U.S. patent application number 10/219549 was filed with the patent office on 2003-09-11 for terephthalic acid producing proteobacteria.
Invention is credited to Bramucci, Michael G., McCutchen, Carol M., Nagarajan, Vasantha, Thomas, Stuart M..
Application Number | 20030170836 10/219549 |
Document ID | / |
Family ID | 26784182 |
Filed Date | 2003-09-11 |
United States Patent
Application |
20030170836 |
Kind Code |
A1 |
Bramucci, Michael G. ; et
al. |
September 11, 2003 |
Terephthalic acid producing proteobacteria
Abstract
This invention relates to a biocatalytic process to produce
terephthalic acid and isophthalic acid from p-xylene and m-xylene,
respectively. Terephthalic acid has been prepared by oxidizing
p-xylene with bacteria belonging to the genus Burkholderia.
Conversion of p-xylene into terephthalic acid is accomplished by a
single bacterial strain that produces all of the requisite enzymes.
In addition, this invention relates to the preparation of
isophthalic acid from a mixture of m- and p-xylene.
Inventors: |
Bramucci, Michael G.;
(Folsom, PA) ; McCutchen, Carol M.; (Dickson,
TN) ; Nagarajan, Vasantha; (Wilmington, DE) ;
Thomas, Stuart M.; (Wilmington, DE) |
Correspondence
Address: |
E I DU PONT DE NEMOURS AND COMPANY
LEGAL PATENT RECORDS CENTER
BARLEY MILL PLAZA 25/1128
4417 LANCASTER PIKE
WILMINGTON
DE
19805
US
|
Family ID: |
26784182 |
Appl. No.: |
10/219549 |
Filed: |
August 15, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10219549 |
Aug 15, 2002 |
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09617854 |
Jul 17, 2000 |
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6461840 |
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09617854 |
Jul 17, 2000 |
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09342579 |
Jun 29, 1999 |
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6187569 |
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60091645 |
Jul 2, 1998 |
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Current U.S.
Class: |
435/142 ;
435/147; 435/156; 435/252.1; 435/253.3 |
Current CPC
Class: |
C12P 7/46 20130101 |
Class at
Publication: |
435/142 ;
435/252.1; 435/253.3; 435/156; 435/147 |
International
Class: |
C12P 007/44; C12P
007/24; C12P 007/22; C12N 001/20 |
Claims
What is claimed is:
1. A TPA producing microorganism isolated by the process
comprising: (i) culturing a sample suspected of containing a TPA
producing microorganism in a suitable growth medium containing at
least one aromatic organic substrate selected from the group
consisting of p-xylene, p-toluic acid, and terephthalic acid; (ii)
selecting those microorganisms which are able to use every
substrate selected individually from the group consisting of
p-xylene, p-toluic acid, and terephthalic acid as a sole carbon
source; (iii) contacting the microorganisms selected in step (ii)
with p-xylene to form a reaction medium; and (iv) monitoring the
reaction medium over time for the presence of terephthalic acid,
whereby the production of terephthalic acid indicates the presence
of a TPA producing microorganism.
2. The TPA producing microorganism of claim 1 wherein the
microorganism comprises the genes encoding the TPA biosynthetic
pathway
3. The TPA producing microorganism of claim 2 wherein the
microorganism is a Proteobacteria.
4. The TPA producing microorganism of claim 3 selected from the
group consisting of, Burkholderia, Alcaligenes, Pseudomonas,
Shphingomonas and Comamonas.
5. The TPA producing microorganism of claim 4 selected from the
group consisting of IR3 (ATCC 202150) and IR10 (ATCC 202151).
6. The TPA producing microorganism of claims 3 or 4 wherein the 16s
RNA of the TPA producing microorganism has at least 95% identity
with the 16s rRNA sequence set forth in SEQ ID NO:1.
7. A process for the isolation of a TPA producing microorganism
comprising: (i) culturing a sample suspected of containing a TPA
producing microorganism in a suitable growth medium containing at
least one aromatic organic substrate selected from the group
consisting of p-xylene, p-toluic acid, and terephthalic acid; (ii)
selecting those microorganisms which are able to use every
substrate selected individually from the group consisting of
p-xylene, p-toluic acid, and terephthalic acid as a sole carbon
source; (iii) contacting the microorganisms selected in step (ii)
with p-xylene to form a reaction medium; and (iv) monitoring the
reaction medium over time for the presence of terephthalic acid,
whereby the production of terephthalic acid indicates the presence
of a TPA producing microorganism.
8. A process for the production of terephthalic acid comprising:
(i) contacting an isolated TPA producing microorganism with an
aromatic organic substrate whereby terephthalic acid accumulates;
and (ii) optionally recovering the terephthalic acid.
9. The process of claim 8 wherein the aromatic organic substrate is
selected from the group consisting of p-xylene, 4-methylbenzyl
alcohol, p-tolualdehyde, p-toluic acid, 4-carboxybenzyl alcohol,
and 4-carboxybenzaldehyde.
10. The process of claim 8 wherein the TPA producing microorganism
is isolated by the process of claim 7.
11. The process of claim 10 wherein the isolated TPA producing
microorganism is a Proteobacteria.
12. The process of claim 11 wherein the isolated TPA producing
microorganism is selected from the group consisting of,
Burkholderia, Alcaligenes, Pseudomonas, Shphingomonas and
Comamonas.
13. The process of claim 12 wherein the 16s RNA of the TPA
producing microorganism has at least 97% identity with the 16s rRNA
sequence set forth in SEQ ID NO:1.
14. The process of claim 13 wherein the isolated TPA producing
microorganism is Burkholderia sp. selected from the group
consisting of IR3 (ATCC 202150) and IR10 (ATCC 202151).
15. A process for the production of 4-carboxybenzyl alcohol
comprising: (i) contacting an isolated TPA producing microorganism
with an aromatic organic substrate whereby 4-carboxybenzyl alcohol
accumulates; and (ii) optionally recovering the 4-carboxybenzyl
alcohol.
16. A process for the production of 4-carboxybenzaldehyde
comprising: (i) contacting an isolated TPA producing microorganism
with an aromatic organic substrate whereby 4-carboxybenzaldehyde
accumulates; and (ii) optionally recovering the
4-carboxybenzaldehyde.
17. A process for the production of 1,4-benzenedimethanol
comprising: (i) contacting an isolated TPA producing microorganism
with an aromatic organic substrate whereby 1,4-benzenedimethanol
accumulates; and (ii) optionally recovering the
1,4-benzenedimethanol.
18. A process for the production of terephthalaldehyde comprising:
(i) contacting an isolated TPA producing microorganism with an
aromatic organic substrate whereby terephthalaldehyde accumulates;
and (ii) optionally recovering the terephthalaldehyde.
19. The process of either of claims 15, 16, 17 or 18 wherein the
isolated TPA producing microorganism is isolated by the process of
claim 7.
20. The process of claim 19 wherein the isolated TPA producing
microorganism is Burkholderia sp. selected from the group
consisting of IR3 (ATCC 202150) and IR10 (ATCC 202151).
21. The process of either of claims 15, 16, 17 or 18 wherein the
aromatic organic substrate is selected from the group consisting of
p-xylene, 4-methylbenzyl alcohol, p-tolualdehyde, p-toluic acid,
and 4-carboxybenzyl alcohol.
22. A process for the production of isophthalic acid comprising:
(i) contacting an isolated TPA producing microorganism with an
aromatic organic substrate and at least one suitable induction
compound whereby isophthalic acid accumulates; and (ii) optionally
recovering the isophthalic acid.
23. The process of claim 22 wherein the isolated TPA producing
microorganism is isolated by the process of claim 7.
24. The process of claim 23 wherein the isolated TPA producing
microorganism is a Proteobacteria.
25. The process of claim 24 wherein the isolated TPA producing
microorganism is selected from the group consisting of,
Burkholderia, Alcaligenes, Pseudomonas, Shphingomonas and
Comamonas.
26. The process of claim 25 wherein the 16s RNA of the TPA
producing microorganism has at least 97% identity with the 16s rRNA
sequence set forth in SEQ ID NO:1.
27. The process of claim 26 wherein the isolated TPA producing
microorganism is Burkholderia sp. selected from the group
consisting of IR3 (ATCC 202150) and IR10 (ATCC 202151).
28. The process of claim 22 wherein the aromatic organic substrate
is selected from the group consisting of m-xylene, 3-methylbenzyl
alcohol, m-tolualdehyde, m-toluic acid, 3-carboxybenzyl alcohol,
and 3-carboxybenzaldehyde.
29. The process of claim 22 wherein the suitable induction compound
is selected from the group consisting of p-xylene, 4-methylbenzyl
alcohol, p-tolualdehyde, p-toluic acid, 4-carboxybenzyl alcohol,
4-carboxybenzaldehyde and terephthalic acid.
30. A process for the production of terephthalic acid comprising:
(i) contacting a mixed population of microorganisms comprising the
genes encoding the TPA biosynthetic pathway, with an aromatic
organic substrate whereby terephthalic acid accumulates; and (ii)
optionally recovering the terephthalic acid.
31. The process of claim 28 wherein the aromatic organic substrate
is selected from the group consisting of p-xylene, 4-methylbenzyl
alcohol, p-tolualdehyde, p-toluic acid, 4-carboxybenzyl alcohol,
and 4-carboxybenzaldehyde.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/091,645, filed Jul. 2, 1998.
FIELD OF INVENTION
[0002] This invention pertains to methods for the production of
terephthalic acid and isophthalic acid by bacteria.
BACKGROUND
[0003] Terephthalic acid and isophthalic acid are two monomers
having utility in the production of polyesters which are
commercially required in large quantities for fibers, films,
paints, adhesives and beverage containers. Isophthalic acid is also
used in condensation reactions with diamines to form polyamides.
Polyesters and polyamides are two extremely important classes of
commercial polymers.
[0004] A variety of chemical routes to terephthalic acid and
isophthalic acid are known. The most notable commercial process to
prepare terephthalic acid involves the liquid-phase oxidation of
p-xylene. The Amoco process involves oxidizing p-xylene with a
molecular oxygen-containing gas in the liquid phase in a lower
aliphatic monocarboxylic acid solvent in the presence of a heavy
metal catalyst and a bromine compound to from terephthalic acid
directly (U.S. Pat. No. 2,833,816). More specifically, the reaction
is catalyzed by Co and Mn in 95% acetic acid with a mixture of
NH.sub.4Br and tetrabromoethane as cocatalysts. The oxidation is
carried out under severe conditions of high temperatures
(109-205.degree. C.) and pressures (15-30 bar). Hence, the rate of
reaction is high and the yield of terephthalic acid based on
p-xylene is as high as 95% or more. However, the reaction apparatus
becomes heavily corroded owing mainly to the use of the bromine
compound and the monocarboxylic acid solvent. Thus, ordinary
stainless steel can not be used to build the reaction apparatus,
and expensive materials such as Hastelloy or titanium are required.
In addition, because the acid solvent is used in large quantity and
the oxidation conditions are severe, combustion of the solvent
itself can not be avoided, and its loss is not negligible. The
Amoco process has also been shown to oxidize m-xylene to
isophthalic acid. Although it is possible to oxidize xylenes by
these methods, they are expensive and generate waste streams
containing environmental pollutants.
[0005] Biological oxidation of methyl groups on aromatic rings,
such as toluene and isomers of xylene, is well known (Dagley et
al., Adv. Microbial Physiol. 6:1-46 (1971)). For example, bacteria
that have the xyl genes for the Tol pathway sequentially oxidize
the methyl group on toluene to afford benzyl alcohol, benzaldehyde
and ultimately benzoic acid. The xyl genes located on the well
characterized Tol plasmid pWWO have been sequenced (Assinder et
al., Adv. Microbial Physiol. 31:1-69 (1990); Burlage et al., Appl.
Environ. Microbiol. 55:1323-1328 (1989)). The xyl genes are
organized into two operons. The upper pathway operon encodes the
enzymes required for oxidation of toluene to benzoic acid. The
lower pathway operon encodes enzymes that convert benzoic acid into
intermediates of the tricarboxylic acid (TCA) cycle.
[0006] In addition to toluene, m-xylene and p-xylene are substrates
of the Tol pathway (JP 9023891). The upper pathway enzymes catalyze
oxidation of one methyl group on m-xylene and p-xylene to produce
the corresponding methylbenzyl alcohol, methylbenzaldehyde and
methylbenzoic acid. Although many bacteria utilize m-xylene and/or
p-xylene as sources of carbon and energy for growth, essentially
all of the known examples oxidize m-xylene or p-xylene to
methylbenzoic acid (i.e., m-toluic acid or p-toluic acid) and then
convert the methylbenzoic acid to methylcatechol. Bacteria with the
Tol pathway have not been shown to produce isophthalic acid or
terephthalic acid as intermediates when p- and m-xylene are used as
substrates. However, certain bacteria are known to oxidize the
methyl group of methylbenzoic acid when this compound is degraded
to provide carbon and energy for growth. For example, Comamonas
testosteroni strain T-2 oxidizes p-methylbenzoic acid (p-toluic
acid) to terephthalic acid (Junker et al., J. Bacteriol.
179:919-927 (1997); Junker et al., Microbiology 142:2419-2427
(1996)). It is important to note that although this strain degrades
methylated aromatics such as p-toluenesulfonic acid, it displays no
activity against toluene or p-xylene.
[0007] In general, biological processes for production of chemicals
are desirable for several reasons. One advantage is that the
enzymes that catalyze biological reactions have substrate
specificity. Accordingly, it is sometimes possible to use a
starting material that contains a complex mixture of compounds to
produce a specific chiral or structural isomer via a biological
process. Another advantage is that biological processes are
commonly perceived as being less harmful to the environment than
chemical manufacturing processes. These advantages, among others,
make it desirable to use p-xylene or m-xylene as the starting
materials for manufacture of terephthalic acid or isophthalic acid,
respectively, by means of a bioprocess.
[0008] SU 419509 claims a method for the cooxidative production of
terephthalic acid by the microbiological transformation of p-xylene
using an active culture from the genus Nocardia, which carries out
the direct oxidation of p-xylene to terephthalic acid. The method
is described as a cooxidative process that involves providing the
bacteria with hexadecane and p-xylene. Since this is a cooxidative
process, the hexadecane is required to induce synthesis of the
appropriate oxidative enzymes.
[0009] Finally, JP 9023891 claims a method for the production of
aromatic carboxylic acids by oxidation of various aromatic
compounds by Mycobacterium sp. strain NS 12523 and similar bacteria
belonging to the genus Mycobacterium. Terephthalic acid and
isophthalic acid are two of the aromatic carboxylic acids claimed
to be formed by the described process. However, there was no
demonstration that the claimed Mycobacterium strains could actually
produce terephthalic acid or isophthalic acid. When p-xylene was
used as a substrate, only p-toluic acid was isolated as the
product. If p-toluic acid could be used as a substrate, as was
claimed, it would be reasonable to expect terephthalic acid to be
isolated with or as the final product.
[0010] A need exists for environmentally friendly, safe and
economical methods to produce compounds of commercial interest. A
method that has broad applicability for the production of
terephthalic acid and isophthalic acid would have great commercial
value. To the best of applicants' knowledge, there is no account of
any bacteria that can oxidize both methyl groups of p-xylene to
form terephthalic acid in the absence of a cooxidized compound.
Furthermore, no such method involving the biological oxidation of
both methyl groups of p- and m-xylene by a single organism was
previously known.
SUMMARY OF THE INVENTION
[0011] The present invention provides a TPA producing microorganism
isolated by the process comprising: (i) culturing a sample
suspected of containing a TPA producing microorganism in a suitable
growth medium containing at least one aromatic organic substrate
selected from the group consisting of p-xylene, p-toluic acid, and
terephthalic acid; (ii) selecting those microorganisms which are
able to use every substrate selected individually from the group
consisting of p-xylene, p-toluic acid, and terephthalic acid as a
sole carbon source; (iii) contacting the microorganisms selected in
step (ii) with p-xylene to form a reaction medium; and (iv)
monitoring the reaction medium over time for the presence of
terephthalic acid, whereby the production of terephthalic acid
indicates the presence of a TPA producing microorganism. Preferred
TPA producing microorganisms are bacteria which comprising the
genes encoding the TPA biosynthetic pathway and are members of
Proteobacteria.
[0012] The invention further provides a process for the isolation
of a TPA producing microorganism comprising: (i) culturing a sample
suspected of containing a TPA producing microorganism in a suitable
growth medium containing at least one aromatic organic substrate
selected from the group consisting of p-xylene, p-toluic acid, and
terephthalic acid; (ii) selecting those microorganisms which are
able to use every substrate selected individually from the group
consisting of p-xylene, p-toluic acid, and terephthalic acid as a
sole carbon source; (iii) contacting the microorganisms selected in
step (ii) with p-xylene to form a reaction medium; and (iv)
monitoring the reaction medium over time for the presence of
terephthalic acid, whereby the production of terephthalic acid
indicates the presence of a TPA producing microorganism.
[0013] In an alternate embodiment the invention provides a process
for the production of terephthalic acid comprising: (i) contacting
an isolated TPA producing microorganism with an aromatic organic
substrate whereby terephthalic acid accumulates; and (ii)
optionally recovering the terephthalic acid. The process for
terephthalic acid production may optioanally use any one of the
aromatic organic substrates p-xylene, 4-methylbenzyl alcohol,
p-tolualdehyde, p-toluic acid, 4-carboxybenzyl alcohol, and
4-carboxybenzaldehyde.
[0014] The invention further provides a process for the production
of various intermediates in the synthesis of TPA such as
4-carboxybenzyl alcohol and 4-carboxybenzaldehyde.
[0015] The invention additionally provides a method for the
production of isophthalic acid comprising: (i) contacting an
isolated TPA producing microorganism with an aromatic organic
substrate and at least one suitable induction compound whereby
isophthalic acid accumulates; and (ii) optionally recovering the
isophthalic acid. The process for isophthalic acid production may
optioanally use any one of the aromatic organic substrates of
m-xylene, 3-methylbenzyl alcohol, m-tolualdehyde, m-toluic acid,
3-carboxybenzyl alcohol, 3-carboxybenzaldehyde and isophthalic
acid.
[0016] In another embodiment the invention provides a process for
the production of terephthalic acid comprising: (i) contacting a
mixed population of microorganisms comprising the genes encoding
the TPA biosynthetic pathway, with an aromatic organic substrate
whereby terephthalic acid accumulates; and (ii) optionally
recovering the terephthalic acid.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 is a HPLC analysis of a biotransfomation of p-xylene
by Burkholderia.
[0018] FIG. 2A is a plot of an UV absorbance spectrum of authentic
4-carboxybenzyl alcohol.
[0019] FIG. 2B is a plot of an UV absorbance spectrum of
presumptive 4-carboxybenzyl alcohol.
[0020] FIG. 3A is a plot of an UV absorbance spectrum of authentic
terephthalic acid.
[0021] FIG. 3B is a plot of an UV absorbance spectrum of
presumptive terephthalic acid.
[0022] FIG. 4A is a plot of an UV absorbance spectrum of authentic
p-toluic acid.
[0023] FIG. 4B is a plot of an UV absorbance spectrum of
presumptive p-toluic acid.
[0024] FIG. 5 shows the synthesis path of terephthalic acid from
p-xylene by Burkholderia isolate IR3.
[0025] FIG. 6 shows the synthesis path of isophthalic acid from
m-xylene by Burkholderia isolate IR3.
TABLE DESCRIPTIONS, SEQUENCE DESCRIPTIONS AND BIOLOGICAL
DEPOSITS
[0026] Table 1 contains a summary of carbon source utilization.
[0027] Table 2 contains a comparison of 16s rRNA genes.
[0028] Table 3 details the production of p-toluic acid and
terephthalic acid from p-xylene by Burkholderia strain IR3.
[0029] Table 4 details the production of p-toluic acid and
terephthalic acid after growth in LB medium
[0030] Table 5 details the production of m-toluic acid and
isophthalic acid from m-xylene by Burkholderia strain IR3.
[0031] Table 6 details the production of terephthalic acid from
p-xylene in mixed cultures of ATCC 33015 and DSM 6577.
[0032] The invention can be more fully understood from the
following detailed description and the accompanying sequence
descriptions which form a part of this application.
[0033] The following sequence descriptions and sequences listings
attached hereto comply with the rules governing nucleotide and/or
amino acid sequence disclosures in patent applications as set forth
in 37 C.F.R. .sctn.1.821-1.825. The Sequence Descriptions contain
the one letter code for nucleotide sequence characters and the
three letter codes for amino acids as defined in conformity with
the IUPAC-IYUB standards described in Nucleic Acids Research
13:3021-3030 (1985) and in the Biochemical Journal 219 (No.
2):345-373 (1984) which are herein incorporated by reference. The
symbols and format used for nucleotide and amino acid sequence data
comply with the rules set forth in 37 C.F.R. .sctn.1.822.
[0034] SEQ ID NO:1 is the nucleotide sequence of a 16s rRNA gene
from isolate IR3.
[0035] SEQ ID NO:2 is the HK12 primer.
[0036] SEQ ID NO:3 is the HK13 primer.
[0037] SEQ ID NO:4 is the HK14 primer.
[0038] Applicants have made the following biological deposits under
the terms of the Budapest Treaty on the International Recognition
of the Deposit of Microorganisms for the Purposes of Patent
Procedure:
1 Depositor Idenitification Internation Depository Reference
Designation Date of Deposit Burkholderia sp. IR3 ATCC 202150 Jul.
2, 1998 Burkholderia sp. IR10 ATCC 202151 Jul. 2, 1998
DETAILED DESCRIPTION OF THE INVENTION
[0039] The present invention is a process for the production
terephthalic acid (TPA) involving the bioconversion of p-xylene to
TPA using a single TPA producing microorganism. The TPA producing
organism comprises the genes encoding enzymes involved in the
enzymatic conversion of p-xylene to TPA as follows: p-xylene is
acted upon by xylene monooygenase to form 4-methylbenzyl alcohol,
which is acted upon by methylbenzyl alcohol dehydrogenase to form
p-tolualdehyde, which is acted upon by tolualdehyde dehydrogenase
to form p-toluic acid, which is acted upon by toluate methyl
monooxygenase to form 4-carboxybenzyl alcohol, which is acted upon
by carboxybenzyl alcohol methyl dehydrogenase to form
4-carboxybenzaldehyde, which is acted upon by caboxybenzaldehyde
methyl dehydrogenase to form terephthalic acid. This enzymatic
pathway is referred to herein as the TPA biosynthetic pathway.
[0040] In another embodiment of the invention, m-xylene is
enzymatically transformed to isophthalate by a TPA producing
microorganism comprising the TPA biosynthetic pathway. The enzymes
involved in the pathway are the same, however the intermediates are
all meta substituted as follows: m-xylene, to 3-methylbenzyl
alcohol, to m-tolualdehyde, to m-toluic acid, to 3-carboxybenzyl
alcohol, to 3-carboxybenzaldehyde and finally to isophthalic
acid.
[0041] The TPA producing microorganism of the present invention is
isolated from activate sludge in the presence of several different
aromatic organic substrates. Those microorganisms having an ability
to grow on these substrates are then screened for their ability to
bioconvert either p-xylene or m-xlyene to terephthalate or
isophthalate respectively.
[0042] It is another aspect of the invention, a mixture of
microorganisms, collectively containing the genes encoding the TPA
biosynthetic pathway may be cultured as a mixed popultation for the
bioconversion p-xylene or m-xylene to their respective
products.
[0043] The present invention is useful for the biological
production of terephthalic acid and isophthalic acid which have
utility in the production of polyesters needed in fibers, films,
paints, adhesives and beverage containers. The present invention
advances the art of the synthesis of terephthalic acid and
isophthalic acid as biological processes are more cost effective
and produce fewer environmentally harmful waste products.
[0044] In this disclosure, a number of terms and abbreviations are
used. The following definitions are provided.
[0045] "Terephthalic acid" is abbreviated TPA.
[0046] "Isophthalic acid" is abbreviated IPA.
[0047] "Dihydroxybenzoic acid" is abbreviated as DHBA.
[0048] "Trimalinic acid" is abbreviated as TMA.
[0049] "Phthalic acid" is abbreviated as PA.
[0050] "4-Carboxybenzyl alcohol" is abbreviated at 4-CBAL.
[0051] "4-Carboxybenzaldehyde" is abbreviated at 4-CBA.
[0052] "Benzoic acid" is abbreviated at BA.
[0053] "p-Toluic acid" is abbreviated as PTA.
[0054] "p-Tolualdehyde" is abbreviated as PTL.
[0055] "Ethylenediaminetetraacetic acid" is abbreviated as
EDTA.
[0056] "Open reading frame" is abbreviated ORF.
[0057] "Polymerase chain reaction" is abbreviated PCR.
[0058] As used herein, "ATCC" refers to the American Type Culture
Collection International Depository located at 10801 University
Boulevard, Manassaa, Va. 20110-2209, U.S.A. The "ATCC No." is the
accession number to cultures on deposit with the ATCC.
[0059] The term "TPA biosynthetic pathway" refers to the seqeunce
of enzymatic steps that will convert either p-xylene to
terephthalic acid or m-xylene to isophthalic acid and consists of
enzymes in the following seqeunce: 1-xylene
monooygenase.fwdarw.2-methylbenzyl alcohol
dehydrogenase.fwdarw.3-tolualdehyde dehydrogenase.fwdarw.4-toluate
methyl monooxygenase.fwdarw.5-carboxybenzyl alcohol methyl
dehydrogenase 6-caboxybenzaldehyde methyl dehydrogenase.fwdarw.7,
where the numbers 1-7 refer to the compounds to be catalyzed.
[0060] The term "TPA producing microorganism" refers to any
microorganism which converts p-xylene to TPA or m-xylene to
isophthalate and which also comprises the enzymes of the TPA
biosynthetic pathway.
[0061] The term "suitable induction compound" refers to any
compound which, when co-fermented with a process starting material,
facilities the bio-conversion of that starting material. Within the
context of the present invention a suitable induction compound for
the bio-conversion of m-xylene to isophthalate is p-xylene.
[0062] The terms "bio-transformation" and "bio-conversion" will be
used interchangeably and will refer to the process of enzymatic
conversion of a compound to another form or compound. The process
of bio-conversion or bio-transformation is typically carried out by
a bio-catalyst.
[0063] As used herein the term "bio-catalyst" refers to a
microorganism which contains an enzyme or enzymes capable of
bio-conversion of a specific compound or compounds.
[0064] The term "percent identity", as known in the art, is a
relationship between two or more polypeptide sequences or two or
more polynucleotide sequences, as determined by comparing the
sequences. In the art, "identity" also means the degree of sequence
relatedness between polypeptide or polynucleotide sequences, as the
case may be, as determined by the match between strings of such
sequences. "Identity" and "similarity" can be readily calculated by
known methods, including but not limited to those described in:
Computational Molecular Biology (Lesk, A. M., ed.) Oxford
University Press, New York (1988); Biocomputing: Informatics and
Genome Projects (Smith, D. W., ed.) Academic Press, New York
(1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M.,
and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence
Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press
(1987); and Sequence Analysis Primer (Gribskov, M. and Devereux,
J., eds.) Stockton Press, New York (1991). Preferred methods to
determine identity are designed to give the largest match between
the sequences tested. Methods to determine identity and similarity
are codified in publicly available computer programs. Preferred
computer program methods to determine identity and similarity
between two sequences include, but are not limited to, the GCG
Pileup program found in the GCG program package, using the
Needleman and Wunsch algorithm with their standard default values
of gap creation penalty=12 and gap extension penalty=4 (Devereux et
al., Nucleic Acids Res. 12:387-395 (1984)), BLASTP, BLASTN, and
FASTA (Pearson et al., Proc. Natl. Acad. Sci. U.S.A. 85:2444-2448
(1988). The BLAST X program is publicly available from NCBI and
other sources (BLAST Manual, Altschul et al., Natl. Cent.
Biotechnol. Inf., Natl. Library Med. (NCBI NLM) NIH, Bethesda, Md.
20894; Altschul et al., J. Mol. Biol. 215:403-410 (1990)). Another
preferred method to determine percent identity, is by the method of
DNASTAR protein alignment protocol using the Jotun-Hein algorithm
(Hein et al., Methods Enzymol. 183:626-645 (1990)). Default
parameters for the Jotun-Hein method for alignments are: for
multiple alignments, gap penalty=11, gap length penalty=3; for
pairwise alignments ktuple=6. As an illustration, by a
polynucleotide having a nucleotide sequence having at least, for
example, 95% "identity" to a reference nucleotide sequence it is
intended that the nucleotide sequence of the polynucleotide is
identical to the reference sequence except that the polynucleotide
sequence may include up to five point mutations per each 100
nucleotides of the reference nucleotide sequence. In other words,
to obtain a polynucleotide having a nucleotide sequence at least
95% identical to a reference nucleotide sequence, up to 5% of the
nucleotides in the reference sequence may be deleted or substituted
with another nucleotide, or a number of nucleotides up to 5% of the
total nucleotides in the reference sequence may be inserted into
the reference sequence. These mutations of the reference sequence
may occur at the 5' or 3' terminal positions of the reference
nucleotide sequence or anywhere between those terminal positions,
interspersed either individually among nucleotides in the reference
sequence or in one or more contiguous groups within the reference
sequence.
[0065] "Gene" refers to a nucleic acid fragment that expresses a
specific RNA molecule (mRNA, rRNA, or tRNA) and which may, in
addition, express a specific protein if the gene initially
expresses a mRNA molecule. The gene includes regulatory sequences
preceding (5' non-coding sequences) and following (3' non-coding
sequences) the coding sequence.
[0066] Standard recombinant DNA and molecular cloning techniques
used here are well known in the art and are described by Sambrook,
J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. (1989) (hereinafter "Maniatis");
and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W.,
Experiments with Gene Fusions, Cold Spring Harbor Laboratory Cold
Press Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al.,
Current Protocols in Molecular Biology, published by Greene
Publishing Assoc. and Wiley-Interscience (1987).
[0067] Isolation of TPA Producing Microorganisms
[0068] The TPA producing microorganisms of the present invention
may be isolated from a variety of sources. Suitable sources include
industrial waste streams, soil from contaminated industrial sites
and waste stream treatment facilities. The microorganisms of the
present invention were isolated from activated sludge from a waste
water treatment plant.
[0069] Samples suspected of containing TPA producing microorganisms
may be enriched by incubation in a suitable growth medium in
combination with at least one aromatic organic substrate. Suitable
substrates may include those intermediates which are
bio-transformed by the enzymes of the TPA biosynthetic pathway.
Suitable aromatic organic substrates for use in the present
invention include, but are not limited to p-xylene, 4-methylbenzyl
alcohol, p-tolualdehyde, p-toluic acid, 4-carboxybenzyl alcohol,
4-carboxybenzaldehyde, terephthalic acid, m-xylene, 3-methylbenzyl
alcohol, m-tolualdehyde, m-toluic acid, 3-carboxybenzyl alcohol,
and 3-carboxybenzaldehyde, wherein p-xylene and p-toluic acid and
terephthalic acid are preferred. It is preferred that the organism
be able to use several different aromatic substrates as a sole
carbon source. So for example, preferred microorganisms will be
able to grow on p-xylene and terephthalic acid and one other
intermediate. The preferred additional intermediate in the present
invention was p-toluic acid.
[0070] Once microorganisms are identified as having the ability to
use the aromatic substrates as the sole carbon/energy source they
are then screened for the ability to bio-convert either p-xylene to
TPA or m-xylene to IPA. This is accomplished by contacting a
suitable amount of either p-xylene or m-xylene with the isolated
organism in the presence of a growth medium and monitoring the
culture for the appearance of the desired end product. Of 20
isolates identified in this manner, 3 had the ability to accomplish
the desired bio-conversions.
[0071] Growth medium and techniques needed in the enrichment and
screening of TPA producing microorganisms are well known in the art
and examples may be found in Manual of Methods for General
Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N.
Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G.
Briggs Phillips, eds), American Society for Microbiology,
Washington, D.C. (1994)); or by Thomas D. Brock in Biotechnology: A
Textbook of Industrial Microbiology, Second Edition, Sinauer
Associates, Inc., Sunderland, Mass. (1989).
[0072] Characterization of TPA Producing Microorganims
[0073] Microorganisms isolated according to the above procedure
have several distinguishing characteristics. All contain the
enzymes of the TPA biosynthetic pathway. All are included in the
group Proteobacteria, of which Burkholderia, Alcaligenes,
Pseudomonas, Sphingomonas, and Comamonas are examples. The
Proteobacteria form a physiologically diverse group of
microorganisms and represent five subdivisions (.alpha., .beta.,
.gamma., .epsilon., .delta.) (Madigan, M. T., et al., Brock Biology
of Microorganisms, 8th edition, Prentice Hall, UpperSaddle River,
N.J. (1997)). All five subdivisions of the Proteobacteria contain
microorganisms that use organic compounds as sources of carbon and
energy. Although the specific microorganisms isolated were all of
the genus Burkholderia (.beta. subdivision) (i.e. IR3 [ATCC 202150]
and IR10 [ATCC 202151]), it is contemplated that other members of
the Proteobacteria isolated according to the above method will be
suitable, e.g. Pseudomonas (.gamma. subdivision) and Sphingomonas
(.alpha. subdivision), because genes for metabolism of p-xylene and
other aromatic compounds are frequently located on plasmids and the
plasmids are frequently capable of transferring between members of
the Proteobacteria (Assinder and Williams, Adv. Microb. Physiol.
31:2-69 (1990); Springael, D. et al. Microbiol. 142:3283-3293
(1996)).
[0074] Another characteristic of the present TPA producing
organisms is that they appear to be genetically similar to members
of the genus Burkholderia as determined by 16s rRNA comparison. 16s
rRNA was isolated from the TPA producing organisms IR3 [ATCC
202150] and IR10 [ATCC 202151] according to standard protocols
(Maniatis, supra) and compared with sequences in public databases.
The comparison revealed that the 16s rRNA sequence most closely
compared to that isolated from Burkholderia (97% identity),
although there was significant identity to 16s rRNA from
Pseudomonas.
[0075] Another distinguishing characteristic of the TPA producing
organisms of the present invention is the presence of all of the
enzymes of the TPA biosynthetic pathway. By the TPA biosynthetic
pathway it is meant that all the enzymes, xylene
monooygenase.fwdarw.methylbenzyl alcohol
dehydrogenase.fwdarw.tolualdehyde dehydrogenase.fwdarw.toluate
methyl monooxygenase.fwdarw.carboxybenzyl alcohol methyl
dehydrogenase.fwdarw.caboxybenzaldehyde methyl dehydrogenase, are
present and functionally linked in the order listed. The presence
of a single organism comprising this complete cadre of linked
enzymes is unique in the art.
[0076] Process for the Production of Terephthalic and Isophthalic
Acids and Intermediates
[0077] TPA producing microorganisms of the present invention may be
used to produce both TPA and IPA. Additionally, a mixture of
microorganisms may be cultured together for the production of TPA
or IPA, where the microorganisms comprising the mixed culture,
collectively, have all the enzymes of the TPA biosynthetic
pathway.
[0078] Where the production of TPA is desired the TPA producing
microorganism is contacted with p-xylene in a suitable growth
medium and the reaction medium is monitored for the production of
TPA. Where the production of IPA is desired it may be necessary to
contact the TPA producing organism with a mixture of aromatic
organic substrates to effect IPA production. For example the
appropriate starting material for the production of IPA using the
TPA biosynthetic enzymes is m-xylene. However, the TPA producers of
the present invention are unable to use m-xylene as a sole carbon
source. It is well known that bacteria sometimes metabolically
transform a compound that can not be used for growth if a second
compound is present that induces synthesis of the appropriate
enzymes (Janke et al., J. Basic Microbiol. 25:603-619 (1985)). In
the context of the present invention m-xylene was co-fed with
p-xylene as an inducer to effect the conversion of m-xylene to IPA.
Although p-xylene is a preferred inducer, it is contemplated that
any of the intermediates converted or acted up by the enzymes of
the TPA pathway in the production of TPA, including but not limited
to p-xylene, 4-methylbenzyl alcohol, p-tolualdehyde, p-toluic acid,
4-carboxybenzyl alcohol, and 4-carboxybenzaldehyde will be
effective inducers.
[0079] The present process is also usful for the production of any
of the intermediates of the TPA biosynthetic pathway that may occur
either in the bioconversion of p-xylene to TPA or of m-xylene to
IPA. For example, 4-carboxybenzyl alcohol is detected in cultures
of isolate IR3 (ATCC 202150) that were given p-xylene as the sole
source of carbon and energy. Thus, it is contemplated that any one
of the intermediates involved in TPA production, such as
4-methylbenzyl alcohol, p-tolualdehyde, p-toluic acid,
4-carboxybenzyl alcohol, and 4-carboxybenzaldehyde for example, and
the intermediates involved in IPA production, such as
3-methylbenzyl alcohol, m-tolualdehyde, m-toluic acid,
3-carboxybenzyl alcohol, and 3-carboxybenzaldehyde could be
produced by the present TPA producing microorganisms.
[0080] These intermediates could be produced by using mutations to
inactivate genes encoding key enzymes in the TPA biosynthetic
pathway by. A mutation is a change in the nucleotide sequence of a
gene. A variety of methods for introducing point mutations,
insertion mutations or deletion mutations into specific genes are
well known and have been described in the art (Maniatis supra). A
mutation frequently inactivates or "knocks out" the protein product
of the mutated gene. If a gene in a biosynthetic pathway is
disrupted by a mutation, there may be accumulation of the pathway
intermediate formed by the enzyme that catalyzes the synthetic step
immediately prior to the step normally catalyzed by the enzyme
knocked out by a mutation (Mengin-Lecreulx, D. and J. van
Heijenoort, J. Bacteriol. 175: 6150-6157 (1993)). For example, a
TPA producer could be used to develop a mutant bacterial strain
that produces 4-methylbenzyl alcohol from p-xylene. The mutant
strain would have a knockout mutation in the gene for the enzyme
4-methylbenzyl alcohol dehydrogenase to prevent conversion of
4-methylbenzyl alcohol to 4-methylbenzaldehyde. Under these
conditions, a person skilled in the art would expect the mutant
strain to accumulate 4-methylbenzyl alcohol because 4-methylbenzyl
alcohol can not be converted to 4-methylbenzaldehyde by the mutant
strain. Since 4-methylbenzyl alcohol will accumulate if the gene
for 4-methylbenzyl alcohol dehydrogenase has a knockout mutation,
the methyl group on 4-methylbenzyl alcohol would be available in
unusually high amounts in the mutant as a possible substrate for
xylene monooxygenase and/or toluate methyl monooxygenase.
Accordingly, the mutant bacterial strain might also produce
1,4-benzenedimethanol as a result of the methyl group on
4-methylbenzyl alcohol being oxidized by xylene monooxygenase
and/or toluate methyl monooxygenase. Similar considerations pertain
to mutants that would accumulate 4-methylbenzaldehyde,
terephthalaldehyde, or other intermediates of the TPA biosynthetic
pathway.
[0081] One way to construct appropriate knockout mutations that
would cause accumulation of a particular intermediate of the TPA
biosynthetic pathway, e.g., 4-methylbenzyl alcohol, would initially
involve inserting a functional antibiotic resistance gene sequence
into a cloned copy of the 4-methylbenzyl alcohol dehydrogenase
gene. The inserted antibiotic resistance gene would prevent the
cloned 4-methylbenzyl alcohol dehydrogenase gene from being
expressed. The 4-methylbenzyl alcohol dehydrogenase gene with
inserted antibiotic resistance gene would be mobilized into the TPA
producer on a plasmid vector that is unable to replicate in the TPA
producer. Genetic selection for the inserted antibiotic resistance
gene will yield recombinants in which the plasmid-borne
4-methylbenzyl alcohol dehydrogenase gene with inserted antibiotic
resistance gene has replaced the original intact copy of the
4-methylbenzyl alcohol dehydrogenase gene. The result would be a
new bacterial strain with a knockout mutation of the 4-methylbenzyl
alcohol dehydrogenase gene which causes 4-methylbenzyl alcohol to
accumulate. Appropriate antibiotic resistance gene sequences and
plasmid vectors are well known in the art (Schweizer, H. P., et
al., pages 229-237, in Molecular Biology of Pseudomonads (Nakazawa
et al., eds.), ASM Press, Washington, D. C. (1996)).
EXAMPLES
[0082] The present invention is further defined in the following
Examples. It should be understood that these Examples, while
indicating preferred embodiments of the invention, are given by way
of illustration only. From the above discussion and these Examples,
one skilled in the art can ascertain the essential characteristics
of this invention, and without departing from the spirit and scope
thereof, can make various changes and modifications of the
invention to adapt it to various usages and conditions.
[0083] General Methods
[0084] Techniques suitable for use in the following examples may be
found in Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular
Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1989) (hereinafter
"Maniatis").
[0085] Materials and methods suitable for the maintenance and
growth of bacterial cultures are well known in the art. Techniques
suitable for use in the following examples may be found as set out
in Manual of Methods for General Bacteriology (Phillipp Gerhardt,
R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A.
Wood, Noel R. Krieg and G. Briggs Phillips, eds), American Society
for Microbiology, Washington, D.C. (1994)); or by Thomas D. Brock
in Biotechnology: A Textbook of Industrial Microbiology, Second
Edition, Sinauer Associates, Inc., Sunderland, Mass. (1989). All
reagents and materials used for the growth and maintenance of
bacterial cells were obtained from Aldrich Chemicals (Milwaukee,
Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL
(Gaithersburg, Md.) or Sigma Chemical Company (St. Louis, Mo.)
unless otherwise specified.
[0086] The meaning of abbreviations is as follows: "h" means
hour(s), "min" means minute(s), "sec" means second(s), "d" means
day(s), ".mu.L" means microliter, "mL" means milliliters, "L" means
liters, ".mu.m" means micrometer, "ppm" means parts per million,
i.e., milligrams per liter.
[0087] Media:
[0088] Synthetic S12 medium was used to establish enrichment
cultures and to culture bacteria for production of terephthalic
acid. S12 medium contains the following: 10 mM ammonium sulfate, 50
mM potassium phosphate buffer (pH 7.0), 2 mM MgCl.sub.2, 0.7 mM
CaCl.sub.2, 50 .mu.M MnCl.sub.2, 1 .mu.M FeCl.sub.3, 1 .mu.M
ZnCl.sub.3, 1.72 .mu.M CuSO.sub.4, 2.53 .mu.M CoCl.sub.2, 2.42
.mu.M Na.sub.2MoO.sub.2, 0.0001% FeSO.sub.4 and 2 .mu.M thiamine
hydrochloride.
[0089] S12 agar was used to isolate bacteria from liquid enrichment
cultures that grow on p-xylene and to test isolates for growth with
p-xylene, p-toluic acid or terephthalic acid supplied a sole source
of carbon and energy. S12 agar was prepared by adding 1.5% Noble
agar (DIFCO) to S12 medium.
[0090] Bacteria growing in S12 medium were supplied with toluene,
m-xylene or p-xylene as vapor by allowing the volatile compound to
evaporate from a sterile 13.times.100 mm glass test tube reservoir
inside of a glass screw cap culture flask. Bacteria growing on S 12
agar were supplied with toluene, m-xylene, p-xylene or other
volatile compounds as vapor by placing 5 .mu.L of a volatile
compound on the interior of the petri dish lid. The petri dish was
sealed with parafilm and incubated with the lid on the bottom.
[0091] Stock solutions of terephthalic acid (Amoco refined 99.9%)
and p-toluic acid (Aldrich) were prepared in 1N NaOH.
[0092] Isolation and Identification of Terephthalic Acid and
Isophthalic Acid:
[0093] The conversion of m-xylene and p-xylene to isophthalic acid
and terephthalic acid, respectively, was monitored by reverse phase
HPLC. Culture supernatants were passed through 0.2 .mu.m filters
(Gelman acrodisc CR PFTE or Millipore millex-gs) prior to analysis.
Analyses were performed on either a Hewlett Packard HPLC model 1050
equipped with a Milton Roy LDC single wavelength detector set at
214 nm or a Hewlett Packard HPLC model 1090 equipped with a diode
array UV-visible detector set at 254 nm (primary wavelength), 230
nm (secondary wavelength), and 450 nm as background reference.
Samples (10 .mu.L) were injected onto a Zorbax C8 column (2.1
mm.times.15 cm). The mobile phase consisted of (A) H.sub.2O
containing 2 mL phosphoric acid/L and (B) acetonitrile. Gradients
were as follow: a) 0 minutes to 25 minutes (B) increased from 10%
to 25%, b) (B) increased to 95% over the next 12 minutes, c) (B)
was held at 95% for 3 minutes, and d) (B) decreased to 10% in 1
min. Standards for HPLC were DHBA (dihydroxybenzoic acid), TMA
(trimalinic acid), PA (phthalic acid), TPA (terephthalic acid), IPA
(isophthalic acid), 4-CBAL (4-carboxybenzyl alcohol), 4-CBA
(4-carboxybenzaldehyde), BA (benzoic acid), 4-MBAL
(4-methylbenzaldehyde), MHT, PTA (p-toluic acid), PTL
(p-tolualdehyde) and p-xylene in 96% tetrahydrofuran and 4%
millipore water. All calibrations and data analysis was done using
Hewlett Packard's Chemstation Software. Preparative HPLC for peak
collection was run on instrument II with either a Zorbax RXC8 9.4
mm.times.25 cm with a 50-250 .mu.L injection volume. The mobile
phase consisted of (A) H.sub.2O containing 2 mL phosphoric acid/L
or 2 mL acetic acid and (B) acetonitrile. For peak collection,
samples were run in 2 mL acetic acid/1L of Milli-Q water mobile
phase. Peaks of interest were collected into 20 mL glass vials.
Samples were then subsequently concentrated in a Savant Speed
Vac.
Example 1
Isolation and Identification of Bacteria that Grow with p-Xylene,
p-Toluic Acid and Terephthalic Acid as Sole Sources of Carbon and
Energy
[0094] Various bacteria were isolated that could grow on p-xylene
as the sole source of carbon and energy. Several of these isolates
also grew on p-toluic acid and terephthalic acid supplied
individually as sole sources of carbon and energy. Since p-toluic
acid is an expected intermediate in the metabolic oxidation of
p-xylene to terephthalic acid by bacteria, a collection of isolates
that grow on p-xylene, p-toluic acid and terephthalic acid is
likely to include bacteria that enzymatically oxidize p-xylene to
terephthalic acid. Analysis of 16s rRNA gene sequences indicated
that the collection of isolates included members of the bacterial
genra Pseudomonas and Burkholderia.
[0095] Bacteria that grow on p-xylene, p-toluic acid and
terephthalic acid were isolated from an enrichment culture. The
enrichment culture was established by inoculating 1 mL of activated
sludge into 10 mL of S 12 medium in a 125 mL screw-cap Erlenmeyer
flask. The activated sludge was obtained from a wastewater
treatment facility. The enrichment culture was supplemented with
100 ppm p-xylene added directly to the culture medium and was
incubated at 25.degree. C. with reciprocal shaking. The enrichment
culture was maintained by adding 100 ppm p-xylene every 2-3 days.
The culture was diluted every 10 days by replacing 9 mL of the
culture with the same volume of S12 medium. After 29 days of
incubation, serial dilutions of the enrichment culture were spread
onto R2A agar (DIFCO) and S12 agar. p-Xylene was placed on the
interior of each petri dish lid. The petri dishes were sealed with
parafilm and incubated upside down at room temperature (25.degree.
C.).
[0096] Representative bacterial colonies were then tested for the
ability to use p-xylene as a sole source of carbon and energy.
Colonies were transferred from the R2A plates and S12 agar plates
to the S12 agar plates and supplied with p-xylene as vapor by
placing 5 .mu.L of p-xylene on the interior of each petri dish lid.
The petri dishes were sealed with parafilm and incubated upside
down at room temperature (25.degree. C.). The isolates that
utilized p-xylene for growth were then tested for growth on S12
agar plates containing either p-toluic acid (6 mM) or terephthalic
acid (100 .mu.g/mL).
[0097] The 16s rRNA genes of each isolate were amplified by PCR and
analyzed as follows. Each isolate was grown on R2A agar. Several
colonies from each culture plate were suspended in 200 mL of lysis
buffer (1% Triton X-100, 20 mM Tris (pH 8.5), 2 mM EDTA). The
mixture was heated to 95.degree. C. for 10 min and then centrifuged
to remove cellular debris. The 16s rRNA gene sequences in the
supernatant were amplified by PCR by using a commercial kit
according to the manufacturer's instructions (Perkin Elmer) with
HK12 primer GAG TTT GAT CCT GGC TCA G (SEQ ID NO:2) and HK13 primer
TAC CTT GTT ACG ACT T (SEQ ID NO:3). PCR was performed in a Perkin
Elmer GeneAMp 9600. The samples were incubated for 5 min at
94.degree. C. and then cycled 35 times at 94.degree. C. for 30 sec,
55.degree. C. for 1 min and 72.degree. C. for 1 min. The amplified
16s rRNA genes were purified using a commercial kit according to
the manufacturer's instructions (QIAquick PCR Purification Kit) and
sequenced on an automated ABI sequencer. The sequencing reactions
were initiated with HK12 primer, HK13 primer and HK14 primer GTG
CCA GCA GYM GCG GT; Y=C or T, M=A or C (SEQ ID NO:4). The 16s rRNA
gene sequence of each isolate was used as the query sequence for a
FastA search (Wisconsin Package Version 9.0, Genetics Computer
Group (GCG), Madison, Wis.) of GenBank for similar sequences.
[0098] The data in Table 1 indicated that 6 out of 20 isolates from
the p-xylene enrichment culture were able to grow on p-xylene,
p-toluic acid and terephthalic acid as sole sources of carbon and
energy. The 16s rRNA genes of these six isolates were sequenced and
compared to other 16s rRNA sequences in the GenBank sequence
database. The 16s rRNA genes from isolates DSK1 and DS6 had high
degrees of homology with bacterial species belonging to the genus
Pseudomonas, whereas isolates IR3 (ATCC 202150), IR10 (ATCC 202151)
and IS3 had a high degree of homology with the genus Burkholderia
(Table 2). Comparison of the 16s rRNA gene sequence of IR3 (ATCC
202150) (SEQ ID NO:1) to the 16s rRNA gene sequence, IR10 (ATCC
202151) and IS3 indicated that the 16s rRNA genes of these isolates
were at least 98% homologous (Table 2).
2TABLE 1 Summary of Carbon Source Utilization Growth on Carbon
Source Isolate p-Xylene p-Toluic Acid Terephthalic Acid DR3 +/- - +
DR7 + + - DR9 + + - DR11 + + - DR13 + - +/- DSK1 + + + DSK2 +/- - +
DS2 + + - DS3 + + - DS6 + + + DS7 + + - IR1 + - + IR3 + + + IR4 + +
+ IR10 + + + IS2 - + +/- IS3 + + + IS4 +/- + - IS6 + + - IS8 + +
-
[0099]
3TABLE 2 Comparison of 16s rRNA genes % homology to % homology to
Isolate Closest Match closest match IR3 DSK1 Pseudomonas graminis
97.6 84.3 DS6 Pseudomonas testosteroni 94.1 89.0 IR3 Burkholderia
sp. 97.6 100 IR10 Burkholderia sp. 97.8 99.3 IS3 Burkholderia sp.
97.2 98.3
Example 2
Conversion of p-Xylene Into Terephthalic Acid by Bacteria of the
Genus Burkholderia
[0100] Example 2 demonstrated that strain IR3 (ATCC 202150) and
other Burkholderia isolates produced terephthalic acid when grown
in S12 medium with p-xylene supplied as the only source of carbon
and energy. Since all of the Burkholderia isolates had a high
degree of homology in the 16s rRNA genes and preliminary
experiments indicated that all four isolates produced terephthalic
acid, isolate IR3 (ATCC 202150) was selected for further
characterization. Isolate IR3 was grown to an optical density at
600 nm (OD.sub.600) of 0.968 in 60 mL of S12 medium in a 250 mL
screw-cap Erlenmeyer flask with 500 .mu.L of p-xylene in a
reservoir. The cells were harvested by centrifugation and
resuspended in S12 medium at an OD.sub.600 of 0.654. The cells were
divided into three 35 mL aliquots. Each aliquot was dispensed into
a sterile 125 mL screw-cap Erlenmeyer flask. One flask was
autoclaved with its contents for 15 min (killed cell control). The
cells in the autoclaved flask were exposed to vapor from 500 .mu.L
of p-xylene in a reservoir. The cells in the second flask were
exposed to vapor from 500 .mu.L of p-xylene in a reservoir. The
cells in the third flask were not exposed to p-xylene. A fourth
flask containing S12 medium exposed to vapor from 500 .mu.L of
p-xylene in a reservoir was established as a control to demonstrate
that the culture medium alone did not convert p-xylene to
terephthalic acid. All of the flasks were incubated at 30.degree.
C. with reciprocal shaking. Samples (1.5 mL) were collected at the
indicated times and passed through 0.22 .mu.m Acrodisc CR PFTE
filters. The first sample was collected immediately after the cells
were suspended in new medium (0 h). The samples were analyzed by
HPLC. Terephthalic acid and p-toluic acid were identified by
retention times.
[0101] The data in Table 3 indicated that p-toluic acid and
terephthalic acid could be detected in a culture of IR3 (ATCC
202150) after 9 h of incubation when viable cells were exposed to
p-xylene vapor. p-Xylene was necessary for production of p-toluic
acid and terephthalic acid since neither compound was detected when
viable cells were incubated without p-xylene. Furthermore, viable
cells were necessary for production of p-toluic acid and
terephthalic acid, since neither compound was detected when
autoclaved cells or medium alone were incubated with p-xylene.
Similar experiments demonstrated that isolates IR4, IR10 (ATCC
202151) and IS3 all produced significant amounts of terephthalic
acid when cultured with p-xylene, whereas isolates DSK1 and DS6 did
not produce detectable amounts of terephthalic acid when cultured
with p-xylene.
4TABLE 3 Production of p-toluic acid and terephthalic acid from
p-xylene by Burkholderia strain IR3 (ATCC 202150) Concentration
(ppm) Time (h) Culture PTA TPA 0 cells alone ND.sup.a ND killed
cells + p-xylene ND ND cells + p-xylene ND ND medium + p-xylene ND
ND 9 cells alone ND ND killed cells + p-xylene ND ND cells +
p-xylene 0.76 0.40 medium + p-xylene ND ND .sup.aNot detected
Example 3
Analysis of Terephthalic Acid Produced by Bacteria of the Genus
Burkholderia
[0102] Example 3 demonstrated that the p-toluic acid,
4-carboxybenzyl alcohol and terephthalic acid produced from
p-xylene by Burkholderia IR3 (ATCC 202150) were identical to
authentic standards by HPLC diode array analysis and mass
spectrometry. These results support the conclusion that isolate IR3
(ATCC 202150) produces terephthalic acid from p-xylene by means of
a previously unknown pathway.
[0103] HPLC Diode Array Analysis:
[0104] Isolate IR3 (ATCC 202150) was grown to an OD.sub.600 of 0.60
in 20 mL of S12 medium in a 250 mL screw-cap Erlenmeyer flask with
200 .mu.L of p-xylene in a reservoir. Samples (1.5 mL) of the
culture were placed in 1.5 mL microfuge tubes and mixed with 15
.mu.L of p-xylene. The samples were incubated without shaking for
72 hours at 25.degree. C. and then filtered. The samples were
analyzed by reverse-phase HPLC with a diode array UV-visible
detector set at 240 nm.
[0105] The chromatogram in FIG. 1 indicated that three major
compounds were present in the IR3 culture. The compounds were
presumptively identified by comparison of HPLC retention times with
authentic standards (Table 4). The UV absorbance spectra of
authentic 4-carboxybenzyl alcohol, terephthalic acid and p-toluic
acid standards were compared to the UV spectra of the presumptive
4-carboxybenzyl alcohol, terephthalic acid and p-toluic acid peaks
(FIGS. 2A and 2B, 3A and 3B, and 4A and 4B, respectively). The
UV-visible spectrum of each compound detected in the IR3 culture
was identical to the UV-visible absorbance spectrum of the
corresponding standard. These results supported two conclusions.
First, each one of the three major peaks that were detected by HPLC
represented a single compound. Second, comparison of the UV spectra
confirmed the presumptive identification of each peak.
5TABLE 4 Production of p-toluic acid and terephthalic acid from
p-xylene by Burkholderia strain IR3 (ATCC 202150) Standard Peak
Peak Retention Time (min) Standard Retention Time (min) 1 6.487
4CBA 6.488 2 9.587 TPA 9.571, 9.568 3 24.188 PTA 24.173
[0106] Mass Spectrometry:
[0107] Isolate IR3 (ATCC 202150) was grown to an OD.sub.600 of 0.60
in duplicate 250 mL screw-cap Erlenmeyer flasks containing 23 mL of
S12 medium with 200 .mu.L of p-xylene in a reservoir. Aliquots (1.0
mL) of the cultures were placed in 1.5 mL microfuge tubes and mixed
with 10 .mu.L of p-xylene. The aliquots were incubated without
shaking for 72 hours at 25.degree. C. and then filtered. The
aliquots from each flask were pooled and a 1 mL sample from each
pool was analyzed by reverse-phase HPLC with a diode array
UV-visible detector at 240 nm. Three major compounds were present
in the IR3 cultures. The compounds were presumptively identified by
retention times as 4-carboxybenzyl alcohol, terephthalic acid and
p-toluic acid.
[0108] The filtered supernatants derived from both IR3 cultures
were combined for preparative HPLC. Preparative HPLC was performed
with a Zorbax RXC8 9.4 mm.times.25 cm (HP part no. 880952.206) with
a 50-250 .mu.L injection volume. The mobile phase consisted of (A)
H.sub.2O containing 2 mL of acetic acid/L and (B) acetonitrile. The
peaks corresponding to 4-carboxybenzyl alcohol, terephthalic acid
and p-toluic acid were manually collected from the HPLC effluent
into 20 mL glass vials. Each of the collected peaks was
concentrated to approximately 1 mL in a Savant Speed Vac.
[0109] GC-MS was performed on a Hewlett Packard 5890 Series II GC
with a HP 5971 Mass Selective Detector. The column was a HP-5 MS 30
m.times.0.25 mm with 0.25 .mu.m film thickness. Samples of HPLC
peaks and standards were dried and derivatized with 1 ampule of
dimethylacetamide dimethyl acetal in a closed vial at 60.degree. C.
for 30 min, venting after 5 and 10 minutes. The vial was washed
with a small amount of reagent-grade methanol, and the vial was
incubated at 60.degree. C. for an additional 20 min. Derivatized
samples (2 .mu.L) were injected, held at 40.degree. C. for 1 min,
ramped to 280.degree. C. at a rate of 10 degrees per minute, and
held at that temperature for 5 min. This analysis confirmed that
isolate IR3 (ATCC 202150) produced p-toluic acid.
[0110] MS/MS was performed on a Finnigan SSQ 7000 mass
spectrometer. Derivatized samples and standards were analyzed by
direct insertion with a probe into the mass spectrometer in
electron impact mode using a single quadrapole. The probe was
ramped from 40.degree. C. to 300.degree. C. at 26 degrees per min.
This analysis confirmed that isolate IR3 (ATCC 202150) produced
p-toluic acid and terephthalic acid.
[0111] Mass spectrometry analysis was performed on a TSQ700 mass
spectrometer from Finnigan of San Jose, Calif. The ion source was
electrospray with a heated capillary (Finnigan). The pooled and
concentrated HPLC peaks of p-toluic acid, 4-carboxybenzyl alcohol
and terephthalic acid were diluted 1:1 in acetonitrile. The samples
were directly infused into the instrument which was run in
electrospray negative ion mode (single quadrapole). The scanning
range was 50 to 350. This analysis confirmed that isolate IR3 (ATCC
202150) produced p-toluic acid, 4-carboxybenzyl alcohol and
terephthalic acid.
[0112] Metabolic Pathway for Conversion of p-Xylene into
Terephthalic Acid:
[0113] p-Toluic acid, 4-carboxybenzyl alcohol and terephthalic acid
were detected in cultures of isolate IR3 (ATCC 202150) that were
given p-xylene as the sole source of carbon and energy. This result
was consistent with sequential oxidation of one methyl group on
p-xylene to form p-toluic acid with subsequent sequential oxidation
of the second methyl group to form terephthalic acid (FIG. 5).
Although 4-methylbenzyl alcohol, 4-methylbenzaldehyde and
4-carboxybenzaldehyde were not detected in cultures of isolate IR3
(ATCC 202150), IR3 utilized all three of these compounds as sole
sources of carbon and energy.
Example 4
Production of Terephthalic Acid from p-Xylene by Isolate IR10 (ATCC
202151) After Growth in Rich Medium
[0114] Example 4 demonstrates that Burkholderia isolate IR10 (ATCC
202151) converted p-xylene to terephthalic acid after initial
growth in LB medium indicating that one type of medium could be
used to generate cell mass and that the cells could be switched to
a second medium for production of terephthalic acid. Isolate IR10
(ATCC 202151) was grown for 9 h in 45 mL of LB medium in a 250 mL
screw-cap Erlenmeyer flask with 250 .mu.L of p-xylene in a
reservoir. The flask was incubated at 28.degree. C. with shaking.
The cells were harvested by centrifugation and resuspended in 40 mL
of S12 medium. A 20 mL aliquot of cells was incubated at 25.degree.
C. without shaking for 30 min in a 250 mL screw-cap Erlenmeyer
flask with 200 .mu.L of p-xylene in a reservoir. Samples (1.5 mL)
were removed from the flask and mixed with 15 .mu.L of p-xylene in
a microfuge tube. The samples were incubated at 25.degree. C.
without shaking for the times indicated in Table 5. The samples
were filtered and analyzed for p-toluic acid and terephthalic acid
by HPLC. P-Toluic acid and terephthalic acid were identified by
retention times.
[0115] The data in Table 5 indicate that cells initially grown in
LB medium and resuspended in S12 medium produced significant
amounts of p-toluic acid and terephthalic acid during a short
incubation. The amounts of p-toluic acid and terephthalic acid
increased with prolonged incubation.
6TABLE 5 Production of p-toluic acid and terephthalic acid after
growth in LB medium Concentration (ppm) Incubation Time PTA TPA 30
min 137.1 24.7 3 days 886.3 89.0
Example 5
Production of Isophthalic Acid from m-Xylene by Isolate IR3
[0116] Example 5 demonstrates that isolate IR3 converted m-xylene
into m-toluic acid and isophthalic acid in the presence of
p-xylene. The conversion follows the pathway as outlined in FIG.
6.
[0117] Isolate IR3 was grown for 18 h in 25 mL of S12 medium in 250
mL screw-cap Erlenmeyer flasks with 200 .mu.L of p-xylene or a
mixture of 100 PL of p-xylene and 100 .mu.L of m-xylene in a
reservoir. The cultures were diluted to an OD.sub.600 of 0.30 and
25 mL aliquots were dispensed into screw-cap Erlenmeyer flasks. The
culture that had been grown with p-xylene alone was given a
reservoir with 200 .mu.L of p-xylene alone. The culture that had
been grown with p-xylene and m-xylene was given a reservoir with a
mixture of 100 .mu.L of p-xylene and 100 .mu.L of m-xylene. The
cultures were incubated without shaking at 28.degree. C. Samples
(1.5 mL) were collected at the indicated times and filtered. The
first sample was collected immediately after the cells were
suspended in new medium (0 h). The samples were analyzed by HPLC
and p-toluic acid, terephthalic acid, m-toluic acid and isophthalic
acid were identified by retention times.
[0118] The data in Table 4 indicate that bacteria initially grown
and then incubated with p-xylene alone produced p-toluic acid and
terephthalic acid. Isophthalic acid was not detected in the
p-xylene culture. A small amount of m-toluic acid was in the
culture at the end of the experiment. Production of m-toluic acid
in this culture was attributed to the presence of a residual amount
of m-xylene (<1%) in the commercial stock of p-xylene. Bacteria
initially grown and then incubated with a mixture of p-xylene and
m-xylene also produced small amounts of p-toluic acid and
terephthalic acid. The bacteria exposed to p-xylene and m-xylene
produced high levels of m-toluic acid after the initial sample
time. A significant level of isophthalic acid was detected in the
culture after 278 h. The UV-visible absorbance spectra of the
presumptive m-toluic acid and isophthalic acid peaks were compared
to the UV-visible spectra of authentic standards. The UV-visible
spectra of m-toluic acid and isophthalic acid detected in the IR3
culture were identical to the UV-visible absorbance spectrum of the
corresponding standards. These results supported two conclusions.
First, the presumptive m-toluic acid and isophthalic acid HPLC
peaks represented single compounds. Second, comparison of
UV-visible spectra confirmed the presumptive identification of each
peak. Therefore, isolate IR3 (ATCC 202150) converted m-xylene into
m-toluic acid and isophthalic acid in the presence of p-xylene even
though m-xylene alone could not be utilized for growth.
7TABLE 5 Production of m-toluic acid and isophthalic acid from
m-xylene by Burkholderia strain IR3 (ATCC 202150) Concentration
(ppm) Time (h) Carbon Source PTA TPA MTA IPA 0 p-xylene .sup.
ND.sup.a ND ND ND p-xylene + m-xylene ND ND ND ND 69 p-xylene 231.8
ND ND ND p-xylene + m-xylene 6.4 5.3 490.5 ND 116 p-xylene 577.0
24.2 ND ND p-xylene + m-xylene ND 56.0 661.1 ND 278 p-xylene 308.8
104.4 2.5 ND p-xylene + m-xylene ND ND 833.1 12.3 .sup.aNot
detected
Example 6
Production of Terephthalic Acid from p-Xylene in Mixed Cultures of
Pseudomonas putida Strain ATCC 33015 and Comamonas testosteroni
Strain DSM 6577
[0119] Biological production of terephthalic acid from p-xylene
requires a set of enzymes that oxidize both methyl groups of
p-xylene. Conversion of p-xylene into terephthalic acid by isolate
IR3 is accomplished by a single cell line or single bacterial
strain that produces all of the requisite enzymes. Example 6
demonstrates that a bacterial strain that converts p-xylene into
p-toluic acid (strain ATCC 33015) could be mixed with a different
bacterial strain that converts p-toluic acid into terephthalic acid
(DSM 6577) with the result that the mixture of bacteria converted
p-xylene into TPA.
[0120] Strain ATCC 33015 was grown for 18 h in 25 mL of S12 medium
in a 250 mL screw-cap Erlenmeyer flask with 100 .mu.L of p-xylene
in a reservoir. The cells were harvested by centrifugation and
resuspended in 15 mL of S12 medium. Strain DSM 6577 was grown 6 h
in 25 mL of LB medium in a 250 mL screw-cap Erlenmeyer flask. The
DSM 6577 cells were harvested by centrifugation and resuspended in
50 mL of S12 medium. One control flask had ATCC 33015 alone in 25
mL of S12 medium at an initial OD.sub.600 of 0.05. A second control
flask had DSM 6577 alone in 25 mL of S12 medium at an initial
OD.sub.600 of 0.01. A third flask was inoculated with 3 mL ATCC
33015 cells and 1 mL of the DSM 6577 cells. Each flask had a
reservoir with 200 .mu.L of p-xylene. The cultures were incubated
without shaking at 28.degree. C. Samples (1.5 mL) were collected at
the indicated times and filtered. The first sample was collected
immediately after the two types of bacteria were mixed (0 h). The
samples were analyzed by HPLC and 4-carboxybenzyl alcohol, p-toluic
acid and terephthalic acid were identified by retention times.
8TABLE 6 Production of terephthalic acid from p-xylene in mixed
cultures of ATCC 33015 and DSM 6577 Concentration (ppm) Time (h)
Culture PTA 4CBAL TPA 0 ATCC 33015 ND.sup.a ND ND DSM 6577 ND ND ND
ATCC 33015 + DSM 6577 ND ND ND 22 ATCC 33015 89.6 ND ND DSM 6577 ND
ND ND ATCC 33015 + DSM 6577 150.4 1.4 ND 68 ATCC 33015 20.4 ND ND
DSM 6577 ND ND ND ATCC 33015 + DSM 6577 226.8 1.6 0.08 .sup.aNot
detected
[0121]
Sequence CWU 1
1
4 1 1451 DNA Burkholderia sp. 1 tggaacgctg ggcggcatgc cttacacatg
caattcaaac ggcagcacgg gtgcttgcac 60 ctggtggcga ttggcgaacg
ggtgattaat acatcggaat gtaccttgta gtgggggata 120 cctcggcaaa
agccggatta ataccgcata cgctctgagg aggaaagcgg gggaccttcg 180
ggcctcgcgc tacaaaagca gccgatgtca aattacctat ttggtggggt aaaagctcac
240 caaggcgaca atctgtacct ggtctgagag gacaaccacc cacactggga
ctgaaacacg 300 gcccaaactc ctacgggagg cagcagtggg gaattttgga
caatgggcga aagcctgatc 360 caccaatgcc gcgtgtgtga aaaaggcctt
cgggttgtaa agcacttttg tccggaaaga 420 aatcctctgg gttaatacct
cggggggatg acggtaccgg aaaaataagc accggctaac 480 tacttgccac
agccgcggta atacttaggg tgcaagcgtt aatcggaatt actgggcgta 540
aagcgtgcgc aggcggtttt gtaagacgga tgtgaaatcc ccgggcttaa cctgggaact
600 gcattcgtga ctgcaaggct agagtatggc agaggggggt agaattccac
gtgtagcagt 660 gaaatgcgta gagatgtgga ggaataccga tggcgaaggc
agccccctgg gccaatactg 720 acgctcatgc acgaaagcgt ggggagcaaa
caggattaga taccctggta gtccacgccc 780 taaacgatgt caactagttg
ttggggattc atttccttag taacgaagct aacgcgtgaa 840 gttgaccgcc
tggggagtac ggtcgcaaga ttaaaactca aaggaattga cggggacccg 900
cacaagcggt ggatgatgtg gattaattcg atgcaacgcg aaaaacctta cctacccttg
960 acatgtacgg aatcttgctg agaggtgaga gtgctcgaaa gagaaccgta
acacaggtgc 1020 tgcatggctg tcgtcagctc gtgtcgtgag atgttgggtt
aagtcccgca acgagcgcaa 1080 cccttgtcct tagttgctac gcaagagcac
tctaaggaga ctgccggtga caaaccggag 1140 gaaggtgggg atgacgtcaa
gtcctcatgg cccttatggg tagggcttca cacgtcatac 1200 aatggtcggt
acagagggct gccaaaccgc gaggtggagc taaccccaga aaaccgatcg 1260
tagtccggat cgcagtctgc aactcgactg cgtgaagctg gaatcgctag taatcgcgga
1320 tcagcatgtc gcggtgaata cgttcccggg tcttgtacac accgcccgtc
acaccatggg 1380 agtgggtttt gccagaagta ggtagcctaa ccgtaaggag
ggcgcttacc acggcaggat 1440 catgactggg g 1451 2 19 DNA Artificial
Sequence Description of Artificial Sequence HK12 primer 2
gagtttgatc ctggctcag 19 3 16 DNA Artificial Sequence Description of
Artificial Sequence HK13 primer 3 taccttgtta cgactt 16 4 17 DNA
Artificial Sequence Description of Artificial Sequence HK14 primer
4 gtgccagcag ymgcggt 17
* * * * *