol and/or acetate by
the thermophilic organism.
Throughout the description and the claims of this specification the word
"comprise" and variations of the word, such as "comprising" and "comprises" is
not intended to exclude other additives, components, integers or steps.
The discussion of documents, acts, materials, devices, articles and the
like is included in this specification solely for the purpose of providing a context
for the present invention. It is not suggested or represented that any or all of
these matters formed part of the prior art base or were common general
knowledge in the field relevant to the present invention as it existed in Australia
before the priority date of each claim of this application.
4 BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows the effect of temperature on growth of strain JW/MS-VS-
td doubling time.
Figure 2 shows effect of pH 2 5C on growth of strain JW/MS-VS-5 at 600C,
td doubling time.
Figure 3 shows an unrooted phylogenetic dendrogram based on a
comparison of the 16S rRNA gene sequences of JW/MS-VS-5 and related
strains. The neighbor-joining tree was reconstructed from distance matrices.
Bootstrap values from the analyses of 1000 data sets (expressed as
percentages) are shown at the branching points. The scale bar represents 
nucleotide substitutions per 100 nucleotides.
X:IGEL SPECIESWO 179-01 .doc

Figure 4 shows a time course assay for the conversion of glycerol to 3-
HPA by anaerobically toluenized JW/MS-VS-5 cells at 600C under anaerobic
conditions.
Figure 5 shows a time course assay for the conversion of 1,2-
propanediol to propionaldehyde by anaerobically toluenized JW/MS-VS-5 cells
at 60 0C under anaerobic conditions.
DETAILED DESCRIPTION OF THE INVENTION
Thermophilic organisms are organisms that can survive and grow at
elevated temperatures where most other organisms mesophiles) would
not be able to survive. This unusual resilience towards high temperatures
results in part from these organisms' use of thermostable enzymes to catalyze
the biological reactions of life. Thermostable enzymes allow thermophilic
organisms to catalyze metabolic reactions at elevated temperatures. Such
organisms can be isolated from ambient temperature
X:\NIGEL\SPECIE S%4079-0.doc

WO 01/21825 PCTUS00/26042
environments, but are more likely isolated from high temperature environments
including, for example, hot springs, thermal vents and laundromat effluents. The
unusual thermostability ofthermophilic bacteria and enzymes allows one to catalyze
economically valuable bioconversions at higher temperatures than could be achieved
using mesophilic organisms and enzymes. In many cases, thermophilic bacteria are
used to catalyze the desired reaction in vivo in a fermentation process. Alternatively,
thermostable enzymes can be used to catalyze in in vitro or "cell-free" bioconversions,
typically by means of enzymes that have been immobilized to facilitate control of the
reaction and recovery of the reaction product.
A number of advantages can be achieved by performing bioconversions at
elevated temperatures. As is the case with any chemical reaction, the rates of
enzymatically catalyzed reactions generally increase dramatically with an increase in
the temperature of the reaction. Obvious efficiency benefits are derived from
increasing the rate at which an industrial bioconversion proceeds. In addition, it is
possible to prevent microbial contamination of a reaction medium by running the
reaction at an elevated temperature where most potential contaminating organisms are
unable to survive.
Another advantage of using a thermophilic organism to catalyze a
bioconversion at high temperatures is that in some cases the high temperature
facilitates the separation and isolation of the desired product from the reaction
medium. For example, U.S. Patent No. 5,182,199 describes the use of thermostable
enzymes and high temperature fermentation to facilitate the separation of ethanol from
a reaction medium.
Beyond being stable towards high temperatures, thermostable enzymes are also
generally found to possess enhanced stability toward other conditions and substances
that normally inactivate enzymes. Thermostable enzymes also tend to have a longer
storage life than enzymes derived from a mesophilic organism. The present invention
provides improved methods and reagents for the enzyme-catalyzed production of 1,3-
propanediol. In part, the present invention provides, for the first time, thermophilic
organisms and thermostable enzymes capable of catalyzing the fermentation of
glycerol into 1,3-propanediol. One novel characteristic of these organisms and
enzymes is their ability to remain viable and catalytically active at elevated
temperatures, temperatures at which previously identified 1,3-propanediol producing

WO 01/21825 PCTUSOO/26042
organisms and enzymes are rapidly inactivated. The methods and compositions of the
invention can be used to biologically convert glycerol to 1,3-propanediol at elevated
temperatures, thereby providing significant advantages over previously described
methods, particularly methods conducted at lower temperatures. The present invention
further includes methods of identifying thermophilic organisms capable of producing
1,3-propanediol from glycerol, as well as methods of cloning polynucleotides that
encode thermostable enzymes that catalyze this conversion.
5.1 Thermophilic Organisms Capable of Fermenting Glycerol to 1.3-
Propanediol at Elevated Temperatures, and Methods of Isolating Such
Organisms
In one embodiment, the instant invention provides a thermophilic organism
capable of fermenting glycerol to 1,3-propanediol at elevated temperatures.
As used herein, the term "thermophilic organism" refers to an organism
capable of growing at a high temperature, preferably at temperatures higher than 
more preferably at temperatures higher than 60*C, still more preferably at
temperatures higher than 70°C, and most preferably at temperatures higher than 
Organisms that are capable of growth at temperatures higher than 85"C are called
hyperthermophilic organisms. Examples of thermophilic organisms can be found, for
instance, among the prokaryotic microorganisms eubacteria and archaebacteria. Such
organisms inhabit, and can be isolated from, hot environments such as hot springs,
volcanic areas, and submarine thermal vents. In addition, thermophilic organisms can
be isolated from sources at ambient temperatures.
In a preferred embodiment of the invention, the thermophilic organism capable
of fermenting glycerol to 1,3-propanediol at elevated temperatures is a strain of
Caloramator viterbiensis. C. viterbiensis is a species of thermophilic bacteria capable
of fermenting glycerol to 1,3-propanediol and acetate. C. viterbiensis is defined
herein as a bacterial species possessing the following identifying characteristics: the
species is thermophilic; capable of fermenting glycerol to 1,3-propanediol; and
shares "substantial genomic sequence identity" with the type strain JW/MS-VS5T
deposited as ATCC designation PTA-584.
In a non-limiting embodiment of the invention, a member of the species C.
viterbiensis will also share the following identifying characteristics of type strain

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T JW/MS-VS5T cells are straight to slightly curved rods, 0.4 to 0.6 jPm in
dimension. Cells occur singly and stain Gram positive. The temperature range for
growth at pH 6.0 is 33 -64°C, the optimum at 580 C. The pH 2SC range for growth is
from 5.0 to 7.6, with an optimum at 6.0-6.5. Growth is observed with glycerol,
glucose, fructose, mannose, galactose, sucrose, cellobiose, lactose, starch, and yeast
extract. The strain does not appreciably ferment xylose, arabinose, acetate, lactate,
formate, methanol, ethanol, n-propanol, i-propanol, n-butanol, propionate, acetone,
succinate, ethylene glycol, 1,2-propanediol, phenol and benzoate. No appreciable
growth occurs under autotrophic conditions in the presence of H:CO2. Fermentation
of glycerol yields acetate and 1,3-propanediol as the primary organic products, with
significant amounts ofH2 produced during growth. Growth is inhibited by ampicillin,
chloramphenicol, erythromycin, rifampicin, and kanamycin (all at 100 pg/ml).
Streptomycin or tetracycline at the same retards growth. The G+C content of the
strain's DNA is 32 mol%, as determined by HPLC.
For the purposes of identifying a putative member of the species C.
viterbiensis, the term "substantial genomic sequence identity" refers to either: at least
identity, preferably at least 95% identity, and more preferably at least 99%
identity of the organism's genome sequence with genome sequence of type strain
or, at least 90% identity, preferably at least 95% identity, and more
preferably at least 99% identity of the organism's 16S rDNA sequence with the 16S
rDNA sequence of type strain 
Substantial sequence identity can be determined by the comparison of the
entire genomic sequences of a putative C. viterbiensis and 
Alternatively, substantial sequence identity can be determined by the comparison of
the 16S rDNA sequences of a putative C. viterbiensis and JW/MS-VS5r. The
sequence of all or a portion of the genome of JW/MS-VS5' and a putative C.
viterbiensis, particularly the sequence of the organisms' 16S rDNA, can be determined
by conventional nucleic acid sequencing techniques well known to those of skill in the
art (see, SAMBROOK ETAL., MOLECULAR CLONING, A LABORATORY MANUAL
(1989) and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY(Ausubel et al., eds.
1989)). The sequences can then be compared, using methods of sequence comparison
well known to the skilled artisan. For example, percent identity can be calculated
using the BLAST computer program, version 2.0, available on the World-Wide Web at

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http://www.ncbi.nlm.nih.gov. For a description of BLAST, see, Altschul et al.,
J. Mol. Biol. 215:403-10 (1990) and Altschul et al., Nucleic Acids Res. 25:3389-3402
(1997).
In a specific embodiment, the invention provides a thermophilic
microorganism capable of fermenting glycerol to 1,3-propanediol, the genome of
which is hybridizable to the genome of type strain JW/MS-VS5T under conditions of
low stringency. In an alternative preferred embodiment, the invention provides a
thermophilic microorganism capable of fermenting glycerol to 1,3-propanediol, the
16S rDNA sequence of which is hybridizable to the 16S rDNA sequence of type strain
JW/MS-VS5T under conditions of low stringency. Procedures using such conditions
of low stringency can be as follows (see also Shilo and Weinberg, Proc. Natl. Acad.
Sci. USA 78:6789-6792 (1981)): Filters containing DNA are pretreated for 6 h at 
in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 5 mM
EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 pg/ml denatured salmon sperm
DNA. Hybridizations are carried out in the same solution with the following
modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 pg/ml salmon sperm DNA,
(wt/vol) dextran sulfate, and 5-20 X 106 cpm "P-labeled probe is used. Filters
are incubated in hybridization mixture for 18-20 h at 40*C, and then washed for 1.5 h
at 55°C in a solution containing 2X SSC, 25 mM Tris-HCl (pH 5 mM EDTA,
and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an
additional 1.5 h at 600C. Filters are blotted dry and exposed for autoradiography. If
necessary, filters are washed for a third time at 65-680 C and reexposed to film. Other
conditions of low stringency which can be used are well known in the art as
employed for cross-species hybridizations).
In another specific embodiment, the invention provides a thermophilic
microorganism capable of fermenting glycerol to 1,3-propanediol, the genome of
which is hybridizable to the genome of type strain JW/MS-VS5T under conditions of
moderate stringency. In a preferred embodiment, the invention provides a
thermophilic microorganism capable of fermenting glycerol to 1,3-propanediol, the
16S rDNA sequence of which is hybridizable to the 16S rDNA sequence of type strain
under conditions of moderate stringency. Procedures using such
conditions of moderate stringency can be as follows: Filters containing DNA are
pretreated for 6 h at 55"C in a solution containing 6X SSC, 5X Denhart's solution,

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SDS and 100 pg/ml denatured salmon sperm DNA. Hybridizations are carried
out in the same solution and 5-20 X 106 cpm 2P-labeled probe is used. Filters are
incubated in hybridization mixture for 18-20 h at 55C, and then washed twice for 
minutes at 60*C in a solution containing IX SSC and 0.1% SDS. Filters are blotted
dry and exposed for autoradiography. Other conditions of moderate stringency which
can be used are well-known in the art. Washing of filters is done at 37°C for 1 h in a
solution containing 2X SSC, 0.1% SDS.
In another specific embodiment, the invention provides a thermophilic
microorganism capable of fermenting glycerol to 1,3-propanediol, the genome of
which is hybridizable to the genome of type strain JW/MS-VS5 r under conditions of
high stringency. In a preferred embodiment, the invention provides a thermophilic
microorganism capable of fermenting glycerol to 1,3-propanediol, the 16S rDNA
sequence of which is hybridizable to the 16S rDNA sequence of type strain JW/MS-
under conditions of high stringency. Procedures using such conditions of high
stringency can be as follows: Prehybridization of filters containing DNA is carried out
for 8 h to overnight at 65"C in buffer composed of 6X SSC, 50 mM Tris-HCl
(pH 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 pg/ml
denatured salmon sperm DNA. Filters are hybridized for 48 h at 65 C in
prehybridization mixture containing 100 Gg/ml denatured salmon sperm DNA and
5-20 X 106 cpm of 2P-labeled probe. Washing of filters is done at 37*C for 1 h in a
solution containing 2X SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is
followed by a wash in 0. IX SSC at 50*C for 45 min before autoradiography. Other
conditions of high stringency which can be used are well known in the art.
The species C. viterbiensis further includes the progeny of the type strain
JW/MS-VS5T including progeny possessing altered genotypes and/or phenotypes
relative to type strain JW/MS-VS5. Phenotypes and/or genotypes can be altered by
mutation, including as the result of directed or random mutagenesis. The invention
provides cells having single or multiple mutations specifically designed to enhance the
production of 1,3-propanediol. For example, it is contemplated that a mutant strain
capable of fermenting glycerol to 1,3-propanediol in a manner that is resistant to
substrate or product repression would be particularly useful in the present invention.
Methods of creating mutants are common and well known in the art. For
example, wild type cells may be exposed to a variety of agents, such as radiation or

WO 01/21825 PCT/US00/26042
chemical mutagens, and then screened for the desired phenotype. When creating
mutations through radiation, either ultraviolet (UV) or ionizing radiation may be used.
Specific methods for creating mutants using radiation or chemical agents are well
documented in the art. See, Brock, Biotechnology: A Textbook of Industrial
Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass., or
Deshpande, Appl. Biochem. Biotechnol., 36: 227-34 (1992), herein incorporated by
reference. Following mutagenic treatement, mutants having the desired phenotype may
be selected by a variety of methods. Random screening is most common where the
mutagenized cells are selected for the desired attribute. Methods of mutant selection
are highly developed and well known in the art of industrial microbiology. See, e.g.,
Brock, supra.
The instant invention further provides methods for isolating and maintaining in
culture thermophilic organisms capable of fermenting glycerol to 1,3-propanediol.
Such organisms are useful in practicing embodiments of this invention, they can
be used to biologically convert glycerol to 1,3-propanediol at an elevated temperature,
or they can be used as a source of a thermostable enzyme that ferments glycerol, or a
polynucleotide encoding the same.
Materials and methods suitable for the maintenance and growth of bacterial
cultures can be found, for example, in MANUAL OF METHODS FOR GENERAL
BACTERIOLOGY (Gerhardt et al., eds), American Society for Microbiology,
Washington, D.C. or Brock, supra. Reagents and materials used for the growth and
maintenance of bacterial cells can be obtained from commercial suppliers, for
example, Aldrich Chemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.),
GIBCO/BRL (Gaithersburg, or Sigma Chemical Company (St. Louis, Mo.).
A thermophilic organism of the invention, C. viterbiensis, can be isolated
from any environment that is conducive to growth of thermophilic organisms, a
thermal vent, hot spring or soil and water and sediments around them, or laundromat
effluent. A sample consisting of, for example, sediment, water or a mixture thereof, is
collected from the environment of interest. It is desirable to determine the pH and
temperature at the sampling point as an indication of optimal conditions for growth of
any organism isolated from the sample. The sample is then used innoculate a basal
growth medium, preferably a medium wherein glycerol is the sole or primary carbon
source. Typically, the basal medium is inoculated with the sample to a final

WO 01/21825 PCT/US00/26042
concentration of approximately 10% The pH of the medium should preferably
approximate that of the sampling point. Alternatively, a plurality of basal growth
mediums at different pH can be inoculated to select for organisms having different pH
growth dependence. The enrichment culture is then incubated at a high temperature,
preferably at least about 500C, more preferably at least about 60"C, and most
preferably at least about 75*C, and is preferably carried out in an anaerobic or
microaerobic environment. Guidance for growing thermophilic organisms can be
found in, for example, Weigel, "Methods for Isolation and Study of Thermophiles",
Chapter 4 in THERMOPHILES: GENERAL, MOLECULAR AND APPLIED MICROBIOLOGY,
T.D. Brock, ed. (John Wiley Sons, pp. 17-37.
When an enrichment culture is found to be able to utilize glycerol as a carbon
source, a homogeneous culture can be isolated by preparing a dilution series of the
culture, plating out the series on a solid basal medium 1.5% agar) using soft agar
overlays basal medium containing 0.8% agar), picking out and subculturing
single colonies in liquid medium of the same composition, and checking for the
formation of 1,3-propanediol and/or acetate. 1,3-Propanediol can be identified directly
by submitting the media to high pressure liquid chromatography (HPLC) analysis. For
example, the fermentation media can be analyzed on an analytical ion exchange
column using a mobile phase of 0.01 N sulfuric acid in an isocratic fashion.
Alternatively, 1,3-propanediol can be identified using other appropriate analytical
techniques, including, but not limited to, gas chromatography (GC) and gas
chromatography-mass spectroscopy (GC-MS).
A strain of a thermophilic organism found capable of converting glycerol to
1,3-propanediol can be maintained in culture using culture maintenance and
preservation techniques known in the art. These methods include refrigeration for
short storage times, freezing in liquid nitrogen for prolonged storage, and
lyophilization to dehydrate the cells. The choice of the preservation method depends
on the nature of the culture and the facilities that are available. When freezing is used,
the rates of freezing and thawing must be carefully controlled to ensure the survival of
the microorganisms, since ice crystals formed during freezing can disrupt membranes.
Glycerol is often employed as an "antifreeze" agent to prevent damage due to ice
crystals and to ensure the ability to recover viable microorganisms when frozen
cultures are thawed.

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5.2 Thermostable Enzymes Capable of Producing 1,3-Propanediol at Elevated
Temperatures
In one embodiment, the instant invention provides a thermostable enzyme that
catalyzes a step in the fermentation of glycerol to 1,3-propanediol at elevated
temperatures, referred to herein as a "thermostable glycerol fermentation enzyme."
As used herein, the term "thermostable glycerol fermentation enzyme" refers to
an enzyme which catalyzes at least one reaction step in the fermentation of a glycerol
to 1,3-propanediol and which is "thermostable," stable and active at high
temperatures, preferably at temperatures higher than 50°C, more preferably at
temperatures higher than 60°C, still more preferably at temperatures higher than
and most preferably at temperatures higher than 85"C. An enzyme is "stable"
at a given temperature if it is able retain at least 50% of its original catalytic activity
after exposure to that temperature for at least 5 minutes, preferably at least 30 minutes,
more preferably at least one hour, still more preferably for at least 8 hours, and most
preferably for at least 24 hours or longer. In many instances, thermostable enzymes
also have increased storage life and are more stable to denaturing conditions, such as
physical agitation or exposure to organic solvents, than non-thermostable enzymes
sharing similar functional characteristics.
In a preferred embodiment, the invention provides a thermostable glycerol
fermentation enzyme that is derived from C. viterbiensis. In a preferred, but non-
limiting embodiment, the invention provides a thermostable dehydratase derived from
C. viterbiensis. In another preferred but non-limiting embodiment, the invention
provides a thermostable 1,3-propanediol oxidoreductase derived from C. viterbiensis.
In a particularly preferred embodiment of the invention, the thermostable glycerol
fermentation enzyme is derived from strain 
As used herein, the term "dehydratase" refers to any enzyme that is capable of
catalyzing the conversion of a glycerol molecule to the product 3-
hydroxypropionaldehyde. Specific examples of dehydratases are glycerol dehydratase
and diol dehydratase, dehydratases having preferred substrates of glycerol and 1,2-
propanediol, respectively.

WO 01/21825 PCT/US00/26042
The term "1,3-propanediol oxidoreductase" refers to an enzymes capable of
catalyzing the conversion 3-hydroxypropionaldehyde to 1,3-propanediol with the
concomitant oxidation of NADH.
The ability to catalyze a reaction step in the fermentation of a glycerol to 1,3-
propanediol can be determined by assaying the activity of the enzyme. Glycerol
fermentation activity can be assayed using techniques known to those skilled in the art.
For example, methods of assaying for the activity of a dehydratase are described by
Poppe et al., Eur. J. ofBiochem 245:398-41 (1997), Honda et al., J. of Bact. 143:1458-
(1980), and Macis et al., FEMSMicrobiology Letters 164:21-28 (1998),
incorporated herein by reference in their entirety. A method of assaying for 1,3-
propanediol oxidoreductase activity is described, for example, in Johnson et al., J of
Bad. 169:2050-54 (1987), incorporated herein by reference in its entirety. Enzyme
activity can be determined in situ using toluene-treated cells, as described for example,
by Honda et al.. The ability of an enzyme to catalyze a reaction step in the
fermentation of a glycerol to 1,3-propanediol can also be determined by expressing the
enzyme in a host cell that is not normally capable of fermenting glycerol, and assaying
whether expression of the enzyme allows the cell to ferment glycerol.
For the purposes of this invention, a thermostable 1,3-propanediol-producing
enzyme is "derived" from C. viterbiensis, or a specified strain of C. viterbiensis, if: (a)
it is endogenously expressed by C. viterbiensis, or the specified strain of C.
viterbiensis, or its amino acid sequence is encoded by a polynucleotide coding
sequence that occurs in C. viterbiensis, or the specified strain ofC. viterbiensis; and
it is useful in practicing the invention. As used herein, an enzyme is "useful in
practicing the invention" if it is a thermostable glycerol fermentation enzyme, an
enzyme that catalyzes at least one reaction step in the fermentation of a glycerol to 1,3-
propanediol and which is stable and active at high temperatures, preferably at
temperatures higher than 50°C, more preferably at temperatures higher than 60°C, still
more preferably at temperatures higher than 70 0 C, and most preferably at temperatures
higher than 85 C.
The invention further provides an enzyme that is homologous to a thermostable
glycerol fermentation enzyme derived from C. viterbiensis. For the purposes of this
invention, an enzyme is "homologous" to a thermostable glycerol fermentation enzyme
derived from C. viterbiensis if: its amino acid sequence is encoded by a

WO 01/21825 PCT/US00/26042
polynucleotide coding sequence that hybridizes to the complement of a polynucleotide
sequence that encodes a thermostable glycerol fermentation enzyme derived from C.
viterbiensis under moderately stringent conditions, hybridization to filter-bound
DNA in 0.5 M NaHPO 4 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 C, and
washing in 0.2xSSC/0.1% SDS at 42*C (Ausubel et al., 1989, Current Protocols in
Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley sons,
Inc., New York, at p. 2.10.3); and it is useful in practicing the invention. In a
preferred embodiment, the homologous enzyme is encoded by a polynucleotide coding
sequence that hybridizes to the complement of a polynucleotide sequence that encodes
a thermostable glycerol fermentation enzyme derived from C. viterbiensis under highly
stringent conditions, hybridization to filter-bound DNA in 0.5 M NaHPO4, 7%
sodium dodecyl sulfate (SDS), 1 mM EDTA at 650 C, and washing in 0.1xSSC/0.1%
SDS at 68 0 C (Ausubel et al., 1989, supra) and is useful in practicing the invention.
An enzyme that is homologous to a thermostable glycerol fermentation enzyme
derived from C. viterbiensis can take various forms. In some embodiments of the
invention, the homologous enzyme is a mutant form of a thermostable glycerol
fermentation enzyme derived from C. viterbiensis, wherein one or more amino acid
residues have been substituted with a different amino acid residue, resulting in an
enzyme useful in practicing the invention. Mutations can be conservative, wherein an
amino acid residue is substituted with another with another amino acid residue sharing
similar functional and structural characteristics, such as acidity, polarity, or bulkiness
of side chains. Mutations can also be non-conservative, where the functional and/or
structural properties of the substituted amino acid residue are not conserved. In some
cases, a mutant form of a thermostable glycerol fermentation enzyme derived from C.
viterbiensis will possess altered functional properties relative to the native enzymes,
such as altered catalytic capability, stability, or pH or temperature optimum. Such
mutants fall within the scope of the invention so long as they are homologous to a
thermostable glycerol fermentation enzyme derived from C. viterbiensis.
Mutant forms of the enzyme can be generated by a variety of techniques well
known to the skilled artisan. A number of these techniques are reviewed in U.S.
Patent No. 5,830,696, incorporated herein by reference in its entirety, which describes

WO 01/21825 PCT/USOO/26042
the directed evolution of thermostable enzymes. Alternatively, mutant enzymes can be
created randomly, or occur spontaneously in an organism.
Furthermore, an enzyme that is homologous to a thermostable glycerol
fermentation enzyme derived from C. viterbiensis can be a deletion mutant or
truncated variant of a native C. viterbiensis enzyme. The use of such enzyme forms
can in some cases be desirable, for example, where a deletion or truncation results in
enhanced stability or ease of purification relative to the corresponding native enzyme.
The invention further provides an enzyme that is homologous to a thermostable
glycerol fermentation enzyme derived from C. viterbiensis that is a chimeric form of a
thermostable glycerol fermentation enzyme derived from C. viterbiensis, a protein
fusion. Chimeras can be useful, for example, for improving the stability of the enzyme
or facilitating purification, by the inclusion of a moiety capable of associating
with an ion exchange, metal chelate, substrate affinity or immunoaffinity matrix. In a
preferred embodiment, the invention provides a homologous enzyme covalently
attached to a solid support, such as cellulose.
Furthermore, the invention provides an enzyme that is homologous to a
thermostable glycerol fermentation enzyme derived from C. viterbiensis and that is
derived from a different microorganism, preferably a thermophilic microorganism. An
example of such an enzyme is a thermostable glycerol fermentation enzyme
"homolog," which is defined as an enzyme encoded by a gene from a different species,
wherein the gene is recognized by those of skill in the art as a homolog of a C.
viterbiensis glycerol fermentation gene based on a degree of nucleotide sequence
identity greater than about 80%. Sequence identity can be determined using the
BLAST program, as described supra.
In one embodiment, the invention provides an isolated thermostable glycerol
fermentation enzyme, where the term "isolated" indicates that enzyme is at least
partially pure, provided in a preparation where the percentage by weight of the
enzyme, relative to other material in the preparation, is higher than would be found in
nature. In a preferred embodiment, the invention provides an isolated thermostable
glycerol fermentation enzyme that is at least 90% pure by weight of contaminating
proteins, and more preferably at least 95% pure by weight of contaminating proteins.
Materials and methods useful in the preparation and isolation of the
aforementioned enzymes are described infra. In one embodiment, an enzyme of the

WO 01/21825 PCT/US00/26042
invention can be isolated from a thermophilic organism, particularly a strain of C
viterbiensis, that expresses the enzyme endogenously. Alternatively, an enzyme of the
invention can be produced recombinantly by introducing a polynucleotide sequence
encoding the enzyme into an appropriate host cell and expressing the encoded gene
product. An enzyme of the invention can also be produced by means of in vitro
translation, or by chemical synthesis techniques.
5.3 Methods of Cloning a Polynucleotide Encoding a Thermostable Enzyme
That Converts Glycerol to 1,3-Propanediol
The instant invention provides a polynucleotide sequences encoding a
thermostable glycerol fermentation enzyme, or a fragment thereof, and methods for
identifying, isolating, and cloning such polynucleotides. These methods involve
providing a sample containing polynucleotide coding sequences derived from a
thermophilic organism or thermophilic organisms, and screening for a polynculeotide
sequence encoding a thermostable glycerol fermentation enzyme, or a fragment
thereof The sample can take any of a variety of forms, and be prepared by a variety of
different techniques known to those of skill in the art. Similarly, screening of the
sample can be accomplished using any of a number of suitable techniques, some non-
limiting examples of which are described infra.
Production and manipulation of the polynucleotide molecules and
oligonucleotide molecules disclosed herein are within the skill in the art and can be
carried out according to recombinant techniques described, among other places, in
SAMBROOK ETAL., MOLECULAR CLONING, A LABORATORY MANUAL (1989); CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel et al. eds., 1989); PCR STRATEGIES
(Innis et al. eds., 1995);and PCR TECHNOLOGY (Erlich ed., 1992), all of which are
incorporated herein by reference.
In some embodiments of the invention, the sample is screened with a probe
having a nucleotide sequence believed to encode a glycerol fermentation enzyme, or a
probe or probes representing a portion thereof A probe sequence can be based on the
amino acid sequence of a known glycerol fermentation enzyme, or some fragment
thereof, or a polynucleotide sequence encoding such an enzyme. For example, the
amino acid sequence of a portion of a glycerol-fermentation enzyme can be
determined, using amino acid sequencing techniques described, in CREIGHTON,

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PROTEIN STRUCTURES AND MOLECULAR PRINCIPLES (1983), and the information used
to generate a set of degenerate probes. In a preferred embodiment of the invention, a
sample is screened using a probe or probes based upon a sequence encoding a
dehydratase or oxidoreductase derived from a thermophilic organism such as C.
viterbiensis. Alternatively, the probe can be based upon a glycerol fermentation
enzyme-encoding gene derived from a non-thermophilic organism K
pasteurianum, C. freundii, or C. pasteurianum), such as the dhaBCE or dhaT genes,
which encode glycerol dehydratase and oxidoreductase, respectively. The nucleotide
sequences of dhaBCE genes have been reported in the literature (see, Macis et
al., FEMS Mocrobiology Letters 164:21-28 (1998) Pasteurianum); Seyfried et al.,
J of Bacteriology 178:5793-96 (1996) freundii); and Tobimatsu et al., J. Biol.
Chem. 271:22352-57 (1996) pneumoniae)). The nucleotide sequences of dhaT
gene have also been reported (see, Luers et al., FEMSMicrobiol. Lett. 154:337-45
(1997) Pasteurianum); and Daniel et al., J. of Bacteriol. 177:2151-56 (1995) (C.
freundii)). Specific probes provided by this invention include primer pairs based on
the dhaB gene of K. pasteurianum as detailed below by way of illustrative
embodiments in dhaB 15' (SEQ ID NO:1); dhaB 13' (SEQ ID NO:2); dhaB25' (SEQ ID
NO:3); dhaB23' (SEQ ID NO:4); dhaB35' (SEQ ID NO:5); and dhaB33' (SEQ ID
NO:6)) which are particularly useful for amplifying a glycerol fermentation enzyme-
encoding polynucleotide, or a fragment thereof, via polymerase chain reaction
Such a primer pair can be used to amplify a segment of a glycerol
fermentation enzyme-encoding polynucleotide from an organism of interest, which can
in turn be used as a probe for identifying and cloning the full-length coding sequence
from the same or a different organism. Probes can be labeled for identifying
homologous sequences, for example in a cDNA library. Alternatively, probes can be
used as primers to amplify a polynucleotide sequence flanked by sequences recognized
by the primers, particularly by means of PCR or RT-PCR. In particular, a sequence of
interest can be amplified from a cDNA or genomic library, from an isolated organism,
or from an environmental sample, as described in more detail below.
In one embodiment of the invention, the sample to be screened is an
environmental sample suspected of harboring a thermophilic organism, such as a
water, soil or mud sample collected from an environment conducive to thermophilic
life. Typically, an environmental sample will be prepared in a manner such that

WO 01/21825 PCT/USOO26042
nucleic acids present in the sample are available for interaction with a complementary
probe, a PCR primer. A glycerol fermentation enzyme-encoding polynucleotide,
or a fragment thereof, can be recognized by its ability to hybridize to a probe as
described above.
A preferred method of isolating and cloning a polynucleotide of the invention
from an environmental sample is by means of PCR. This technique takes advantage of
the sequence homology between polynucleotides encoding homologous enzymes, or
enzymes with similar activity. The method employs a set of primers based on a
known glycerol fermentation enzyme-encoding polynucleotide sequence, or a portion
thereof, to amplify a homologous polynucleotide sequence, or a fragment thereof, from
an environmental sample. The homoiogous polynucleotide can be amplified from
DNA in the sample, particularly genomic DNA of microorganims in the sample, using
conventional PCR methodology well known in the art. Alternatively, the homologous
polynucleotide can be amplified from RNA in the sample, particularly mRNA or total
RNA, by synthesizing a first-strand cDNA copy of RNA and amplifying the cDNA via
PCR, RT-PCR. Using techniques known to those of skill in the art, one can
adjust the annealing conditions of the reaction to allow hybridization of primers at a
desired level of homology, thereby permitting the amplification of sequences sharing
more or less homology with the sequence upon which the primers are based. This
method allows for the isolation of a complete glycerol fermentation enzyme-encoding
open reading frame or a fragment thereof. One or more fragments of a
glycerol fermentation enzyme-encoding ORF can be cloned and spliced together so as
to produce a complete ORF. The amplified glycerol fermentation enzyme-encoding
ORF can then be used to express the encoded glycerol fermentation enzyme in the
practice of various aspects of the invention. For example, the sequence can be
introduced into a host cell, thereby enabling the host cell to ferment glycerol.
Alternatively, the expressed enzyme can be purified for applications such as cell-free
fermentation. Alternatively, the ORF sequence can itself be used to design novel
primers and probes for the further detection and isolation of novel thermostable
glycerol fermentation enzyme-encoding sequences.
In another embodiment of the invention, the sample is a polynucleotide library,
preferably a genomic or cDNA library prepared from a thermophilic organism,
especially an organism known to be capable of fermenting glycerol to 1,3-propanediol.

WO 01/21825 PCTIUS00/26042
For general background on molecular biology techniques and on how to prepare a
cDNA library and a genomic library, see, Ausubel et al., supra; Sambrook et al.,
supra; and U.S. Patent No. 5,650,148. The library can be screened using nucleic acid
hybridization screening techniques in conjunction with the appropriate labeled probes,
as described above. Appropriate screening techniques are set forth, among other
places, in Benton and Davis, Science 196:180 (1977) bacteriophage libraries) and
Grunstein and Hogness, Proc. Nail. Acad. Sci. USA 72:3961-65 (1975) (plasmid
libraries), which publications are incorporated herein by reference.
In still another embodiment of the invention, the sample comprises an isolated
organism or organisms, or a polynucleotide library generated from an isolated
organism or organisms, from which a polynucleotide of the invention can be amplified
using PCR or RT-PCR. Preferably, the isolated organism or organisms used are
thermophilic and capable of fermenting glycerol.
In one embodiment of the invention, an expression library is constructed which
is able to direct the expression of the gene products encoded by the polynucleotide
constituents of the library.
In a particularly preferred embodiment of the invention, an expression library is
functionally screened by testing its constituents for the ability to encode a gene product
that catalyzes a step in the fermentation of glycerol. In a preferred embodiment, an
organism that is normally incapable of fermenting glycerol to 1,3-propanediol is
tranformed with a constituent of a DNA library as described above, preferably a library
prepared from a thermophilic organism known to be capable of fermenting glycerol. The
transformed organisms are then screened for the ability to ferment glycerol to 1,3-
propanediol by their anaerobic growth on glycerol. The transformed microorganisms can
be screened for the production of 1,3-propanediol and/or acetate. The ability to grow
anaerobically on glycerol, or to produce 1,3-propanediol and/or acetate, indicates that the
library constituent used to transform the organism encodes an enzyme that is able to
provide an enzymatic activity required for the fermentation of glycerol. The library
constituent can then be cloned and characterized by standard techniques and used in the
practice of various aspects of the instant invention.
The microorganism transformed in the functional cloning strategy described
above can be one that inherently lacks the ability to ferment glycerol, or a
microorganism that has lost the ability to ferment glycerol as the result of a mutation,

WO 01121825 PCT/US00/26042
a null mutation in one of the genes required for glycerol fermentation. Such a
mutation can be engineered into an organism using techniques well known in the art.
The microorganism can, but need not necessarily be, thermophilic. It is preferable that
the test organism possess all but one of the enzymatic activities required for glycerol
fermentation, so that the ability to ferment glycerol can be recovered by the introduction
of a single enzymatic activity.
Alternatively, the expressed gene products can be immunologically screened
using standard techniques in conjunction with antibodies raised against a glycerol
fermentation enzyme. For such screening techniques, see, ANTIBODIES: A
LABORATORY MANUAL (Harlow and Lane, eds., 1988).
5.4 Recombinant Organisms Capable of Fermenting Glycerol to 1,3-Propanediol
The instant invention also provides a recombinant organism that has been
genetically engineered to express one or more exogenous, heterologous, thermostable
glycerol fermentation enzymes. The term "exogenous," when used in this context,
indicates that enzyme is encoded by an heterologous polynucleotide coding sequence,
a polynucleotide sequence that is not native, or endogenous, to the host organism.
In a preferred embodiment, the recombinant organism is engineered to express a
thermostable glycerol fermentation enzyme derived from C. viterbiensis, or an enzyme
that is homologous to such an enzyme. In a particularly preferred embodiment, the
recombinant organism is engineered to express a dehydratase or 1,3-propanediol
oxidoreductase derived from a strain ofC. viterbiensis, such as 
A recombinant organism of the invention can be useful in the practice of various
aspects of the instant invention. For example, the recombinant organism can be used as a
catalyst in a process for biologically converting glycerol to 1,3-propanediol. In another
non-limiting embodiment, the recombinant organism can serve as the source of a
thermostable glycerol fermentation enzyme, which can be isolated using protein
purification techniques familiar to the skilled artisan.
The invention provides a variety of vectors and transformation and expression
cassettes suitable for cloning, transformation and expression of a polynucleotide
sequence encoding a thermostable glycerol fermentation enzyme, or a fragment thereof.
Suitable vectors will be those which are compatible with the host microorganism and the
intended use of the resulting recombinant microorganism. For example, a suitable vector

WO 01/21825 PCT/US00/26042
can be derived from a plasmid, a virus (such as bacteriophage T7 or a M 13 derived
phage), or a cosmid. Protocols for obtaining and using such vectors are known to those
in the art. See, Sambrook et al., supra.
Typically, the vector or cassette contains sequences directing transcription and
translation of the relevant gene, a selectable marker, and sequences allowing autonomous
replication or chromosomal integration. Suitable vectors comprise a region 5' of the
coding sequence that harbors transcriptional initiation controls and a region 3' of the
coding sequence that controls transcriptional termination. In a preferred embodiment,
control regions are derived from genes homologous to the transformed host cell.
Recombinant vectors of the present invention, particularly expression vectors, are
preferably constructed so that the coding sequence is in operative association with one or
more regulatory elements necessary for transcription and translation of the coding
sequence. As used herein, the term "regulatory element" includes but is not limited to
nucleotide sequences that encode inducible and noninducible promoters, enhancers,
operators and other elements known in the art to drive and/or regulate expression of
polynucleotide coding sequences. Also, as used herein, the coding sequence is in
"operative association" with one or more regulatory elements where the regulatory
elements effectively regulate or allow for the transcription of the coding sequence or the
translation of its mRNA, or both.
The regulatory elements of these vectors can vary in their strength and
specificities. Depending on the host/vector system utilized, any of a number of suitable
transcription and translation elements can be used. Non-limiting examples of
transcriptional regulatory regions or promoters for bacteria include the P-gal promoter,
the T7 promoter, the TAC promoter, lambda left and right promoters, trp and lac
promoters, and the trp-lac fusion promoters.
General methods of expressing recombinant proteins are well known in the art, as
exemplified in R. Kaufman, Methods in Enzymology 185:537-566 (1990). Optionally,
the heterologous sequence can encode a fusion protein including an N-terminal
identification peptide imparting desired characteristics, stabilization or simplified
purification of expressed recombinant product.
To direct the glycerol fermentation enzyme into the secretory pathway of the host
cells, a secretory signal sequence may be provided in the expression vector. The
secretory signal sequence is joined to the polynucleotide sequence encoding the glycerol
WO 01/21825 PCT/US00/26042
fermentation enzyme in the correct reading frame. Secretory signal sequences are
commonly positioned 5' to the coding sequence. The secretory signal sequence may be
that normally associated with a protein secreted by the host organism.
Once a suitable transformation or expression vector has been constructed, it can
be used to transform an appropriate host cell, using known procedures such as, e.g.,
calcium-permeabilized cells, electroporation, protoplast transformation, or by
transfection using a recombinant phage virus. Suitable expression hosts include E. coli,
Bacillus subtilis, Salmonella typhimurium and various species within the genera Bacillus,
Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed
as a matter of choice.
In a preferred embodiment, the invention provides a thermophilic recombinant
microorganism that has been genetically engineered to express one or more exogenous
thermostable glycerol fermentation enzymes. Such microorganims are particularly useful
in the biological conversion of glycerol to 1,3-propanediol, as described infra. Examples
of thermophilic microorganisms include members of the genera Bacillus, Thermus,
Sulfolobus, Thermoanaerobacter, Thermobrachium, and Caloramator.
The selected host microorganism will preferably be able to produce the co-factor
adenosyl-cobalamin (coenzyme If necessary, B1 2 synthesis genes can be introduced
the microorganism and/or the media can be supplemented with vitamin B 2
Methods for Isolating Thermostable Glycerol Fermentation Enzymes
The instant invention provides a method for preparing and isolating a
thermostable glycerol fermentation enzyme. In a preferred embodiment, the enzyme is
provided in a substantially purified form. In a non-limiting embodiment, the enzyme is
isolated from a thermophilic microorganism that expresses a native thermostable glycerol
fermentation enzyme. In another non-limiting embodiment, the enzyme is isolated from
a recombinant organism engineered to express an exogenous polynucleotide sequence
encoding a thermostable glycerol fermentation enzyme. Alternatively, a thermostable
enzyme of the invention can be prepared using conventional solution or solid phase
peptide syntheses procedures, as described in, CREIGHTON, SUPRA.
Host cells expressing the thermostable glycerol fermentation enzyme of interest
are typically cultivated in a nutrient medium suitable for production of the enzyme, using
methods known in the art. For example, the cell may be cultivated by shake flask

WO 01/21825 PCT/USO0/26042
cultivation, small-scale or large-scale fermentation (including continuous, batch,
fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed
in a
suitable medium and under conditions allowing the enzyme to be expressed and/or
isolated. A suitable nutrient medium typically comprises carbon and nitrogen sources and
inorganic salts, using procedures known in the art. Suitable media are available from
commercial suppliers, or may be prepared according to published compositions in
catalogs of the American Type Culture Collection).
Depending on the nature of the host cell and expression vector used, the enzyme
may be retained in the cytoplasm, often as insoluble granules (known as inclusion
bodies), or may be directed to the periplasmic space by a bacterial secretion sequence. In
the former case, the cells are lysed and the granules are recovered and denatured, after
which the enzyme is refolded by diluting the denaturing agent. In the latter case, the
enzyme may be recovered from the periplasmic space by disrupting the cells, by
sonication or osmotic shock, to release the contents of the periplasmic space and
recovering the enzyme. If the enzyme is secreted into the nutrient medium, it can be
recovered directly from the medium.
Enzyme produced by the cells may then be recovered by means of conventional
techniques known in the art, including, but not limited to, centrifugation, filtration,
extraction, spray-drying, evaporation, differential solubility ammonium sulfate
precipitation), chromatography ion exchange, affinity, hydrophobic,
chromatofocusing, and size exclusion) or electrophoretic procedures preparative
isoelectric focusing (IEF) (see, PROTEIN PURIFICATION (Janson and Ryden eds.,
1989); SCOPES, PROTEIN PURIFICATION: PRINCIPLES AND PRACTICE (1994); Sambrook el
al. supra; and Ausubel et al. supra.
In a preferred embodiment, the enzyme is purified as a fusion protein including a
fusion element that facilitates rapid affinity purification. Useful purification fusion
elements include histidine and glutathione S-transferase (GST) tags. A protease
recognition site, a thrombin or factor Xa recognition site, can be included to permit
cleavage of the desired protein product from the fusion element. Vectors useful for the
construction of gene fusion expression systems are commercially available, for example
from Amersham Pharmacia Biotech, Inc. (Piscataway, NJ).

WO 01/21825 PCT/US00/26042
Specific protein purification protocols useful for isolating glycerol fermentation
enzymes have been described in the literature (see, Seyfried et al., J. ofBact.
178:5793-96 (1996) (glycerol dehydratase); Tobimatsu etal., J. ofBact. 181:4110-13
(1996) (glycerol dehydratase); and Johnson et al., J of Bact. 169:2050-54 (1987) (1,3-
propanediol oxidoreductase). For example, Seyfried et al. describe the use of a vitamin
B, 2-agarose resin as an affinity matrix for the purification of glycerol dehydratase.
5.6 Methods of Biologically Converting Glycerol to 1,3-Propanediol
The present invention provides biological methods for converting glycerol to 1,3-
propanediol. As used herein, a "biological" method of converting glycerol to 1,3-
propanediol is a method that employs a biological catalyst, a glycerol fermentation
enzyme, either as a constituent of a microorganism or in a cell-free enzyme catalyzed
reaction system. In particular, the invention provides biological conversion processes
that can be carried out at high temperatures, catalyzed by a thermophilic organism and/or
thermostable enzyme. In a preferred embodiment, the method is carried out at
temperatures higher than 500C, more preferably at temperatures higher than 60°C, and
employs a thermophilic microorganism capable of fermenting glycerol to 1,3-
propanediol.
In a non-limiting embodiment, the process employs a thermophilic
microorganism that naturally expresses a full complement of thermostable glycerol
fermentation enzymes, sufficient to catalyze all reaction steps in the fermentation of
glycerol to 1,3-propanediol, 1,3-propanediol oxidoreductase, glycerol dehydratase
and/or diol dehydratase. In a particularly preferred embodiment, a strain of C.
viterbiensis, strain JW/MS-VS5', is employed as the biological catalyst.
In an alternative embodiment, the process employs a recombinant microorganism,
preferably a thermophilic recombinant microorganism, that has been genetically
engineered to express one or more exogenous thermostable glycerol fermentation
enzymes. Examples of thermophilic microorganisms include members of the genera
Bacillus, Thermus, Sulfolobus, Thermoanaerobacter, Thermobrachium, and
Caloramator. The term "exogenous," when used in this context, indicates that enzyme is
encoded by an heterologous polynucleotide coding sequence, a polynucleotide
sequence that is not native, or endogenous, to the thermophilic host organism. In a
preferred embodiment, the heterologous polynucleotide sequence encodes a thermostable

WO 01/21825 PCT/USOO/26042
glycerol fermentation enzyme derived from C. viterbiensis, or an enzyme that is
homologous to such an enzyme. In a particularly preferred embodiment, the
heterologous polynucleotide sequence is derived from a strain of C. viterbiensis.
Optionally, a thermophilic microorganism used in the process of the invention can
produce the co-factor adenosyl-cobalamin (coenzyme B, 2 If necessary, synthesis
genes can be introduced the microorganism and/or the media can be supplemented with
vitamin B12
The invention provides a fermentation process for the bioconversion of glycerol
to 1,3-propanediol using a thermophilic organism of the invention. The fermentation
conditions should be optimized, balancing cost and convenience with product yield and
efficiency. The development of a commercial fermentation process typically occurs in a
stepwise fashion, initially using small flasks, then small fermentors (under 10 gallons),
intermediate size fermentors (up to several hundred gallons), and finally, large scale
fermentors (thousands of gallons). At each stage of production, development conditions
are adjusted to produce maximal yields at minimal costs. The organic and inorganic
composition of the medium, as well as the pH, temperature, and oxygen concentration,
are the main factors that are varied to maximize the efficiency of the production process.
Even in a batch process, conditions are often varied during fermentation to achieve the
maximal product yield, and conditions are monitored during the fermentation process to
ensure that critical parameters remain within the allowable limits. The reaction chambers
and substrate solutions should be sterilized prior to the addition of the microbial strain
being used in the production process. When fermenting at lower temperature, infection
of the reaction with microbial contaminants can easily lead to a competitive displacement
of the strain being employed to produce the product, with obviously deleterious results.
An important advantage of the present invention is that by using thermophilic organisms
and/or thermostable enzymes, the reaction can be run at high temperatures where most
potential contaminants are not viable.
The composition of the fermentation medium must include the nutrients essential
to support the growth of the microorganism and formation of the desired product.
Essential nutrients include sources of carbon, nitrogen and phosphorous. The choice of a
particular nutritive source is made on economic as well as biological grounds.
Depending on the nature of the fermentation process, all of the raw ingredients may be

WO 01/21825 PCT/US00/26042
added at the beginning of the fermentation, or nutrients may be fed to the microorganisms
gradually throughout the process.
The pH of the reaction can substantially affect fermentation. The enzymes
involved in forming the desired product all have optimal pH ranges for maximal activity
and limited pH ranges in which activity is maintained. The rapid growth of organisms in
a fermentor can quickly alter the pH of the reaction medium. For example, the
accumulation of acid, acetic acid, can cause the pH of a nonbuffered medium to
decline precipitously, inhibiting or halting production of the desired fermentation
product. To prevent such changes, the fermentation medium should be buffered to
dampen pH changes. Additionally, the pH of the reaction solution normally is
continuously monitored, and acid or base is added as need to maintain it within
acceptable tolerance limits. In fermentation processes employing strain JW/MS-VS5T
the pH of the medium should be maintained between about 5.0 to 7.8, preferably between
about pH 6.0 and 
The temperature of the reaction should also be carefully regulated to achieve
optimal yield of product. Heating coils can be used to maintain elevated temperatures so
as to achieve optimal rates of product formation and to inhibit infection by microbial
contaminants. The heating coils can also be used for periodic sterilization of the
fermentor chamber. Fermentation can be carried out at any temperature consistent with
activity and viability of the microorganism. In a preferred embodiment the invention is
carried out at a temperature of at least 40"C, preferably at a temperature of at least 
and most preferably at a temperature of 60*C or greater. In fermenation processes
employing strain JW/MS-VS5T the temperature should be maintained between 33 and
64"C, preferably between 50 and 64°C, and most preferably between 57 and 64"C.
In addition to its nutritional and environmental parameters, a fermentation process
may be designed as a batch process, which is analogous to inoculating a flask containing
a broth with a microbial culture, or as a continuous flow process, which is analogous to
that of a chemostat. The choice of the process design depends on the economics of both
production and recovery of the desired product. Compared to batch processes, flow-
through fermentors are more prone to contamination with undesired organisms, which
can make quality control difficult to maintain, particularly if the fermentation is
conducted at a moderate temperature. The flow-through design, however, has the
advantage of producing a continuous supply of product that can be recovered at a

WO 01/21825 PCT/US00/26042
constant rate for commercial distribution. By their very nature, batch processes require
significant startup time to initiate the fermentation process, incubation times to allow
fermentation products to accumulate, and recovery times during which the product is
separated from the spent medium and microbial cells.
In another embodiment of the invention, the process can be used to convert a
fermentable carbon substrate other than glycerol to 1,3-propanediol. In a non-limiting
embodiment, a thermophilic microorganism capable of converting a fermentable carbon
substrate, for example, a monosaccharide such as glucose or a polysaccharide, to glycerol
can be engineered to produce 1,3-propanediol by recombinantly introducing into the
microorganism the complement of thermostable 1,3-propanediol producing enzymes
necessary for converting glycerol into 1,3-propanediol. These enzymes can be derived
from a thermophilic organism capable of converting glycerol to 1,3-propanediol, for
example C. viterbiensis. Techniques for recombinantly engineering the enzymatic
activity required to ferment glycerol into 1,3-propanediol into a non-thermophilic
organism are described in U.S. Patent No. 5,686,276, incorporated herein by reference in
its entirety.
Alternatively, a recombinant microorganism of the invention can be engineered to
ferment carbon sources other than glycerol to 1,3-propanediol. The use of such
organisms can be advantageous, particularly for the production of 1,3-propanediol from
inexpensive carbon sources such as glucose or fermentable polysaccharides. For
example, a microorganism capable of fermenting glycerol to 1,3-propanediol can be
engineered to convert a glycolytic intermediate, dihydroxyacetone phosphate, into
glycerol, thereby permitting the introduction of carbon from non-glycerol sources into the
fermentation pathway. WO 98/21340, incorporated herein by reference in its entirety,
describes methods for the production of glycerol from a recombinant organism by
transforming a suitable host cell with an expression cassette comprising either or both a
gene encoding a glycerol-3-phosphate dehydrogenase enzyme and a gene encoding a
glycerol-3-phosphate phosphatase enzyme. Such organisms are able to ferment simple
sugars, glucose, into glycerol. These organisms, or indeed any organism genetically
engineered to produce these enzymes or alternative glycerol synthetic pathway enzymes,
can be used as host cells for the further fermentation of glycerol to 1,3-propanediol using
the compositions and methods of the invention. Further, WO 98/21339, incorporated
herein by reference in its entirety, describes recombinant microorganisms engineered to

WO 01/21825 PCT/US00/26042
express genes encoding glycerol-3-phosphate dehydrogenase, glycerol-3-phosphatase,
glycerol dehydratase and 1,3-propanediol oxidoreductases, and thereby enabled to
convert glucose and other sugars to 1,3-propanediol. Thus, this strategy can be applied
by one skilled in the art to produce 1,3-propanediol from any carbon substrate that can be
converted to either glycerol, dihydroxyacetone, C3 compounds at the oxidation state of
glycerol glycerol 3-phosphate), or C3 compounds at the oxidation state of
dihydroxyacetone dihydroxyacetone phosphate or glyceraldehyde 3-phosphate)
(see, WO 98/21339).
In another embodiment of the invention, the recombinant thermophilic organism
used can be engineered to express, or express at enhanced levels, a protein capable of
suppressing the inactivation of a glycerol fermentation enzyme. For example, techniques
described in WO 98/21341, incorporated herein by reference in its entirety, can be used
to introduce the expression of an enzyme capable of reversing the substrate-mediated
inactivation of glycerol dehydratase.
The process of the invention is carried out in a medium that contains the carbon
substrate to be converted into 1,3-propanediol, preferably as the sole or primary carbon
source. In a preferred embodiment, a basal medium is used wherein glycerol is present as
the sole or primary carbon source. In addition to an appropriate carbon source,
fermentation media must contain suitable minerals, salts, cofactors, buffers and other
components, known to those skilled in the art, suitable for the growth of the cultures and
promotion of the enzymatic activities necessary for the production of 1,3-propanediol.
Particular attention should be given to Co(II) salts and/or vitamin B, 2 or precursors
thereof. A specific non-limiting example of a suitable medium for use in the conversion
of glycerol to 1,3-propanediol by C. viterbiensis is provided below in the Examples.
The process of the invention can be carried out under aerobic or anaerobic
conditions, where anaerobic or microaerobic conditions are preferred. In a particularly
preferred embodiment the process is carried out under a nitrogen or argon atmosphere,
for example, under an atmosphere of N/CO2 at ratio of 80:20. Chemicals may also be
added that react with and remove molecular oxygen from the growth medium. For
example, sodium thioglycollate will react with free oxygen and remove it from solution.
Similarly, the amino acid cysteine and other compounds containing sulthydryl groups can
also be used to scavenge molecular oxygen from a growth medium. For liquid cultures,

WO 01/21825 PCT/USOO/26042
nitrogen may be bubbled through the medium to remove air and traces of oxygen, after
which the culture vessel is tightly sealed to prevent oxygen from reentering.
Additionally, an anaerobic culture chamber may be employed to exclude oxygen
from the atmosphere. Common forms of anaerobic chambers, such as the Gas Pak
system, generate hydrogen, which reacts with the oxygen as a catalyst within the chamber
to produce water. Carbon dioxide is also generated in this system to replace the volume
of gas depleted by the conversion of oxygen to water.
1,3-Propanediol may be identified directly by submitting the media to high
pressure liquid chromatography (HPLC) analysis. For example, fermentation media can
be analyzed on an analytical ion exchange column using a mobile phase of 0.01 N
sulfuric acid in an isocratic fashion. Alternatively, 1,3-propanediol can be identified
using other appropriate analytical techniques, including, but not limited to, gas
chromatography (GC) and gas chromatography-mass spectroscopy (GC-MS).
1,3-propanediol from can be purified from the fermentation media using
techniques known to the skilled artisan, including distillation, centrifugation, filtration
and chromatographic separation. For example, propanediols can be obtained from cell
media by subjecting the reaction mixture to extraction with an organic solvent,
distillation and column chromatography, as described, for example, in U.S. Patent No.
5,356,812, incorporated herein by reference in its entirety. A particularly good organic
solvent for this process is cyclohexane, as described in U.S. Patent No. 5,008,473.
Alternatively, 1,3-propanediol can be produced in a fermentation system
employing enzymes and/or microbial cells adsorbed or bonded to a solid support, such as
cellulose. The bonded and thus immobilized enzymes act as a solid-surface catalyst. A
solution containing the reaction substrate, glycerol, is then passed across the solid
surface. Temperature, pH and oxygen concentration are set at optimal levels to achieve
maximal rates of conversion. This type of process is particularly appropriate when the
desired transformation involves a single metabolic step, the conversion of glycerol
to an intermediate in the formation of 1,3-propanediol, or the conversion of such an
intermediate to 1,3-propanediol. The process is more complex when multiple enzymatic
activities are required to convert an initial substrate into a desired product. Generally the
process medium must include any ancillary factors required for catalysis, such as enzyme
cofactors vitamin B, 2 and reducing equivalents. The desired product can be

WO 01/21825 PCT/US00/26042
purified from the fermentation media using the techniques described supra. When using
such immobilized systems, it is essential to provide conditions that maintain enzymatic
activity and minimize the inactivation or loss of enzyme. When whole cells, rather than
cell-free enzymes, are'employed in such immobilized systems, it is important to maintain
viability of the microorganisms during the process. This process using immobilized cells
generally involves adding necessary growth substrates.
The process according to the invention enables glycerol to be converted
substantially stoichiometrically into 1,3-propanediol. The yield of 1,3-propanediol is
often of the order of 2 moles of 1,3-propanediol from 3 moles of glycerol. In the present
context, substantially is understood to mean glycerol consumption of at least 80%, and
preferably at least 
The 1,3-propanediol produced by the methods and compositions of the invention
is useful in the production of polyesters and films. For example, poly(l,3-propylene
terephthalate) (PPT) has been synthesized by Whinfield and Dickson using
1,3-propandiol as the starting material. The physical properties of PPT are superior than
those of the generally commercially available polyester poly(ethylene terephthalate)
(PET). Other processes for synthesizing PPT are described in, for example, U.S. Pat.
Nos. 5,340,909 and 5,872,204, incorporated by reference herein. Other polymers can
also be synthesized.
The following examples are provided to illustrate, but not limit, the instant
invention.
6 EXAMPLE: Isolation of C viterbiensis Type Strain 
6.1 Materials and Methods
6.1.1 Source of organism
The strain was isolated from a mixed sediment/water sample collected from a
freshwater hot spring in the Bagnaccio Spring area near Viterbo, Italy, in June 1997.
Temperatures at sampling points were 63"C and the pH was 6.6-6.7. Conditions are
described more fully in Canganella et Basic Microhiol. 35:9-19 (1995),
incorporated by reference herein in its entirety.
6.1.2 Media and cultivation
A basal medium used for enrichment, isolation and cultivation was prepared by the
modified Hungate technique under N,/CO, (80:20) gas phase, as described in Ljungdahl et

WO 01/21825 PCTIUS00/26042
al., MANUAL OF INDUSTRIAL MICROBIOLOGY AND BIOTECHNOLOGY (Demain and Solomon
eds., 1896). The basal medium contained (per liter of deionized water): 0.5 g (NH4)SO, 4 
g NHIC1, 2.0 g KIHPO,. 0.04 g MgCl 2*6HO, 0.04 g CaCI,*2H20, 4.2 g NaHCO3, 0.13 g
Na 2S*9H 20, 0.13 g cystein-HCI, 0.3 gyeast extract, 0.001g resazurin, 3.0 g glycerol, 2 ml
vitamins solution (described in Woli et al., J. Biol. Chem. 238:2882-86 (1963)) and 1 ml
trace elements solution. The trace elements solution contained (mmol/l): 
(NH,),Fe(SO 4 1.0 CoCI,*6H,0, 1.0 (NH4 2Ni(SO,) *6H,0, 0.1 
0.1 NaWO0*2HO, 0.5 ZnSO4*7H,O, 0.01 CuCI,*2HO, 0.5 NaSeO,. 0.1 H3BO3.0.5
MnCI,*4H,O, and 0.01 AIK(S0 4),*12H0O. The pH was adjusted to 6.0 (at 25 0C)
Enrichments and pure cultures were grown usually in 10 ml medium in Hungate tubes under
an atmosphere of N,/CO, (80:20). All incubations were at 60 0C unless noted otherwise.
6.1.3 Determination of growth
Growth of bacteria was determined by measuring the increase in optical density at a
wavelength of 600 nm (Spectronic 21, Bausch Lomb, Rochester, NY).
6.1.4 pH and temperature ranges
For the determination of the pH range for growth, the pH was determined at 25 0C
using a model 815 MP pH meter (Fisher Scientific, Pittsburg, PA). Temperature range for
growth was determined using a temperature gradient incubator (Scientific Industries, Inc.,
Bohemia, under shaking (15 spm) in basal medium at pfH2c 
6.1.5 Substrate utilization
The ability of the organism to utilize different substrates was tested using the basal
medium supplemented with autoclaved or filter-sterilized substrates instead of glycerol. The
cultures were incubated for two weeks and monitored for growth by measuring optical
density.
6.1.6 Electron acceptors
The potential use of different electron acceptors was studied in the basal medium
containing glycerol (3g/l) as substrate. The different electron acceptors were added from
autoclaved stock solutions. Cultures grown in basal medium were used as an inoculum 
The use of the electron acceptors (10mM) was monitored by determination of nitrite
production (for nitrate), sulfide production (for sulfate and elemental sulfur) or change in
color (AQDS).

WO 01/21825 PCT/US00/26042
6.1.7 Antibiotic susceptibility
Susceptibility to antibiotics was determined by transferring an exponentially growing
culture into fresh basal media containing 100 g/ml of filter-sterilized antibiotics. The
cultures were incubated for two weeks.
6.1.8 Microscopy
Routine examinations were performed using light microscopy (model PM 
Olympus Optical Co., Ltd, Tokyo, Japan, equipped with phase-contrast optics).
Transmission electron microscopy was performed with a model 100CX electron microscope
(JEOL, Tokyo, Japan). The samples used for ultrathin sectioning were prepared by using
uranyl acetate and lead citrate for poststaining as described by Spurr, J. Ultrastruc. Res.
26:31-43 (1969). Gram staining was performed by the method of Hucker (Doetsch in
MANUAL OF METHODS FOR GENERAL MICROBIOLOGY (Gerhardt et al., eds. 1981)).
6.1.9 Analytical techniques
Determination of glycerol, glucose, short-chain organic acids and alcohols was
performed by high-performance liquid chromatography (HPLC) as previously described in
Svetlitshnyi et al., Int Syst. Microbiol. 46:1131-37 (1996). Molecular hydrogen was
analyzed by gas chromatography (Svetlitshnyi et Production of nitrite was measured
using an enzymatic analysis kit from Boehringer Mannheim (catalog no. 905608). Sulfide
was determined by the method of Cord-Ruwisch, J. Microbiol. Methods. 4:33-36 (1985).
6.1.10 G+C content of DNA
The DNA was isolated and purified using the QIAGEN Genomic DNA purification
protocol according to the manufacturer's instructions. The DNA was digested enzymatically,
and the guanine-plus-cytosine content was determined by separating the nucleosides
by HPLC as described by Whitman et al., Syst. Appl. Microbiol. 7:235-240 (1986) and
Mesbah et al., Int. J Syst. Bacteriol. 39:159-167 (1989).
6.1.11 16S rRNA gene sequence determination and phylogenetic analyses
The extraction ofgenomic DNA, PCR amplification of the 16S rRNA gene and
sequencing of the purified PCR products were carried out as described in Rainey et al.,
nt. J. Syst. Bacteriol. 46:28-96 (1996). Sequence reaction products were purified by
ethanol precipitation and electrophoresed with a model 310 Genetic Analyzer (Applied
Biosystems, Foster City, Calif). The 16S rRNA gene sequences obtained in this study
were aligned against the previously determined low G+C Gram positive sequences
available from the public databases using the ae2 editor Maidak et al., Nucleic Acids Res

WO 01/21825 PCTUSOO/26042
27:171-173 (1999). The programs of the PHYLLP package including DNADIST and
NEIGHBOR were used for the phylogenetic analyses (Felsenstein (1993), phylogenetic
inference package, version 3.5. 1. Department of Genetics, University of Washington,
Seattle). The method of Jukes el al in MAMMALIAN PROTEIN METABOLISM (Munro, ed.,
1969), was used to calculate evolutionary distances. The tree topology was reanalyzed
using 1000 bootstrapped data sets and the programs SEQBOOT, DNADIST and
CONSENSE of the PHYLIIP package Felsenstein, supra.
6.1.12 Nucleotide sequence accession numbers
The 16S rRNA gene sequence determined in this study is deposited with
GenBank under accession number AF18 1848. The accession numbers and strain
designations of the reference 16S rRNA gene sequences used in the phylogenetic
analyses are as follows: Anacrobranca horikoshii DSM 9786 (U2 1809),
Caldicellzzlosirupt'or saccarolyficus ATCC 4 3 4 9 4 T (1-09 178), Caloramalor coolhaasii
DSM 12679 AF104215, Caloramatorfervidus ATCC 43 2 04 5 T (L09187), Caloramalor
indicus ACM 3 9 8 2 T (X75788), Caloramatorproteoclaviiculs DSM 1 0 1 2 4 T (X90488),
Clostridium butyricum ATCC 19398"T (M59085), Clostridium perftingensv ATCC 13 12 4 T
(M59 103), Moorella glycerini DSM 1 12 5 4 (U82327), Moore/ia therinoacetica LJD T
(M59 12 Moorella therrnoautotrophica DSM 1 9 7 4 T (1-09 168), Oxobaci'er pfennigii
DSM 3222"T (X7783 Thermoanaerobacter ethanolicuts ATCC 31 5 5 0 T (L09162),
Thermnoanaerobacturium thermostfrigenes ATCC 3374"T (L09 171), Thermobrachium
celere DSM 8682 X9923 8, Thermosyntropho lipolytica DSM 1 1003"T (X99980).
6.2 Results
6.2.1 Enrichment and isolation
The basal medium containing glycerol was inoculated with approx. 10% of the
sample and incubated at 600C. In addition medium of the same composition exhibiting pH
and pH 9.0 was inoculated with the sample (10% each). Only at pH 6.0 an
enrichment culture was obtained that utilized glycerol. After subsequent transfers (10% v/v),
dilution series of this enrichment culture were prepared and plated out on solid basal
medium 5 agar) using soft agar overlays, which consisted of basal medium containing
0.8 agar. Single colonies were picked, subcultured in liquid medium of the same
composition and formation of 1,3-propanediol was determined by 1{PLC. For subsequent
platings, the procedure was repeated several times using basal medium as well as defined

WO 01/21825 PCT/US00/26042
complex medium described by Kell et al., Biochem. Biophys. Res. Commum. 99:81-88
(1981). Growth in liquid medium was largely enhanced when the cultures were incubated
under shaking. After transferring colonies into liquid basal media at 60 0C under shaking, a
glycerol-fermenting culture was obtained, which was considered as pure and was designated
as strain 
6.2.2 Colony and cell morphology
On plates containing basal medium the colonies appeared after 7-10 days. Once
adapted to growth on plates, subsequent streaks on fresh plates yielded colonies after 2-3
days. The colonies were uniformly round, white and 1.0 to 1.5 mm in diameter. Cells of
strain JW/MS-VS5' were straight to slightly curved rods, 0.4 to 0.6 m in diameter and 2.0 to
m in length. The cells occurred mostly single. No indication of motility was obtained by
using light microscopy. Electron microscopy analysis performed after negative staining did
not reveal the presence of flagella, using cells from different growth stages. The occurrence
of spores was not observed at any growth stage.
6.2.3 Gram-staining reaction and Gram type
The cells stained Gram positive only in the early exponential growth phase.
Ultrathin sections of strain JW/MS-VS5T revealed a thick peptidoglycan, thus the organism
was regarded as Gram type positive Wiegel, Int. J. Syst. Bacteriol., 47:651-56 (1997). This
placement is consistent with the 16S rRNA sequencing data which placed the organism in
the Clostridium-Bacillus branch. In addition to the peptidoglycan, another outer layer was
observed, which might represent an S-layer, however EM micrographs failed to reveal any
geometrical arrays.
6.2.4 Temperature and pH ranges
The temperature range at pWH" 6.0 for growth of strain JW/MS-VS5T was from 33 to
640C with an optimum at 58°C (Fig. No growth was detected at 670 C or at temperatures
lower than 330C. The strain grew in a pH c range from 5.0 to 7.8, with an optimum at pH{ c
6.0-6.5 (Fig. No growth was detected at pIF4.7 and pH"S 8.0. The shortest doubling
time under optimal conditions was 2.8 h.
6.2.5 Substrate utilization and fermentation products
The substrates utilized included glycerol, glucose, fructose, sucrose, cellobiose,
lactose, galactose, mannose (20 mM), starch and yeast extract (5 Strain 
did not use xylose, arabinose, acetate, lactate, formate, methanol, ethanol, n-propanol, i-
propanol, n-butanol, propionate, acetone, succinate, ethylene glycol, 1,2-propanediol,
phenol, benzoate and H,/CO,.

WO 01/21825 PCT/USOO/26042
As shown in Table 1, fermentation of glycerol yielded acetate and 1,3-propanediol as
the only organic metabolic products. No alcohols, diols other than 1,3-propanediol or
organic acids other than acetate were detected in measurable amounts. Significant amounts of
H2 were produced in these cultures.
The obtained fermentation pattern suggests a conversion of glycerol according to the
following equation: 3 Glycerol 2 1,3-Propanediol Acetate CO, H,.
6.2.6 Electron acceptors
In the presence of glycerol as a substrate, strain JW/MS-VS5r did not reduce nitrate
mM), amorphous Fe(I) oxide (90 mM), 9 ,10-anthraquinone-2,6-disulfonic acid
(AQDS), sulfate (10 mM), or precipitated or sublimed S. (30 mM). Production of 1,3-
propanediol was not effected in the presence of any of these electron acceptors. Strain
was not capable of growth with 02 (20% v/v) in the gas phase.
6.2.7 Antibiotic susceptibility
Ampicillin, chloramphenicol, erythromycin, rifampicin, and kanamycin completely
inhibited growth at a concentration of 100 mg/ml of medium. Addition of streptomycin and
tetracyclin of the same concentration resulted in retardation of growth.
6.2.8 DNA base composition
The G+C content of the genomic DNA was 32 mol (HPLC).
6.2.9 16S rRNA gene sequence comparison
An almost complete 16S rRNA gene sequence of strain 
comprising 1480 nucleotides in length was determined. Two data sets were used for
the phylogenetic analyses. Data set one contained the sequence determined in this
study and a selection of reference sequences from the low G+C Gram positive bacteria
while data set two contained the new sequence and the sequences available in the
databases for the five species of the genera Caloramator and Thermobrachium.
Phylogenetic analyses based on data set one comprising 1206 unambiguous nucleotides
between positions 98 and 1469 coli positions, Brosius et al., Proc. Natl. Acad Sci.
USA 75:4801-4805 (1978)) showed the new isolate to cluster together as distinct
lineage within the radiation of the previously described genera Caloramator and
Thermobrachium (Fig. Strain JW/MS-VS5 shares 91.7 to 94.1% 16S rRNA gene
sequence similarity with species of the previously described genera Caloramator and
Thermobrachium and between 83.4 and 87.1% similarity to the sequences of the other
taxa included in data set one and shown in (Fig The second data set containing the

WO 01/21825 PCT/USOO/26042
sequences of the new isolate and those of the five other Caloramator and
Thermobrachium species and comprising 1393 unambiguous nucleotides between
positions 38 and 1469 coli positions, Brosius et al., above) was used to calculate
pairwise similarity values between the taxa of this cluster. The 16S rRNA gene
sequence similarities between strain JW/MS-VS5 and the previously described taxa
were in the range 91.3 to 93.7%. Within the Caloramator/Thermobrachium cluster the
highest sequence similarity value was found between strain JW/MS-VS5 and
Thermobrachium celere. Bootstrap analyses on the branches recovered in Figure 
clearly give support to the Caloramator/Thermobrachium cluster and the relationship
of this group to the genus Clostridium cluster I as defined in Collins et Int. J. Swiss
P. Bacteriol. 44:812-826 (1994).
As is apparent from the phylogenetic analysis (Fig. the strain is closest related
to 7hermobrachium celere and three Caloramator species that form a distinct cluster
within the Gram-type positive Bacillus-Clostridium branch of the phylogenetic tree.
These four species are thermophilic, chemoorganoheterotrophic anaerobic bacteria
exhibiting a low G+C content, which applies for strain JW/MS-VS5" as well. Table 2
shows a comparison of morphological and physiological traits of the five species found
in this cluster.
Table I. Fermentation of glycerol by strain 
Carbon
Substrate Product formed [mM] recovery
Glycero 1,3- Acetate H2
I consumed Propanediol
[mM]
16.2 11.2 4.8 6.5 99
The initial concentration of glycerol used for cultivation of strain JW/MS-VS5 in
basal medium was 33 mM.

WO 0 1/21825 PCTIUSOOI26042
Table 2. Selected properties of strains used in this study: JWIMS-VS5 T
Thermobrachium celere, and Caloramator species C fervidus, C indicus, and C
proleoclasticus.
Property
Strain Spore Tcrmnperature Temperature pH Utilrzatio Shortest Gr+C
Formation Range Optimum optimum n doubling content
of time [hi [mol
glycrol 
JWIMS- A33 -!564 58 6.0-6.5 2.80 32
T. 62-67 8.0-8.5 -0.17 30-31
Meere* <(70-80)
C >37-<80 60-65 8.1 NR 0.33 
indicus
C >37-<80 68 7.0-7.5 NR 0.75 39
fervidus
C !00-<68 55 7.0-7.5 NR 0.50 31
proteo-
clasticus
NR: not reported
*:different strains
7 EXAMPLE: Detection of a dhaB-like gene in 
7.1 Materials and Methods
was characterized by a PCR method using the K pneumoniae dhaB
gene as a positive control. Four sets of primers were used: dhaBl1, consisting of
dhaB I5' GGA AflT GAG ATC TCA OCA ATG AAA AGA TCA AAA CG (SEQ ID
NO: 1) and dhaB 13'GCT GTA GAT TAT TCA ATG GTG TCG GG (SEQ ID NO:2),-
dhaB2, consisting of
GGA ATA GAG ATC TCA GCA ATG CAA GAG ACA ACC C (SEQ ID
NO:3) and dhaB23' GGT CTA GAT GAG TCC GTT ACT AAG TCG (SEQ ED NO:4);
dhaB3, consisting of
dhaIB3S' GGA ATT GAG ATG TCA GGA ATG AGC GAG AAA AGG ATG C (SEQ
ID NO:5) and dhaB33'GCT CTA GAT TAG CTT GGT TTA GG AG-C (SEQ ID
NO: and

WO 01/21825 PCT/US00/26042
dhaX, consisting of
AGG TGG TGC GGA TCC TGT CGA ATC CCT A (SEQ ID NO:7) and
dhaX3' GAT ACG AGA TCT TTA ATT CGC CTG ACC GGC CAG TAG CAG (SEQ
ID NO:8). All primers were purchased from GIBCO BRL. The PCR reaction was carried
out in Hot Start PCR tubes (Molecular Bio-Products, Inc.) with 100 pl of PCR reaction
reagents. Each 100l of PCR reaction contains: 2u of bacteria culture, 20u1
ImMdNTPs, 10ul of 10X PCR buffer (100mMKCL, 100mM (NH4)2S04, 200mMTris-
CL (pl-18.75), 20mM MgSO4. l%Triton, 1 mg/ml BSA)-[Stratagene, La Jolla, CA], 3ul
of each 2 opposing primer set (10 and lul Pfu DNA Polymerase (2.5U/ul) 
[Stratagene, La Jolla, CA.] PCR amplifications were performed in an automated thermal
MiniCycler (MJ Research) at three different annealing temperature to test the best
condition. The final condition is as following: Five initial cycles of denaturation (94C,
low stringency annealing (37C, 1.5 and extension (72C, 2.5 After
initial cycles. 25 additional cycles at higher annealing stringency were conducted:
denaturation (94C, 1.5 annealing (55C, 1.5 and extension (72C, 2.5 mm.).
Tengl of 10X DNA loading dye was added to each sample and 15Ml of each PCR
products were electrophoresed on an agarose gel.
7.2 Results
The positive control give the proper bands as expected. The unknown
microorganism showed faint DNA bands with same size of dhaB 1 and dhab3 but not on
dhaB2 and dhaX. The faint bands may be due to low homology of PCR primers and
target genes. These results suggested that JW/MS-VS-5 r may harbor a dhaB-like gene
which serves the same or similar function as dhaB in K. pneumoniae.
8 EXAMPLE: In vitro determination of JW/MS-VS5 glycerol dehydratase
activity
Toluene-treated cells of strain JW/MS-VS5 T were tested for the ability to convert
glycerol to 3-hydroxypropionaldehye (3-HPA) and 1,2-propanediol to propioinaldehyde.

WO 01/21825 PCTIUS00/26042
8.1 Materials and Methods
Cells were grown anaerobically at 60*C in a mineral salts medium supplemented
with 0.5 g/l yeast extract and 3 g/l glycerol, pH 6.8. Cells were harvested anaerobically
and stored at -700C under nitrogen.
Frozen cells were thawed and treated with toluene in a glove box under anaerobic
conditions. Toluene treatment consisted of the following steps: washing the cells in
1 ml 50 mM KPO, pH 8.0; spinning the cells 6 minutes at 14,000 RPM in an 
Eppendorf 5415C centrifuge; washing the cells in 1.5 ml 50 mM KPO4 pH 8.0; (d)
again spinning the cells 6 minutes at 14,000 RPM in an Eppendorf 5415C centrifuge; (e)
redissolving the cells in 2 ml 50 mM KPO4 pH 8.0; adding 20 pl toluene; shaking
vigorously for 15 minutes; spinning the cells 10 minutes at 14,000 RPM in an
Eppendorf 5415C centrifuge; washing the cells in 2 ml 50 mM KPO4 pH 8.0; (j)
spinning the cells 6 minutes at 14,000 RPM in an Eppendorf 5415C centrifuge; (k)
repeating steps and redissolving the toluene-treated cells in 50 mM KPO, pH
8.0; measuring the absorbance of the dissolved cells at 600 nm; and storing the
cells at 
Reaction mixtures contained in 1.0 ml: 15 mM KPO,, pH 8.0; 0.25 M KCI; 200
mM glycerol or 1,2-propanediol; toluene-treated cells, 0.38 OD600; and 12 pM
coenzyme B12 Reactions were incubated at 60°C anaerobically and samples were taken
at times indicated on Figure 4. 100lp samples were added to 50 pl methyl benzyl
thiazolinone hydrazone (MBTH) solution (6 mg/ml in 375 mM glycine-HCI pH 2.7),
heated at 100*C for 3 minutes, cooled on ice for 30 seconds, and clarified by
centrifugation at 14K RPM for 5 minutes.
The clarified supernatants were then analyzed by detection of product on a C,-
reversed phase HPLC system as follows: the column was a 15 x 4.6 cm Supelco,
Supelcosil LC-8-DB, 3 pM particle size; solvent A was 0.1% TFA; solvent B was
acetonitrile, 0.08% TFA; the gradient was (time[min.]/%B):0/0, 7/45, 17/65,
22/100, 24/100, 25/0, 30/0; ejection volume was 20 pl; injector draw speed was
833.3 pl/min; detection was at 305 nm with a 500 nm reference; and the
bandwidth was 4 for sample wavelength and 80 for reference wavelength. Using this
system, the retention time for 3-HPA was 8.7 min and for propionaldehyde was 10.7 min.

WO 01/21825 PCT/US00/26042
8.2 Results
The time courses for the conversion of glycerol to 3-HPA and 1,2-propanediol to
propionaldehyde at 60*C are shown in Figure 4. The results indicate that glycerol
dehydratase is active at 60 0 C, the growth temperature of the organism. The enzyme is
inactivated by glycerol, but not 1,2-propanediol. Similar inactivation has been reported
in the literature for other glycerol dehydratases. However, the glycerol dehydratase from
Klebsiella is inactive at 60°C. Indeed, no glycerol dehydratase has been reported as
active at 60 0 C.
9 BRIEF DESCRIPTION OF BIOLOGICAL DEPOSIT AND SEQUENCE
LISTING
C. viterbiensis type strain JW/MS-VS5T was deposited on August 27, 1999 with
the ATCC under the terms of the Budapest Treaty on the International Recognition of the
Deposit of Micro-organisms for Purpose of Patent Procedure and is designated as ATCC
PTA-584. "ATCC" refers to the American Type Culture Collection international
depository located at 12301 Parklawn Drive, Rockville, MD 20852 U.S.A. The
designations refer to the accession number of the deposited material.
The 16S rDNA sequence of strain JW/MS-VS5T was deposited in the Genbank
database under accession number AF 181848.
EQUIVALENTS
The foregoing written specification is sufficient to enable one skilled in the art to
practice the invention. Indeed, various modifications of the above-described means for
carrying out the invention which are obvious to those skilled in the field of molecular
biology, biotechnology or related fields are intended to be within the scope of the
following claims.

The claims defining the invention are as follows:
1. A method of converting glycerol to 1,3-propanediol in Caloramator
viterbiensis, the method comprising culturing Caloramator viterbiensis that
ferments glycerol to 1,3 -propanediol under conditions such that 1,3-propanediol
is produced, wherein said Caloramator viterbiensis is the organism deposited as
ATCC designation PTA-584.
2. The method of claim 1, wherein the Caloramator viterbiensis is cultured
at a pH between about 5.0 to 7.8.
3. The method of claim 1, wherein the Caloramator viterbiensis is cultured
at a pH between about 6.0 to 
4. The method of any one of claims 1 to 3, wherein the Caloramator
viterbiensis is cultured at a temperature between about 330 C and about 640 C.
The method of any one of claims 1 to 3, wherein the Caloramator
viterbiensis is cultured at a temperature between about 500C and about 640C.
6. The method of any one of claims 1 to 3, wherein the Caloramator
viterbiensis is cultured at a temperature between about 570C and about 640C.
:l 7. The method of claim 1, wherein the Caloramator viterbiensis is cultured
at a pH of about 6.0 and a temperature of about 580C.
8. The method of any one of claims 1 to 7, wherein the Caloramator
viterbiensis is cultured under anaerobic conditions.
9. The method of claim 8, wherein the Caloramator viterbiensis is cultured
under nitrogen.

V UU vn. VtI 1. UX 0 0 .Jt A.V fYLjLCI l LJ- i-j w
-44-
The method of claim 8, wherein the Caloramator viterbiensis is cultured
under argon.
11. The method of claim 8, wherein the Caloramator viterbiensis is cultured
under a mixture of nitrogen and carbon dioxide in a ratio of nitrogen to carbon
dioxide about 80 to about 
12. The method of any one of claims 8 to 11, wherein the Caloramator
viterbiensis is cultured in the presence of an oxygen scavenger.
13. The method of claim 8, wherein the Caloramator viterbiensis is cultured
in an anaerobic chamber.
14. The method of any one of claims 1 to 7, wherein the Caloramator
viterbiensis is cultured under microaerobic conditions.
15. The method of any one of claims 1 to 14, wherein the Caloramator
viterbiensis is adsorbed on a solid support.
16. The method of any one of claims 1 to 7, wherein the Caloramator
viterbiensis is cultured under aerobic conditions.
l
17. A method of converting glycerol to 1,3-propanediol in Caloramator
viterbiensis, the method comprising culturing Caloramator viterbiensis, wherein
said Caloramator viterbiensis is the organism deposited as ATCC designation
PTA-584 that ferments glycerol to 1,3-propanediol at a temperature between 33
and 640C and the pH of the medium being maintained between pH 6.0 and 
such that 1,3-propanediol is produced.
18. A method of converting glycerol to 1,3-propanediol in Caloramator
viterbiensis, the method comprising culturing Caloramator viterbiensis, wherein
said Caloramator viterbiensis is the organism deposited as ATCC designation
01/02 2005 TUE 15:07 [TX/RX NO 6157] 1005

VJ KJ lA U- J V 
PTA-584 that ferments glycerol to 1,3-propanediol at a temperature between 
and 640 C and the pH of the medium being maintained between pH 6.0 and 
such that 1,3-porpanediol is produced.
19. A method of converting glycerol to 1,3-propanediol in Caloramator
viterbiensis, the method comprising culturing Caloramator viterbiensis, wherein
said Caloramator viterbiensis is the organism deposited as ATCC designation
PTA-584 that ferments glycerol to 1,3-propanediol at a temperature between 57
and 640C and the pH of the medium being maintained between pH 6.0 and 
such that 1,3-propanediol is produced.
20. A method according to claim 1 substantially as hereinbefore described
with reference to the Examples.
o
DATED: 26 July 2004
PHILLIPS ORMONDE FITZPATRICK
Attorneys for:
UNIVERSITY OF GEORGIA RESEARCH FOUNDATION and
GENENCOR INTERNATIONAL, INC
01/02 2005 TUE 15:07 [TX/RX NO 6157] 0006

Thermobrochium ce/ere
cn Co/oromotor indicus
w Coloroaaor proleoclasticus
strain 
Cluoomotor fervidus Ooalrpen~
C/s riim Costridim butyricum C
m Closb~idu hermocel/um
q Moorel/a thermoacetico
m Moore/Ia thermocutotrophica
rmi Moore/Ia glycerini
Thermoonoerobacterium thermosulfurigenes
The rmoonaerobocter ethonolicus
T9 hermosywitropho Iipooytic
Anoerobronco horikoshli
FIG.3

WO 01/21825 WO 0121825PCTIUSOO/26042
IME COURSE ASSAY FOR THE CONVERSION OF GLYCEROL TO 3-HPA BY
ANAEROBICALLY TOLUENIZED JW/MS-VS-5 CELL AT 60 IC UNDER ANAEROBIC
CONDTONS
=E 0.4-
E0.35-
0.3-
I 0.25-
0.2-
10 1'5 
TIME [MIN.]
FIG.4
SUBSTITUTE SHEET (RULE 26)

WO 01/21925 WOO1/182SPCTIUS0O126042
IME COURSE ASSAY FOR THE
CONVERSION OF 1 .2-PROPANEDIOL, TO PROPIONALDEHfYDE BY ANAEROBICALLY
TOLUENIZED JW/MS-VS-5 CELLS AT 60 -C UNDER ANAEROBIC CONDITONS
cn 4000
3500-
3000-
2500-
2000-
1500-
1000-
E 500
5 10 15 
IME [MIN.]
SUBSTITE SHEET (RULE 

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