Novel Enzymes Which Dehydrate Glycerol

  • Published: Apr 24, 2001
  • Earliest Priority: Sep 24 1999
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AU 40179/01 A1
(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 
(19) World Intellectual Property Organization 
International Bureau 1111111111111111 E 11111 iii 1111  I N 
(43) International Publication Date (10) International Publication Number 
29 March 2001 (29.03.2001) PCT W O 01/21825 A2 
(51) International Patent Classification 7: C12P Sciences, Athens, GA 30602 (US). WHITED, Gregory; 
304 South Road, Belmont, CA 94002 (US).  
(21) International Application Number: PCT/US00/26042 
(74) Agents: FRIEBEL, Thomas, E. et al.; Pennie & Edmonds (22) International Filing Date: LLP, 1155 Avenue of the Americas, New York, NY 10036 
22 September 2000 (22.09.2000) (US).  
(25) Filing Language: English (81) Designated States (national): AE, AG, AL, AM, AT, AU.  
(26) Publication Language: English AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, 
DE, DK, DM, DZ, EE, ES, Fl, GB, GD, GE, Gil, GM, HR, 
(30) Priority Data: HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, 
09/405,692 24 September 1999 (24.09.1999) US LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, 
NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, 
(71) Applicants: GENENCOR INTERNATIONAL, INC. TR, TT,17, UA, UG, UZ, VN, YU, ZA, ZW.  
[US/US]; 925 Page Mill Road, Palo Alto, CA 94304 
(US). UNIVERSITY OF GEORGIA RESEARCH (84) Designated States (regional): ARIPO patent (Gi, GM, 
FOUNDATION, INC. [US/US]; Boyd Graduate Studies KE, LS, MW, MZ, SD, SL, SZ, 17, UG, ZW), Eurasian 
Research Center, Athens, GA 30602 (US). patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European 
patent (AT, BE, CH, CY, DE, DK, ES, ES, FR, GB, GR, IE, 
(72) Inventors: SEYFRIED, Markus; 8403 16th Street. Silver IT, LU, MC, NL, PT, SE), OAPI patent (BF, B , CF, CG, 
NSprings, MD 20910 (US). WIEGEL, Juergen; 215 Biol. C, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG).  
(a([Continued on next page] 
e(54) Title: NOVEL ENZYMES WHICH DEHYDRATE GLYCEROL 
1/td [1/h] .* 
0.15-, 
0.1 -4 
0.05
0 10 20 30 40 50 60 70 
TEMPERATURE ['C] 
(57) Abstract: 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 
C 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.

W O 0 1/2 1825 A 2 111111I||||||1111111111111|||||| || |111 111 ||||| i|||  | 111  
Published: For two-letter codes and other abbreviations, refer to the "Guid
Without international search report and to be republished ance Notes on Codes andAbbreviations" appearing at the begin
upon receipt of that report. ning ofeach regular issue of the PCT Gazette.

WO 01/21825 PCTIUSOO/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: (1) by the conversion of ethylene oxide over a 
catalyst in the presence of phosphine, water, carbon monoxide, hydrogen and an acid; 
(2) by the catalytic solution phase hydration of acrolein, followed by reduction; or (3) 
by reacting a hydrocarbon (e.g., 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/USOO/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 (i.e., in a 
microorganism) or in vitro, to catalyze the fermentation of glycerol to 1,3-propanediol.  
See, e.g., 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 1). In the second step, 3-HP is reduced 
to 1,3-propanediol by a NAD*-linked oxidoreductase (Equation 2).  
Glycerol - 3-HP + H 20 (Equation 
1) 
3-1HP + 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 of glycerol 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, e.g., 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 NADP*-) linked glycerol dehydrogenase (Equation 3). The 
DHA, following phosphorylation to dihydroxyacetone phosphate (DHAP) by a DHA 
kinase (Equation 4), becomes available for biosynthesis and for supporting ATP 
generation via, for example, glycolysis.  
WO 01/21825 PCTUSOO/26042 
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 (dha7), 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 PCTUSOO/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 of C. viterbiensis.  
In a non-limiting embodiment, the genome of the culture or cell is at least 85% 
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/USOO/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 95% 
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 
WO 01/21825 PCT/USOO/26042 
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 40*C to 
about 1000 C 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 100 C, and more preferably from about 50' to about 100*C. In a 
preferred embodiment, the method further comprises the step of detecting production 
of 1,3-propanediol and/or acetate by the thermophilic organism.  
4 BRIEF DESCRIPTION OF THE FIGURES 
Figure 1 shows the effect of temperature on growth of strain JW/MS-VS-5, td 
= doubling time.  
Figure 2 shows effect of pH2 sc on growth of strain JW/MS-VS-5 at 60'C, 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 5 nucleotide substitutions per 100 nucleotides.  
Figure 4 shows a time course assay for the conversion of glycerol to 3-HPA by 
anaerobically toluenized JW/MS-VS-5 cells at 60*C 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*C under anaerobic 
conditions.  
DETAILED DESCRIPTION OF THE INVENTION 
Thermophilic organisms are organisms that can survive and grow at elevated 
temperatures where most other organisms (e.g., 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 
WO 01/21825 PCTUSOO/26042 
environments, but are more likely isolated from high temperature environments 
including, for example, hot springs, thermal vents and laundromat effluents. The 
unusual thermostability of thermophilic 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 PCTIUSOO/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 50 C, 
more preferably at temperatures higher than 60'C, still more preferably at 
temperatures higher than 70 C, and most preferably at temperatures higher than 85'C.  
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 (1) thermophilic; (2) capable of fermenting glycerol to 1,3-propanediol; and 
(3) shares "substantial genomic sequence identity" with the type strain JW/MS-VS 5 T, 
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 
WO 01/21825 PCTUSOO/26042 
JW/MS-VS5T. JW/MS-VS5T cells are straight to slightly curved rods, 0.4 to 0.6 Pm 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 58'C. The pH2sc 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 H2 :CO2.. Fermentation 
of glycerol yields acetate and 1,3-propanediol as the primary organic products, with 
significant amounts of H2 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 
90% 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 
JW/MS-VS5 T; 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 JW/MS-VS5T.  
Substantial sequence identity can be determined by the comparison of the 
entire genomic sequences of a putative C. viterbiensis and JW/MS-VS 5T.  
Alternatively, substantial sequence identity can be determined by the comparison of 
the 16S rDNA sequences of a putative C. viterbiensis and JW/MS-VS5T. The 
sequence of all or a portion of the genome of JW/MS-VS5T 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, e.g., 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, e.g., 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 40'C 
in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM 
EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 ptg/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 tg/ml salmon sperm DNA, 
10% (wt/vol) dextran sulfate, and 5-20 X 106 cpm "2P-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 7.4), 5 mM EDTA, 
and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an 
additional 1.5 h at 60 C. Filters are blotted dry and exposed for autoradiography. If 
necessary, filters are washed for a third time at 65-68 C and reexposed to film. Other 
conditions of low stringency which can be used are well known in the art (e.g., 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 
JW/MS-VS5T 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, 
WO 01/21825 PCT/USOO/26042 
0.5% 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 55 *C, and then washed twice for 30 
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-VS5T 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
VS5T 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 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 ptg/ml 
denatured salmon sperm DNA. Filters are hybridized for 48 h at 65 0 C in 
prehybridization mixture containing 100 ptg/ml denatured salmon sperm DNA and 
5-20 X 106 cpm of "2P-labeled probe. Washing of filters is done at 370 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 0.1X SSC at 500C 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-VS5 T, including progeny possessing altered genotypes and/or phenotypes 
relative to type strain JW/MS-VS5T. 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 PCTUSOO/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, e.g., 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, e.g., 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, Md.), or Sigma Chemical Company (St. Louis, Mo.).  
A thermophilic organism of the invention, e.g., C. viterbiensis, can be isolated 
from any environment that is conducive to growth of thermophilic organisms, e.g., 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/USOO/26042 
concentration of approximately 10% (w/v). 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 50*C, 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, J., "Methods for Isolation and Study of Thermophiles", 
Chapter 4 in THERMOPHILES: GENERAL, MOLECULAR AND APPLIED MICROBIOLOGY, 
T.D. Brock, ed. (John Wiley & Sons, N.Y.), 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 (e.g., 1.5% agar) using soft agar 
overlays (e.g., 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," i.e., 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 
C, 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 JW/MS-VS5T.  
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, i.e., dehydratases having preferred substrates of glycerol and 1,2
propanediol, respectively.  
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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. of Biochem 245:398-41 (1997), Honda et al., J. of Bact. 143:1458
(1980), and Macis et aL., FFAISMicrobiology 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 
Bact. 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 of C. viterbiensis; and 
(b) 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, i.e., 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'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: (a) its amino acid sequence is encoded by a 
WO 01/21825 PCTIUSOO/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, i.e., 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 (b) 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, i.e., hybridization to filter-bound DNA in 0.5 M NaHP0 4, 7% 
sodium dodecyl sulfate (SDS), I mM EDTA at 65'C, and washing in 0.1xSSC/0.1% 
SDS at 68 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, i.e., a protein 
fusion. Chimeras can be useful, for example, for improving the stability of the enzyme 
or facilitating purification, e.g., 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, i.e., 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/USOO/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, e.g., 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 (e.g., 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, e.g., Macis et 
al., FEMSMocrobiology Letters 164:21-28 (1998) (C. Pasteurianum); Seyfried et al., 
J. of Bacteriology 178:5793-96 (1996) (C. freundii); and Tobimatsu et al., J. Biol.  
Chem. 271:22352-57 (1996) (K. pneumoniae)). The nucleotide sequences of dhaT 
gene have also been reported (see, e.g., Luers et al., FEMSMicrobiol. Lett. 154:337-45 
(1997) (C. 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 (e.g., as detailed below by way of illustrative 
embodiments in dhaB15' (SEQ ID NO:1); dhaB13' (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 
("PCR"). 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 PCTUSOO/26042 
nucleic acids present in the sample are available for interaction with a complementary 
probe, e.g., 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 homologous 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, e.g., 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 ("ORF"), 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 PCT/USOO/26042 
For general background on molecular biology techniques and on how to prepare a 
cDNA library and a genomic library, see, e.g., Ausubel et a., 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. Natl. 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 01/21825 PCT/USOO/26042 
e.g., 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, e.g., 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, i.e., heterologous, thermostable 
glycerol fermentation enzymes. The term "exogenous," when used in this context, 
indicates that enzyme is encoded by an heterologous polynucleotide coding sequence, 
i.e., 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 of C. viterbiensis, such as JW/MS-VS5T.  
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/USOO/26042 
can be derived from a plasmid, a virus (such as bacteriophage T7 or a M13 derived 
phage), or a cosmid. Protocols for obtaining and using such vectors are known to those 
in the art. See, e.g., 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, e.g., stabilization or simplified 
purification of expressed recombinant product.  
To direct the glycerol fermentation enzyme into the secretory pathway of the host 
cells, a secretary signal sequence may be provided in the expression vector. The 
secretory signal sequence is joined to the polynucleotide sequence encoding the glycerol 
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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 B, 2). If necessary, B1 2 synthesis genes can be introduced 
the microorganism and/or the media can be supplemented with vitamin B12.  
5.5 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, e.g., 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 
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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 (e.g., 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, e.g.. 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 (e.g., ammonium sulfate 
precipitation), chromatography (e.g., ion exchange, affinity, hydrophobic, 
chromatofocusing, and size exclusion) or electrophoretic procedures (e.g., preparative 
isoelectric focusing (IEF) (see, e.g., 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, e.g., 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, e.g., Seyfried et al., J. ofBact.  
178:5793-96 (1996) (glycerol dehydratase); Tobimatsu et al., J. of Bact. 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 
B12-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, e.g., 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 50'C, 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, e.g., 1,3-propanediol oxidoreductase, glycerol dehydratase 
and/or diol dehydratase. In a particularly preferred embodiment, a strain of C.  
viterbiensis, e.g., strain JW/MS-VS5T, 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, i.e., 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 B12). If necessary, B12 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/USOO/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, e.g., 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-VS5 T, 
the pH of the medium should be maintained between about 5.0 to 7.8, preferably between 
about pH 6.0 and 6.5.  
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 50'C, 
and most preferably at a temperature of 60*C or greater. In fermenation processes 
employing strain JW/MS-VS5 T, 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 PCTUSOO/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, e.g., 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, e.g., 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/USOO/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 (e.g., glycerol 3-phosphate), or C3 compounds at the oxidation state of 
dihydroxyacetone (e.g., dihydroxyacetone phosphate or glyceraldehyde 3-phosphate) 
(see, e.g., 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 B12 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 N2/CO 2 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 sulfhydryl groups can 
also be used to scavenge molecular oxygen from a growth medium. For liquid cultures, 
WO 01/21825 PCTIUSOO/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, e.g., 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, e.g., 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 (e.g., vitamin B12) and reducing equivalents. The desired product can be 
WO 01/21825 PCT/USOO/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 95%.  
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(1,3-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 JW/MS-VS5 T 
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 al., J. Basic Microbiol. 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 N2/CO2 (80:20) gas phase, as described in Ljungdahl et 
WO 01/21825 PCT/USOO/26042 
al., MANUAL OF INDUSTRIAL MICROBIOLOGY AND BIOTECHNOLOGY (Demain and Solomon 
eds., 1896). The basal medium contained (per liter of demonized water): 0.5 g (NH4)2SO, 0.5 
g NH4 Cl, 2.0 g KH 2PO, 0.04 g MgCl 2*6HO, 0.04 g CaCl.*2H 20, 4.2 g NaHCO 3, 0.13 g 
Na2S*9H 20, 0.13 g cystein-HCl, 0.3 g yeast extract, 0.001g resazurin, 3.0 g glycerol, 2 ml 
vitamins solution (described in Wolin et al., J Biol. Chem. 238:2882-86 (1963)) and I ml 
trace elements solution. The trace elements solution contained (mmol/l): 2.0 
(NH4)2Fe(SO 4)2*6H20, 1.0 CoCl2*6H20, 1.0 (NH 4)2Ni(S0 4)2*6H20, 0.1 NaMoO 4*2H20, 
0.1 Na1WO4*2HO, 0.5 ZnSO 4*7H 20, 0.01 CuCl2*2HO, 0.5 Na2Se03, 0.1 H3B0 3, 0.5 
MnCl2*4H.0, and 0.01 AIK(SO 4)2*12H 20. The pH was adjusted to 6.0 (at 250C).  
Enrichments and pure cultures were grown usually in 10 ml medium in Hungate tubes under 
an atmosphere of N2 /CO, (80:20). All incubations were at 600C 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'C 
using a model 815 MiP pH meter (Fisher Scientific, Pittsburg, PA). Temperature range for 
growth was determined using a temperature gradient incubator (Scientific Industries, Inc., 
Bohemia, N.Y.) under shaking (15 spm) in basal medium at pHWsc 6.0.  
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 (10 
% v/v). 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).  
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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 1 0AD, 
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, . 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. J.. Syst. Microbiol. 46:1131-37 (1996). Molecular hydrogen was 
analyzed by gas chromatography (Svetlitshnyi et al.). 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 (G+C) content was determined by separating the nucleosides 
by HPLC as described by Whitman et al., Syst. Apple. 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 of genomic DNA, PCR amplification of the 16S rRNA gene and 
sequencing of the purified PCR products were carried out as described in Rainey et al., 
Int. 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 PCTIUSOO/26042 
27:171-173 (1999). The programs of the PHYLIP 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 et 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 PHYLIP 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 AF181848. The accession numbers and strain 
designations of the reference 16S rRNA gene sequences used in the phylogenetic 
analyses are as follows: Anaerobranca horikoshii DSM 9 7 8 6T (U21809), 
Caldicellulosiruptor saccaro!yticus ATCC 4 34 9 4T (109178), Caloramator coolhaasii 
DSM 12679 AF104215, Caloramatorfervidus ATCC 4 3 2 045T (L09187), Caloramator 
indicus ACM 3 9 82T (X75788), Caloramatorproteoc/asticus DSM 10 12 4T (X90488), 
Clostridium butyricum ATCC 19 3 9 8T (M59085), Clostridiumperfringens ATCC 1312 4T 
(M59103), Moorella glycerini DSM 112 54T (U82327), Moorella thermoacetica LJDT 
(M59121), Moorella therrnoautotrophica DSM 19 74T (L09168), Oxobacter pfennigii 
DSM 32 2 2T (X77838), Thermoanaerobacter ethanolicus ATCC 31 5 50 T (L09162), 
Thernoanaerobacterium thermosulfurigenes ATCC 337 4T (L09171), Thermobrachium 
celere DSM 8682 X99238, Thermosyntropho lipolytica DSM 110 0 3T (X99980).  
6.2 Results 
6.2.1 Enrichment and isolation 
The basal medium containing glycerol was inoculated with approx. 10% (w/v) of the 
sample and incubated at 60 0C. In addition medium of the same composition exhibiting pH 
7.5 and pH 9.0 was inoculated with the sample (10% (w/v) 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 (1.5 % 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 HPLC. For subsequent 
platings, the procedure was repeated several times using basal medium as well as defined 
WO 01/21825 PCTUSOO/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'C under shaking, a 
glycerol-fermenting culture was obtained, which was considered as pure and was designated 
as strain JW/MS-VS5T.  
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-VS5T were straight to slightly curved rods, 0.4 to 0.6 m in diameter and 2.0 to 
3.0 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 pH2 sc 6.0 for growth of strain JW/MS-VS5T was from 33 to 
640C with an optimum at 580C (Fig. 1). No growth was detected at 670C or at temperatures 
lower than 33 C. The strain grew in a pHsc range from 5.0 to 7.8, with an optimum at pH 5 c 
6.0-6.5 (Fig. 2). No growth was detected at pH2c 4.7 and pWc 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 g/l). Strain JW/MS-VS5T 
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 H2/CO2.  
WO 01/21825 PCTUSOO/26042 
As shown in Table 1, fermentation of glycerol yielded acetate and 1,3-propanediol as 
the only organic metabolic products. No C1-C3 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 2 + 112
6.2.6 Electron acceptors 
In the presence of glycerol as a substrate, strain JW/MS-VS5T did not reduce nitrate 
(10 mM), amorphous Fe(II) 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 
JW/MS-VS5T 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 C+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 JW/MS-VS5 
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 (E. 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 Caloranator and 
Thermobrachium (Fig. 3). 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 3). 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 (E. 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 (93.7%) was found between strain JW/MS-VS5 and 
Thermobrachium celere. Bootstrap analyses on the branches recovered in Figure 5 
clearly give support to the Caloramator/Thermobrachiuni cluster and the relationship 
of this group to the genus Clostridium cluster I as defined in Collins et al., Int. J. Swiss 
P. Bacteriol. 44:812-826 (1994).  
As is apparent from the phylogenetic analysis (Fig. 3), the strain is closest related 
to Thermobrachium 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-VS5T as well. Table 2 
shows a comparison of morphological and physiological traits of the five species found 
in this cluster.  
Table 1. Fermentation of glycerol by strain JW/MS-VS5T 
Carbon 
Substrate Product formed [mM] recovery 
____ ____ ___ ____ ____ 
___[%] 
Glycero 1,3- Acetate H2 1 consumed Propanediol 
[mM] 
16.2 11.2 4.8 6.5 99 
The initial concentration of glycerol used for cultivation of strain JW/MS-VS5T in 
basal medium was 33 mM.  

WO 01/21825 PCT/US00/26042 
Table 2. Selected properties of strains used in this study: JW/MS-VS 5T 
Thermobrachium celere, and Caloramator species C. fervidus, C. indicus, and C.  
proteoclasticus.  
Property 
Strain Spore Termperature Temperature pH Utilizatio Shortest G+C 
Formation Range Optimum optimum n doubling content 
of time [h] [mol 
glycerol %] 
JW/MS- - 233 - 64 58 6.0-6.5 + 2.80 32 
VS5T 
T. - >(37-45)- 62-67 8.0-8.5 - 0.17 30-31 
celere* <(70-80) 
C. - >37-<80 60-65 8.1 NR 0.33 25 
indicus 
C. + >37-<80 68 7.0-7.5 NR 0.75 39 
fervidus 
C. + 30-<68 55 7.0-7.5 NR 0.50 31 
proteo
clasticus 
NR: not reported 
*: different strains 
7 EXAMPLE: Detection of a dhaB-ike gene in JW/MS-VS5T 
7.1 Materials and Methods 
JW/MS-VS5T was characterized by a PCR method using the K. pneumoniae dhaB 
gene as a positive control. Four sets of primers were used: (a) dhaB1, consisting of 
dhaB 15' GGA ATT CAG ATC TCA GCA ATG AAA AGA TCA AAA CG (SEQ ID 
NO:1) and dhaB 13' GCT CTA GAT TAT TCA ATG GTG TCG GG (SEQ ID NO:2); 
(b) dhaB2, consisting of 
dhaB25' GGA ATA CAG ATC TCA GCA ATG CAA CAG ACA ACC C (SEQ ID 
NO:3) and dhaB23' GCT CTA GAT CAC TCC CTT ACT AAG TCG (SEQ ID NO:4); 
(c) dhaB3, consisting of 
dhaB35' GGA ATT CAG ATC TCA GCA ATG AGC GAG AAA ACC ATG C (SEQ 
ID NO:5) and dhaB33' GCT CTA GAT TAG CTT CCT TTA CGC AGC (SEQ ID 
NO:6); and 
WO 01/21825 PCT/USOO/26042 
(d) dhaX, consisting of 
dhaX5' 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 100,ul of PCR reaction contains: 2ul of bacteria culture, 20ul 
1mMdNTPs, 1Oul of lOX PCR buffer (1OOmMKCL, 100mM (NH4)2SO4, 200mMTris
CL (pH8.75), 20mM MgSO4. 1%Triton, 1 mg/ml BSA)-[Stratagene, La Jolla, CA], 3ul 
of each 2 opposing primer set (10 O.D./ul), 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, 
1.5 mm.), low stringency annealing (37C, 1.5 mm.), and extension (72C, 2.5 mm.). After 
initial cycles. 25 additional cycles at higher annealing stringency were conducted: 
denaturation (94C, 1.5 mm.). annealing (55C, 1.5 mm.), and extension (72C, 2.5 mm.).  
Tenyl of I OX DNA loading dye was added to each sample and 15pl 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-5T 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-VS5T glycerol dehydratase 
activity 
Toluene-treated cells of strain JW/MS-VS5T were tested for the ability to convert 
glycerol to 3-hydroxypropionaldehye (3-HPA) and 1,2-propanediol to propioinaldehyde.  

WO 01/21825 PCT/USOO/26042 
8.1 Materials and Methods 
Cells were grown anaerobically at 60 0 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 -70 C under nitrogen.  
Frozen cells were thawed and treated with toluene in a glove box under anaerobic 
conditions. Toluene treatment consisted of the following steps: (a) washing the cells in 
I ml 50 mM KPO 4 pH 8.0; (b) spinning the cells 6 minutes at 14,000 RPM in an 
Eppendorf 5415C centrifuge; (c) washing the cells in 1.5 ml 50 mM KPO 4 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 KPO 4 pH 8.0; (f) adding 20 pl toluene; (g) shaking 
vigorously for 15 minutes; (h) spinning the cells 10 minutes at 14,000 RPM in an 
Eppendorf 5415C centrifuge; (i) washing the cells in 2 ml 50 mM KPO 4 pH 8.0; (j) 
spinning the cells 6 minutes at 14,000 RPM in an Eppendorf 5415C centrifuge; (k) 
repeating steps (i) and (j); (1) redissolving the toluene-treated cells in 50 mM KPO 4 pH 
8.0; (in) measuring the absorbance of the dissolved cells at 600 nm; and (n) storing the 
cells at -70*C.  
Reaction mixtures contained in 1.0 ml: 15 mM KPO 4, pH 8.0; 0.25 M KCl; 200 
mM glycerol or 1,2-propanediol; toluene-treated cells, 0.38 OD600; and 12 pM 
coenzyme B, 2. Reactions were incubated at 60'C anaerobically and samples were taken 
at times indicated on Figure 4. 100pl samples were added to 50 [1 methyl benzyl 
thiazolinone hydrazone (MBTH) solution (6 mg/ml in 375 mM glycine-HCl 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: (a) the column was a 15 x 4.6 cm Supelco, 
Supelcosil LC-8-DB, 3 p.M particle size; (b) solvent A was 0.1% TFA; (c) solvent B was 
90% acetonitrile, 0.08% TFA; (d) the gradient was (time[min.]/%B):0/0, 7/45, 17/65, 
22/100, 24/100, 25/0, 30/0; (e) ejection volume was 20 pl; (f) injector draw speed was 
833.3 p.l/min; (g) detection was at 305 nm with a 500 nm reference; and (h) 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 PCTIUSOO/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*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'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 AF181848.  
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.  
WO 01/21825 PCT/USOO/26042 
WHAT IS CLAIMED IS: 
1. A method of converting glycerol to 1,3-propanediol in a thermophilic organism, 
the method comprising: 
providing a thermophilic organism that ferments glycerol to 1,3-propanediol; and 
culturing the thermophilic organism under conditions such that 1,3-propanediol is 
produced.  
2. The method of Claim 1, further comprising the step of collecting 1,3-propanediol 
produced by the thermophilic organism.  
3. The method of Claim 2, further comprising the step of polymerizing the 1,3
propanediol into a polymer.  
4. The method of Claim 3, wherein the polymer is poly(1,3-propylene terephthalate) 
(PPT).  
5. The method of Claim 1, wherein the thermophilic organism is Caloramator 
viterbiensis.  
6. The method of Claim 5, wherein the thermophilic organism is derived from the 
organism deposited as ATCC designation PTA-584.  
7. 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 
adding a reducing agent capable of reducing 3-hydroxypropionaldehyde to 1,3
propanediol.  
8. The method of Claim 7, wherein the reduction of the 3-hydroxypropionaldehyde 
to 1,3-propanediol is catalyzed by a thermostable 1,3-propanediol oxidoreductase.  
WO 01/21825 PCT/USOO/26042 
9. The method of Claim 7 or 8, further comprising the step of collecting 1,3
propanediol.  
10. The method of Claim 9, further comprising the step of polymerizing the 1,3
propanediol into a polymer.  
11. The method of Claim 10, wherein the polymer is poly(1,3-propylene 
terephthalate) (PPT).  
12. The method of Claim 8, wherein the thermostable dehydratase enzyme is derived 
from a thermophilic organism.  
13. The method of Claim 12, wherein the thermophilic organism is Caloramator 
viterbiensis.  
14. The method of Claim 12, wherein the thermophilic organism is derived from the 
organism deposited as ATCC designation PTA-584.  
15. An isolated thermostable glycerol fermentation enzyme that is derived from C.  
viterbiensis.  
16. An isolated thermostable glycerol fermentation enzyme that is derived from the 
organism deposited as ATCC designation PTA-584.  
17. An isolated thermostable glycerol fermentation enzyme that is homologous to a 
thermostable glycerol fermentation enzyme derived from C. viterbiensis.  
18. The isolated thermostable glycerol fermentation enzyme of Claim 11, 12 or 13 
that is a dehydratase.  
19. The enzyme of Claim 18 that is glycerol dehydratase.  
20. The enzyme of Claim 15, 16 or 17 that is 1,3-propanediol oxidoreductase.  
WO 01/21825 PCT/US00/26042 
21. An isolated culture or cell of Caloramator viterbiensis..  
22. The isolated culture or cell of Claim 21, wherein the genome of the culture or cell 
is at least 95% identical to the genome of the organisms deposited as ATCC designation 
PTA-584.  
23. The isolated culture or cell of Claim 21, wherein the genome of the culture or cell 
is at least 99% identical to the genome of the organisms deposited as ATCC designation 
PTA-584.  
24. The isolated culture or cell of Claim 21, wherein the 16S rDNA sequence of the 
culture or cell is at least 95% identical to the 16S rDNA of the organisms deposited as 
ATCC designation PTA-584.  
25. The isolated culture or cell of Claim 21, wherein the 16S rDNA sequence of the 
culture or cell is at least 99% identical to the 16S rDNA of the organisms deposited as 
ATCC designation PTA-584.  
26. The isolated culture or cell of Claim 21 that is a progeny of the organisms 
deposited as ATCC designation PTA-584.  
27. A method of cloning a polynucleotide sequence that encodes a thermostable 
glycerol fermentation enzyme, the method comprising: 
hybridizing a polynucleotide probe 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 the polynucleotide probe.  
28. The method of Claim 27, wherein a polymerase chain reaction using a second 
polynucleotide probe is used to amplify the polynucleotide sequence that binds to the 
polynucleotide probes.  
WO 01/21825 PCTUSOO/26042 
29. The method of Claim 27 or 28, wherein the thermostable glycerol fermentation 
enzyme is derived from a thermophilic organism identified as fermenting glycerol to 1,3
propanediol.  
30. The method of Claim 29, wherein the thermophilic organism is Caloramator 
viterbiensis.  
31. The method of Claim 30, wherein the polynucleotide probe is homologous to a 
portion of a known dhaB gene.  
32. The method of Claim 31, wherein the dhaB gene is from Klebsiella.  
33. The method of Claim 29, wherein at least one polynucleotide probe is selected 
from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5 and SEQ ID 
NO:6.  
34. The method of Claim 33, wherein the polynucleotide probe and the second 
polynucleotide probe are SEQ ID NO: 1 and SEQ ID NO:2.  
35. The method of Claim 33, wherein the polynucleotide probe and the second 
polynucleotide probe are SEQ ID NO:5 and SEQ ID NO:6.  
36. 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.  

WO 01/21825 PCT/USOO/26042 
37. The method of Claim 36, wherein the thermostable glycerol fermentation 
enzyme is derived from a thermophilic organism identified as fermenting glycerol to 1,3
propanediol.  
38. The method of Claim 37, wherein the thermophilic organism is Caloramator 
viterbiensis.  
39. The method of Claim 38, wherein the Caloramator viterbiensis is derived from 
the organism deposited as ATCC designation PTA-584.  
40. 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.  
41. The method of Claim 40, wherein the sample is incubated at a temperature in the 
range of about 40*C to about 100'C.  
42. The method of Claim 40, wherein the sample is incubated under anaerobic 
conditions.  
43. The method of Claim 40, wherein the sample is obtained from a natural source 
having a temperature of between about 50' to about 100 C.  
44. The method of Claim 40, further comprising the step of detecting production of 
1,3-propanediol by the thermophilic organism.  
45. The method of Claim 40, further comprising the step of determining the 
production of acetate by the thermophilic organism.  

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