U.S. patent application number 14/148820 was filed with the patent office on 2014-07-10 for extracellular polyhydroxyalkanoates produced by genetically engineered microorganisms.
The applicant listed for this patent is Wolf-Rainer Abraham, Manuel Ferrer, Peter Golyshin, Heinrich Lunsdorf, Julia Sabirova, Kenneth Timmis. Invention is credited to Wolf-Rainer Abraham, Manuel Ferrer, Peter Golyshin, Heinrich Lunsdorf, Julia Sabirova, Kenneth Timmis.
Application Number | 20140193868 14/148820 |
Document ID | / |
Family ID | 35285615 |
Filed Date | 2014-07-10 |
United States Patent
Application |
20140193868 |
Kind Code |
A1 |
Sabirova; Julia ; et
al. |
July 10, 2014 |
EXTRACELLULAR POLYHYDROXYALKANOATES PRODUCED BY GENETICALLY
ENGINEERED MICROORGANISMS
Abstract
The present invention is in the field of biosynthesis of
polyhydroxyalkanoates (PHA). The invention relates to a genetically
engineered microorganism having at least one gene involved in the
metabolism, preferably in the production, of polyhydroxyalkanoates
(PHA). This microorganism is useful in commercial production of
polyhydroxyalkanoates. The present invention further relates to a
method for the production of polyhydroxyalkanoates (PHA).
Inventors: |
Sabirova; Julia;
(Brauschweig, DE) ; Golyshin; Peter;
(Wolfenbuttel, DE) ; Ferrer; Manuel; (Madrid,
ES) ; Lunsdorf; Heinrich; (Erkerode, DE) ;
Abraham; Wolf-Rainer; (Hillerse, DE) ; Timmis;
Kenneth; (Wolfenbuttel, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sabirova; Julia
Golyshin; Peter
Ferrer; Manuel
Lunsdorf; Heinrich
Abraham; Wolf-Rainer
Timmis; Kenneth |
Brauschweig
Wolfenbuttel
Madrid
Erkerode
Hillerse
Wolfenbuttel |
|
DE
DE
ES
DE
DE
DE |
|
|
Family ID: |
35285615 |
Appl. No.: |
14/148820 |
Filed: |
January 7, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11989909 |
Jul 30, 2010 |
8623632 |
|
|
PCT/EP2006/007888 |
Aug 9, 2006 |
|
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14148820 |
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Current U.S.
Class: |
435/135 ;
435/193; 435/196; 435/252.3; 435/252.34; 435/320.1; 536/23.2 |
Current CPC
Class: |
C12N 9/13 20130101; C12N
9/16 20130101; C12N 9/1205 20130101; C12N 9/88 20130101; C12N
9/1029 20130101; C12P 7/625 20130101 |
Class at
Publication: |
435/135 ;
536/23.2; 435/193; 435/252.3; 435/252.34; 435/320.1; 435/196 |
International
Class: |
C12P 7/62 20060101
C12P007/62 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 9, 2005 |
EP |
05017308.7 |
Claims
1. A genetically engineered microorganism having at least one
modification in at least one gene encoding a protein involved in
the metabolism of polyhydroxyalkanoate(s) (PHA), or in at least one
gene encoding a protein, which interferes in the metabolism of the
microorganism with the production of PHA, wherein the at least one
modification causes deposition and/or overproduction of medium or
long chain polyhydroxyalkanoate(s) PHA.
2. The genetically engineered microorganism of claim 1, wherein the
at least one gene encodes a protein, which interferes in the
metabolism of the microorganism with the production of PHA, wherein
the protein is an enzyme, which competes with enzymes of the PHA
synthesis pathway for intermediates of the PHA synthesis
pathway.
3. The genetically engineered microorganism of claim 2, wherein the
enzyme is a thioesterase.
4. The genetically engineered microorganism of claim 2, wherein the
thioesterase acts on (R)-3-OH-acyl-CoA as a substrate.
5. The microorganism of claim 2, wherein the thioesterase is
acyl-CoA thioesterase tesB-like.
6. The genetically engineered microorganism of claim 2, wherein the
enzyme is encoded by a nucleic acid sequence comprising a
(modified) nucleic acid sequence as shown in any of FIGS. 11 and
12, or a homolog thereof.
7. The genetically engineered microorganism of claim 2, wherein the
enzyme is encoded by a nucleic acid sequence comprising a (native)
nucleic acid sequence as shown in any of FIGS. 14, 16 and 18 to 25,
or a homolog thereof, wherein the native nucleic acid sequence is
modified by the at least one modification.
8. The genetically engineered microorganism of claim 1, wherein the
at least one modification is causes a complete or partial
inactivation of the modified gene.
9. The genetically engineered microorganism of claim 1, wherein the
at least one modification is effected by a transposon insertion
selected from the group consisting of Tn5 and Tn10 transposons,
preferably by a Tn5 insertion, more preferably on the gene
downstream to the modified gene.
10. The genetically engineered microorganism of claim 1, wherein
the at least one modification is effected by transposon
mutagenesis, preferably based on miniTn5 Km element, more
preferably based on miniTn5 Str/Sp element.
11. The genetically engineered microorganism of claim 1, wherein
the gene is integrated into the chromosome of the
microorganism.
12. The genetically engineered microorganism of claim 1, wherein
the at least one gene encodes a protein selected from the group
consisting of PHA synthase, PHB synthase, acyl-CoA transferase,
enyol-CoA hydratase or reductase.
13. The genetically engineered microorganism of claim 1, wherein
the at least one modification causes an extracellular deposition or
an overproduction of polyhydroxyalkanoate(s) PHA, preferably medium
or long chain polyhydroxyalkanoates (PHA), produced by the
microorganism.
14. The genetically engineered microorganism of claim 13, wherein
the extracellular deposition or overproduction of
polyhydroxyalkanoate(s) PHA, results in a PHA production which is
at least 5 times, preferably at least 10 times, more preferably at
least 15 times, more preferably at least 25 times, even more
preferably at least 40 times, even more preferably at least 50
times, most preferably at least 60 times, most preferably at least
80 times, even most preferably at least 100 times higher than the
PHA production of the corresponding wild type microorganism.
15. The genetically engineered microorganism of claim 1, wherein
the microorganism is selected from the group consisting of
Alcanivorax borkumensis, Pseudomonas putida, Pseudomonas
aeruginosa, Pseudomonas syringae, Pseudomonas fluorescens,
Pseudomonas acitophila, Pseudomonas oleovarans, Idiomarina
loihiensis, Acinetobacter sp., Caulobacter crescentus, Alcaligenes
eutrophus, Alcaligenes latus, Azotobacter vinlandii, Rhodococcus
eutropha, Chromobacterium violaceum, and Chromatium vinosum.
16. The genetically engineered microorganism of claim 1, wherein
the microorganism is selected from the group consisting of
Alcanivorax, preferably Alcanivorax borkumensis, more preferably
Alcanivorax borkumensis SK2.
17. A nucleic acid sequence which constitutes at least one gene
encoding a protein involved in the metabolism of
polyhydroxyalkanoate(s) (PHA), or in at least one gene encoding a
protein, which interferes in the metabolism of the microorganism
with the production of PHA, wherein the at least one modification
causes deposition and/or overproduction of medium or long chain
polyhydroxyalkanoate(s) PHA.
18. The nucleic acid sequence of claim 17, wherein the gene is
selected from the group consisting of PHA synthase, PHB synthase,
acyl-CoA transferase, and enyol-CoA.
19. The nucleic acid sequence of claim 17 wherein the gene is
acyl-CoA thioesterase tesB-like, preferably acyl-CoA thioesterase
tesB-like of Alcanivorax borkumensis, Pseudomonas putida,
Pseudomonas aeruginosa, Pseudomonas syringae, Pseudomonas
fluorescens, Pseudomonas acitophila, Pseudomonas oleovarans,
Idiomarina loihiensis, Acinetobacter sp., Caulobacter crescentus,
Alcaligenes eutrophus, Alcaligenes latus, Azotobacter vinlandii,
Rhodococcus eutropha, Chromobacterium violaceum, or Chromatium
vinosum.
20. The nucleic acid sequence of claim 17 wherein the gene is
acyl-CoA thioesterase tesB-like, preferably acyl-CoA thioesterase
tesB-like of Alcanivorax borkumensis, more preferably acyl-CoA
thioesterase tesB-like of Alcanivorax borkumensis SK2.
21. The nucleic acid sequence of claim 17, wherein the at least one
modification is effected by a transposon insertion, preferably Tn-5
insertion, on the gene downstream to the modified gene.
22. The nucleic acid sequence of claim 17, wherein the at least one
modification is achieved by transposon mutagenesis, preferably
based on miniTn5 Km element, more preferably based on miniTn5
Str/Sp element.
23. The nucleic acid sequence of claim 17, wherein the nucleic acid
comprises a (modified) nucleic acid sequence as shown in any of
FIGS. 11 and 12, or a homolog thereof.
24. The nucleic acid sequence of claim 17, wherein the nucleic acid
comprises a (native) nucleic acid sequence as shown in any of FIGS.
14, 16 and 18 to 25, or a homolog thereof, wherein the native
nucleic acid sequence is modified by the at least one
modification.
25. Polypeptide encoded by a nucleic acid sequence of claim 17.
26. Polypeptide of claim 25, wherein the polypeptide comprises one
of the amino acid sequences shown in FIG. 11 or 12.
27. A vector comprising the nucleic acid sequence of claim 17.
28. A vector comprising a PHA producing gene cluster comprising a
gene having at least one modification in at least one gene encoding
for a protein involved in the metabolism of polyhydroxyalkanoate(s)
(PHA), or in at least one gene encoding for a protein, which
interferes in the metabolism of the genetically engineered
microorganism of claim 1 with the production of PHA, wherein the at
least one modification causes extracellular deposition of medium or
long chain polyhydroxyalkanoate(s) (PHA).
29. A cell comprising the vector of claim 27 and/or the nucleic
acid sequence of claim 17.
30. A method for producing polyhydroxyalkanoates (PHA) comprising
the following steps: a. cultivating a microorganism of claim 1 or a
cell of claim 29 and b. recovering polyhydroxyalkanoates (PHA) from
the culture medium.
31. (canceled)
32. (canceled)
Description
[0001] The present invention is in the field of biosynthesis of
polyhydroxyalkanoates (PHA). The invention relates to a genetically
engineered microorganism having at least one gene involved in the
metabolism, preferably in the production, of polyhydroxyalkanoates
(PHA). This microorganism is useful in commercial production of
polyhydroxyalkanoates. The present invention further relates to a
method for the production of polyhydroxyalkanoates (PHA).
[0002] Polyhydroxyalkanoates (PHA) are polymers that are
biodegradable and biocompatible thermoplastic materials (polyesters
of 3-hydroxy fatty acids), produced from renewable resources, with
a broad range of industrial and biomedical applications (Williams
and Peoples, 1996). Polyhydroxyalkanoates (PHA) are synthesized by
a broad range of bacteria and have been extensively studied due to
their potential use to substitute conventional petrochemical
plastics to protect the environment from harmful effects of plastic
wastes.
[0003] PHA can be divided into two groups according to the length
of their side chains and their biosynthetic pathways. Those with
short side chains, such as PHB, a homopolymer of
(R)-3-hydroxybutyric acid units, are crystalline thermoplastics,
whereas PHA with long side chains are more elastomeric. The former
have been known for about seventy years (Lemoigne and Roukhelman,
1925), whereas the latter materials were discovered relatively
recently (deSmet et al., 1983, J. Bacteriol. 154: 870-78). Before
this designation, however, PHA of microbial origin containing both
(R)-3-hydroxybutyric acid units and longer side chain
(R)-3-hydroxyacid units from 5 to 16 carbon atoms had been
identified (Wallen, Rohweder, 1974, Environ. Sci. Technol. 8:
576-79). A number of bacteria which produce copolymers of
(R)-3-hydroxybutyric acid and one or more long side chain
hydroxyacid units containing from 5 to 16 carbon atoms have been
identified (Steinbuchel, Wiese, 1992, Appl. Microbiol. Biotechnol.
37: 691 97; Valentin et al., 1992, Appl. Microbiol. Biotechnol. 36:
507-14; Valentin et al., Appl. Microbiol. Biotechnol. 1994, 40:
710-16; Abe et al., 1994, Int. J. Biol. Macromol. 16: 115-19; Lee
et al., 1995, Appl. Microbiol. Biotechnol. 42: 901-09; Kato et al.,
1996, Appl. Microbiol. Biotechnol. 45: 363-70; Valentin et al.,
1996, Appl. Microbiol. Biotechnol. 46: 261-67; U.S. Pat. No.
4,876,331). These copolymers can be referred to as PHB-co-HX
(wherein X is a 3-hydroxyalkanoate or alkanoate or alkenoate of 6
or more carbons). A useful example of specific two-component
copolymers is PHB-co-3-hydroxyhexanoate (PHB-co-3HH) (Brandl et
al., 1989, Int. J. Biol. Macromol. 11: 49-55; Amos & McInerey,
1991, Arch. Microbiol. 155: 103-06; U.S. Pat. No. 5,292,860).
[0004] However, although PHAs have been extensively studied because
of their potential use as renewable resource for biodegradable
thermoplastics and biopolymers (as mentioned above) and have been
commercially developed and marketed (Hrabak, O. 1992), their
production costs are much higher than those of conventional
petrochemical-based plastics, thus presenting a major obstacle to
their wider use (Choi and Lee 1997). As described above, many
bacteria produce PHA, e.g. Alcaligenes eutrophus, Alcaligenes
latus, Azotobacter vinlandii, Pseudomonas acitophila, Pseudomonas
oleovarans, Eschericha coli, Rhodococcus eutropha, Chromobacterium
violaceum, Chromatium vinosum, Alcanivorax borkumensis etc.. All
PHA producing bacteria known in the art produce intracellular PHA
and accumulate it in PHA granules (Steinbuchel, 1991). The main
aspect which renders PHA production expensive and therefore
unfavorable as compared to petrochemical-based plastic, is the
difficulty to recover produced PHA from within the bacterial cells
where it is accumulated. In order to reduce the total production
costs of PHA the development of an efficient recovery process was
considered to be necessary generally aiming at cell disruption (Lee
1996) by [0005] i) an appropriate solvent, [0006] ii) hypochlorite
extraction of PHA and/or [0007] iii) digestion of non-PHA cellular
materials.
[0008] However, at an industrial scale, all methods known in the
art require large amounts of chemical reagents and/or enzymes which
is an obstacle to reducing the recovery cost. Therefore,
alternative strategies for PHA recovery are in urgent need.
[0009] The object of the present invention is to provide a system
permitting production of commercially significant levels of PHA
while reducing the costs for recovering produced PHA.
[0010] This technical problem is solved by the invention,
especially in providing a microorganism that complies with the
aforementioned requirements and is applicable in said system.
[0011] The invention relates in its first embodiment to a
genetically engineered microorganism having at least one
modification in at least one gene encoding for a protein involved
in the metabolism of polyhydroxyalkanoate(s) (PHA) or, preferably,
in at least one gene encoding for a protein, which interferes in
the metabolism of the microorganism with the production of PHA,
wherein the at least one modification causes extracellular
deposition, e.g. excretion, of polyhydroxyalkanoate(s) (PHA),
preferably medium or long chain polyhydroxyalkanoate(s) (PHA),
produced by the microorganism, preferably in/into the culture
medium. Preferably, the microorganism comprises one of the nucleic
acid sequences shown in FIGS. 11, 12, 14, 16 und 18 to 25 or a
functional fragment or variant thereof. More preferably, the
microorganism may comprise nucleic acids No. 180 to No. 680,
preferably No. 230 to No. 640, more preferably No. 310 to No. 550,
most preferably No. 350 to No. 510, according to one of the nucleic
acid sequences shown in FIGS. 11, 12, 14, 16 und 18 to 25, or a
functional fragment or variant thereof.
[0012] It has been found by the inventors that a genetically
engineered microorganism according to the invention produces
extracellular PHA by extracellularly depositing, e.g. excreting PHA
(produced intracellularly) in/into the culture medium. The
deposition, e.g. excretion of PHA by a microorganism has not been
described before in the art. Preferably, the microorganism of the
present invention produces large amounts of PHA, preferably
overproduces PHA, and deposits a large proportion of its PHA
product in the extracellular medium, unlike the microorganisms
known in the art. Extracellular deposition, e.g. excretion, and
overproduction of large amounts PHA into the culture medium was
achieved by modifying at least one gene encoding a polypeptide
involved in the metabolism of PHA or, preferably by introducing at
least one modification in a gene encoding a polypeptide (enzyme)
interfering with the production of PHA. The term "polypeptide"
according to the invention also encompasses the terms "peptide",
"protein" or "enzyme".
[0013] Various genes encoding polypeptides are involved in the
metabolism of PHA. Several of these genes are specified in Table 1
as represented by FIG. 5. Thus, a gene according to the invention
encompasses any gene encoding a polypeptide which is involved in
PHA metabolism, preferably in PHA production. Preferably, such gene
encodes (without being restricted to) PHA synthase,
poly(3-hydroxyalkanoate)synthase, enyol-CoA hydratase, and PHB
synthase.
[0014] Other enzymes, which are also involved in the fatty acid
metabolism, e.g. fatty acid synthesis or beta-oxidation
(poly(3-hydroxybutyrate)depolymerase, acyl-CoA transferase,
reductase, or thiolase), may be modified as well. However, these
enzymes do not specifically influence PHA synthesis and are,
therefore, less preferred for a modification allowing to increase
PHA synthesis.
[0015] Preferably, the set of genes required for PHA production is
not compromised in a microorganism of the invention. Any
modification introduced into any of the PHA production genes (see
above) is preferably intended not to reduce, but to enhance their
enzymatic activity. Thereby, the microorganism according to the
invention is allowed to provide a higher yield of PHA than
microorganisms known in the art.
[0016] In contrast, a microorganism according to the invention
contains most preferably modifications in any genes encoding for
polypeptides which compromise the production of PHA. In particular,
a microorganism according to the invention is modified in a gene
encoding_enzymes which cleave Acyl-CoA molecules by a thioesterase
function, e.g. Acyl-CoA thioesterases. Depending on the specific
microorganisms one or more Acyl-CoA thioesterases are acting on the
fatty acid metabolism. Acyl-CoA thioesterases were especially
studied in E. coli possessing two of such enzymes called acyl-CoA
thioesterase I (encoded by tesA gene) and acyl-CoA thioesterase II
(encoded by tesB gene). Thioesterase I exhibits specificity towards
C12 to C18 acyl-CoA esters (Bonner, W M et al., 1972, J. Biol.
Chem. 247, 3123-3133), while thioesterase II cleaves C6 to C18
acyl-CoA esters as well as .beta.-hydroxyacyl-CoA esters of chain
length C12 to C18 (Barnes et al., 1970, The Journal of Biological
Chemistry, vol. 245, No. 12, issue of June 25, 3122-3128). TesA is
implicated in chain termination of de novo biosynthesis of fatty
acids and mediates acyl-ACP intermediates from the fatty acid de
novo biosynthesis pathway to fatty acid .beta.-oxidation in E. coli
(Klinke, S Q et al., 1999, Appl. Environ. Microbiol. 65: 540-548).
Up to now, little was known on physiological role of tesB in
bacterial metabolism.
[0017] A very recent report described that thioesterase II plays an
important role in 3-hydroxyalkanoic acid (3-HAA) production (Zweng,
Z et al., 2004, Appl. Environ. Microbiol. 70(7): 3807-3813) by
cleaving 3-hydroxyacyl-CoA thioester bonds thereby converting them
into free 3-HAA. According to the invention, it was found that
various microorganisms express thioesterases which act with a high
specific activity on the cleavage of 3-hydroxyacyl-CoAs which forms
the building block of PHA synthesis. These thioesterases allow to
release free 3-HAAs. However, the conversion to 3-HAA is a reaction
which competes with the synthesis of PHA by PHA-synthase, which
acts on the same cellular intermediate (namely 3-hydroxyacyl-CoAs).
According to the invention, it was found that (i) the release of
free 3-HAAs and the synthesis of PHA are interfering metabolic
pathways and (ii) that functional knock-out of the specific
thioesterase, herein termed tesB-like thioesterase, provides for
deposition of PHA in the extracellular medium. A functional
knock-out mutation in a tesB-like gene (as described below in more
detail) was identified to increase the intracellular amount of
3-hydroxyacyl-CoA in a number of microorganism, thereby guiding the
metabolism of 3-hydroxyacyl-CoA (as PHA precursor) towards PHA
synthesis (see FIG. 8).
[0018] As explained above, the present invention is based on the
general finding that (knock-out) modifications of thioesterases
using (R)-3-OH-acyl-CoA as substrate allow PHA producing
microorganisms to deposit PHA in the extracellular medium. While
e.g. in Alkanivorax, Pseudomonas putida, Pseudomonas aeruginosa,
Pseudomonas syringae, Pseudomonas fluorescens, Acinetobacter sp.,
Caulobacter crescentus the thioesterase found to act specifically
on (R)-3-OH-acyl-CoA, is acyl-CoA thioesterase tesB-like, other PHA
producing microorganism being based on a slightly different
metabolism use other thioesterase, e.g. acyl-CoA thioesterase tesB
or tesA, to produce 3-HAA.
[0019] In any case, a modification of an thioesterase specifically
using (R)-3-OH-acyl-CoA to produce 3-HAA is most preferably
expected to either reduce the thioesterase activity, e.g. of
acyl-CoA thioesterase tesB-like, considerably, e.g. by at least
50%, more preferably by at least 60%, even more preferably by at
least 80%, more preferably by at least 90%. In a particularly
preferred embodiment, the enzymatic activity of this thioesterase,
e.g. acyl-CoA thioesterase tesB-like, is completely switched
off.
[0020] The present invention also encompasses tesB-like homologs of
proteins as disclosed herein (and as well their encoding nucleic
acids), particularly of thioesterases. In the context of the
present invention tesB-like homologs of proteins as disclosed
herein is meant to include any protein or peptide sequence,
occurring in a different microorganism than a tesB-like homolog of
proteins as disclosed herein, which preferably show a (significant)
homology to this tesB-like protein and display similar or even
identical biological function. A (significant) homology to a
tesB-like protein as shown herein may be easily determined by a
skilled person using method well known in the art, e.g. methods for
determining sequence identities as also disclosed herein or by
activity assays.
[0021] According to the finding of the present invention
Alkanivorax is characterized by two tesB thioesterases, namely
acyl-CoA thioesterase tesB-like specifically acting on
(R)-3-OH-acyl-CoA to produce 3-HAA and acyl-CoA thioesterase tesB,
which is involved in the production of fatty acids. By performing
homology searches (BLAST searches) in several PHA producing
bacteria the inventors have screened for other microorganisms
showing the same or a similar metabolic structure as in Alkanivorax
(with a homologue of the highly specific acyl-CoA thioesterase
tesB-like and another thioesterase (tesB)).
[0022] Both types of thioesterases being present in Alcanivorax
borkumensis SK2 are also expressed in a number of closely related
PHA producing gamma-proteobacteria (e.g., Pseudomonas putida, P.
aeruginosa, P. aeruginosa, P. syringae, P. fluorescens, Idiomarina
loihiensis, Acinetobacter sp., Caulobacter crescentus). The
homologues of the Alkanivorax tesB-like protein are named
differently in the other microorganisms, e.g. as tesB -like
thioesterase, putative acyl-CoA thioesterase II or hypothetical
protein. However, it is to be understood that the term "tesB-like"
is intended to encompass all these thioesterases of the
aforementioned microorganism with deviating nomenclature. These
homologues of the Alkanivorax tesB-like thioesterase are presented
in Table 4 (see Table 4 of FIG. 10). The presence of two
thioesterases in Alkanivorax and the other aforementioned
microorganisms, namely the tesB and tesB-like proteins, is
explained by the present inventors by different functions of tesB
and tesB-like proteins. Most likely, the tesB protein exclusively
acts on C6 to C18 acyl-CoA derivatives, whereas the tesB-like
protein exclusively cleaves hydroxyacyl-CoAs. This conclusion was
supported by an earlier study of tesB protein showing that unlike
similar thioesterase II in E.coli (Barnes et al., supra), tesB
thioesterase II of PHA-producing Rhodobacter sphaeroides
(Wieczorek, R A et al., 1996, FEMS Microbiology Letters 135: 23-30)
is not able to hydrolyze 3-hydroxyacyl-CoA substrates (Seay, T et
al., 1982, Biochemistry May 6, 25(9): 2480-2485).
[0023] In a preferred embodiment, the microorganism of the present
invention typically contains at least one modified gene as outlined
above, wherein the modified gene is integrated into its
chromosome.
[0024] The modification of at least one of said genes encoding a
protein involved in PHA metabolism and/or, preferably, the
degradation of (R)-3-OH-acyl-CoA is achieved by inserting a
mutation in the nucleic acid sequence of the gene using genetic
engineering techniques. The term "genetically engineered" (or
genetically modified) means the artificial manipulation of a
microorganism of the invention, its gene(s) and/or gene product(s)
(polypeptide). Subsequently, the modification (mutation) was
confirmed by sequence analysis (see e.g. nucleic acid and amino
acid sequences of FIGS. 11 to 14 and 15 to 33 as well as
Examples).
[0025] The term "modification" encompasses any manipulation and
mutation of a microorganism of the invention, especially of at
least one gene of said microorganism of the invention. Preferably,
the modification results in an alteration of the nucleic acid
sequence(s) of said at least one gene and is typically expressed on
the amino acid sequence level accordingly or may be due to a
modification in the regulatory regions, e.g. promotor regions of
the gene. Preferably, the modification resulting in an alteration
of said nucleic acid sequence(s) is carried out by addition,
substitution, deletion or insertion of one or more nucleotide(s).
Furthermore, the modification may encompass one or more additional
copies of a gene in a microorganism and/or (complete) deletion of a
gene. Deletion may also be due to a disruption of the gene by
recombination or insertion of e.g. a transposon. In a preferred
embodiment, a modification within a microorganism according to the
present invention causes a complete or partial inactivation of at
least one gene encoding a protein which is involved in the
metabolism interfering with the production of PHA (e.g. by
biochemically converting intermediates of the PHA synthesis
pathway), more preferably a thioesterase, even more preferably a
thioesterase, which degrades intermediates of the PHA synthesis
pathway, and most preferably a thioesterase, which converts
(specifically) (R)-3-OH-acyl-CoA to 3-HAA. In the most preferred
embodiment of the present invention the microorganism is defective
with regard to the tesB-like thioesterase (Alkanivorax) or a
homologue thereof (in other microorganisms). The defective
character may be due to various modification on the genetic level
or may be due to posttranscriptional modifications reducing or
abolishing the enzymatic activity of the relevant thioesterase.
[0026] Additionally, modifications in one or more of the genes
involved in the PHA synthesis may occur in the microorganism
according to the invention. These modifications may be directed to
PHA synthase, poly(3-hydroxyalkanoate)synthase, enyol-CoA
hydratase, and/or PHB synthase and/or other enzymes involved in the
fatty acid metabolism. Genes encoding for enoyl-CoA hydratase in
Alcanivorax are e.g. ABO 2240; ABO.sub.--0526; ABO.sub.--1238;
ABO.sub.--0987; ABO.sub.--0148; or ABO.sub.--1645. Enoyl-CoA
hydratase links beta-oxidation with PHA biosynthesis catalyzing
production of 3-hydroxy-acyl-CoA from enoyl-CoA, an intermediate of
beta-oxidation. 3-hydroxy-acyl-CoA is a precursor of PHA and is
incorporated by phaC synthase to PHA. There are two genes encoding
PHA synthases in Alcanivorax: ABO.sub.--2214 and ABO.sub.--1418.
PHA synthases catalyze the crucial last step in biosynthesis of
PHA. Generally, the enzymatic activity of these enzymes is enhanced
due to the modification(s) introduced.
[0027] According to the invention the genetically engineered
microorganism has at least one modification in at least one gene
encoding a protein which is involved in the metabolism interfering
with the production of PHA and, optionally, at least one
modification of a gene involved in PHA metabolism. Thus, it is
possible that the microorganism has only one modification in one
gene encoding a protein which is involved in the metabolism
interfering with the production of PHA. However, it is also
possible that the microorganism has more than one (two or more)
modification(s) in the same gene or in two (or more) different
genes involved in the relevant metabolism of the microorganism
according to the invention. In this case, it is possible that the
more than one modification causes different results or phenotypes.
For example, one of these modifications results in PHA excretion
whereas another modification results in PHA overproduction (as
mentioned below).
[0028] Moreover, it is also possible that the microorganism
according to the present invention has more than one modification
in different genes with differing functions, namely (at least) one
modification occurs in (at least) one gene encoding a protein which
is involved in the metabolism interfering with the production of
PHA, whereas (at least) one other modification occurs in (at least)
one gene which is involved in PHA metabolism. In addition, further
genes may be modified, e.g. genes encoding for a protein involved
in secretion mechanisms. In such a case, it is also possible that
(at least) one of these modifications results in (increased) PHA
excretion whereas (at least) one other modification results in PHA
overproduction (as mentioned below).
[0029] Several suitable genetic engineering technique known in the
art can be used to generate a microorganism of the invention. In
general, genes from any source can be broken into pieces and
modified in various ways, using microorganisms and their enzymes or
transposable elements as molecular tools. According to the present
invention, it is even possible to construct at least one completely
artificial gene which either involved in the metabolism of PHA by a
microorganism of the invention (e.g. increasing the amount of PHA
produced) and/or involved e.g. in the inhibition of 3-HAA
production using genetic engineering techniques. Once the desired
gene has been selected or created, it can be inserted into a
microorganism of the invention where it can be expressed to produce
the desired gene products. For example, a wide range of genetic
engineering methods are based on molecular cloning. In molecular
cloning, a DNA fragment from essentially any type of genetic
element composed of double-stranded DNA is recombined with a vector
and introduced into a suitable host. Commonly employed cloning
vectors include, e.g., plasmids and bacteriophages (e.g., plasmid
pBR322, bacteriophage lambda, also see below). Molecular cloning
can be divided in single steps:
[0030] 1. isolation and fragmentation of the source DNA (e.g.
genomic DNA, cDNA, synthetic DNA etc.)
[0031] 2. joining DNA fragments to a cloning vector with DNA ligase
and
[0032] 3. introduction and maintenance in a host organism
(microorganism), e.g., by transformation.
[0033] Thereby, the microorganism of the invention may e.g. contain
an artificial or native gene (operably coupled to a strong
promoter), which expresses a protein of the PHA synthesis pathway
in excess, thereby increasing the amount of PHA pathway
intermediates. As a result, PHA is produced in larger amounts than
naturally. The additional gene is inserted into the cell and may be
located on a separate DNA molecule, e.g. a vector (e.g. a plasmid)
or my be incorporated into the chromosome of the cell.
[0034] Another preferred technique to alter a nucleic acid sequence
relates to oligonucleotide site-directed mutagenesis whereby a
specific base pair in a gene can be changed to another base pair
(see, e.g., Comack B, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,
8.01-8.5.9, Ausubel F, et al., eds. 1991). In this technique an
oligonucleotide, whose sequence contains a mutation of interest, is
synthesized as described supra. This oligonucleotide is then
hybridized to a template containing the wild-type nucleic acid
sequence. In a preferred embodiment of this technique, the template
is a single-stranded template. Particularly preferred are plasmids
which contain regions such as the f1 intergenic region. This region
allows the generation of single-stranded templates when a helper
phage is added to the culture harboring the phagemid. After
annealing of the oligonucleotide to the template, a DNA-dependent
DNA polymerase is used to synthesize the second strand from the
oliognucleotide, complementary to the template DNA. The resulting
product is a heteroduplex molecule containing a mismatch due to the
mutation in the oligonucleotide. After DNA replication by the host
cell a mixture of two types of plasmid are present, the wild-type
and the newly constructed mutant. This technique permits the
introduction of convenient restriction sites such that the coding
nucleic acid sequence may be placed immediately adjacent to
whichever transcriptional or translational regulatory elements are
employed by the practitioner. The construction protocols utilized
for E. coli can be followed to construct analogous vectors for
other organisms, merely by substituting, if necessary, the
appropriate regulatory elements using techniques well known to
skilled artisans.
[0035] An especially preferred technique according to the invention
relates to transposon mutagenesis, a type of random recombination.
This procedure usually involves breakage reactions at the ends of
the mobile DNA segments embedded in chromosomes and the attachment
of those ends at one of many different non-homologous target DNA
sites. It does not involve the formation of heteroduplex DNA.
Transposons can be used as mutagenic agents without the use of
chemical or physical agents. Transposons (also called transposable
elements) can be integrated in the chromosome (e.g. of a bacterium)
at various locations and causes mutations (mutations are defined as
inherited or artificial changes in the base sequence of nucleic
acids and/or in the amino acid sequence of peptides or
polypeptides), wherein an insertion within a gene generally results
in the loss of gene function. Thus, they provide facile means of
creating mutants throughout the chromosome. The most convenient
element for transposon mutagenesis is one containing an antibiotic
resistance gene. Clones containing the transposon can then be
selected by isolation of antibiotic-resistance colonies. Two
transposons widely used for mutagenesis are Tn10, which contains a
marker for tetracycline resistance, and Tn5, which confers neomycin
and kanamycin resistance. Accordingly, a preferred embodiment
relates to a microorganism of the invention, wherein the at least
one modification is performed by transposon mutagenesis, preferably
based on miniTn5 kanamycin element (miniTn5 Km element) (sequences
see FIGS. 14 and 15), more preferably miniTn5 streptomycin element
(miniTn Str/Sp element) (see Example 1).
[0036] In a preferred embodiment of the present invention, the
microorganism contains a modification, which causes polar effects
subsequent to transposon insertion, preferably Tn-5 insertion, on
the gene downstream to the modified gene as described below.
[0037] Another useful technique relates to conservative
site-specific recombination involving the production of very short
heteroduplex joint and therefore requiring a short DNA sequence
that is the same on both donor and recipient DNA molecules. In this
pathway, breakage and joining occur at two special sites, one on
each participating DNA molecule. The double-stranded molecule
obtained is inserted into a cloning host by transformation and
mutants are selected. Depending on the orientation of the two
recombination sites, DNA integration, DNA excision or DNA inversion
can occur. Conservative site-specific recombination is especially
usable to turn genes on or off.
[0038] As mentioned above, the at least one modification of at
least one gene encoding a protein which is involved in the
metabolism interfering with the production of PHA and/or a gene
encoding a protein involved in PHA metabolism leads to extracelluar
deposition, e.g. excretion, of polyhydroxyalkanoate(s) (PHA),
preferably medium or long chain polyhydroxyalkanoate(s) (PHA),
produced by the microorganism, preferably into the surrounding
medium. Thus, microorganism of the invention typically deposits,
e.g. excretes, polyhydroxyalkanoates (PHA), preferably medium or
long chain polyhydroxyalkanoates (PHA), preferably into the
surrounding natural or culture medium. According to the invention
the term "deposition" or "deposited" means that the microorganism
releases the (intracellular) produced PHA, preferably into the
surrounding medium which is a culture medium containing all
necessary components and suitable conditions (nutrients, buffer,
pH, temperature) for existence and growth of the microorganism. The
deposition may be due to an active process of living cells
("excretion") and/or due to the release of PHA from microorganisms
dying subsequent to PHA (over)production. A typically polyester of
hydroxyacid units (PHA) contains side chain hydroxyacid units
[(R)-3-hydroxyacid units] from 5 to 16 carbon atoms. The term "long
chain PHA" is intended to encompass PHA containing at least 12,
preferably at least 14 carbon atoms per monomer (molecule), whereas
5 to 12 carbon atoms are intended to be meant by "medium chain
PHA".
[0039] According to the invention, overproduction of PHA by the
microorganism of the invention has been shown (see FIGS. 1, 4, 6
and 7). Thus, microorganisms having at least one modification of at
least one gene encoding a protein which is involved in the
metabolism interfering with the production of PHA, in particular a
modification leading to a (partially) defective thioesterase, more
particularly to a (partially) thioesterase using (R)-3-OH-acyl-CoA
as a substrate with high specificity show generally (in addition to
deposition of PHA in the culture medium) an overproduction of
polyhydroxyalkanoate(s) PHA, preferably medium and/or long chain
polyhydroxyalkanoates (PHA). The term "overproduction" means a PHA
production by a microorganism of the invention which is at least 5
times, preferably at least 10 times, more preferably at least 15
times, more preferably at least 25 times, even more preferably at
least 40 times, even more preferably at least 50 times, most
preferably at least 60 times, most preferably at least 80 times,
even most preferably at least 100 times higher than the PHA
production of the corresponding wild type microorganism. Wild type
microorganism means a PHA producing microorganism which is not
genetically engineered and which genes are not artificially
modified (mutated). Wild type microorganisms produce normal levels
of PHA, but do not show deposition properties. In addition, the
microorganism according to invention may contain at least one
modification in a gene encoding a protein which is involved in PHA
synthesis. This modification may be due to e.g. overexpression of
the protein by a modified promoter or another modified regulatory
element of the PHA synthesis gene or by additional copies of the
PHA synthesis gene (achieved by e.g. transformation of the
microorganism) or by a mutation within the coding region of the
gene, which increases the activity and/or specificity of the PHA
synthesis enzyme.
[0040] In general, the term "microorganism" means a large and
diverse group of microscopic organisms that exist as single cells
or cell clusters of procaryotic (e.g. bacteria, cyanobacteria) and
eucaryotic (e.g. protozoa, fungi) microorganisms. A preferred
microorganism according to the present invention is a PHA producing
bacterium. The inventive microorganism to be used according to the
present invention is preferably selected from the group consisting
of PHA producing bacteria Alcanivorax borkumensis, Pseudomonas
putida, Pseudomonas aeruginosa, Pseudomonas syringae, Pseudomonas
fluorescens, Idiomarina loihiensis, Acinetobacter sp., Caulobacter
crescentus. Nevertheless, any other PHA producing microorganism,
e.g., Alcaligenes eutrophus, Alcaligenes latus, Azotobacter
vinlandii, Pseudomonas acitophila, Pseudomonas oleovarans,
Rhodococcus eutropha, Chromobacterium violaceum, Chromatium vinosum
may be used as well. Moreover, any microorganism, which does not
naturally produce PHA, may be used according to the invention, if
such microorganism comprises an expression vector which comprises a
gene cluster or a corresponding expression cassette, which allows
to express the enzymes needed for PHA production, in particular PHA
synthase, poly(3-hydroxyalkanoate)synthase, enyol-CoA hydratase,
and/or PHB synthase, and contains at least one modification as
disclosed above. Such an expression vector can be introduced into a
microorganism, in particular into a cell of said microorganism,
e.g., Escherichia coli, by any suitable method, e.g. by
transfection, transduction, transformation etc. (see below).
[0041] An especially preferred microorganism according to the
present invention is an oligotrophic bacterium, more preferably
halophilic oligotroph bacterium, even more preferably marine
oil-degrading bacterium, especially of the strainAlcanivorax,
preferably Alcanivorax borkumensis, more preferably Alcanivorax
borkumensis SK2. A. borkumensis is a marine oil-degrading bacterium
widely distributed in the aquatic environments. It is a moderately
halophilic oligotroph bacterium which is able to use essentially
only petroleum hydrocarbons as carbon and energy sources. A.
borkumensis is of particular interest because it is mostly found as
predominant species in oil polluted sea water (Harayama et al.,
1999; Kasai et al., 2001; 2002; Syutsubo et al., 2001) and,
accordingly, has key applications for bioremediation purposes. For
oligotrophic marine bacteria including Alcanivorax oil pollution
constitutes temporary conditions of nutrient abundance,
characterized by high C/N ratio. Under such conditions the
microorganism will store excess of carbon for the use as energy
source when lack of nitrogen will not more be a limiting factor.
These conditions of high C/N ratio indeed favor microbial
intracellular storage inclusions. Correspondingly, carbon excess
allows PHA producing bacterial species to be accumulated in form of
polyhydroxyalkanoate (PHA) granules (Steinbuchel 1991). Previously,
it was described that A. borkumensis was not able to produce PHA
(Yakimov et all. 1998). However, culturing conditions were not
favorable for PHA accumulation due to relatively high nitrogen
concentration in the culture medium (5 g/l). According to the
invention, the complete genome of A. borkumensis was sequenced.
Functional analysis of A. borkumensis genome revealed the presence
of genes encoding proteins for polyhydroxyalkanoate (PHA)
production (as shown by homology studies with other organisms).
Therefore, according to the invention, it was shown that A.
borkumensis is a PHA producing bacterium. Further amino acid
sequence alignments of the proteins revealed low sequence homology
with the proteins involved in PHA synthesis metabolism in other
bacterial species (see FIG. 5, Table 1) suggesting a characteristic
metabolic root of PHA production in A. borkumensis.
[0042] As mentioned above, a microorganism of the present invention
may typically contain at least one modified gene encoding a protein
interfering with PHA production, preferably a thioesterase, more
preferably a thioesterase synthesizing 3-HAA encoding a protein
involved in the metabolism of PHA synthesis. However, the invention
provides also a nucleic acid sequence which constitutes a gene
encoding a protein involved in the metabolism of
polyhydroxyalkanoate(s) (PHA), wherein the gene has at least one
modification causing excretion of polyhydroxyalkanoate(s) PHA,
preferably long chain polyhydroxyalkanoates (PHA), produced by the
microorganism of the present invention. Preferably, the modified
nucleic acid sequence of the invention is based on a wild type gene
selected from the group consisting of PHA synthase, PHB synthase,
acyl-CoA transferase, enyol-CoA hydratase, reductase, thiolase and
acyl-CoA thioesterase. On the other hand, the present invention
provides a gene modified by at least one modification, preferably
rendering the enzymatic activity of the encoded enzyme defective,
whereby the gene is acyl-CoA thioesterase tesB-like, preferably
acyl-CoA thioesterase tesB-like of Alcanivorax borkumensis, more
preferably acyl-CoA thioesterase tesB-like of Alcanivorax
borkumensis SK2 or homologues of this enzyme in other
microorganisms, especially as shown by table 4 (FIG. 10).
[0043] An especially preferred microorganism of the present
invention has been deposited according to the Budapest Treaty on
the International Recognition of the Deposit of
[0044] Microorganisms for the Purpose of Patent Procedure at
Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ),
Mascheroder Weg 1b, 38124 Braunschweig, Germany with the
identification reference SK2 C9 mutant and the Accession Number
DSM17483.
[0045] A particularly preferred embodiment relates to a nucleic
acid sequence comprising or consisting of one of the nucleic acid
sequences shown in FIGS. 11, 12, 16 and 18 to 25 or a functional
fragment or variant thereof.
[0046] The nucleic acid sequence of the invention can be DNA
comprising the coding sequence and eventually further sequences.
The nucleic acid molecule can be double stranded or single
stranded; single stranded RNA or DNA can be either the coding
(sense) strand or the non-coding (antisense) strand. If desired,
the nucleic acid sequence can include additional non-coding
sequences such as non-coding 3'- and 5'- sequences (including
regulatory sequences, for example). All nucleic acid sequences,
unless designated otherwise, are written in the direction from the
5' end to the 3' end. The term "nucleic acid sequence" also
includes a fragment or variant thereof as described below.
Furthermore, the nucleic acid sequence of the invention can be
fused to a nucleic acid sequence comprising or consisting of, for
example, a marker sequence, a leader sequence or a nucleic acid
sequence which encodes a polypeptide to assist, e.g., in isolation
or purification of the polypeptide. Representative sequences
include, but are not limited to those which encode a
glutathione-S-transferase (GST) fusion protein, a poly-histidine
(e.g., His6), hemagglutinin, HSV-Tag, for example.
[0047] The term "functional" fragment or variant of a nucleic acid
sequence relates to a nucleic acid sequence of the invention which
is able to constitute a typically modified gene encoding for a
protein involved in the metabolism of PHA, or a gene, encoding for
a protein, which interferes in the metabolism of the microorganism
with the production of PHA, and/or encodes a biologically active
(e.g., a protein involved in the metabolism of PHA, or a protein,
which interferes in the metabolism of the microorganism with the
production of PHA) polypeptide of the invention as described
below.
[0048] Additionally, in the context of the present invention,
nucleic acid sequences may be used herein, which encode a protein,
which provides for deposition of PHA in the extracellular medium,
wherein the protein competes with the synthesis of PHA by
PHA-synthase in a microorganism. These nucleic acid sequences may
thus be termed herein "dysfunctional" nucleic acid sequences.
"Dysfunctional" nucleic acid sequences according to the invention
may comprise native nucleic acid sequences according to FIGS. 16
and 18 to 25 or encode native polypeptides sequences according to
FIGS. 17 and 26 to 33, wherein these sequences have been
interrupted by transposon inserts (e.g. as exemplified in FIGS. 14
and 15) as disclosed above for nucleic acid sequences and the
corresponding genes. Furthermore, "dysfunctional" nucleic acid
sequences according to the invention may comprises nucleic acid
sequences according to FIGS. 11 and 12, which have been interrupted
by transposon inserts.
[0049] The term "fragment" of a nucleic acid sequence is intended
to encompass a portion of a nucleic acid sequence described herein
which is from at least about 25 contiguous nucleotides to at least
about 50 contiguous nucleotides, preferably at least about 60
contiguous nucleotides, more preferably at least about 120
contiguous nucleotides, more preferably at least about 180
contiguous nucleotides, more preferably at least about 250
contiguous nucleotides, more preferably at least about 410
contiguous nucleotides or longer in length. In this context,
shorter fragments according to the invention are useful as probes
and also as primer. Particularly preferred primers and probes
selectively hybridize to the nucleic acid sequence encoding the
polypeptides described herein. A primer is a fragment of a nucleic
acid sequence which functions as an initiating substrate for
enzymatic or synthetic elongation. A probe is a nucleic acid
sequence which hybridizes with a nucleic acid sequence of the
invention, a fragment or a complementary nucleic acid sequence
thereof. Fragments which encode polypeptides according to the
invention that retain function as described above are particularly
useful.
[0050] Hybridization can be used herein to analyze whether a given
fragment or gene corresponds to the microorganism of the invention
and thus falls within the scope of the present invention.
Hybridization describes a process in which a strand of a nucleic
acid sequence joins with a complementary strand through base
pairing. The conditions employed in the hybridization of two
non-identical, but very similar, complementary nucleic acids varies
with the degree of complementary of the two strands and the length
of the strands. Such conditions and hybridization techniques are
well known by a person skilled in the art and can be carried out
following standard hybridization assays (see e.g., Sambrook J,
Maniatis T (2001) supra). Consequently, all nucleic acid sequences
which hybridize to the nucleic acid sequence or the functional
fragments or functional variants thereof according to the invention
are encompassed by the invention.
[0051] A variant of a nucleic acid sequence means a nucleic acid
sequence which is derived from a nucleic acid sequence of the
invention by addition, substitution, deletion or insertion of one
or more nucleic acid(s) retaining the characteristic function of
said nucleic acid sequence as described above. Such nucleic acid
sequence can exhibit altered properties in some specific aspect
(e.g. increased or decreased expression rate). Beside that, skilled
artisans will recognize that the amino acids of polypeptides of the
invention, as described below, can be encoded by a multitude of
different nucleic acid triplets because most of the amino acids are
encoded by more than one nucleic acid triplet due to the degeneracy
of the genetic code. Because these alternative nucleic acid
sequences would encode the same amino acid sequence, the present
invention also comprises those alternate nucleic acid
sequences.
[0052] A variant of a nucleic acid sequence according to the
invention has substantial identity with the nucleic acid sequences
described herein. Particularly preferred are nucleic acid sequences
which have at least about 30%, preferably at least about 40%, more
preferably at least about 50%, even more preferably at least about
60%, yet more preferably at least about 80%, still more preferably
at least about 90%, and even more preferably at least about 95%
identity with nucleic acid sequences described herein.
[0053] To determine the percent identity of two nucleic acid
sequences in the above mentioned context, the sequences can be
aligned for optimal comparison purposes (e. g., gaps can be
introduced in the sequence of a first nucleic acid sequence). The
nucleotides at corresponding nucleotide positions can then be
compared. When a position in the first sequence is occupied by the
same nucleotide as the corresponding position in the second
sequence, then the molecules are identical at that position. The
percent identity between two sequences is a function of the number
of identical positions shared by the sequences. Therefore, the
determination of percent identity of two sequences can be
accomplished using a mathematical algorithm. A preferred,
non-limiting example of a mathematical algorithm utilized for the
comparison of two sequences is the algorithm of Karlin et al.
(1993), PNAS USA, 90:5873-5877. Such algorithm is incorporated into
the NBLAST program which can be used to identify sequences having
the desired identity to nucleic acid sequences of the invention. To
obtain gapped alignments for comparison purposes, Gapped BLAST can
be utilized as described in Altschul et al. (1997), Nucleic Acids
Res, 25:3389-3402. When utilizing BLAST and Gapped BLAST programs,
the default parameters of the respective programs (e. g., NBLAST)
can be used. The described method of determination of the percent
identity of two can be also applied to amino acid sequences.
[0054] The production of fragments or variants of a nucleic acid
sequence of the invention can be carried out following standard
methods which are well known by a person skilled in the art (see
e.g., Sambrook J, Maniatis T (2001) supra). In general, the
preparation of such functional fragments or variants of a nucleic
acid sequence can be achieved by modifying (altering) a DNA
sequence which encodes a polypeptide of the invention and
amplifying the DNA sequence with suitable means, e.g., by PCR
technique. Those modifications (mutations) of the nucleic acid
sequences may be generated by genetic engineering techniques as
described above. The isolation of a functional fragment or
functional variant of a nucleic acid sequence can be carried out
using standard methods as screening methods (e.g., screening of a
genomic DNA library) followed by sequencing or hybridization (with
a suitable probe, e.g., derived by generating an oligonucleotide of
desired sequence of the target nucleic acid) and purification
procedures, if appropriate.
[0055] The present invention also encompasses gene products of the
nucleic acid sequence of the invention. Gene products according to
the invention not only relate to the transcripts, accordingly RNA,
preferably mRNA, but also to alleles, polypeptides or proteins or
enzymes, particularly, in purified form. Preferably the gene
product is a polypeptide encoded by a nucleic acid sequence of the
invention. Preferably, the inventive polypeptide comprises one of
the amino acid sequences shown in FIGS. 13, 17 and 26 to 33.
[0056] A "functional" polypeptide according to the invention means
that the polypeptide can be used to produce deposited, e.g.
excreted, PHA, preferably in combination with other polypeptides
involved in the metabolism of PHA. Preferably an overproduction of
PHA is also achieved by functional polypeptides of the PHA
synthesis pathway. Methods for measuring and analyzing production,
excretion and/or overproduction of substances, like PHA, are well
known in the art (see e.g., Sambrook J, Maniatis T (2001) Molecular
Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press,
Cold Spring Habor, N.Y.) and are also described by this invention,
e.g. Examples 3 to 8, FIGS. 1, 3, 4 to 6). Without being limited
thereto, "functional" polypeptides according to the invention may
comprises native polypeptides sequences according to FIGS. 17 and
26 to 33.
[0057] Additionally, in the context of the present invention,
polypeptides may be used herein, which provide for deposition of
PHA in the extracellular medium, particularly by competing with the
synthesis of PHA by PHA-synthase in a microorganism, and thus may
be termed herein "dysfunctional" polypeptides, e.g. tesB-like
proteins as defined herein. Such competition may occur, as
explained above, due to thioesterases which act with a high
specific activity on the cleavage of 3-hydroxyacyl-CoAs which forms
the building block of PHA synthesis. These thioesterases allow to
release free 3-HAAs. However, the conversion to 3-HAA is a reaction
which competes with the synthesis of PHA by PHA-synthase, which
acts on the same cellular intermediate (namely 3-hydroxyacyl-CoAs).
As explained above, it was found according to the invention, that
(i) the release of free 3-HAAs and the synthesis of PHA are
interfering metabolic pathways and (ii) that functional knock-out
of the specific thioesterase, herein termed tesB-like thioesterase,
provides for deposition of PHA in the extracellular medium.
"Dysfunctional" polypeptides according to the invention may
comprises native polypeptides sequences according to FIGS. 17 and
26 to 33 or as encoded by nucleic acid sequences according to FIGS.
16 and 18 to 25, wherein these sequences have been interrupted by
transposon inserts (e.g. as exemplified in FIGS. 14 and 15) as
disclosed above for nucleic acid sequences and the corresponding
genes. Furthermore, polypeptides according to the invention may
comprises polypeptides sequences as encoded by the nucleic acid
sequences according to FIGS. 11 and 12, which have been interrupted
by transposon inserts.
[0058] The production of polypeptides of the invention is well
known and can be carried out following a number of different
standard methods which are well known by a person skilled in the
art (see e.g., Sambrook J, Maniatis T (2001) supra), e.g., by solid
phase peptide synthesis or recombinant methods. Both methods are
described in U.S. Pat. No. 4,617,149, the entirety of which is
herein incorporated by reference. The principles of solid phase
chemical synthesis of polypeptides are well known in the art and
are described by, e.g., Dugas H. and Penney C. (1981), Bioorganic
Chemistry, pages 54-92. For examples, peptides may be synthesized
by solid-phase methodology utilizing an Applied Biosystems 430A
peptide synthesizer (commercially available from Applied
Biosystems, Foster City, Calif.) and synthesis cycles supplied by
Applied Biosystems. Essential protected amino acids, such as
t-butoxycarbonyl-protected amino acids, and other reagents are
commercially available from many chemical supply houses. The
isolation of polypeptides of the invention can be carried out using
standard methods like separation from the culture medium by
centrifugation, filtration or chromatography and precipitation
procedures (see, e.g., Sambrook J, Maniatis T (2001) supra).
[0059] The polypeptide(s) of the invention can also be fused to at
least one second moiety. The at least second moiety can be an amino
acid, oligopeptide or polypeptide and can be linked to the
polypeptide of the invention at a suitable position, for example,
the N-terminus, the C-terminus or internally. Linker sequences can
be used to fuse the polypeptide of the invention with at least one
other moiety/moieties. According to one embodiment of the
invention, the linker sequences preferably form a flexible sequence
of 5 to 50 residues, more preferably 5 to 15 residues. In a
preferred embodiment the linker sequence contains at least 20%,
more preferably at least 40% and even more preferably at least 50%
Gly residues. Appropriate linker sequences can be easily selected
and prepared by a person skilled in the art. Additional moieties
may be linked to the inventive sequence, if desired. If the
polypeptide is produced as a fusion protein, the fusion partner
(e.g, HA. HSV-Tag, His6) can be used to facilitate purification
and/or isolation. If desired, the fusion partner can then be
removed from polypeptide of the invention (e.g., by proteolytic
cleavage or other methods known in the art) at the end of the
production process.
[0060] The invention also provides a vector comprising the nucleic
acid sequence of the invention. The terms "construct", "recombinant
construct" and "vector" are intended to have the same meaning and
define a nucleotide sequence which comprises beside other sequences
one or more nucleic acid sequences (or functional fragments or
functional variants thereof) of the invention. A vector can be
used, upon transformation into an appropriate cell (host cell) to
cause expression of the nucleic acid. The vector may be a plasmid,
a phage particle or simply a potential genomic insert. Once
transformed into a suitable host, the vector may replicate and
function independently of the host genome, or may, under suitable
conditions, integrate into the genome itself.
[0061] The aforementioned term "other sequences" of a vector
relates to the following: In general, a suitable vector includes an
origin of replication, for example, Ori p, colE1 Ori, sequences
which allow the inserted nucleic acid sequence to be expressed
(transcribed and/or translated) and/or a selectable genetic marker
including, e.g., a gene coding for a fluorescence protein, like
GFP, or a gene which confer resistance to antibiotics, such as the
p-lactamase gene from Tn3, the kanamycin-resistance gene from Tn903
or the chloramphenicol-resistance gene from Tn9.
[0062] The term "plasmid" means an extrachromosomal usually
self-replicating genetic element. Plasmids are generally designated
by a lower "p" preceded and/or followed by letters and numbers. The
starting plasmids herein are either commercially available,
publicly available on an unrestricted basis or can be constructed
from available plasmids in accordance with the published
procedures. In addition, equivalent plasmids to those described are
known to a person skilled in the art. The starting plasmid employed
to prepare a vector of the present invention may be isolated, for
example, from the appropriate E. coli containing these plasmids
using standard procedures such as cesium chloride DNA
isolation.
[0063] A vector according to the invention encompasses a
(recombinant) DNA cloning vector as well as a (recombinant)
expression vector. Preferred vectors according to the invention are
E.coli pBR322, XL-Blue MRF' and pBK-CMV, bacteriophage lambda etc..
A DNA cloning vector refers to an autonomously replicating agent,
including, but not limited to, plasmids and phages, comprising a
DNA molecule to which one or more additional nucleic acid sequences
of the invention have been added. An expression vector relates to
any DNA cloning vector recombinant construct comprising a nucleic
acid sequence of the invention operable linked to a suitable
control sequence capable of effecting the expression and to control
the transcription of the inserted nucleic acid sequence of the
invention in a suitable host. Operable linked means that the
nucleic acid sequence is linked to a control sequence in a manner
which allows expression (e. g., transcription and/or translation)
of the nucleic acid sequence. Transcription means the process
whereby information contained in a nucleic acid sequence of DNA is
transcribed to complementary RNA sequence.
[0064] Aforementioned control sequences are well known in the art
and are selected to express the nucleic acid sequence of the
invention and to control the transcription. Such control sequences
include, but are not limited to a polyadenylation signal, a
promoter (e.g., natural or synthetic promotor) or an enhancer to
effect transcription, an optional operator sequence to control
transcription, a locus control region or a silencer to allow a
tissue-specific transcription, a sequence encoding suitable
ribosome-binding sites on the mRNA, a sequence capable to stabilize
the mRNA and sequences that control termination of transcription
and translation. These control sequences can be modified, e.g., by
deletion, addition, insertion or substitution of one or more
nucleic acids, whereas saving their control function. Other
suitable control sequences are well known in the art and are
described, for example, in Goeddel (1990), Gene Expression
Technology:Methods in Enzymology 185, Academic Press, San Diego,
Calif.
[0065] Promoters for use in bacterial systems also will contain a
Shine-Dalgarno sequence operably linked to the DNA encoding the
desired polypeptides.
[0066] Useful expression vectors, for example, may consist of
segments of chromosomal, non-chromosomal and synthetic DNA
sequences such as various known variants of SV40 and known
bacterial plasmids, e.g., plasmids from E. coli including col E1,
pBK, pCR1, pBR322, pMb9, pUC 19 and their variants, wider host
range plasmids, e.g., RP4, phage DNAs e.g., the numerous variants
of phage lambda, e.g., NM989, and other DNA phages, e.g., M13 and
filamentous single stranded DNA phages and vectors derived from
combinations of plasmids and phage DNAs, such as plasmids which
have been modified to employ phage DNA or other expression control
sequences. Expression techniques using the expression vectors of
the present invention are known in the art and are described
generally, for example, in Sambrook J, Maniatis T (2001) supra.
[0067] Preferably, a vector of the invention, especially an
expression vector, comprises a gene cluster comprising a modified
gene as defined above, e.g. a gene having at least one modification
in at least one gene encoding for a protein involved in the
metabolism of polyhydroxyalkanoate(s) (PHA) or, preferably, in at
least one gene encoding for a protein, which interferes in the
metabolism of the microorganism with the production of PHA, wherein
the at least one modification causes extracellular deposition, e.g.
excretion, of polyhydroxyalkanoate(s) (PHA), preferably medium or
long chain polyhydroxyalkanoate(s) (PHA), produced by the
microorganism, preferably in/into the culture medium. Such an
expression vector can be introduced in any suitable microorganism,
as mentioned above, to generate an inventive microorganism
producing and excreting PHA. Such gene cluster typically comprises
all genes which are necessary or relevant in the metabolism of PHA.
Consequently, also encompassed by the invention is an inventive
microorganism comprising aforementioned (expression) vector which
comprises said gene cluster.
[0068] The invention also provides a cell (also: host cell)
comprising a vector or a nucleic acid (or a functional fragment, or
a functional variant thereof) according to the invention. A cell
(host cell) means a cell of any microorganism described herein
which is useful according to the present invention). Furthermore, a
cell or host cell means a cell which has the capacity to act as a
host and expression vehicle for a nucleic acid or a vector
according to the present invention. Preferably, the cell is a
prokaryotic cell. Cells comprising (for example, as a result of
transformation, transfection or tranduction) a vector or nucleic
acid as described herein include, but are not limited to, bacterial
cells (e.g., A. borkumensis, E. coli). The choice of a particular
cell depends to some extent on the particular expression vector
used to drive expression of the nucleic acids of the present
invention.
[0069] A vector can be introduced into a cell (host cell) using any
suitable method (e.g., transformation, electroporation,
transfection using calcium chloride, rubidium chloride, calcium
phosphate, DEAE dextran or other substances, microprojectile
bombardment, lipofection, infection or transduction).
Transformation relates to the introduction of DNA (nucleic acid
sequence) into an organism so that the DNA is replicable, either as
an extrachromosomal element or by chromosomal integration. Methods
of transforming bacterial hosts are well known in the art. Numerous
methods, such as nuclear injection, protoplast fusion or by calcium
treatment are summarized in Sambrook J, Maniatis T (2001) supra.
Transfection refers to the taking up of a vector by a cell (host
cell) whether or not any coding sequences are in fact expressed.
Successful transfection is generally recognized when any indication
or the operation or this vector occurs within the cell.
[0070] Another aspect of the invention relates to a method for
producing polyhydroxyalkanoates (PHA) comprising the following
steps: [0071] cultivating a microorganism or a cell of the
invention and [0072] recovering polyhydroxyalkanoates (PHA) from
the culture medium.
[0073] Standard methods for cultivating a microorganism or a cell
under suitable conditions are well known in the art. See, e.g.
below under Examples, Materials and also Sambrook J, Maniatis T
(2001) supra.
[0074] PHA can be isolated from the culture medium by conventional
procedures including separating the cells from the medium by
centrifugation or filtration, precipitating or filtrating the
components (PHA) of the supernatant, followed by purification,
e.g., by chromatographic procedures, e.g., ion exchange
chromatography. affinity chromatography or similar art recognized
procedures (see also Example 4).
[0075] Another aspect of the invention relates to the use of a
microorganism, a polypeptide, a nucleic acid, a vector and/or a
cell of the invention for the production and deposition, e.g.
excretion, and/or overproduction of polyhydroxyalkanoates (PHA),
especially medium and/or long chain polyhydroxyalkanoates
(PHA).
[0076] In summary, the invention relates to a genetically
engineered microorganism having at least one modification in at
least one gene encoding for a protein involved in the metabolism of
polyhydroxyalkanoate(s) (PHA) or, preferably, in at least one gene
encoding for a protein, which interferes in the metabolism of the
microorganism with the production of PHA, wherein the at least one
modification causes extracellular deposition, e.g. excretion, of
polyhydroxyalkanoate(s) (PHA), preferably medium and/or long chain
polyhydroxyalkanoate(s) (PHA), produced by the microorganism,
preferably in/into the culture medium. Transposon mutagenesis,
based on miniTn5 Str/Sp element (see Example 1) as well as miniTn5
Km element, was used for introducing the at least one modification
into at least one gene encoding for a protein, which interferes in
the metabolism of the microorganism with the production of PHA.
Subsequently, a screening for miniTn5 mutants deficient in biofilm
formation, as measured by a deficiency of attachment of mutant
cells to plastic surfaces, was performed (see Example 2). A mutant
(hereinafter indicated with "C9" or "C9 mutant") was isolated
showing a clear deficiency in biofilm formation due to excessive
production of secreted polymeric material, later identified as PHA
(see Example 4).
[0077] According to the invention production and deposition, e.g.
excretion, of PHA, by applying an inventive genetically engineered
microorganism, e.g., as derived from A. borkumensis, as well as
intracellular PHA accumulation in form of granula by these
microorganisms, especially by A. borkumensis, was confirmed by
chemical analysis. It has been analyzed that by use of these
genetically engineered microorganism microorganisms, especially A.
borkumensis, grown e.g., on octadecane under conditions of a high C
to N ratio, a mixture of different PHA (hydroxyhexanoate,
hydroxyoctanoate, hydroxydecanoate, hydroxydodecanoate) can be
produced in form of intracellular granula as storage material.
Beyond that, the isolation and characterization of genetically
engineered microorganisms having at least one modification,
particularly in at least one gene encoding for a protein, which
interferes in the metabolism of the microorganism with the
production of PHA, wherein the at least one modification causes
extracellular deposition, was achieved. This invention will bypass
the problem of costly recovery of intracellular produced PHA and
will lead to a higher yield of PHA.
[0078] The following Figures and Examples are thought to illustrate
the invention. They should not be constructed to limit the scope of
the invention thereon. All references cited by the disclosure of
the present application are hereby incorporated in their entirety
by reference.
[0079] In another embodiment enzymes are provided, which provide
for production of 3-HAA as defined above. Such enzymes include,
without being limited thereto, any enzymes derived from
Alkanivorax, Pseudomonas putida, Pseudomonas aeruginosa,
Pseudomonas syringae, Pseudomonas fluorescens, Acinetobacter sp.,
Caulobacter crescentus the thioesterase and being capable of
producing 3-HAA.
[0080] The present invention furthermore provides the use of these
enzymes, which provide for production of 3-HAA as defined above,
and/or their encoding nucleic acids, for producing PHA, preferably
medium or long chain polyhydroxyalkanoate(s) PHA. Such enzymes may
be transfected into microorganisms as outlined above, e.g. as a
vector or as a (naked)nucleic acid, or in the form of a protein,
e.g. as a fusion with cell penetrating peptides, using methods,
known in the art.
FIGURES
[0081] FIG. 1 shows PHA production in wild type A. borkumensis SK2
(hereinafter also referred to as "SK2" or "SK2 wild type") and
mutant A. borkumensis SK2 (hereinafter also referred to as "C9
mutant") on either 2% pyruvate or 1.5% octadecane as carbon source.
Cells and corresponding supernatants were checked for PHA content,
indicated in g/l. The production of Pha was in the following order:
C9 mutant supernatant octadecane>C9 mutant supernatant
pyruvate>SK2 wild type cells+supernatant octadecane>SK2 wild
type cells+supernatant pyruvate. The amount of Pha isolated from C9
mutant cells grown on either pyruvate or octadecane was too low for
quantification (third and fifth probe in the diagram). C9 mutants
grown on pyruvate produced almost 10 times more PHA than SK2 wild
type grown on pyruvate. Moreover, C9 mutants grown on octadecane
produced almost 10 times more PHA than SK2 wild type grown on
octadecane. Therefore, according to the invention, a genetically
engineered microorganism is provided that deposites, e.g. excretes,
and overproduces PHA.
[0082] FIG. 2 shows electron microscopic images of ultrathin
sections of A. borkumensis SK2 strain. Cells were cultivated in
ONR7a containing 1.5% (w/v) octadecane and 0.27 g/l of NH.sub.4Cl
(storage conditions) and harvested in stationary phase of growth.
These ultrathin sections prepared from the cells confirmed presence
of intracellular granula.
[0083] FIG. 3 shows scanning electron microscopy images (FIG. 3A
and 3B) and electron microscopic images (FIG. 3C and D) of SK2 wild
type cells and C9 mutant cells, grown on Permanox hydrophobic
slides covered with octadecane. FIG. 3A and 3C show results for SK2
wild type cells. FIG. 3B and 3D show results for C9 mutant cells
confirming the production of extracellular PHA and the excretion of
PHA into the surrounding culture medium, respectively. Although
both images contain rod-shaped cells, it is obvious that C9 mutant
cells are embedded in extracellular network of some deposited, e.g.
excreted material, whereas the SK2 wild type cells are not. These
results support the hypothesis that C9 mutant strain is involved in
the production of some polymeric extracellular material proved by
chemical analysis to be PHA. Further EM images of shadow casting
technique of SK2 wild type and C9 mutant cells grown on octadecane
in storage conditions provided additional support for the
hypothesis that the PHA produced by the mutant strain is deposited,
e.g. excreted in the culture medium. The EM pictures for the
shadow-casted cells of the C9 mutant strain revealed the
perforations on the surface of the C9 mutant cells which are
probably involved in excretion of intracellular produced PHA.
[0084] FIG. 4 shows in a diagram the comparison of growth
characteristics of C9 mutants and SK2 wild type. As can be seen. C9
mutant did have growth characteristics comparable with SK2 wild
type when pyruvate was used as a carbon source. In case of
octadecane C9 mutant even showed better growth compared with SK2
wild type. SK2 wild type had some deficiency in growth on
octadecane in these conditions compared with C9 mutant. The
explanation for this could be that the high intracellular PHA
content inhibits cell division of SK2 wild type. Cell counts also
show the domination in growth on octadecane for C9 mutant (data not
shown) which under the conditions of high C/N ratio releases the
synthesized PHA into the medium and is still able for cell
division. In conclusion, the microorganism according to the
invention is absolutely usable for biotechnological, commercial and
industrial purposes.
[0085] FIG. 5 shows Table 1 representing the results of in silico
analysis of sequences data of the genes for polyhydroxyalkanoate
production and mobilization in A. borkumensis SK2.
[0086] FIG. 6 shows Table 2 representing the analysis of PHA and
its composition.
[0087] To confirm that the substance deposited, e.g. excreted by
wild type A. borkumensis SK2 (hereinafter also referred to as "SK2"
or "SK2 wild type") and mutant A. borkumensis SK2 (hereinafter also
referred to as "C9 mutant") is in fact PHA, further chemical
analysis were conducted and revealed the presence of PHA. The cells
were grown on either pyruvate or octadecane as carbon and energy
source in PHA accumulating conditions (PHA storage conditions),
i.e., high C/N ratio (C:N ratio 100:1) and were separated from the
medium. Cells and corresponding supernatants were checked for PHA
content. Pha from C9 mutant and SK2 wild type cells or supernatants
were purified by sodium hypochlorite digestion and subsequent
solvent extraction with acetone/diethylether (Solaiman et al.,
1999). The production of Pha was in the following order:
C9 mutant supernatant octadecane>C9 mutant supernatant
pyruvate>SK2 wild type cells+supernatant octadecane>SK2 wild
type cells+supernatant pyruvate.
[0088] The amount of Pha isolated from C9 mutant cells grown on
either pyruvate or octadecane was too low for quantification (see
also FIG. 1). SK2 wild type cells grown on octadecane produced
almost 3 times more PHA compared with the cells grown on pyruvate
(18 mg/l vs. 6.5 mg/l). The amount of PHA isolated from C9 mutant
supernatant grown on octadecane produced 22 times more PHA compared
with the cells grown on pyruvate (2,960 mg/l vs. 134 mg/l).
[0089] As shown in Table 2, the amount of PHA produced by SK2 wild
type was rather low (6.5 mg/l on pyruvate and 18 mg/l on
octadecane) and was dependent on the carbon source with more PHA
produced on alkanes. The PHA produced by SK2 wild type was
consisting of hydroxyhexanoate (C6), hydroxyoctanoate (C8),
hydroxydecanoate (C10) and hydroxydodecanoate (C 12) with
hydroxydecanoate as the dominant monomer compound. Thus, it was
shown despite previous findings (Yakimov et al. 1998) that under
conditions of high C/N ratio Alcanivorax borkumensis SK2 strain
produces a mixture of polyhydroxyalkanoates mostly from alkanes.
Moreover, later growth of A. borkumenis SK2 under high C/N ratio
indeed showed granula visible under light microscope consisting of
PHA (see also FIG. 2).
[0090] Table 2 also shows that PHA of SK2 wild type were
accumulated inside the cells (no extracellular production), whereas
the all PHA produced by C9 mutant was deposited, e.g. excreted to
the medium cells (extracellular production). The monomer
repeat-unit composition and molecular mass of the polymer (from all
the batches) were determined by gas chromatography/mass
spectroscopic analysis and gel permeation chromatography,
respectively.
[0091] FIG. 7 shows Table 3 representing the molecular weights of
PHA from A. borkumensis. Results for C9 mutant supernatants growing
on pyruvate and octadecane and for SK2 wild type growing on
pyruvate and octadecane are shown. The A. borkumensis SK2 polymer
had repeat unit composition ranging from C6 to C12. The molecular
weight of the polymers varied from 180,000 to 540,000 Da (up to
2500 monomers per molecule). This corresponds to a monomer
repetition of 1,027 to 2,246 units, depending on the polymer.
Similar composition and/or molecular weight was observed for C9
mutant, although the PHA molecular weights of the components were
slightly lower in SK2 wild type cells. The molecular weight of the
monomers also was not dependent on the carbon source.
[0092] These data strongly suggest that the mutation affected the
way of PHA production, but not the composition of the produced
polymer.
[0093] FIG. 8 shows a scheme of the hypothetical pathway of PHA
biosynthesis in A.borkumensis SK2 grown on hydrocarbons/pyruvate
(Modified version of Klinke et al.
[0094] 1999 Hypothetical pathway of MCL PHA biosynthesis of PHA
polymerase- and thioesterase I-containing E. coli JMU 193 grown on
gluconate). Hydrocarbons are degraded via terminal oxidation step
by consequent action of a monooxygenase, alcohol dehydrogenase and
aldehyde dehydrogenase resulting in free fatty acids which are
activated by acyl-CoA synthase and enter b-oxidation in form of
acyl-CoA. The (S)-3-OH-acyl-CoA produced in beta oxidation are
isomerised into (R)-3-OH-acyl-CoAs by action of an isomerase.
Pyruvate enters fatty acid biosynthesis in form of acetyl-CoA.
Acyl-ACP produced in fatty acid biosynthesis is converted to free
fatty acids by action of tesA and tes B. Tree fatty acids produced
in fatty acid biosynthesis are activated by acyl-CoA synthase and
entered beta-oxidation cycle. (R)-3-OH-acyl-CoAs produced in
beta-oxidation are used for biosynthesis for either
3-hydroxyalkanoic acids (3-HAA) via action of tesB-like acyl-CoA
thioesterase and/or polyhydroxyalkanoate acids (PHA) via action of
phaC snthase. The mutation in the tesB-like acyl-CoA thioesterase
abolishes production of 3-HAA and results in uncontrolled
production of PHA.
[0095] Thus, in other words, according to the invention, the
mutation of tesB-like gene abolishing release of free 3-HAA from
3-HAA-CoA, would contribute to increasing the pool of the PHA
precursor 3-hydroxyacyl-CoA, leading to uncontrolled PHA formation
and its following excretion.
[0096] FIG. 9 shows the operon structure of the two genes tesB-like
and "putative acetyltransferase" forming a single operon.
[0097] The determination of the site of insertion of the transposon
(see Example 1) revealed that mini-Tn5 is inserted in acyl-CoA
thioesterase tesB-like gene (Abo.sub.--1044), which is one of the
genes which encodes a protein which interferes with
metabolism/production/synthesis of PHA, and likely to disrupt
function of the gene, thus inactivating the gene. The tesB-like
gene is followed by a downstream 1-acyl-sn-glycerol-3-phosphate
acyltransferase "putative acetyltransferase" gene (Abo_1045). The
ORF of "putative acetyltransferase" gene comprises 645 bp,
exhibiting an overlap of 3 by with the last codon of the preceding
ORF of tesB-like gene and is predicted to encode a 214-amino-acid
protein with a predicted molecular mass of 23.7 kDa. TesB-like gene
and "putative acetyltransferase" gene are arranged in one operon as
shown in FIG. 9.
[0098] To assess that the PHA excretion and overproduction
phenotype, as described herein, was the result of the modification
(mutation) in a gene encoding a protein which interferes with
metabolism/production/synthesis of PHA, especially tesB-like
acyl-CoA thioesterase gene, and to estimate the possibility of
polar effect of the transposon mutation on the downstream gene, a
site-directed mutant of the downstream gene was constructed and its
phenotype compared with C9 mutant (see Example 3). To construct the
knockout mutant the wild type copy of this gene was amplified and
cloned in a vector that does not replicate in A.borkumensis. A Str
resistance cassette was inserted into a unique site inside of this
gene and the resulted construct was used to replace the wild type
copy of the gene. The results were confirmed by light microscopy
and chemical analysis of the culture medium (data not shown). They
showed that the knockout mutant was deposited, e.g. excreted PHA in
the medium which means that the mini-Tn5 insertion had polar effect
on "putative acetyltransferase" gene. Therefore, taking into
account the known positive contribution of tesB-like into PHA
production, the PHA excretion and overproduction phenotype can
beside tesB-like inactivation also be caused by a polar effect of
the Tn-5 insertion on the downstream "putative acetyltransferase"
gene.
[0099] FIG. 10 represents Table 4 showing acyl-CoA thioesterase
proteins of several closely related gram-negative bacteria encoded
by genes homologous of the tesB-like gene of Alcanivorax
borkumensis SK2 (Abo.sub.--1044). These data are results of
corresponding homology searches (BLAST searches) of putative
acyl-CoA thioesterase proteins. As can be seen from Table 4 the
tesB-like protein nominated differently, namely as putative
acyl-CoA thioesterase II, hypothetical protein or tesB-like
thioesterase. Bacteria of these searches were the species
Pseudomonas putida KT2440, Pseudomonas aeruginosa PA01, Pseudomonas
syringae pv B728a, Pseudomonas fluorescens PfO-1, Idiomarina
loihiensis L2TR, Acinetobacter sp. ADP 1 and Caulobacter crescentus
CB 15. However, it is to be understood that the terms "tesB-like",
"tesB-like gene" and/or "tesB-like protein" and description thereof
is intended to relate to all these mentioned different nominations.
The proteins presented in Table 4 show a high homology to the
tesB-like gene of A. borkumensis SK2 (Abo.sub.--1044).
[0100] FIG. 11 shows nucleic acid sequences of the two genes
tesB-like (Abo.sub.--1044). (underlined) and "putative
acetyltransferase" (Abo.sub.--1045) forming a single operon. The
first line is the region upstream from the tesB-like gene. The
start codon of the tesB-like gene is atg and marked in bold
letters. The Tn5 insertion took place at position 527 within
tesB-like gene and marked with//. Downstream from the tesB-like
gene is the "putative acetyltransferase" gene. There is overlap in
3 by of the end of tesB-like (Abo.sub.--1044). and the start site
of "putative acetyltransferase" (Abo.sub.--1045).
[0101] FIG. 12 shows the nucleic acid sequence of tesB-like of
A.borkumensis. The Tn5 insertion took place at position 557. and is
indicated with//. Start and stop codons are marked in bold
letters.
[0102] FIG. 13 shows the amino acid sequence of tesB-like of A.
borkumensis.
[0103] FIG. 14 shows the nucleic acid sequence of miniTn5 Km
element.
[0104] FIG. 15 shows the amino acid sequence of neomycin
phosphotransferase responsible for neomycin and kanamycin
resistance.
[0105] FIG. 16 shows the nucleic acid sequence of "putative
acetyltransferase" of A.borkumensis. Start and stop codons are
marked in bold letters.
[0106] FIG. 17 shows the amino acid sequence of "putative
acetyltransferase" of A.borkumensis.
[0107] FIG. 18 shows the nucleic acid sequence of putative acyl-CoA
thiosterase II of Pseudomonas putida KT2440.
[0108] FIG. 19 shows the nucleic acid sequence of hypothetical
protein PA2871 of Pseudomonas aeruginosa PA01.
[0109] FIG. 20 shows the nucleic acid sequence of acyl-CoA
thioesterase II, putative, of Pseudomonas syringae pv B728a.
[0110] FIG. 21 shows the nucleic acid sequence of acyl-CoA
thiosterase of Pseudomonas fluorescens PfO-1.
[0111] FIG. 22 shows the nucleic acid sequence of tesB-like
acyl-CoA thiosterase of Idiomarina loihiensis L2TR.
[0112] FIG. 23 shows the nucleic acid sequence of putative acyl-CoA
thioesterase II of Acinetobacter sp. ADP1.
[0113] FIG. 24 shows the nucleic acid sequence of hypothetical
protein CC2472 of Caulobacter crescentus CB15.
[0114] FIG. 25 shows the nucleic acid sequence of hypothetical
protein ELI0992 of Erythrobacter litoralis HTCC2594.
[0115] FIG. 26 shows the amino acid sequence of putative acyl-CoA
thiosterase II of Pseudomonas putida KT2440.
[0116] FIG. 27 shows the amino acid sequence of hypothetical
protein PA2871 of Pseudomonas aeruginosa PA01.
[0117] FIG. 28 shows the amino acid sequence of acyl-CoA
thioesterase II, putative, of Pseudomonas syringae pv B728a.
[0118] FIG. 29 shows the amino acid sequence of acyl-CoA
thiosterase of Pseudomonas fluorescens PfO-1.
[0119] FIG. 30 shows the amino acid sequence of tesB-like acyl-CoA
thiosterase of Idiomarina loihiensis L2TR.
[0120] FIG. 31 shows the amino acid sequence of putative acyl-CoA
thioesterase II of Acinetobacter sp. ADP 1.
[0121] FIG. 32 shows the amino acid sequence of hypothetical
protein CC2472 of Caulobacter crescentus CB15.
[0122] FIG. 33 shows the amino acid sequence of hypothetical
protein ELI0992 of Erythrobacter litoralis HTCC2594.
EXAMPLES
Materials: Bacterial Strains, Media and Growth Conditions
[0123] A.borkumensis SK2 was used as the wild type for all
experiments. The bacteria were grown at 30.degree. C. in modified
ONR7a medium (Yakimov et al 1998) where 0.27 g/l of NH.sub.4Cl was
used as the source of nitrogen instead of KNO.sub.3. Pyruvate (2%)
or octadecane (1.5%) were used as a carbon and energy source.
[0124] E.coli was grown at 37.degree. C. in Luria-Bertani medium
supplemented with streptomycin (50 .mu.g/ml), chloramphenicol (12.5
.mu.g/ml), kanamycin (50 .mu.g/ml), nalidixic acid (10 .mu.g/ml) as
necessary. Plasmids were introduced into A.borkumensis by
conjugation with E.coli strain S17-1.
Example 1
[0125] Mini-Tn5 Mutagenesis
[0126] Transposon mutagenesis was based on miniTn5 Str/Sp element
(and miniTn5 Km element) developed by de Lorenzo et al (1998).
A.borkumensis SK2 was grown at 30.degree. C. on ONR7a media till
stationary phase of growth, the cells were centrifuged. The donor
and helper cultures of E.coli were grown overnight at 37.degree. C.
on LB with either streptomycin or chloramphenicol respectively,
washed with fresh LB and centrifuged. The pellets of A.borkumensis
and E.coli donor and helper strains were mixed in proportion 4:1:1
and placed on a membrane filter on a plate with LB agar and salts
(Na.sub.2HPO.sub.4.2H.sub.2O, 0.45 g/l; NaNO.sub.3, 2.5 g/l; NaCl,
11.5 g/l; KCl, 0.38 g/l; CaCl.sub.2.2H.sub.2O, 0.7 g/l) and 2%
pyruvate as carbon and energy source. The plate was incubated for
24 hours at 30.degree. C. The cells were then washed with 10 mM
MgSO.sub.4 and the transconjugants were selected on ONR7a with
nalidixic acid and streptomycin.
[0127] mini-Tn5 insertion sites of the strains were determined by
inverse PCR as described previously (Ochman et al 1988). Shortly,
total DNA of mutants was isolated and digested with ClaI which does
not cut mini-Tn5 element. The resulted DNA fragments were
circularised and the flanking regions of the inserted mini-Tn5 were
amplified with two primers: OTR End (GGC CGC ACT TGT GTA TAA GAG
TCA G) and 1TR End (GCG GCC AGA TCT GAT CAA GAG ACA G). The
conditions for the PCR were: 94.degree. C. for 1.5 min; 48.degree.
C. for 1 min; 70.degree. C. for 4 min, 30 cycles. The PCR products
were gel purified and used for automate DNA sequencing with BigDye
terminators on an ABI Prism 377 sequencer (AP Biosystems). To
determine the precise site of transposon mutation for the tesB-like
mutant we designed primers which would read the flanking regions of
the disrupted gene: 1086F (TTA CTG GCT TCG CAG GAA TGG) and
IntSM160 (CTT GGC ACC CAG CAT GCG CGA GCA GG9).
Example 2
[0128] Biofilm Formation Assay
[0129] In order to screen for mini-Tn5 mutants defective in biofilm
formation, an assay described by O'Toole and Kolter (1998) was
employed. This assay scores the ability of bacterial cells to
adhere to the wells of 96-well microtitre dishes made of
polyvinylchloride (PVC) plastic. The ONR7A medium (100 .mu.l/well)
was inoculated using replicator device. After inoculation, plates
were incubated at 30.degree. C. for 48 h, then 25 .mu.l of a 1%
solution of crystal violet (CV) was added to each well (this dye
stains the cells but not the PVC), the plates were incubated at
room temperature for approximately 15 min, rinsed thoroughly and
repeatedly with water and scored for the formation of a
biofilm.
[0130] Biofilm formation was quantified by the addition of
2.times.200 .mu.l of 95% ethanol to each CV-stained microtitre dish
well. CV-stained biofilm was solubilized in 200 .mu.l of 95%
ethanol, of which 125 ml were transferred to a new polysterene
microtitre dish, and the adsorbance was determined with a plate
reader at 600 nm (series 700 microplate reader; Cambridge
Technology).
Example 3
[0131] Site-Directed Mutagenesis of "Putative Acetyltransferase"
Gene
[0132] To analyze, if the phenotype observed with strain C9 was the
result of tesB-like inactivation or of polar effect of the
mini-transposon mutation on the downstream gene, a targeted
"putative acetyltransferase" mutant was constructed. A 769 by
fragment of the downstream "putative acetyltransferase" gene was
amplified from A. borkumensis SK2 DNA with primers 1087F:
(CAGTGATGGCTATGGTCAAAG) and 1087R: (CTTTGATCAGTCCGGCAAAAC) and
cloned into pCR 2.1 TOPO Cloning vector (Invitrogen) containing
ampicillin resistance gene for counterselection. A Str-resistance
cassette was excised from Tn5 Str/Sp plasmid (de Lorenzo et al.,
1998) and inserted into the unique site inside of the gene. The
non-functional "putative acetyltransferase" gene was then
re-introduced into A.borkumensis SK2 genome by homologous
recombination via conjugation. To confirm loss of vector associated
DNA Str.sup.R colonies were then plated in parallel on ONR7A agar
containing streptomycin, nalidixic acid and ONT7A agar containing
ampicillin to identify isolates that had lost the Amp.sup.R marker
associated with the TopoCloning vector. The growth characteristics
and PHA accumulation of the knockout mutant were measured and
compared with the mini-Tn5 mutant and the wild type.
Example 4
[0133] Chemical Analysis of PHA
[0134] To analyze the PHA released into the medium by the wild type
A.borkumensis SK2 and by the mutant strain, the bacteria were
cultured in ONR7a medium containing either 2% of pyruvate or 1.5%
octadecane as carbon sources (storage conditions). The bacteria
were incubated in a rotary shaker (100 rpm) at 30.degree. C. until
late stationary phase of growth. The bacteria were harvested by
centrifugation (60' 12,000) and the supernatant and the pellet were
collected separately, lyophilized and used for the subsequent
chemical analysis of PHA.
Example 5
[0135] PHA Quantification
[0136] Aliquots of the cells and of the supernatant were washed
with ice-cold water and dried overnight at 80.degree. C. under
vacuum. Polyhydroxyalkanoates (PHA) were purified from the cells by
sodium hypochlorite digestion and subsequent solvent extraction
with acetone/diethylether (Solamain et al., 1999).
Example 6
[0137] Gas Chromatography/Mass Spectroscopy
[0138] To determine the PHA composition, approximately 2 mg of PHA
were let to react in a small flask with a mixture containing
chloroforms:methanol:sulphuric acid (1:0.85:0.15 ml) for 2 h at
100.degree. C. in thermostatically regulated bath. This method
degrades PHA by methanolysis to its constituent
.beta.-hydroxycarboxylic acid methyl esters (FAME). After the
reaction, 0.5 ml of destilled water was added and the tube was
shaken for 1 min. After phase separation, the organic phase was
removed, transferred into a vial and used for analysis. FAMEs were
analyzed with gas chromatograph-mass spectrometer (GC/MS, model
Varian 3400CX, Varian Chromatography Systems, Sugar Land, Tex., and
VG Autospec spectrometer), equipped with a 30 m.times.0.25 mm HP-5
(5% diphenyl and 95% dimethylpolysiloxane) fused silica capillary
column; flow rate 1 ml/min; sample input temperature to 230.degree.
C. at a rate of 8.degree. C./min; interface temperature 250.degree.
C.; ion source temperature 175.degree. C.; electron impact mode 70
eV; scanning from 45 to 450 amu at 0.5 s/scan. The degree of purity
of the PHA samples taken for investigation was up to 99.5%. No
trace amounts of proteins, carbohydrates and lipids were registered
in them. See data in Table 1.
Example 7
[0139] Gel Permeation Chromatography
[0140] The samples of Example 6 were analyzed in a HPLC system with
a Spectra-Physics pump and an Aminex HPX-87H column (Bio-Rad,
Hercules, Calif.) under the following conditions: column
temperature, 50.degree. C.; gradient, isocratic; mobile phase, 5 mM
sulfuric acid; flow rate, 0.5 ml/min; detector, light scattering.
See data in Table 2.
Example 8
[0141] Electron Microscopy
[0142] Cells were cultivated in ONR7a containing 1.5% (w/v)
octadecane and NH.sub.4Cl (storage conditions) and were harvested
in the stationary phase of growth. Cells for scanning electron
microscopy were grown in the same conditions with the only
difference that octadecane was embedded on Permanox cell culture
slides (Nalge Nunc). The cells were shadow-casted according to
Golyshina et al. (2000), embedding and ultrathin sections were done
as described by Yakimov et al. (1998) and scanning electron
microscopy was performed as described by Lunsdorf et al.
(2001).
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Sequence CWU 1
1
3111546DNAAlcanivorax borkumensisNucleotide sequences of the genes
tesB-like (Abo_1044) and plsC (Abo_1045) consisting of an operon in
Alcanivorax borkumensis (fig. 11) 1ggcttcgcag gaatggtaaa taaagcaccg
caaattcaaa acacgcagaa gtgagagacc 60atgacattcg atgagattct agccacgatt
gatggccagg gtaacgccac gtttccagaa 120gggtgggggc agggccggac
cttatttggt ggcctggtgg gggcggtgct gtttgaacat 180ttagaaaaaa
ccgtggctcg cgggaggttt ctacgtagtt tttctctctc ttttgtcgcc
240cctgcggtgc cgggtccggt ggcactagac gagactgtgt ttcgggaagg
caaatccgtt 300atgcaggcca tggtctccgc ccgtcaagga gggcaggtgg
tggcggttat gttggccagc 360tttggggcca gtcgccaatc cagtgtagtg
gtagaagggc catcggcccc agtgatgaaa 420tctccagagc agagtatttc
ggtaccgttt atcaaaggat tgacgccgga ttttttttcc 480cattttaata
tccattacgc agaaggcatg ccgccgttca gtggcagttc tgagcccgat
540tacggtggtt acatggggtt tactgtgccg ccggaaacca tgagcaccgc
agcgctgatt 600gcattggtgg atacctgggc gccttcagtt ttacctctgc
tcaaagggcc agcacccgcc 660agttccttga cctggaccat ggagctcttg
gatgacccta gtgtccactc gccggagact 720ttatggcaat atcgggtaaa
cacggaccaa tgcagtgatg gctatggtca aagtcaggcg 780gtggtttggg
atgctgcggg taaggctgtg gcgttgagtc gacaaacctt tacggtattt
840gcatgaaatc ggaactggtg ccactcacgg cgcggacttt aggtaagcaa
gtgcctcgtc 900gtgggcactg gctgttggcg gcgctagggc ggctgatttt
gactgttatg gggtggcgta 960ttgttggtga tttgcccgat acgcctcggg
cagttttggc ggtggcgcca cacacgtcga 1020acatagacgg ggtcattggt
atcagtgcta ttcagtcttt gcgcttggat gtgcgcttca 1080tgggtaagca
cacgttgttt aaaggtcgtc ttgggcggtt catgtactgg ctgggcggca
1140tccctgtgaa tcgagaaagt gccagggatg tggtggacca gacgacgtcg
gtgatggggg 1200aaacaccatt ttggcttggg ctaacgccgg aggggacgcg
taaaggcgcc aagcgttgga 1260aaaccgggtt ttaccgtatt gctgagcaaa
tgcaggtgcc gattgtcgtg ttaggtttct 1320gttaccggcg ccggcaggtc
cggattgtag attgctttct gccgacgggc gatattgatg 1380ctgatatggc
gcgaatgacc gcgtcgttgg cggatattgt tccgcgcaaa cctgcgcagt
1440tatccgcccc gctgaaagcg gaaaaagctg ctcgcggcat tgattgattc
agacgttggc 1500agttttgccg gactgatcaa ggagatgctg gcggtgctgg gcggat
15462786DNAAlcanivorax borkumensisNucleotide sequence of tesB-like
acyl-CoA thioesterase from Alcanivorax borkumensis (Abo_1044) (fig.
12) 2atgacattcg atgagattct agccacgatt gatggccagg gtaacgccac
gtttccagaa 60gggtgggggc agggccggac cttatttggt ggcctggtgg gggcggtgct
gtttgaacat 120ttagaaaaaa ccgtggctcg cgggaggttt ctacgtagtt
tttctctctc ttttgtcgcc 180cctgcggtgc cgggtccggt ggcactagac
gagactgtgt ttcgggaagg caaatccgtt 240atgcaggcca tggtctccgc
ccgtcaagga gggcaggtgg tggcggttat gttggccagc 300tttggggcca
gtcgccaatc cagtgtagtg gtagaagggc catcggcccc agtgatgaaa
360tctccagagc agagtatttc ggtaccgttt atcaaaggat tgacgccgga
ttttttttcc 420cattttaata tccattacgc agaaggcatg ccgccgttca
gtggcagttc tgagcccgat 480tacggtggtt acatggggtt tactgtgccg
ccggaaacca tgagcaccgc agcgctgatt 540gcattggtgg atacctgggc
gccttcagtt ttacctctgc tcaaagggcc agcacccgcc 600agttccttga
cctggaccat ggagctcttg gatgacccta gtgtccactc gccggagact
660ttatggcaat atcgggtaaa cacggaccaa tgcagtgatg gctatggtca
aagtcaggcg 720gtggtttggg atgctgcggg taaggctgtg gcgttgagtc
gacaaacctt tacggtattt 780gcatga 7863261PRTAlcanivorax
borkumensisAmino acid sequence of tesB-like acyl-CoA thioesterase
(figure 13) 3Met Thr Phe Asp Glu Ile Leu Ala Thr Ile Asp Gly Gln
Gly Asn Ala1 5 10 15 Thr Phe Pro Glu Gly Trp Gly Gln Gly Arg Thr
Leu Phe Gly Gly Leu 20 25 30 Val Gly Ala Val Leu Phe Glu His Leu
Glu Lys Thr Val Ala Arg Gly 35 40 45 Arg Phe Leu Arg Ser Phe Ser
Leu Ser Phe Val Ala Pro Ala Val Pro 50 55 60 Gly Pro Val Ala Leu
Asp Glu Thr Val Phe Arg Glu Gly Lys Ser Val65 70 75 80 Met Gln Ala
Met Val Ser Ala Arg Gln Gly Gly Gln Val Val Ala Val 85 90 95 Met
Leu Ala Ser Phe Gly Ala Ser Arg Gln Ser Ser Val Val Val Glu 100 105
110 Gly Pro Ser Ala Pro Val Met Lys Ser Pro Glu Gln Ser Ile Ser Val
115 120 125 Pro Phe Ile Lys Gly Leu Thr Pro Asp Phe Phe Ser His Phe
Asn Ile 130 135 140 His Tyr Ala Glu Gly Met Pro Pro Phe Ser Gly Ser
Ser Glu Pro Asp145 150 155 160 Tyr Gly Gly Tyr Met Gly Phe Thr Val
Pro Pro Glu Thr Met Ser Thr 165 170 175 Ala Ala Leu Ile Ala Leu Val
Asp Thr Trp Ala Pro Ser Val Leu Pro 180 185 190 Leu Leu Lys Gly Pro
Ala Pro Ala Ser Ser Leu Thr Trp Thr Met Glu 195 200 205 Leu Leu Asp
Asp Pro Ser Val His Ser Pro Glu Thr Leu Trp Gln Tyr 210 215 220 Arg
Val Asn Thr Asp Gln Cys Ser Asp Gly Tyr Gly Gln Ser Gln Ala225 230
235 240 Val Val Trp Asp Ala Ala Gly Lys Ala Val Ala Leu Ser Arg Gln
Thr 245 250 255 Phe Thr Val Phe Ala 260 42356DNAArtificial
SequenceNucleic acid sequence of miniTn5 Km element, coding repeats
functional in transposition and neomycin phosphotransferase (figure
14) 4ctgtctcttg atcagatctg gccacctagg ccgaattccc ggggatccgg
tgattgattg 60agcaagcttt atgcttgtaa accgttttgt gaaaaaattt ttaaaataaa
aaaggggacc 120tctagggtcc ccaattaatt agtaatataa tctattaaag
gtcattcaaa aggtcatcca 180ccggatcacc ttaccaagcc ctcgctagat
tgttaatgcg gatgttgcga ttacttcgcc 240caactattgc gataacaaga
aaagcgcctt tcatgatata tctcccaatt ttgtgtaggg 300cttattatgc
acgcttaaaa ataataaaag cgacttgacc tgatagtttg gctgtgagca
360attatgtgct tagtgcatct aacgcttgag ttaaccgcgc cgcgaagcgg
cgtcggcttg 420aacgaattgt tagacattat ttgccgacta ccttggtgat
tcgcctttca cgtagtggac 480aaaatcaacc aactgatctg cgcgagcttc
acgctgccgc aagcatcagg gcgcaagggc 540tgctaaagga agcggaacac
gtagaaagcc agtccgcaga aacggtgcta ccccggatga 600atgtcagcta
ctgggctatc tggacaaggg aaaacgcaag cgcaaagaga aagaggtagc
660ttgcagtggg cttacatgac gatagctaga ctgggcggtt ttatggacag
caagcgaacc 720ggaattgcca gctggggcgc cctctggtaa ggttgggaag
ccctgcaaag taaactggat 780ggctttcttg ccgccaagga tctgatggcg
caggggatca agatctgatc aagagacagg 840atgaggatcg tttcgcatga
ttgaacaaga tggattgcac gcaggttctc cggccgcttg 900ggtggagagg
ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
960cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
acctgtccgg 1020tgccctgaat gaactgcagg acgaggcagc gcggctatcg
tggctggcca cgacgggcgt 1080tccttgcgca gctgtgctcg acgttgtcac
tgaagcggga agggactggc tgctattggg 1140cgaagtgccg gggcaggatc
tcctgtcatc tcaccttgct cctgccgaga aagtatccat 1200catggctgat
gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
1260ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
ttgtcgatca 1320ggatgatctg gacgaagagc atcaggggct cgcgccagcc
gaactgttcg ccaggctcaa 1380ggcgcgcatg cccgacggcg aggatctcgt
cgtgacccat ggcgatgcct gcttgccgaa 1440tatcatggtg gaaaatggcc
gcttttctgg attcatcgac tgtggccggc tgggtgtggc 1500ggaccgctat
caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
1560atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
agcgcatcgc 1620cttctatcgc cttcttgacg agttcttctg agcgggactc
tggggttcga aatgaccgac 1680caagcgacgc ccaacctgcc atcacgagat
ttcgattcca ccgccgcctt ctatgaaagg 1740ttgggcttcg gaatcgtttt
ccgggacgcc ggctggatga tcctccagcg cggggatctc 1800atgctggagt
tcttcgccca ccccgggctc gatcccctcg cgagttggtt cagctgctgc
1860ctgaggctgg acgacctcgc ggagttctac cggcagtgca aatccgtcgg
catccaggaa 1920accagcagcg gctatccgcg catccatgcc cccgaactgc
aggagtgggg aggcacgatg 1980gccgctttgg tcgacccgga cgggacggat
cagtgagggt ttgcaactgt gggtcaagga 2040tctggatttc gatcacggca
cgatcatcgt cgggagggca agggctccaa ggatcgggcc 2100ttgatgttac
cgagagcttg gtacccagtc tgtgtgagca ggggaattga tccggtggat
2160gaccttttga atgaccttta atagattata ttactaatta attggggacc
ctagaggtcc 2220ccttttttat tttaaaaatt ttttcacaaa acggtttaca
agcataaagc ttgctcaatc 2280aatcaccgga tccccgggaa ttcgtcgaca
agctgcggcc gcctaggccg tggccgaact 2340tgtgtataag agtcag
23565264PRTArtificial SequenceAmino acid sequence of neomycin
phosphotransferase responsible for neomycin and kanamycin
resistance (figure 15) 5Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser
Pro Ala Ala Trp Val1 5 10 15 Glu Arg Leu Phe Gly Tyr Asp Trp Ala
Gln Gln Thr Ile Gly Cys Ser 20 25 30 Asp Ala Ala Val Phe Arg Leu
Ser Ala Gln Gly Arg Pro Val Leu Phe 35 40 45 Val Lys Thr Asp Leu
Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala 50 55 60 Ala Arg Leu
Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val65 70 75 80 Leu
Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu 85 90
95 Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys
100 105 110 Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu
Asp Pro 115 120 125 Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg
Ile Glu Arg Ala 130 135 140 Arg Thr Arg Met Glu Ala Gly Leu Val Asp
Gln Asp Asp Leu Asp Glu145 150 155 160 Glu His Gln Gly Leu Ala Pro
Ala Glu Leu Phe Ala Arg Leu Lys Ala 165 170 175 Arg Met Pro Asp Gly
Glu Asp Leu Val Val Thr His Gly Asp Ala Cys 180 185 190 Leu Pro Asn
Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp 195 200 205 Cys
Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala 210 215
220 Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg
Phe225 230 235 240 Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln
Arg Ile Ala Phe 245 250 255 Tyr Arg Leu Leu Asp Glu Phe Phe 260
6645DNAAlcanivorax borkumensisNucleotide sequence of putative
acyltransfrase plsC (Abo_1045) (figure 16) 6atgaaatcgg aactggtgcc
actcacggcg cggactttag gtaagcaagt gcctcgtcgt 60gggcactggc tgttggcggc
gctagggcgg ctgattttga ctgttatggg gtggcgtatt 120gttggtgatt
tgcccgatac gcctcgggca gttttggcgg tggcgccaca cacgtcgaac
180atagacgggg tcattggtat cagtgctatt cagtctttgc gcttggatgt
gcgcttcatg 240ggtaagcaca cgttgtttaa aggtcgtctt gggcggttca
tgtactggct gggcggcatc 300cctgtgaatc gagaaagtgc cagggatgtg
gtggaccaga cgacgtcggt gatgggggaa 360acaccatttt ggcttgggct
aacgccggag gggacgcgta aaggcgccaa gcgttggaaa 420accgggtttt
accgtattgc tgagcaaatg caggtgccga ttgtcgtgtt aggtttctgt
480taccggcgcc ggcaggtccg gattgtagat tgctttctgc cgacgggcga
tattgatgct 540gatatggcgc gaatgaccgc gtcgttggcg gatattgttc
cgcgcaaacc tgcgcagtta 600tccgccccgc tgaaagcgga aaaagctgct
cgcggcattg attga 6457214PRTAlcanivorax borkumensisAmino acid
sequence of putative acyltransferase plsC (figure 17) 7Met Lys Ser
Glu Leu Val Pro Leu Thr Ala Arg Thr Leu Gly Lys Gln1 5 10 15 Val
Pro Arg Arg Gly His Trp Leu Leu Ala Ala Leu Gly Arg Leu Ile 20 25
30 Leu Thr Val Met Gly Trp Arg Ile Val Gly Asp Leu Pro Asp Thr Pro
35 40 45 Arg Ala Val Leu Ala Val Ala Pro His Thr Ser Asn Ile Asp
Gly Val 50 55 60 Ile Gly Ile Ser Ala Ile Gln Ser Leu Arg Leu Asp
Val Arg Phe Met65 70 75 80 Gly Lys His Thr Leu Phe Lys Gly Arg Leu
Gly Arg Phe Met Tyr Trp 85 90 95 Leu Gly Gly Ile Pro Val Asn Arg
Glu Ser Ala Arg Asp Val Val Asp 100 105 110 Gln Thr Thr Ser Val Met
Gly Glu Thr Pro Phe Trp Leu Gly Leu Thr 115 120 125 Pro Glu Gly Thr
Arg Lys Gly Ala Lys Arg Trp Lys Thr Gly Phe Tyr 130 135 140 Arg Ile
Ala Glu Gln Met Gln Val Pro Ile Val Val Leu Gly Phe Cys145 150 155
160 Tyr Arg Arg Arg Gln Val Arg Ile Val Asp Cys Phe Leu Pro Thr Gly
165 170 175 Asp Ile Asp Ala Asp Met Ala Arg Met Thr Ala Ser Leu Ala
Asp Ile 180 185 190 Val Pro Arg Lys Pro Ala Gln Leu Ser Ala Pro Leu
Lys Ala Glu Lys 195 200 205 Ala Ala Arg Gly Ile Asp 210
8798DNAPseudomonas putida KT2440Nucleotide sequence of putative
acyl-CoA thioesterase II (GeneID1045294) (figure 18) 8atgactttca
accaactgct cgacgccgtg cgggccaacc cggattccgt cagcattccg 60cccagctggg
cccaggggcg cgctgccttt ggcgggctga tggcggccat ggtctatgaa
120accatgcgcc tcaagatcag cgacaaccgc ccggtacgct cattggccat
cagcttcgtg 180gcacccgcgg cggcggatgt gcccatccgc ttcgaggtgg
aggttttgcg cgaaggcaag 240gcggttagca cgttgctggg ccgcgctgtt
caggatggcc aggtggtgac tttggtgcag 300ggcaatttcg gtgcgggccg
cccttcggtg gtcgaagtgc cggcgttgcc ggccatcgaa 360atgcctgcgc
tcgatgaggc ggccccggag ttgccctata tcaaaggcgt tacccctgag
420ttcatgcggc acgtggccct gcgctgggca gtaggtgggc tgccgttcag
tggcaatcag 480tcgcgcaaga tgggcggctg ggtgcgcttg cgggatgtgg
tggaagaaca ggtgaacgag 540gcgcacctgt tggcgttggt cgatgcctgg
ccgcccagcc tcatgccgtt tctcaagcag 600cccgctgcgg gcagtacgtt
gacctggacc atcgagttca tccagccaac ggcgaagctg 660tcgaccctgg
attggtgccg gtactgtgtg gagaccgagc atgcgcggga tggctatggg
720catgctgctg cggcgttgtg gacggcgcag ggcgagttgt tggcgttgag
ccggcagacc 780gtcaccgttt tcgcctga 7989798DNAPseudomonas aeruginosa
PA01Nucleotide sequence of hypothetical protein PA2871
(GeneID882599) (figure 19) 9atgaattttt ccgaattgat ccaggcggtc
cgccgcgacc cttcctcggt ggtagtaccg 60gccagttggg gccagggccg cgccaccttc
ggtggcctgg tggtggcgtt ggcctacgag 120gccatgcttg cggtggtcga
ggcggggcgt ccgttgcgct ccatcggcgt cagcttcgtc 180ggaccgctgg
cccccgagca gccggcgagc ttcagcgccc ggttgttgcg cgagggcaag
240gcggtgagcc aggtccaggt cgaggtccgt cagggcgagc aggtggtgac
gctggtccag 300gccagtttcg gcgtcgcccg cgcatcggcg gtggcggtgg
aagcgttgcc ggcggccggg 360atgaagggcc ccgaagagag ccaggagctg
ccctatatcc gtaacgtgac cccggagttc 420actcgctaca tcgccatgcg
ctgggcagtg ggcggcctgc cgttctcttc gaacaagtcg 480cgccagatgg
gcggctggat gcgtttccgc gacgaacccg agggagagcc catggaggtt
540tcccacctgc tggcgctgct cgactcctgg ccgccggcgc tgttgccgca
cctgggcacc 600ccggcgatgg ccagctcgct gacctggacc gccgagttcc
tccagccgct gccgcagcaa 660ggcagcggcg actggtgccg ttacctggcg
gagatcgagg aggcgcgtga cggctacggc 720cacgtggcgg cgcggatgtg
gagcgccgac ggccagttgc tggcgatcag ccggcagatg 780gtcacggtgt tcggctga
79810798DNAPseudomonas syringae pv B728aNucleotide sequence of
acyl-CoA thioesterase II, putative of p. syringae pv B728a
(GeneID3367571) (figure 20) 10atgacctttt ctgaactgat cgatgcgctg
cgtcgcgatc cgcgctctgt cacgatccct 60gccgaatggt ctcaagggcg tgcctgcttc
ggcgggctga tggctgcgct gacctacgaa 120gcgatgcgtg cacaggtgcc
ggaagggcgg ccggttcgtt cgttggcgat cacctttgtc 180gggccggccg
cgcccggtgt gccgattgct ttcgaggtcg acaccctgcg ccatggcaag
240gcagtcagtc aggtgctggg acgcgccatg cagaacggtc aggtcatgac
cctgatacaa 300ggcagcttcg gtgcccctcg ggaatcgatg atcaccgttg
ccgcagaggc cgcgccggtc 360cttaaaccgg ttgatcaatg tccggagctg
ccgttcgcca gcggcgtgat gcctgattac 420ctgcgcttca tggacatccg
ctgggcgttg ggcggcatgc cattcagtaa tacccgatca 480ccggcgattg
gcggctacgt gcgctttcgc gatacgccgc acgccacgcc catgagcgaa
540gcgcacattc tggcgctggt ggacacctgg ccgcctgcgg tactgccgca
cctggacaaa 600ccggcccccg gcagctcact gacctggacc attgagttcg
tccagcccca gccgtcgctc 660gataccctgc agtggtgcag ctaccgcgca
gtcatcgagc atgcccgcga tggctatggc 720cataccgccg cggcattgtg
gagccccgac ggcgagctga tcgcaatcag ccgccagacg 780gttaccgtat ttggctga
79811795DNAPseudomonas fluorescens PfO-1Nucleotide sequence of
acyl-CoA thioesterase (locus_tag=Pflu02003109) (figure 21)
11atgcgctttt gcgatctgat cgatgctgtc cgtcgtcaac cggaggtcac gattccggcg
60gagtggggcc agggccgggc cagttttggc gggctggtgg ccgcgctgca atttgaagtg
120atgcgcacca aggttccgac cgatcggccg gtacgttcgc tggcgatcac
cttcgtcggc 180ccggtcgagc ccgaagtgcc ggtgagtttt gaagtcgagg
tactacgcga aggcaaagcg 240gtcagccagg tgctggggcg tgctgtgcag
aacggtcagg tggtgacgat ggtgcaaggc 300agcttcgggg cttcgcggcc
atcggaagtg gcggttgaag cctatcccgc gccggaaatg 360aagcactggg
acgattgcca ggaactgccg tacatcaaag gcgtaacccc cgagttcatg
420cgtcatctgg cgatgcgctg gagcgttggc gggatgccgt tcaccggcaa
tcaatcgcgg 480ctgatgggtg gctgggtgcg cctgcgtggg gatgtgaagg
aagagtcggt caacgaagcg 540cacctgctgg cgctggtcga tgcctggcca
ccagcgctgt tgccgtacct gaagaaaccg 600gcaccgggca gtacgctgac
ctggaccatc gaattcgttc agccgttacg cgatttgagt 660acgctggatt
tttgccaata cctggcggac atcgagtatg ccgccgacgg ttacggccac
720gtcgccgcca agctgtggag tgcgaagggt gaactgattg ccatgagtcg
gcagacggtg 780acgatcttcg cctga 79512795DNAIdiomarina loihiensis
L2TRNucleotide sequence of tesB-like acyl-CoA thioesterase
(locus_tag="IL0656") (figure 22) 12atgaacttcc acacagctgt agaacaaatc
gttgcggata agaacaatca agtcattgaa 60ctaccttccg gctgggctca gggacgcgcg
tttttcggcg gattcagcgg agcattggct 120gctcagtttt tgttgaaaca
atttccgatt gaatatcatc ttcgttccat gagtatctct 180tttgtcgcgc
ctgctgaacc gggtgaggct gagttaaatt accgaatttt gcgcgaggga
240aaatcggtta ttcaggttgc tgtcgaactg cagcagcaag ggcagattat
gttgtcttgt 300ctggcgagcc tgggcaaagg ccgaagttca acggttacag
tggtaagtga aacgccaccc 360gatcttaaaa ccatcaacga cggaccaggt
ttacctgaag cggatattgt cccagagttt 420gcgaaaaact tcgattaccg
tattacgtca ggtggtatgc catttagcgg acaaccggga 480agaacttttg
gtggctggat acgttttcgt gaagaacaac agccgctgac aacggcaact
540atactggctt tagttgacgc ctggccaccg gcagtcttgc ctcatctgga
cagccctgcg 600ccggcctcgt ctttaacctg gactattgag tttcccgata
ttcctctaca aagtttcagt 660agccacgact ggtttcagta cgaagctttt
attgagcatg ctgaaaatgg ttatgggcac 720agccgcgcgg gtctgtggag
tgagaagggc gagttattag ctataagtcg acagactttt 780acggtatttg cgtaa
79513804DNAAcinetobacter sp. ADP1Nucleotide sequence of putative
acyl-CoA thioesterase II (locus_tag="ACIAD0341) (figure 23)
13atgagtgatg ctatgccttt agatcagaca ttggaacaac tgacgcaaga tgaatggatt
60gatattcccc aaggctggtc gcagggaaga acgatttatg gtggattggt tgcaggcttg
120ctcatgcata aagcattaag tgtgatgaat gatgagtcca aaaatctgtt
aagtaccagc 180attacttttg tgggaccagt gaatgaaggg cgggtacgac
taacagttga aatcttgcgg 240cagggtaaat ctgtcaccac aattgaggca
cgtttatggc aggaccaggc tgttcaaacc 300atcttgatcg caagtttcgg
acagccgcgt tcttctgaaa tttttgtgct taacttaccc 360gaggcaccag
actatttgtc tcccgagcag ttctcaagaa tgccttttgt aaaaatgatg
420ccagaatgct atcaacaatt cgatctgcga tgggcagaag gtcattatcc
catgactcaa 480caagctccag attttggtgg ctggtgccgt tacgatatcc
aaaaacattc accgcgagca 540ttgaatgtgg ctgatttgtt aattttgatg
gatatctggc caccaggggt actgcccatg 600tttcaaacca tcgcacctgc
cagttctcta acatggcatc tcacttttgt tcgtcccgtt 660gcttatgagt
tacatgattg gtttaagtat caggtcgtca cgcagcatgc tgcctttggc
720tatgctacag aatatgcgca tttatgggat gctcaaaacc gtctgattgc
tatttcgagg 780cagacagtta ctgtttttgc ctag 80414789DNACaulobacter
crescentus CB15Nucleotide sequence of hypothetical protein CC2472
(GeneID943632) (figure 24) 14atgacgctct acaccgacct cgtcgcggcc
atcgcctcga ccgaaactgg cttttccgcc 60catgtctccg acgactggaa gcaaggccgc
accacctatg gcggcttgag cggcgccttg 120tgcgtcgagg ccgccctgcg
agcctttccc gaggcccctc ctctgcgctc ggcgcaattc 180gcctttgtcg
gcccggcggc cggcgagttg gcgatctcgg tgcggccgct gcggcagggc
240aagtcgacgc ttttcgtcgc cgtcgatctg atcggcgaac agggcgtggc
cacccacggc 300gtgctgacct tcggcgcggc gcggacctca gccatctcgt
acgaagaagt cctctgcccg 360cccgtggcgc cggccggcgc ctgtgagctg
ttcttccccg aaaatcgcca gggcgcgccg 420cacttctcgg cgcagttcga
ggtgcgcaag gccggcggca cgcgccccct ggccgggggt 480gagccggaat
atctgctgtg gatccgccac cgcgatccgg ccgcgacctc gatctcggcc
540ctggtggcgt tggccgacat gccgccgccc ccggccatgg cgctgttccc
gcagtttggg 600ccgatctcga ccatgacctg gtcgctggat atcgtgggcc
tgcccgaggc ggacgacgac 660ggctggcggc tgctgcgcac ccgggccgag
accatcggcg acggctactc gacccaggag 720atgcatctgt gggacgccaa
gggccgcccg ctggtcctgg cgcgacagaa cgtggcgatc 780ttcgtctga
78915777DNAErythrobacter litoralis HTCC2594Nucleotide sequence of
hypothetical protein ELI0992 (locus_tag=ELI0992) (figure 25)
15atgtccgttt ccgatcttcg tgcgccgatc acgtccgaag gtggcgctgt cactcttcca
60gccgacaaat ggctgcaggg ccgcacgctc tttggcggag cctcggcgct tgtcgcctac
120accgccgcgg tgcgcgcttt ccccgatctc ccgcccttgc gcgcagcgca
gatcggattt 180gtcgcgccgg tcggaaagga cgtggagacg cgggccgcaa
tggtccgaca gggtcgcaac 240gtcgcgcagg tgcgcagcga actgctggtc
gaaggcaagg tcgcgctcac cgcattctgg 300ctgttcggaa ccggtcgcga
ggccaacgcc gtacatgccg ctgccaaggc cgatccctgg 360cccggcgcac
cggaagagaa cgattccgcg atgaccgaca agggcccgcc tttcatcgtc
420aacaatttcg acattcgccg cgcgcaggaa acgcaaggcc ccggcgaacc
gatcgtccgg 480cgctggttca ggctgaccga tcggggcgag ctcgatcgcg
tatcggagct gatcctggtg 540ggcgatacgc tgcccccggg cgccatgcgc
gcgatgcagc gccagggccc gatcagctcg 600atcaactggt cgttcaatat
tctcgatgcg gaactcggca cgcgcgacgg ctggtggctc 660ggcgagaccg
ccagccagca tgccggtgca ggctattcga gcgagcggct acggctctgg
720aatgccgacg gcgtgcaggt gatggacgga ttgcaatccg ttgccgtctt cggctga
77716265PRTPseudomonas putida KT2440Amino acid sequence of putative
acyl-CoA thioesterase II (protein_id="NP_744457.1) (figure 26)
16Met Thr Phe Asn Gln Leu Leu Asp Ala Val Arg Ala Asn Pro Asp Ser1
5 10 15 Val Ser Ile Pro Pro Ser Trp Ala Gln Gly Arg Ala Ala Phe Gly
Gly 20 25 30 Leu Met Ala Ala Met Val Tyr Glu Thr Met Arg Leu Lys
Ile Ser Asp 35 40 45 Asn Arg Pro Val Arg Ser Leu Ala Ile Ser Phe
Val Ala Pro Ala Ala 50 55 60 Ala Asp Val Pro Ile Arg Phe Glu Val
Glu Val Leu Arg Glu Gly Lys65 70 75 80 Ala Val Ser Thr Leu Leu Gly
Arg Ala Val Gln Asp Gly Gln Val Val 85 90 95 Thr Leu Val Gln Gly
Asn Phe Gly Ala Gly Arg Pro Ser Val Val Glu 100 105 110 Val Pro Ala
Leu Pro Ala Ile Glu Met Pro Ala Leu Asp Glu Ala Ala 115 120 125 Pro
Glu Leu Pro Tyr Ile Lys Gly Val Thr Pro Glu Phe Met Arg His 130 135
140 Val Ala Leu Arg Trp Ala Val Gly Gly Leu Pro Phe Ser Gly Asn
Gln145 150 155 160 Ser Arg Lys Met Gly Gly Trp Val Arg Leu Arg Asp
Val Val Glu Glu 165 170 175 Gln Val Asn Glu Ala His Leu Leu Ala Leu
Val Asp Ala Trp Pro Pro 180 185 190 Ser Leu Met Pro Phe Leu Lys Gln
Pro Ala Ala Gly Ser Thr Leu Thr 195 200 205 Trp Thr Ile Glu Phe Ile
Gln Pro Thr Ala Lys Leu Ser Thr Leu Asp 210 215 220 Trp Cys Arg Tyr
Cys Val Glu Thr Glu His Ala Arg Asp Gly Tyr Gly225 230 235 240 His
Ala Ala Ala Ala Leu Trp Thr Ala Gln Gly Glu Leu Leu Ala Leu 245 250
255 Ser Arg Gln Thr Val Thr Val Phe Ala 260 265 17265PRTPseudomonas
aeruginosa PA01Amino acid sequence of hypothetical protein PA2871
(protein_id="NP_251561.1) (figure 27) 17Met Asn Phe Ser Glu Leu Ile
Gln Ala Val Arg Arg Asp Pro Ser Ser1 5 10 15 Val Val Val Pro Ala
Ser Trp Gly Gln Gly Arg Ala Thr Phe Gly Gly 20 25 30 Leu Val Val
Ala Leu Ala Tyr Glu Ala Met Leu Ala Val Val Glu Ala 35 40 45 Gly
Arg Pro Leu Arg Ser Ile Gly Val Ser Phe Val Gly Pro Leu Ala 50 55
60 Pro Glu Gln Pro Ala Ser Phe Ser Ala Arg Leu Leu Arg Glu Gly
Lys65 70 75 80 Ala Val Ser Gln Val Gln Val Glu Val Arg Gln Gly Glu
Gln Val Val 85 90 95 Thr Leu Val Gln Ala Ser Phe Gly Val Ala Arg
Ala Ser Ala Val Ala 100 105 110 Val Glu Ala Leu Pro Ala Ala Gly Met
Lys Gly Pro Glu Glu Ser Gln 115 120 125 Glu Leu Pro Tyr Ile Arg Asn
Val Thr Pro Glu Phe Thr Arg Tyr Ile 130 135 140 Ala Met Arg Trp Ala
Val Gly Gly Leu Pro Phe Ser Ser Asn Lys Ser145 150 155 160 Arg Gln
Met Gly Gly Trp Met Arg Phe Arg Asp Glu Pro Glu Gly Glu 165 170 175
Pro Met Glu Val Ser His Leu Leu Ala Leu Leu Asp Ser Trp Pro Pro 180
185 190 Ala Leu Leu Pro His Leu Gly Thr Pro Ala Met Ala Ser Ser Leu
Thr 195 200 205 Trp Thr Ala Glu Phe Leu Gln Pro Leu Pro Gln Gln Gly
Ser Gly Asp 210 215 220 Trp Cys Arg Tyr Leu Ala Glu Ile Glu Glu Ala
Arg Asp Gly Tyr Gly225 230 235 240 His Val Ala Ala Arg Met Trp Ser
Ala Asp Gly Gln Leu Leu Ala Ile 245 250 255 Ser Arg Gln Met Val Thr
Val Phe Gly 260 265 18265PRTPseudomonas syringae pv B728aAmino acid
sequence of acyl-CoA thioesterase II, putative of p. syringae pv
B728a (protein_id="YP_235138.1) (figure 28) 18Met Thr Phe Ser Glu
Leu Ile Asp Ala Leu Arg Arg Asp Pro Arg Ser1 5 10 15 Val Thr Ile
Pro Ala Glu Trp Ser Gln Gly Arg Ala Cys Phe Gly Gly 20 25 30 Leu
Met Ala Ala Leu Thr Tyr Glu Ala Met Arg Ala Gln Val Pro Glu 35 40
45 Gly Arg Pro Val Arg Ser Leu Ala Ile Thr Phe Val Gly Pro Ala Ala
50 55 60 Pro Gly Val Pro Ile Ala Phe Glu Val Asp Thr Leu Arg His
Gly Lys65 70 75 80 Ala Val Ser Gln Val Leu Gly Arg Ala Met Gln Asn
Gly Gln Val Met 85 90 95 Thr Leu Ile Gln Gly Ser Phe Gly Ala Pro
Arg Glu Ser Met Ile Thr 100 105 110 Val Ala Ala Glu Ala Ala Pro Val
Leu Lys Pro Val Asp Gln Cys Pro 115 120 125 Glu Leu Pro Phe Ala Ser
Gly Val Met Pro Asp Tyr Leu Arg Phe Met 130 135 140 Asp Ile Arg Trp
Ala Leu Gly Gly Met Pro Phe Ser Asn Thr Arg Ser145 150 155 160 Pro
Ala Ile Gly Gly Tyr Val Arg Phe Arg Asp Thr Pro His Ala Thr 165 170
175 Pro Met Ser Glu Ala His Ile Leu Ala Leu Val Asp Thr Trp Pro Pro
180 185 190 Ala Val Leu Pro His Leu Asp Lys Pro Ala Pro Gly Ser Ser
Leu Thr 195 200 205 Trp Thr Ile Glu Phe Val Gln Pro Gln Pro Ser Leu
Asp Thr Leu Gln 210 215 220 Trp Cys Ser Tyr Arg Ala Val Ile Glu His
Ala Arg Asp Gly Tyr Gly225 230 235 240 His Thr Ala Ala Ala Leu Trp
Ser Pro Asp Gly Glu Leu Ile Ala Ile 245 250 255 Ser Arg Gln Thr Val
Thr Val Phe Gly 260 265 19264PRTPseudomonas fluorescens PfO-1Amino
acid sequence of acyl-CoA thioesterase (protein_id="ZP_00264181.1)
(figure 29) 19Met Arg Phe Cys Asp Leu Ile Asp Ala Val Arg Arg Gln
Pro Glu Val1 5 10 15 Thr Ile Pro Ala Glu Trp Gly Gln Gly Arg Ala
Ser Phe Gly Gly Leu 20 25 30 Val Ala Ala Leu Gln Phe Glu Val Met
Arg Thr Lys Val Pro Thr Asp 35 40 45 Arg Pro Val Arg Ser Leu Ala
Ile Thr Phe Val Gly Pro Val Glu Pro 50 55 60 Glu Val Pro Val Ser
Phe Glu Val Glu Val Leu Arg Glu Gly Lys Ala65 70 75 80 Val Ser Gln
Val Leu Gly Arg Ala Val Gln Asn Gly Gln Val Val Thr 85 90 95 Met
Val Gln Gly Ser Phe Gly Ala Ser Arg Pro Ser Glu Val Ala Val 100 105
110 Glu Ala Tyr Pro Ala Pro Glu Met Lys His Trp Asp Asp Cys Gln Glu
115 120 125 Leu Pro Tyr Ile Lys Gly Val Thr Pro Glu Phe Met Arg His
Leu Ala 130 135 140 Met Arg Trp Ser Val Gly Gly Met Pro Phe Thr Gly
Asn Gln Ser Arg145 150 155 160 Leu Met Gly Gly Trp Val Arg Leu Arg
Gly Asp Val Lys Glu Glu Ser 165 170 175 Val Asn Glu Ala His Leu Leu
Ala Leu Val Asp Ala Trp Pro Pro Ala 180 185 190 Leu Leu Pro Tyr Leu
Lys Lys Pro Ala Pro Gly Ser Thr Leu Thr Trp 195 200 205 Thr Ile Glu
Phe Val Gln Pro Leu Arg Asp Leu Ser Thr Leu Asp Phe 210 215 220 Cys
Gln Tyr Leu Ala Asp Ile Glu Tyr Ala Ala Asp Gly Tyr Gly His225 230
235 240 Val Ala Ala Lys Leu Trp Ser Ala Lys Gly Glu Leu Ile Ala Met
Ser 245 250 255 Arg Gln Thr Val Thr Ile Phe Ala 260
20264PRTIdiomarina loihiensis L2TRAmino acid sequence of tesB-like
acyl-CoA thioesterase (protein_id=AAV81497.1) (figure 30) 20Met Asn
Phe His Thr Ala Val Glu Gln Ile Val Ala Asp Lys Asn Asn1 5 10 15
Gln Val Ile Glu Leu Pro Ser Gly Trp Ala Gln Gly Arg Ala Phe Phe 20
25 30 Gly Gly Phe Ser Gly Ala Leu Ala Ala Gln Phe Leu Leu Lys Gln
Phe 35 40 45 Pro Ile Glu Tyr His Leu Arg Ser Met Ser Ile Ser Phe
Val Ala Pro 50 55 60 Ala Glu Pro Gly Glu Ala Glu Leu Asn Tyr Arg
Ile Leu Arg Glu Gly65 70 75 80 Lys Ser Val Ile Gln Val Ala Val Glu
Leu Gln Gln Gln Gly Gln Ile 85 90 95 Met Leu Ser Cys Leu Ala Ser
Leu Gly Lys Gly Arg Ser Ser Thr Val 100 105 110 Thr Val Val Ser Glu
Thr Pro Pro Asp Leu Lys Thr Ile Asn Asp Gly 115 120 125 Pro Gly Leu
Pro Glu Ala Asp Ile Val Pro Glu Phe Ala Lys Asn Phe 130 135 140 Asp
Tyr Arg Ile Thr Ser Gly Gly Met Pro Phe Ser Gly Gln Pro Gly145 150
155 160 Arg Thr Phe Gly Gly Trp Ile Arg Phe Arg Glu Glu Gln Gln Pro
Leu 165 170 175 Thr Thr Ala Thr Ile Leu Ala Leu Val Asp Ala Trp Pro
Pro Ala Val 180 185 190 Leu Pro His Leu Asp Ser Pro Ala Pro Ala Ser
Ser Leu Thr Trp Thr 195 200 205 Ile Glu Phe Pro Asp Ile Pro Leu Gln
Ser Phe Ser Ser His Asp Trp 210 215 220 Phe Gln Tyr Glu Ala Phe Ile
Glu His Ala Glu Asn Gly Tyr Gly His225 230 235 240 Ser Arg Ala Gly
Leu Trp Ser Glu Lys Gly Glu Leu Leu Ala Ile Ser 245 250 255 Arg Gln
Thr Phe Thr Val Phe Ala 260 21267PRTAcinetobacter sp. ADP1Amino
acid sequence of putative acyl-CoA thioesterase II
(protein_id=CAG67294.1) (figure 31) 21Met Ser Asp Ala Met Pro Leu
Asp Gln Thr Leu Glu Gln Leu Thr Gln1 5 10 15 Asp Glu Trp Ile Asp
Ile Pro Gln Gly Trp Ser Gln Gly Arg Thr Ile 20 25 30 Tyr Gly Gly
Leu Val Ala Gly Leu Leu Met His Lys Ala Leu Ser Val 35 40 45 Met
Asn Asp Glu Ser Lys Asn Leu Leu Ser Thr Ser Ile Thr Phe Val 50 55
60 Gly Pro Val Asn Glu Gly Arg Val Arg Leu Thr Val Glu Ile Leu
Arg65 70 75 80 Gln Gly Lys Ser Val Thr Thr Ile Glu Ala Arg Leu Trp
Gln Asp Gln 85 90 95 Ala Val Gln Thr Ile Leu Ile Ala Ser Phe Gly
Gln Pro Arg Ser Ser 100 105 110 Glu Ile Phe Val Leu Asn Leu Pro Glu
Ala Pro Asp Tyr Leu Ser Pro 115 120 125 Glu Gln Phe Ser Arg Met Pro
Phe Val Lys Met Met Pro Glu Cys Tyr 130 135 140 Gln Gln Phe Asp Leu
Arg Trp Ala Glu Gly His Tyr Pro Met Thr Gln145 150 155 160 Gln Ala
Pro Asp Phe Gly Gly Trp Cys Arg Tyr Asp Ile Gln Lys His 165 170 175
Ser Pro Arg Ala Leu Asn Val Ala Asp Leu Leu Ile Leu Met Asp Ile 180
185 190 Trp Pro Pro Gly Val Leu Pro Met Phe Gln Thr Ile Ala Pro Ala
Ser 195 200 205 Ser Leu Thr Trp His Leu Thr Phe Val Arg Pro Val Ala
Tyr Glu Leu 210 215 220 His Asp Trp Phe Lys Tyr Gln Val Val Thr Gln
His Ala Ala Phe Gly225 230 235 240 Tyr Ala Thr Glu Tyr Ala His Leu
Trp Asp Ala Gln Asn Arg Leu Ile 245 250 255 Ala Ile Ser Arg Gln Thr
Val Thr Val Phe Ala 260 265 22262PRTCaulobacter crescentus
CB15Amino acid sequence of hypothetical protein CC2472
(protein_id="NP_421275.1) (figure 32) 22Met Thr Leu Tyr Thr Asp Leu
Val Ala Ala Ile Ala Ser Thr Glu Thr1 5 10 15 Gly Phe Ser Ala His
Val Ser Asp Asp Trp Lys Gln Gly
Arg Thr Thr 20 25 30 Tyr Gly Gly Leu Ser Gly Ala Leu Cys Val Glu
Ala Ala Leu Arg Ala 35 40 45 Phe Pro Glu Ala Pro Pro Leu Arg Ser
Ala Gln Phe Ala Phe Val Gly 50 55 60 Pro Ala Ala Gly Glu Leu Ala
Ile Ser Val Arg Pro Leu Arg Gln Gly65 70 75 80 Lys Ser Thr Leu Phe
Val Ala Val Asp Leu Ile Gly Glu Gln Gly Val 85 90 95 Ala Thr His
Gly Val Leu Thr Phe Gly Ala Ala Arg Thr Ser Ala Ile 100 105 110 Ser
Tyr Glu Glu Val Leu Cys Pro Pro Val Ala Pro Ala Gly Ala Cys 115 120
125 Glu Leu Phe Phe Pro Glu Asn Arg Gln Gly Ala Pro His Phe Ser Ala
130 135 140 Gln Phe Glu Val Arg Lys Ala Gly Gly Thr Arg Pro Leu Ala
Gly Gly145 150 155 160 Glu Pro Glu Tyr Leu Leu Trp Ile Arg His Arg
Asp Pro Ala Ala Thr 165 170 175 Ser Ile Ser Ala Leu Val Ala Leu Ala
Asp Met Pro Pro Pro Pro Ala 180 185 190 Met Ala Leu Phe Pro Gln Phe
Gly Pro Ile Ser Thr Met Thr Trp Ser 195 200 205 Leu Asp Ile Val Gly
Leu Pro Glu Ala Asp Asp Asp Gly Trp Arg Leu 210 215 220 Leu Arg Thr
Arg Ala Glu Thr Ile Gly Asp Gly Tyr Ser Thr Gln Glu225 230 235 240
Met His Leu Trp Asp Ala Lys Gly Arg Pro Leu Val Leu Ala Arg Gln 245
250 255 Asn Val Ala Ile Phe Val 260 23258PRTErythrobacter litoralis
HTCC2594Amino acid sequence of hypothetical protein ELI0992
(protein_id=ZP_00375750.1) (figure 33) 23Met Ser Val Ser Asp Leu
Arg Ala Pro Ile Thr Ser Glu Gly Gly Ala1 5 10 15 Val Thr Leu Pro
Ala Asp Lys Trp Leu Gln Gly Arg Thr Leu Phe Gly 20 25 30 Gly Ala
Ser Ala Leu Val Ala Tyr Thr Ala Ala Val Arg Ala Phe Pro 35 40 45
Asp Leu Pro Pro Leu Arg Ala Ala Gln Ile Gly Phe Val Ala Pro Val 50
55 60 Gly Lys Asp Val Glu Thr Arg Ala Ala Met Val Arg Gln Gly Arg
Asn65 70 75 80 Val Ala Gln Val Arg Ser Glu Leu Leu Val Glu Gly Lys
Val Ala Leu 85 90 95 Thr Ala Phe Trp Leu Phe Gly Thr Gly Arg Glu
Ala Asn Ala Val His 100 105 110 Ala Ala Ala Lys Ala Asp Pro Trp Pro
Gly Ala Pro Glu Glu Asn Asp 115 120 125 Ser Ala Met Thr Asp Lys Gly
Pro Pro Phe Ile Val Asn Asn Phe Asp 130 135 140 Ile Arg Arg Ala Gln
Glu Thr Gln Gly Pro Gly Glu Pro Ile Val Arg145 150 155 160 Arg Trp
Phe Arg Leu Thr Asp Arg Gly Glu Leu Asp Arg Val Ser Glu 165 170 175
Leu Ile Leu Val Gly Asp Thr Leu Pro Pro Gly Ala Met Arg Ala Met 180
185 190 Gln Arg Gln Gly Pro Ile Ser Ser Ile Asn Trp Ser Phe Asn Ile
Leu 195 200 205 Asp Ala Glu Leu Gly Thr Arg Asp Gly Trp Trp Leu Gly
Glu Thr Ala 210 215 220 Ser Gln His Ala Gly Ala Gly Tyr Ser Ser Glu
Arg Leu Arg Leu Trp225 230 235 240 Asn Ala Asp Gly Val Gln Val Met
Asp Gly Leu Gln Ser Val Ala Val 245 250 255 Phe
Gly2425DNAArtificial SequenceDescription of Artificial Sequence
Primer (OTR End) used in Example 1 24ggccgcactt gtgtataaga gtcag
252525DNAArtificial SequenceDescription of Artificial Sequence
Primer (1TR End) used in Example 1 25gcggccagat ctgatcaaga gacag
252621DNAArtificial SequenceDescription of Artificial Sequence
Primer 1086F used in Example 1 26ttactggctt cgcaggaatg g
212726DNAArtificial SequenceDescription of Artificial Sequence
Primer IntSM160 used in Example 1 27cttggcaccc agcatgcgcg agcagg
262821DNAArtificial SequenceDescription of Artificial Sequence
Primer 1087F used in Example 3 28cagtgatggc tatggtcaaa g
212921DNAArtificial SequenceDescription of Artificial Sequence
Primer 1087R used in Example 3 29ctttgatcag tccggcaaaa c
21303902DNAAlcanivorax borkumensisNucleotide sequences of the genes
tesB-like (Abo_1044) and plsC (Abo_1045) consisting of an operon in
Alcanivorax borkumensis, with Tn5 inserted at position 588.
30ggcttcgcag gaatggtaaa taaagcaccg caaattcaaa acacgcagaa gtgagagacc
60atgacattcg atgagattct agccacgatt gatggccagg gtaacgccac gtttccagaa
120gggtgggggc agggccggac cttatttggt ggcctggtgg gggcggtgct
gtttgaacat 180ttagaaaaaa ccgtggctcg cgggaggttt ctacgtagtt
tttctctctc ttttgtcgcc 240cctgcggtgc cgggtccggt ggcactagac
gagactgtgt ttcgggaagg caaatccgtt 300atgcaggcca tggtctccgc
ccgtcaagga gggcaggtgg tggcggttat gttggccagc 360tttggggcca
gtcgccaatc cagtgtagtg gtagaagggc catcggcccc agtgatgaaa
420tctccagagc agagtatttc ggtaccgttt atcaaaggat tgacgccgga
ttttttttcc 480cattttaata tccattacgc agaaggcatg ccgccgttca
gtggcagttc tgagcccgat 540tacggtggtt acatggggtt tactgtgccg
ccggaaacca tgagcacctg tctcttgatc 600agatctggcc acctaggccg
aattcccggg gatccggtga ttgattgagc aagctttatg 660cttgtaaacc
gttttgtgaa aaaattttta aaataaaaaa ggggacctct agggtcccca
720attaattagt aatataatct attaaaggtc attcaaaagg tcatccaccg
gatcacctta 780ccaagccctc gctagattgt taatgcggat gttgcgatta
cttcgcccaa ctattgcgat 840aacaagaaaa gcgcctttca tgatatatct
cccaattttg tgtagggctt attatgcacg 900cttaaaaata ataaaagcga
cttgacctga tagtttggct gtgagcaatt atgtgcttag 960tgcatctaac
gcttgagtta accgcgccgc gaagcggcgt cggcttgaac gaattgttag
1020acattatttg ccgactacct tggtgattcg cctttcacgt agtggacaaa
atcaaccaac 1080tgatctgcgc gagcttcacg ctgccgcaag catcagggcg
caagggctgc taaaggaagc 1140ggaacacgta gaaagccagt ccgcagaaac
ggtgctaccc cggatgaatg tcagctactg 1200ggctatctgg acaagggaaa
acgcaagcgc aaagagaaag aggtagcttg cagtgggctt 1260acatgacgat
agctagactg ggcggtttta tggacagcaa gcgaaccgga attgccagct
1320ggggcgccct ctggtaaggt tgggaagccc tgcaaagtaa actggatggc
tttcttgccg 1380ccaaggatct gatggcgcag gggatcaaga tctgatcaag
agacaggatg aggatcgttt 1440cgcatgattg aacaagatgg attgcacgca
ggttctccgg ccgcttgggt ggagaggcta 1500ttcggctatg actgggcaca
acagacaatc ggctgctctg atgccgccgt gttccggctg 1560tcagcgcagg
ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
1620ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
ttgcgcagct 1680gtgctcgacg ttgtcactga agcgggaagg gactggctgc
tattgggcga agtgccgggg 1740caggatctcc tgtcatctca ccttgctcct
gccgagaaag tatccatcat ggctgatgca 1800atgcggcggc tgcatacgct
tgatccggct acctgcccat tcgaccacca agcgaaacat 1860cgcatcgagc
gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
1920gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
gcgcatgccc 1980gacggcgagg atctcgtcgt gacccatggc gatgcctgct
tgccgaatat catggtggaa 2040aatggccgct tttctggatt catcgactgt
ggccggctgg gtgtggcgga ccgctatcag 2100gacatagcgt tggctacccg
tgatattgct gaagagcttg gcggcgaatg ggctgaccgc 2160ttcctcgtgc
tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
2220cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa
gcgacgccca 2280acctgccatc acgagatttc gattccaccg ccgccttcta
tgaaaggttg ggcttcggaa 2340tcgttttccg ggacgccggc tggatgatcc
tccagcgcgg ggatctcatg ctggagttct 2400tcgcccaccc cgggctcgat
cccctcgcga gttggttcag ctgctgcctg aggctggacg 2460acctcgcgga
gttctaccgg cagtgcaaat ccgtcggcat ccaggaaacc agcagcggct
2520atccgcgcat ccatgccccc gaactgcagg agtggggagg cacgatggcc
gctttggtcg 2580acccggacgg gacggatcag tgagggtttg caactgtggg
tcaaggatct ggatttcgat 2640cacggcacga tcatcgtcgg gagggcaagg
gctccaagga tcgggccttg atgttaccga 2700gagcttggta cccagtctgt
gtgagcaggg gaattgatcc ggtggatgac cttttgaatg 2760acctttaata
gattatatta ctaattaatt ggggacccta gaggtcccct tttttatttt
2820aaaaattttt tcacaaaacg gtttacaagc ataaagcttg ctcaatcaat
caccggatcc 2880ccgggaattc gtcgacaagc tgcggccgcc taggccgtgg
ccgaacttgt gtataagagt 2940cagcgcagcg ctgattgcat tggtggatac
ctgggcgcct tcagttttac ctctgctcaa 3000agggccagca cccgccagtt
ccttgacctg gaccatggag ctcttggatg accctagtgt 3060ccactcgccg
gagactttat ggcaatatcg ggtaaacacg gaccaatgca gtgatggcta
3120tggtcaaagt caggcggtgg tttgggatgc tgcgggtaag gctgtggcgt
tgagtcgaca 3180aacctttacg gtatttgcat gaaatcggaa ctggtgccac
tcacggcgcg gactttaggt 3240aagcaagtgc ctcgtcgtgg gcactggctg
ttggcggcgc tagggcggct gattttgact 3300gttatggggt ggcgtattgt
tggtgatttg cccgatacgc ctcgggcagt tttggcggtg 3360gcgccacaca
cgtcgaacat agacggggtc attggtatca gtgctattca gtctttgcgc
3420ttggatgtgc gcttcatggg taagcacacg ttgtttaaag gtcgtcttgg
gcggttcatg 3480tactggctgg gcggcatccc tgtgaatcga gaaagtgcca
gggatgtggt ggaccagacg 3540acgtcggtga tgggggaaac accattttgg
cttgggctaa cgccggaggg gacgcgtaaa 3600ggcgccaagc gttggaaaac
cgggttttac cgtattgctg agcaaatgca ggtgccgatt 3660gtcgtgttag
gtttctgtta ccggcgccgg caggtccgga ttgtagattg ctttctgccg
3720acgggcgata ttgatgctga tatggcgcga atgaccgcgt cgttggcgga
tattgttccg 3780cgcaaacctg cgcagttatc cgccccgctg aaagcggaaa
aagctgctcg cggcattgat 3840tgattcagac gttggcagtt ttgccggact
gatcaaggag atgctggcgg tgctgggcgg 3900at 3902313142DNAAlcanivorax
borkumensisNucleotide sequence of tesB-like acyl-CoA thioesterase
from Alcanivorax borkumensis (Abo_1044), with Tn5 inserted at
position 558. 31atgacattcg atgagattct agccacgatt gatggccagg
gtaacgccac gtttccagaa 60gggtgggggc agggccggac cttatttggt ggcctggtgg
gggcggtgct gtttgaacat 120ttagaaaaaa ccgtggctcg cgggaggttt
ctacgtagtt tttctctctc ttttgtcgcc 180cctgcggtgc cgggtccggt
ggcactagac gagactgtgt ttcgggaagg caaatccgtt 240atgcaggcca
tggtctccgc ccgtcaagga gggcaggtgg tggcggttat gttggccagc
300tttggggcca gtcgccaatc cagtgtagtg gtagaagggc catcggcccc
agtgatgaaa 360tctccagagc agagtatttc ggtaccgttt atcaaaggat
tgacgccgga ttttttttcc 420cattttaata tccattacgc agaaggcatg
ccgccgttca gtggcagttc tgagcccgat 480tacggtggtt acatggggtt
tactgtgccg ccggaaacca tgagcaccgc agcgctgatt 540gcattggtgg
atacctgctg tctcttgatc agatctggcc acctaggccg aattcccggg
600gatccggtga ttgattgagc aagctttatg cttgtaaacc gttttgtgaa
aaaattttta 660aaataaaaaa ggggacctct agggtcccca attaattagt
aatataatct attaaaggtc 720attcaaaagg tcatccaccg gatcacctta
ccaagccctc gctagattgt taatgcggat 780gttgcgatta cttcgcccaa
ctattgcgat aacaagaaaa gcgcctttca tgatatatct 840cccaattttg
tgtagggctt attatgcacg cttaaaaata ataaaagcga cttgacctga
900tagtttggct gtgagcaatt atgtgcttag tgcatctaac gcttgagtta
accgcgccgc 960gaagcggcgt cggcttgaac gaattgttag acattatttg
ccgactacct tggtgattcg 1020cctttcacgt agtggacaaa atcaaccaac
tgatctgcgc gagcttcacg ctgccgcaag 1080catcagggcg caagggctgc
taaaggaagc ggaacacgta gaaagccagt ccgcagaaac 1140ggtgctaccc
cggatgaatg tcagctactg ggctatctgg acaagggaaa acgcaagcgc
1200aaagagaaag aggtagcttg cagtgggctt acatgacgat agctagactg
ggcggtttta 1260tggacagcaa gcgaaccgga attgccagct ggggcgccct
ctggtaaggt tgggaagccc 1320tgcaaagtaa actggatggc tttcttgccg
ccaaggatct gatggcgcag gggatcaaga 1380tctgatcaag agacaggatg
aggatcgttt cgcatgattg aacaagatgg attgcacgca 1440ggttctccgg
ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
1500ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
tctttttgtc 1560aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
aggcagcgcg gctatcgtgg 1620ctggccacga cgggcgttcc ttgcgcagct
gtgctcgacg ttgtcactga agcgggaagg 1680gactggctgc tattgggcga
agtgccgggg caggatctcc tgtcatctca ccttgctcct 1740gccgagaaag
tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
1800acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
tcggatggaa 1860gccggtcttg tcgatcagga tgatctggac gaagagcatc
aggggctcgc gccagccgaa 1920ctgttcgcca ggctcaaggc gcgcatgccc
gacggcgagg atctcgtcgt gacccatggc 1980gatgcctgct tgccgaatat
catggtggaa aatggccgct tttctggatt catcgactgt 2040ggccggctgg
gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
2100gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
cgccgctccc 2160gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
tcttctgagc gggactctgg 2220ggttcgaaat gaccgaccaa gcgacgccca
acctgccatc acgagatttc gattccaccg 2280ccgccttcta tgaaaggttg
ggcttcggaa tcgttttccg ggacgccggc tggatgatcc 2340tccagcgcgg
ggatctcatg ctggagttct tcgcccaccc cgggctcgat cccctcgcga
2400gttggttcag ctgctgcctg aggctggacg acctcgcgga gttctaccgg
cagtgcaaat 2460ccgtcggcat ccaggaaacc agcagcggct atccgcgcat
ccatgccccc gaactgcagg 2520agtggggagg cacgatggcc gctttggtcg
acccggacgg gacggatcag tgagggtttg 2580caactgtggg tcaaggatct
ggatttcgat cacggcacga tcatcgtcgg gagggcaagg 2640gctccaagga
tcgggccttg atgttaccga gagcttggta cccagtctgt gtgagcaggg
2700gaattgatcc ggtggatgac cttttgaatg acctttaata gattatatta
ctaattaatt 2760ggggacccta gaggtcccct tttttatttt aaaaattttt
tcacaaaacg gtttacaagc 2820ataaagcttg ctcaatcaat caccggatcc
ccgggaattc gtcgacaagc tgcggccgcc 2880taggccgtgg ccgaacttgt
gtataagagt cagggcgcct tcagttttac ctctgctcaa 2940agggccagca
cccgccagtt ccttgacctg gaccatggag ctcttggatg accctagtgt
3000ccactcgccg gagactttat ggcaatatcg ggtaaacacg gaccaatgca
gtgatggcta 3060tggtcaaagt caggcggtgg tttgggatgc tgcgggtaag
gctgtggcgt tgagtcgaca 3120aacctttacg gtatttgcat ga 3142
* * * * *