U.S. patent number 10,385,327 [Application Number 13/344,062] was granted by the patent office on 2019-08-20 for enhanced e. coli for the production of fatty acids and method of producing the same.
This patent grant is currently assigned to University of Puerto Rico. The grantee listed for this patent is Abel Baerga-Ortiz, Delise Oyola-Robles. Invention is credited to Abel Baerga-Ortiz, Delise Oyola-Robles.
United States Patent |
10,385,327 |
Baerga-Ortiz , et
al. |
August 20, 2019 |
Enhanced E. coli for the production of fatty acids and method of
producing the same
Abstract
The invention analyzed a protein sequence using the
Udwary-Merski algorithm and identified a tetradomain fragment
(DH1-DH2-UMA) which consists of two predicted DH-like domains and
two pseudodomains N-terminal to them. This arrangement of domains
and pseudodomains is fundamentally the opposite of what is
typically observed in the DH cassettes of actinobacterial
polyketide synthases or mammalian fatty acid synthases, both of
which feature C-terminal pseudodomains. The invention modified E.
coli by over expressing DH1-DH2-UMA in E. coli resulting in an
increase in the overall production of all the fatty acids normally
present in the E. coli fatty acid profile.
Inventors: |
Baerga-Ortiz; Abel (San Juan,
PR), Oyola-Robles; Delise (San Juan, PR) |
Applicant: |
Name |
City |
State |
Country |
Type |
Baerga-Ortiz; Abel
Oyola-Robles; Delise |
San Juan
San Juan |
PR
PR |
US
US |
|
|
Assignee: |
University of Puerto Rico (San
Juan, PR)
|
Family
ID: |
48744166 |
Appl.
No.: |
13/344,062 |
Filed: |
January 5, 2012 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20130177989 A1 |
Jul 11, 2013 |
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N
15/70 (20130101); C12N 9/88 (20130101) |
Current International
Class: |
C12N
9/88 (20060101); C12N 15/70 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Udwary et al. 2002 (A Method for Prediction of the Locations of
Linker Regions within Large Multifunctional Proteins, and
Application to a Type I Polyketide Synthase; JMB, 323:585-598).
cited by examiner .
Allen et al. 2002 (Structure and regulation of the omega-3
polyunsaturated fatty acid synthase genes from the deep-sea
bacterium Photobacterium profundum strain SS9; Microbiology;
148:1903-1913). cited by examiner .
Oyola-Robles et al. 2013 (Identification of novel protein domains
required for the expression of an active dehydratase fragment from
a polyunsaturated fatty acid synthase; Protein Science 22:954-963).
cited by examiner .
Oyola-Robles et al. 2014 (Expression of dehydratase domains from a
polyunsaturated fatty acid syntase increase the production of fatty
acids in Escherichia coli; Enzyme and Microbial Technology,
55:133-139). cited by examiner.
|
Primary Examiner: Lyons; Mary Maille
Attorney, Agent or Firm: Hoglund & Pamias, PSC Rios;
Roberto J.
Government Interests
GOVERNMENT INTEREST
The claimed invention was made with U.S. Government support under
grant number CHE-0953254 awarded by the US National Science
Foundation (NSF). The government has certain rights in this
invention.
Claims
We claim:
1. A gene vector for modifying Escherichia coli (E. coli)
comprising: the dehydratase tetradomain gene fragment DH1-DH2-UMA
from Photobacterium profundum of SEQ ID:17 cloned into the plasmid
vector of SEQ ID:15.
2. The gene vector of claim 1, wherein said dehydratase tetradomain
gene fragment encodes a DH1-DH2 protein.
3. The gene vector of claim 1, wherein said gene vector modifies E.
coli to produce about 4-5 times more free saturated and
monounsaturated fatty acids than wild-type E. coli.
4. The gene vector of claim 1, wherein said dehydratase tetradomain
gene fragment is over-expressed at about room temperature.
5. A method for increasing the production of free saturated and
monounsaturated fatty acids in E. coli comprising: cloning the
dehydratase tetradomain gene fragment DH1-DH2-UMA from
Photobacterium profundum of SEQ ID: 17 into the plasmid vector of
SEQ ID:15; and inserting said cloned vector into E. coli.
6. The method of claim 5, wherein said dehydratase tetradomain gene
fragment is over-expressed at about room temperature.
7. The method of claim 5, wherein said dehydratase tetradomain gene
fragment encodes a DH1-DH2 protein.
Description
SEQUENCE LISTING
The sequence listing submitted via EFS, in compliance with 37 CFR
.sctn. 1.52(e) (5), is incorporated herein by reference. The
sequence listing text file submitted via EFS contains the file
"Sequence Listing 13344062", created on Jun. 25, 2012, which is
32,350 bytes in size.
BACKGROUND OF THE INVENTION
Long-chain polyunsaturated fatty acids (PUFAs) have been implicated
in human brain development as well as in the maintenance of
cardiovascular health. Although animals have the enzymes necessary
to form long-chain PUFAs through the elongation of plant-derived
PUFAs, this oxygen-dependent process is not efficient. An efficient
pathway for the biosynthesis of PUFAs in deep-sea bacteria utilizes
a polyketide synthase-like (PKS-like) multienzyme complex. A total
of five genes from this pathway have been found to be sufficient
for the production of polyunsaturated fatty acids in an otherwise
non-producing Escherichia coli. These genes are pfaA, pfaB, pfaC,
pfaD, encoding PUFA synthases containing enzyme domains for acyl
tranferases (AT), keto-acyl synthase (KS), acyl carrier protein
(ACP), keto-acyl reductase (KR), enoyl reductase (ER) and
dehydratase (DH) activities and also pfaE, which encodes a required
phosphopantetheine transferase (PPTase) essential for the
activation of ACP domains through chemical modification as shown in
FIG. 1. While some of the required enzymatic activities are housed
in independent stand-alone proteins (pfaB, pfaD and pfaE: FIG. 1)
others are assembled into multidomains (pfaA and pfaC: FIG. 1). No
thioesterase activity has been observed in the PUFA synthase
cluster and no dedicated thioesterase protein from the producing
organism is required for heterologous production of PUFAs in E.
coli.
Dehydratase (DH) domains are responsible for the formation of the
cis double bonds in the structure of PUFAs. They can be easily
identified by their sequence similarity to FabA and FabZ, the two
DH enzymes involved in fatty acid biosynthesis in E. coli. FabA/Z
catalyze the dehydration of 3Rhydroxyacyl-ACP via a syn elimination
mechanism which has also been reported in the DH domain from the
erythromycin PKS.
The structure of FabA, and more recently FabZ, revealed an obligate
homodimeric arrangement in which both DH subunits contribute key
residues to the active site. This distinct architectural feature
has been found to extend to DH domains from the animal Fatty Acid
Synthase (FAS), and more recently to the erythromycin PKS, although
with the following variation on the E. coli arrangement. While the
E. coli FabA and FabZ form homodimers of identical subunits, the DH
domains from FAS and PKS systems form a heterodimeric double hotdog
arrangement in which two contiguous pseudosubunits are housed
within the same polypeptide and separated by a 25-residue amino
acid stretch. Thus, the required dimerization of the DH domain in
the context of a multienzyme complex does not necessarily involve
interactions between different polypeptides, but rather within the
same polypeptide.
In both the FAS and PKS DH, the protein region that is homologous
to FabA is followed by a necessary C-terminal pseudodomain with no
previously known function and no known sequence homologue. In the
case of the FAS DH, the C-terminal pseudodomain was found to
contribute to dehydratase activity in in vitro enzyme assays. The
structure of the PKS DH showed that the Cterminal pseudodomain
forms the other half of the double hotdog in the three-dimensional
structure. In that work, the protein construct that was
crystallized, and whose structure was determined, contained the
pseudodomain but lacked dehydratase activity in vitro, although
mutations made elsewhere did show an effect on overall polyketide
production by the full-length multienzyme.
The PUFA synthase multienzyme contains two putative DH domains in
tandem. They have been identified as DH domains based on their
sequence similarity to FabA/Z, but their activity or specificity
has not been confirmed biochemically. The tandem arrangement, while
not previously observed in other biosynthetic enzyme systems, is a
well-conserved feature of PUFA synthases. However, it is unknown
how these tandem domains act to generate the combination of double
and single C--C bonds in the final PUFA structure.
SUMMARY OF THE INVENTION
According to an aspect of the invention, a protein fragment
consisting of the two tandem putative DH domains and the two
corresponding pseudodomains from the PUFA synthase was designed
using the Udwary-Merski Algorithm (UMA) developed at Johns Hopkins
University.
According to another aspect of the invention, the resulting
tetradomain fragment showed some dehydratase activity against an
acyl-CoA soluble substrate. Examination of the three dimensional
models for the individual domains reveal that while two domains
contain all the conserved residues expected for a functional DH
domain, the other two domains contain other residues present on
other hot-dog proteins.
According to still another aspect of the invention, the analysis of
the tetradomain sequence anticipates an "inverted" double hotdog
arrangement in which the pseudodomain is actually located
N-terminal to the FabA homology domain, thus providing an
alternative topological solution which suggests evolutionary
convergence of the DH architecture in PUFA synthase
multienzymes.
BRIEF DESCRIPTION OF THE DRAWINGS
Further features and advantages of the invention will become
apparent from the following detailed description taken in
conjunction with the accompanying figures showing illustrative
embodiments of the invention, in which:
FIG. 1 shows a gene cluster for the anaerobic production of
eicosapentaenoic acid (EPA) in Photobacterium profundum according
to the present invention.
FIG. 2 shows a scheme summarizing the construction of different
protein fragments for the isolation of dehydratase activity
according to the present invention.
FIG. 3 shows graphs for Dehydratase activity of DH1-DH2-UMA towards
crotonyl-CoA and the specific activity of DH1-DH2-UMA toward
crotonyl-NAC in the hydration reaction and towards
.beta.-hydroxybutyryl-CoA in the dehydration reaction according to
the present invention.
FIG. 4 shows a saturation curve obtained by measuring the activity
of DH1-DH2-UMA towards crotonyl-CoA at 235 nm according to the
present invention.
FIG. 5 shows a graph illustrating the over-expression of
DH1-DH2-UMA in E. coli resulting in an increase in the total
production of free fatty acids in liquid bacterial culture
according to the present invention.
FIG. 6 illustrates a comparison of the three-dimensional models
obtained for the FabA-homology regions (DH1 and DH2) and for the
uncharacterized pseudodomains (DH1.' and DH2.') according to the
present invention.
Throughout the figures, the same reference numbers and characters,
unless otherwise stated, are used to denote like elements,
components, portions or features of the illustrated embodiments.
The subject invention will be described in detail in conjunction
with the accompanying figures, in view of the illustrative
embodiments.
DETAILED DESCRIPTION OF THE INVENTION
Experimental Procedures
Cloning, Expression and Purification
Different DH fragments were cloned from fosmid 8E1. All restriction
endonucleases, polynucleotide kinase, T4 DNA ligase, and alkaline
phosphatase were purchased. The primers used to make the different
fragments are summarized in Table 1 below. For cloning into
pGEX4T-3 vector, the amplified DNA was phosphorylated using
polynucleotide kinase and cloned into pUC19 which was previously
digested with SmaI and treated with alkaline phosphatase. The
ligation mixture was used to transform DH10B cells and clones were
selected in LB-agar containing ampicillin (100 .mu.g/mL). Insertion
of the DH fragment into pUC19 was confirmed by agarose gel
electrophoresis. The resulting plasmid pUC19:DH was digested with
BamHI and SmaI and the resulting excised DNA fragment was cloned
into the corresponding sites in pGEX4T-3.
TABLE-US-00001 TABLE 1 Dehydratase construct Oligonucleotide
sequence DH1S (SEQ.ID1) - Fwd:
5'-CATGCATGGGATCCAACTTGCTAGACGCAAATATCGCA-3' (SEQ.ID2) - Rv:
5'-CATGCATGCCCGGGTCATGATTCTTCTTTGATCATCACG-3' DH1L (SEQ.ID3) - Fwd:
5'-CACCTTCTCTTACGAATGTTTCGTTGGC-3' (SEQ.ID4) - Rv:
5'-CATGCATGCCCGGGTCATGATTCTTCTTTGATCATCACG-3' DH2S (SEQ.ID5) - Fwd:
5'-CATGCATGGGATCCAACTTACTGGATAAAGAAAGCCGTT-3' (SEQ.ID6) - Rv:
5'-TCAGGCTTCTTCAATACAGATTGC-3' DH2L (SEQ.ID7) - Fwd:
5'-CATGCATGGGATCCTTCAGCTTCGAACTCAGTACCGA-3' (SEQ.ID8) - Rv:
5'-TCAGGCTTCTTCAATACAGATTGC-3' DH1-DH2-S (SEQ.ID9) - Fwd:
5'-CACCAACTTGCTAGACGCAAATATCGCA-3' (SEQ.ID10) - Rv:
5'-TCAGGCTTCTTCAATACAGATTGC-3' DH1-DH2-L (SEQ.ID11) - Fwd:
5'-CACCTTCTCTTACGAATGTTTCGTTGGC-3' (SEQ.ID12) - Rv:
5'-TCAGGCTTCTTCAATACAGATTGC-3' DH1-DH2 UMA (SEQ.ID13) - Fwd:
5'-CACCCGCAAACCTTGTATCTGGGATTA-3' (SEQ.ID14) - Rv:
5'-TCAGGCTTCTTCAATACAGATTGC-3'
For the cloning of fragments into pET200TOPO SEQ.ID15, the
amplified DNA was gel purified using a Gel Extraction Kit and
incubated with pET200TOPO. The resulting clones were selected in
LB-agar containing kanamycin (100 .mu.g/mL). All resistant clones
were introduced into E. coli strain SEQ.ID16 BL21-DE3-Codon
Plus-RIL and grown in liquid LB at 37.degree. C. until the
OD600=0.4 at which time the temperature was decreased to 22.degree.
C..degree. until the OD600=0.6 at which time protein expression was
induced with 1 mM IPTG. After 16 h, the cells were collected and
resuspended in lysis buffer (50 mM Na3HPO4 pH 7.2, 150 mM NaCl, 1
mM DTT, 10% glycerol, 0.1 mg/mL lysozyme and DNAse) for 1 hr,
sonicated and centrifuged at a speed of 14,000 rpm at 4.degree. C.
for 30 min in a J2-21 Beckman centrifuge in a JA17 rotor. Samples
were collected for the total, supernatant and pellet to assess
solubility of the protein products.
For His-tagged soluble proteins, the lysate was collected and
poured through a column filled with Ni-NTA resin (Qiagen)
equilibrated in 25.0 mM Tris pH 8.0, 150 mM NaCl, 10% glycerol, 1.0
mM DTT. The DH fragment was eluted with the same buffer containing
300 mM imidazole.
Eluted protein was infused into an ion exchanger column operated at
room temperature and equilibrated in 25 mM Tris pH 8.0, 150 mM
NaCl, 1.0 mM DTT and 10% glycerol. The proteins were eluted in a
40-minute gradient 0.15 M-2 M NaCl. The fraction containing the
protein was concentrated and stored at -80.degree. C. Typical
yields for all proteins were 1.0 mg of protein per liter of
culture, purity .about.99% by 8% SDS-PAGE.
UMA Parameters.
The UMA program was used and UMA calculations were done as in
(Udwary et al., 2002) using the sequence of SEQ.ID17 pfaC from
Photobacterium profundum (GenBank Accession no. AF409100.1). A
multiple alignment of homologues of pfaC was performed in CLUSTALW
in ".pir" format and a secondary structure prediction for pfaC was
performed using the PSIPRED Server (University College London). The
output for the secondary structure prediction was used to generate
an ".ss" file. Finally, both the "pir." alignment and the "ss."
secondary structure prediction were used as inputs for the
"uma19.pl" application with the input parameters in Table 2 below.
Results in the output file were visualized using Keleidagraph for
Windows.
TABLE-US-00002 TABLE 2 Parameter Value Homology matrix blosum 30
Gap to gap penalty 0 Gap to aa penalty -4 Component averaging (k) 5
Final averaging (gamma) 20 Sim score weight 10 Struc score weight 1
Hydro score weight 5
Dehydratase Assays.
Dehydratase activity was measured in a hydration assay by using
Crotonyl-CoA and Crotonyl-NAC as substrates. Crotonyl-NAC was
synthesized from crotonic acid and N-acetylcysteamine using a DCC
coupling strategy as describes by the prior art and purified by
flash column chromatography on silica gel using 1:1 ethyl
acetate:ethyl ether. For the dehydration assay
.beta.-hydroxybutyryl-CoA was used as the substrate. Enzymatic
reactions were followed spectrophotometrically by monitoring the
absorbance at 260 nm in a 96-well plate format on. The total volume
was 200 .mu.L (25 mM Tris, 150 mM NaCl, 10% glycerol, pH 8.0, 3.20
.mu.M DH1-DH2-UMA, and 117 .mu.M of substrate). The values for the
absorbance slope (given in mAU/min) were converted to units of
.mu.mole of product per minute by using the following equation:
.mu.mole of
product/min=[Slope/.epsilon..times.b].times.Vol.sub.total (Eq1)
in which the slope is given by the instrument in units of
milliabsorbance (mAU) per minute, b is the path length measured to
be 0.89 cm for a Vol.sub.total=200 .mu.l in our 96-well plates. The
.epsilon. is the molar extinction coefficient resulting from the
loss of a double bond as defined by the difference in absorbance
between crotonyl-CoA and .beta.-hydroxybutyryl-CoA at a particular
wavelength. The extinction coefficient was calculated to be
.epsilon.=969.9 M.sup.-1 cm.sup.-1 for the reaction monitored at
260 nm and .epsilon.=790.7 M.sup.-1 cm.sup.-1 for the reaction
monitored at 235 nm.
For the kinetic assays the reaction was monitored at a wavelength
of 235 nm and using a range of substrate concentrations between 0
and 600 .mu.M. The data was fit to a simple Michaelis-Menten
Equation (Eq 2) using Kaleidagraph v4.03. V.sub.o=V
max[S]/([S]+K.sub.m) (Eq2)
Fatty Acid Profiles.
E. coli BL21-DE3-CodonPlus (RIL) SEQ.ID16 cells expressing
DH1-DH2-UMA SEQ.ID14 in the pET200Topo vector were cultured in LB
media and the expression was induced as described for protein
production. Protein expression was confirmed by SDS-PAGE. Cells
were collected by centrifugation at 4,400 rpm, 10 min, 4.degree. C.
and freeze-dried. The fatty acid components of the cell culture
were obtained as their methyl esters by the reaction of 0.05 g of
dried cell pellet with 10.0 mL of methanolic HCl, refluxed for 2 hr
followed by workup with hexane twice. The organic layer was dried
over MgSO.sub.4 and concentrated in vacuo. The fatty acid methyl
esters were analyzed by GC-MS (at 70) equipped with a 30
m.times.0.25 mm special performance capillary column (HP-5MS) of
polymethylsiloxane crossed-linked with 5% phenyl
methylpolysiloxane. The temperature program was as follows:
130.degree. C. for one minute, increase at a rate of 3.degree.
C./min to a 270.degree. C., where the temperature is maintained for
30 min. Methyl heneicosanoate was used as an internal standard for
quantification of fatty acid methyl esters.
Results
Design and Expression of Putative DH Domains from the PUFA
Synthase.
The pfaC protein of the PUFA synthase complex harbors two
homologues of FabA/Z dehydratases as shown in FIG. 1. Initially, a
number of protein constructs were designed on the basis of FabA
homology and sequence conservation alone as summarized in FIG. 2A.
Two "short" fragments, DH1S (H1318-51491) and DH2S (I1787-C-term)
were designed to include only the sequence homologous to FabA. Two
"longer" fragments, DH1L (F1249-51491) and DH2L (S1733-C-term),
were designed to include additional conserved sequence N-terminal
to the FabA-homology region. Finally, to explore the possibility
that the two FabA-homology regions stabilized one another, we also
generated protein fragments which contained both Fab-homology
regions, DH1-DH2S (H1318-C-Term) and DH1-DH2L (F1249-C-Term). All
of these protein fragments were expressed as GST fusion proteins
and as His-tagged proteins in E. coli and all were found to be
insoluble as evidenced by their presence in the lysis pellet (data
not shown).
In order to more accurately define the boundaries for the putative
DH domains from pfaC so as to increase the likelihood of generating
a functional enzyme fragment, we analyzed the sequence using the
Udwary-Merski Algorithm (UMA) which assigns a numerical score to
each amino acid based on the probability that it is located within
a structured domain, as opposed to it being located in an
unstructured linker region. UMA analysis of the pfaC sequence
revealed six domain regions as defined by their high UMA score as
shown in FIG. 2B. Four of the six domains had been previously
identified based on sequence alignments: two KS domains in the
N-terminal portion of the protein (KS1 and KS2), and the two FabA
homologs (DH1 and DH2). The two other areas of high UMA score were
located directly N-terminal to the putative DH domains. The
predicted secondary structure for the two new pseudodomains (termed
DH1' and DH2') was that of a hot-dog fold, much like the predicted
secondary structure for the Fabhomology domains.
Based on the UMA analysis and on the secondary structure
prediction, fragment DH1-DH2-UMA (I1096-N-Term) was designed and
expressed as a His-tagged protein in soluble form. After nickel
resin purification and anion exchange chromatography, a total yield
of 1.0 mg of pure protein was obtained per liter of culture. Gel
filtration chromatography of this protein revealed an equilibrium
between a monomer and a dimer in equal proportions (data not
shown).
Preliminary Activity of DH1-DH2-UMA.
Incubation of DH1-DH2-UMA with crotonyl-CoA resulted in a decrease
in the absorbance at 260 nm, consistent with the hydration of the
double bond as shown in FIG. 3. The N-acetyl cysteamine (NAC)
thioester of crotonic acid was not hydrated suggesting the
importance of the pantetheine carrier for substrate recognition.
The dehydration of the .beta.-hydroxybutyryl-CoA was also monitored
but no activity was detected in the forward reaction, probably due
to the fact that the chemical equilibrium favors the reverse
reaction. Initial efforts to measure the Michaelis-Menten kinetic
parameters were frustrated by the fact that the amount of substrate
required to saturate the enzyme was too high for spectrophotometric
determination at a wavelength of 260 nm. In order to lower the
absorption intensity of the acyl-CoA substrate, the reaction was
monitored at a wavelength of 235 nm and the initial velocity was
measured at different substrate concentration. From the saturation
curve for crotonyl-CoA, the kinetic parameters were extracted (FIG.
4: Vmax=0.0001 .mu.mol product/min; Km=156 .mu.M). The measured
activity towards NAC-loaded substrates or .beta.-hydroxybutyryl-CoA
was too low to yield a reliable saturation curve for the
determination of the kinetic parameters.
Effect of DH1-DH2-UMA Overexpression on the Fatty Acid Profile of
E. coli.
The overexpression of enzymes has been employed as a strategy to
enhance fatty acid production or to alter the normal fatty acid
profile of E. coli. In order to investigate whether DH1-DH2-UMA
would interact with the fatty acid biosynthesis machinery of E.
coli and result in the formation of polyunsaturated fatty acids, we
measured the production of fatty acids in a strain overexpressing
DH1-DH2-UMA. No polyunsaturated fatty acids were detected in any of
the bacterial extracts, indicating that the expression of
DH1-DH2-UMA is not sufficient to catalyze the formation of multiple
cis double bonds in the fatty acids normally made by E. coli. It
was observed, however, a 4-fold to 5-fold increase in the total
production of free saturated and monounsaturated fatty acids
without a change in the percentage composition of fatty acids as
shown in FIG. 5. The fact that the expression of DH1-DH2-UMA
affected the production of all fatty acids in equal proportions
suggests that the protein is capable of interacting with the E.
coli machinery for fatty acid biosynthesis in a way that does not
discriminate based on fatty acid chain length.
Three-Dimensional Models of DH Domains and Pseudodomains.
In order to verify the presence of amino acid residues normally
associated with dehydratase activity, we built three-dimensional
models of all domains and pseudo-domains using the Phyre Server
from Imperial College London as shown in FIG. 6. The 3D models
generated for the actual Fab-homology domains (DH1 and DH2) feature
an active site His70 and the conserved Glu84 typical of
dehydratases (amino acid sequence numbers are based on the FabA
numbering). Interestingly, even though the newly identified
N-terminal pseudodomains (DH1' and DH2') do not have a high enough
sequence similarity with any known protein, their secondary
structure prediction in the Phyre server was found to be consistent
with the formation of a hotdog fold, possibly the first half of a
double hotdog. Instead of the expected His70 conserved in
dehydratases, DH1' featured an Asp72 and DH2' featured a Glu70 in
the corresponding region as shown in FIG. 6. These acidic residues
in the active site are not typically observed in the hotdog
dehydratases but they are a defining feature of the hotdog
hydrolases, suggesting a possible involvement of DH1' and DH2' in
hydrolysis.
Discussion
The biosynthesis of PUFAs in deep-sea bacteria is carried by a
family of enzymes that contain a unique and conserved arrangement
of enzyme domains. PUFA synthases have been found in metagenomic
DNA from marine samples collected throughout the world, indicating
that anaerobic PUFA biosynthesis is a widely selected mechanism for
microbial adaptation to high-pressure and low temperature
environments. Despite much interest in elucidating how the PUFA
synthase carries out its function, published work on the enzymatic
activities of PUFA synthases has been sparse. Bumpus et al., 2008
showed for the first time the in vitro activity of the enoyl
reductase (pfaD) enzyme from Shewanella oneidensis PUFA synthase
and Jiang et al., 2008 interrogated the role of the tandem ACP
arrangement, which is a hallmark of PUFA synthases. The present
invention addressed another conserved feature of PUFA synthases, a
pair of conserved DH domains arranged in tandem near the C-terminus
of the multidomain protein, pfaC.
Analysis of the sequence of pfaC protein using the Udwary-Merski
Algorithm revealed the presence of two new pseudodomains located
directly N-terminal to the regions of FabA homology. These
pseudodomains were found to be essential for the proper expression
of protein fragments, since only the protein fragments that
included both pseudodomains were soluble, stable and active. This
result alone would suggest that DH' pseudodomains are important
components of the three-dimensional structure of dehydratase
domains. This finding also confirms the general applicability of
the Udwary-Merski Algorithm for the identification of functional
units within multidomain proteins with unknown functions or from
unexplored lineages.
The predicted secondary structure for both DH' pseudodomains was
that of a hotdog fold, which is also the expected three-dimensional
topology of the FabA-homology DH domains. This predicted
arrangement of contiguous hotdog folds points towards an overall
double hotdog structure, which has become the widely accepted model
for embedded dehydratases based on structural and biochemical
evidence. However, several differences exist between the PUFA
arrangement and its FAS and PKS evolutionary cousins. While in
FAS/PKS DH, the pseudodomains are located C-terminal to the
Fabhomology domain, in the PUFA DH, the pseudodomains are located
N-terminal to the Fab-homology domain. This alternative gene
structure of the PUFA DH suggests a tandem gene duplication event
that took place independently in terrestrial FAS/PKS and marine
PUFA synthase for the generation of functional DH dimers, resulting
in two alternative convergent topological solutions. Another
difference between FAS/PKS DH and PUFA DH is that, while FAS/PKS DH
domains consist of didomains (one FabA homology domain plus one
pseudodomain), the PUFA DH complex invariably consists of a
tetradomain (two FabA homology plus two pseudodomains). This
invention does not address the question of how the four protein
domains are paired in the functional assembly. Additional
structural characterization of DH1-DH2-UMA will have to be carried
out in order to elucidate how the different domains are arranged in
a functional complex.
Substantial work has been dedicated to determining the specific
role of pseudodomains in the activity of FAS DH domains beyond
stabilizing the dimeric structure by partnering with the
FabA-homology domain. Amino acids in the DH pseudodomain have been
implicated in the partial activity of the FAS ketoreductase domain.
Additionally, an Asp residue in the FAS pseudodomain has been found
to be essential and a Gln residue in the pseudodomain has been
found to be important for dehydratase activity. In the PUFA DH in
this report, multiple sequence alignment of the pseudodomains
reveal levels of sequence conservation (67% and 71% for DH1' and
DH2', respectively) that were comparable to the sequence
conservation of the FabA homology domains (61% and 75% for DH1 and
DH2, respectively). This high level of sequence similarity among
the pseudodomains is suggestive of a role in DH function beyond
that of a structural scaffold for dimerization.
The soluble DH1-DH2-UMA fragment was competent to catalyze the
hydration of crotonyl-CoA with a specific activity of 0.009 .mu.mol
product/(min*mg enzyme). When this number is converted to the units
of specific activity employed in Pasta et al., 2007, it becomes
0.83 mol product/(min*mol enzyme), at least two orders of magnitude
lower than the specific activity reported for the FAS 1-1168
construct (204 mol product/(min*mol enzyme)). It has been shown
that dehydratase activity decreases dramatically with decreasing
length of the acyl chain. Although that report does not include the
activity toward crotonyl-ACP (3:1), the difference between the
specific activity against octenoyl-ACP(8:1) and butenoyl-ACP (4:1)
was about one order of magnitude. In addition a similar dramatic
effect was observed when comparing ACP-linked substrate to
pantetheine-linked substrates. The PUFA DH in this report was
assayed for activity against crotonyl-CoA (3:1). Thus, it is not
surprising that the specific activity is low considering that the
acyl chain is even shorter that the shortest one in Pasta et al.,
2007 and that the substrate in this report is not loaded on an ACP.
Further work will need to be carried out to determine the substrate
preference for PUFA DH domains in a more physiological context.
Additional confirmation of the activity of DH1-DH2-UMA came from
measuring the effect of its overexpression on the production of
fatty acids in E. coli. According to the invention, a significant
increase in the production of fatty acids was observed in the BL21
E. coli strain expressing the DH1-DH2-UMA protein. Previous work by
others has shown that overexpression of the E. coli FabA
dehydratase does not increase the production of fatty acids in E.
coli. Thus, it is hard to argue that the observed increase in fatty
acid production in this report is due to the dehydratase activity
of DH1-DH2-UMA although it cannot be entirely ruled out. It has
been well established that the overexpression of thioesterases and
other hydrolases results in the enhancement of the production of
fatty acids and other high-energy biofuel precursors. Therefore, it
is possible that an adventitious or unphysiological hydrolase
activity, possibly an artifact arising from high enzyme
concentration inside overexpressing bacterial cells, could be
responsible for the observed enhancement of fatty acid production
in E. coli.
Inspection of the homology model made for the DH' pseudodomains
reveals a hotdog fold similar to that expected for the
FabA-homology regions, although with a different amino acid
occupying the active site position as shown in FIG. 6. While the
model for the FabA-homology region contains a His residue in
position 70 and a Glu in position 84, consistent with dehydratase
function, the homology model for the DH' pseudodomains reveals a
Glu70 and a Glu84, which are more commonly found in hotdog
hydrolases than in dehydratases. There have been reports of bona
fide DH domains with the His70 and Glu84, that have hydrolase
activity. Moriguchi et al., 2010 reported a hidden thioesterase
function in what appeared by sequence homology to be an embedded
dehydratase domain in the 6-MSA Synthase fungal multienzyme. The
thioesterase activity in that domain was abolished when the
conserved active site His70 residue was replaced by Ala, thus
showing that an apparent DH domain could catalyze either
dehydration or hydrolysis. Therefore, based on our results and on
the three-dimensional models for the DH domains according to the
invention, it cannot be ruled out that the DH tetradomain of PUFA
synthases houses a hydrolase or esterase activity in addition to
the reported dehydratase activity.
Although the present invention has been described herein with
reference to the foregoing exemplary embodiment, this embodiment
does not serve to limit the scope of the present invention.
Accordingly, those skilled in the art to which the present
invention pertains will appreciate that various modifications are
possible, without departing from the technical spirit of the
present invention.
SEQUENCE LISTINGS
1
17138DNAArtificial SequenceSequence source PCR primer, 38 bases
1catgcatggg atccaacttg ctagacgcaa atatcgca 38239DNAArtificial
SequenceSequence source PCR primer, 39 bases 2catgcatgcc cgggtcatga
ttcttctttg atcatcacg 39328DNAArtificial SequenceSequence source PCR
primer, 28 bases 3caccttctct tacgaatgtt tcgttggc 28439DNAArtificial
SequenceSequence source PCR primer, 39 bases 4catgcatgcc cgggtcatga
ttcttctttg atcatcacg 39539DNAArtificial SequenceSequence source PCR
primer, 39 bases 5catgcatggg atccaactta ctggataaag aaagccgtt
39624DNAArtificial SequenceSequence source PCR primer, 24 bases
6tcaggcttct tcaatacaga ttgc 24737DNAArtificial SequenceSequence
source PCR primer, 37 bases 7catgcatggg atccttcagc ttcgaactca
gtaccga 37824DNAArtificial SequenceSequence source PCR primer, 24
bases 8tcaggcttct tcaatacaga ttgc 24928DNAArtificial
SequenceSequence source PCR primer, 28 bases 9caccaacttg ctagacgcaa
atatcgca 281024DNAArtificial SequenceSequence source PCR primer, 24
bases 10tcaggcttct tcaatacaga ttgc 241128DNAArtificial
SequenceSequence source PCR primer, 28 bases 11caccttctct
tacgaatgtt tcgttggc 281224DNAArtificial SequenceSequence source PCR
primer, 24 bases 12tcaggcttct tcaatacaga ttgc 241327DNAArtificial
SequenceSequence source PCR primer, 27 bases 13cacccgcaaa
ccttgtatct gggatta 271424DNAArtificial SequenceSequence source PCR
primer, 24 bases 14tcaggcttct tcaatacaga ttgc 24155741DNAArtificial
SequencePlasmid vector; pET200TOPO 15caaggagatg gcgcccaaca
gtcccccggc cacggggcct gccaccatac ccacgccgaa 60acaagcgctc atgagcccga
agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat 120ataggcgcca
gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
180gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga
attgtgagcg 240gataacaatt cccctctaga aataattttg tttaacttta
agaaggagat atacatatgc 300ggggttctca tcatcatcat catcatggta
tggctagcat gactggtgga cagcaaatgg 360gtcgggatct gtacgacgat
gacgataagg atcatccctt caccaagggc gagctcaacg 420atccggctgc
taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
480aactagcata accccttggg gcctctaaac gggtcttgag gagttttttg
ctgaaaggag 540gaactatatc cggatatccc gcaagaggcc cggcagtacc
ggcataacca agcctatgcc 600tacagcatcc agggtgacgg tgccgaggat
gacgatgagc gcattgttag atttcataca 660cggtgcctga ctgcgttagc
aatttaactg tgataaacta ccgcattaaa gcttatcgat 720gataagctgt
caaacatgag aattaattct tgaagacgaa agggcctcgt gatacgccta
780tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
cacttttcgg 840ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
tacattcaaa tatgtatccg 900ctcatgagac aataaccctg ataaatgctt
caataatatt gaaaaaggaa gagtatgatt 960gaacaagatg gattgcacgc
aggttctccg gccgcttggg tggagaggct attcggctat 1020gactgggcac
aactgacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
1080gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
actgcaggac 1140gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
cttgcgcagc tgtgctcgac 1200gttgtcactg aagcgggaag ggactggctg
ctattgggcg aagtgccggg gcaggatctc 1260ctgtcatctc accttgctcc
tgccgagaaa gtatccatca tggctgatgc aatgcggcgg 1320ctgcatacgc
ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
1380cgggcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
cgaagagcat 1440caggggctcg cgccagccga actgttcgcc aggctcaagg
cgcgcatgcc cgacggcgag 1500gatctcgtcg tgacacatgg cgatgcctgc
ttgccgaata tcatggtgga aaatggccgc 1560ttttctggat tcatcgactg
tggccggctg ggtgtggcgg accgctatca ggacatagcg 1620ttggctaccc
gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
1680ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
tcttgacgag 1740ttcttctgag cgggactctg gggttcgaaa tgaccgacca
agcgacgcct aactgtcaga 1800ccaagtttac tcatatatac tttagattga
tttaaaactt catttttaat ttaaaaggat 1860ctaggtgaag atcctttttg
ataatctcat gaccaaaatc ccttaacgtg agttttcgtt 1920ccactgagcg
tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
1980gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
tttgtttgcc 2040ggatcaagag ctaccaactc tttttccgaa ggtaactggc
ttcagcagag cgcagatacc 2100aaatactgtc cttctagtgt agccgtagtt
aggccaccac ttcaagaact ctgtagcacc 2160gcctacatac ctcgctctgc
taatcctgtt accagtggct gctgccagtg gcgataagtc 2220gtgtcttacc
gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
2280aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
aactgagata 2340cctacagcgt gagctatgag aaagcgccac gcttcccgaa
gggagaaagg cggacaggta 2400tccggtaagc ggcagggtcg gaacaggaga
gcgcacgagg gagcttccag ggggaaacgc 2460ctggtatctt tatagtcctg
tcgggtttcg ccacctctga cttgagcgtc gatttttgtg 2520atgctcgtca
ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
2580cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
ctgattctgt 2640ggataaccgt attaccgcct ttgagtgagc tgataccgct
cgccgcagcc gaacgaccga 2700gcgcagcgag tcagtgagcg aggaagcgga
agagcgcctg atgcggtatt ttctccttac 2760gcatctgtgc ggtatttcac
accgcaatgg tgcactctca gtacaatctg ctctgatgcc 2820gcatagttaa
gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
2880gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
gcatccgctt 2940acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
gaggttttca ccgtcatcac 3000cgaaacgcgc gaggcagctg cggtaaagct
catcagcgtg gtcgtgaagc gattcacaga 3060tgtctgcctg ttcatccgcg
tccagctcgt tgagtttctc cagaagcgtt aatgtctggc 3120ttctgataaa
gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
3180tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
aggatgctca 3240cgatacgggt tactgatgat gaacatgccc ggttactgga
acgttgtgag ggtaaacaac 3300tggcggtatg gatgcggcgg gaccagagaa
aaatcactca gggtcaatgc cagcgcttcg 3360ttaatacaga tgtaggtgtt
ccacagggta gccagcagca tcctgcgatg cagatccgga 3420acataatggt
gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
3480agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
cttcacgttc 3540gctcgcgtat cggtgattca ttctgctaac cagtaaggca
accccgccag cctagccggg 3600tcctcaacga caggagcacg atcatgcgca
cccgtggcca ggacccaacg ctgcccgaga 3660tgcgccgcgt gcggctgctg
gagatggcgg acgcgatgga tatgttctgc caagggttgg 3720tttgcgcatt
cacagttctc cgcaagaatt gattggctcc aattcttgga gtggtgaatc
3780cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc
atgcaccgcg 3840acgcaacgcg gggaggcaga caaggtatag ggcggcgcct
acaatccatg ccaacccgtt 3900ccatgtgctc gccgaggcgg cataaatcgc
cgtgacgatc agcggtccaa tgatcgaagt 3960taggctggta agagccgcga
gcgatccttg aagctgtccc tgatggtcgt catctacctg 4020cctggacagc
atggcctgca acgcgggcat cccgatgccg ccggaagcga gaagaatcat
4080aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc
ccagcgcgtc 4140ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa
cgtttggtgg cgggaccagt 4200gacgaaggct tgagcgaggg cgtgcaagat
tccgaatacc gcaagcgaca ggccgatcat 4260cgtcgcgctc cagcgaaagc
ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg 4320tcctacgagt
tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
4380cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
gagatcccgg 4440tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
tcactgcccg ctttccagtc 4500gggaaacctg tcgtgccagc tgcattaatg
aatcggccaa cgcgcgggga gaggcggttt 4560gcgtattggg cgccagggtg
gtttttcttt tcaccagtga gacgggcaac agctgattgc 4620ccttcaccgc
ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
4680ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg
tcttcggtat 4740cgtcgtatcc cactaccgag atatccgcac caacgcgcag
cccggactcg gtaatggcgc 4800gcattgcgcc cagcgccatc tgatcgttgg
caaccagcat cgcagtggga acgatgccct 4860cattcagcat ttgcatggtt
tgttgaaaac cggacatggc actccagtcg ccttcccgtt 4920ccgctatcgg
ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
4980gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
cccaatgcga 5040ccagatgctc cacgcccagt cgcgtaccgt cttcatggga
gaaaataata ctgttgatgg 5100gtgtctggtc agagacatca agaaataacg
ccggaacatt agtgcaggca gcttccacag 5160caatggcatc ctggtcatcc
agcggatagt taatgatcag cccactgacg cgttgcgcga 5220gaagattgtg
caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
5280ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
tgcgacggcg 5340cgtgcagggc cagactggag gtggcaacgc caatcagcaa
cgactgtttg cccgccagtt 5400gttgtgccac gcggttggga atgtaattca
gctccgccat cgccgcttcc actttttccc 5460gcgttttcgc agaaacgtgg
ctggcctggt tcaccacgcg ggaaacggtc tgataagaga 5520caccggcata
ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
5580gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
tcgatggtgt 5640ccgggatctc gacgctctcc cttatgcgac tcctgcatta
ggaagcagcc cagtagtagg 5700ttgaggccgt tgagcaccgc cgccgcaagg
aatggtgcat g 57411614994DNAEscherichia coli 16aaattgaaga gtttgatcat
ggctcagatt gaacgctggc ggcaggccta acacatgcaa 60gtcgaacggt aacaggaaac
agcttgctgt ttcgctgacg agtggcggac gggtgagtaa 120tgtctgggaa
actgcctgat ggagggggat aactactgga aacggtagct aataccgcat
180aacgtcgcaa gaccaaagag ggggaccttc gggcctcttg ccatcggatg
tgcccagatg 240ggattagcta gtaggtgggg taacggctca cctaggcgac
gatccctagc tggtctgaga 300ggatgaccag ccacactgga actgagacac
ggtccagact cctacgggag gcagcagtgg 360ggaatattgc acaatgggcg
caagcctgat gcagccatgc cgcgtgtatg aagaaggcct 420tcgggttgta
aagtactttc agcggggatg aagggagtaa agttaatacc tttgctcatt
480gacgttaccc gcagaagaag caccggctaa ctccgtgcca gcagccgcgg
taatacggag 540ggtgcaagcg ttaatcggaa ttactgggcg taaagcgcac
gcaggcggtt tgttaagtca 600gatgtgaaat ccccgggctc aacctgggaa
ctgcatctga tactggcaag cttgagtctc 660gtagaggggg gtagaattcc
aggtgtagcg gtgaaatgcg tagagatctg gaggaatacc 720ggtggcgaag
gcggccccct ggacgaagac tgacgctcag gtgcgaaagc gtggggagca
780aacaggatta gataccctgg tagtccacgc cgtaaacgat gtcgacttgg
aggttgtgcc 840cttgaggcgt ggcttccgga gctaacgcgt taagtcgacc
gcctggggag tacggccgca 900aggttaaaac tcaaatgaat tgacgggggc
ccgcacaagc ggtggagcat gtggtttaat 960tcgatgcaac gcgaagaacc
ttacctggtc ttgacatcca cggaagtttt cagagatgag 1020aatgtgcctt
cgggaaccgt gagacaggtg ctgcatggct gtcgtcagct cgtgttgtga
1080aatgttgggt taagtcccgc aacgagcgca acccttatcc tttgttgcca
gcggtccggc 1140cgggaactca aaggagactg ccagtgataa actggaggaa
ggtggggatg acgtcaagtc 1200atcatggccc ttacgaccag ggctacacac
gtgctacaat ggcgcataca aagagaagcg 1260acctcgcgag agcaagcgga
cctcataaag tgcgtcgtag tccggattgg agtctgcaac 1320tcgactccat
gaagtcggaa tcgctagtaa tcgtggatca gaatgccacg gtgaatacgt
1380tcccgggcct tgtacacacc gcccgtcaca ccatgggagt gggttgcaaa
agaagtaggt 1440agcttaacct tcgggagggc gcttaccact ttgtgattca
tgactggggt gaagtcgtaa 1500caaggtaacc gtaggggaac ctgcggttgg
atcacctcct taccttaaag aagcgtactt 1560tgcagtgctc acacagattg
tctgatgaaa atgagcagta aaacctctac aggcttgtag 1620ctcaggtggt
tagagcgcac ccctgataag ggtgaggtcg gtggttcaag tccactcagg
1680cctaccaaat ttgcacggca aatttgaaga ggttttaact acatgttatg
gggctatagc 1740tcagctggga gagcgcctgc tttgcacgca ggaggtctgc
ggttcgatcc cgcatagctc 1800caccatctct gtagtgatta aataaaaaat
acttcagagt gtacctgcaa aggttcactg 1860cgaagttttg ctctttaaaa
atctggatca agctgaaaat tgaaacactg aacaacgaaa 1920gttgctcgtg
agtctctcaa attttcgcaa ctctgaagtg aaacatcttc gggttgtgag
1980gttaagcgac taagcgtaca cggtggatgc cctggcagtc agaggcgatg
aaggacgtgc 2040taatctgcga taagcgtcgg taaggtgata tgaaccgtta
taaccggcga tttccgaatg 2100gggaaaccca gtgtgtttcg acacactatc
attaactgaa tcaaattgaa gagtttgatc 2160atggctcaga ttgaacgctg
gcggcaggcc taacacatgc aagtcgaacg gtaacaggaa 2220gcagcttgct
gcttcgctga cgagtggcgg acgggtgagt aatgtctggg aaactgcctg
2280atggaggggg ataactactg gaaacggtag ctaataccgc ataacgtcgc
aagaccaaag 2340agggggacct tagggcctct tgccatcgga tgtgcccaga
tgggattagc tagtaggtgg 2400ggtaacggct cacctaggcg acgatcccta
gctggtctga gaggatgacc agccacactg 2460gaactgagac acggtccaga
ctcctacggg aggcagcagt ggggaatatt gcacaatggg 2520cgcaagcctg
atgcagccat gccgcgtgta tgaagaaggc cttcgggttg taaagtactt
2580tcagcgggga ggaagggagt aaagttaata cctttgctca ttgacgttac
ccgcagaaga 2640agcaccggct aactccgtgc cagcagccgc ggtaatacgg
agggtgcaag cgttaatcgg 2700aattactggg cgtaaagcgc acgcaggcgg
tttgttaagt cagatgtgaa atccccgggc 2760tcaacctggg aactgcatct
gatactggca agcttgagtc tcgtagaggg gggtagaatt 2820ccaggtgtag
cggtgaaatg cgtagagatc tggaggaata ccggtggcga aggcggcccc
2880ctggacgaag actgacgctc aggtgcgaaa gcgtggggag caaacaggat
tagataccct 2940ggtagtccac gccgtaaacg atgtcgactt ggaggttgtg
cccttgaggc gtggcttccg 3000gagctaacgc gttaagtcga ccgcctgggg
agtacggccg caaggttaaa actcaaatga 3060attgacgggg gcccgcacaa
gcggtggagc atgtggttta attcgatgca acgcgaagaa 3120ccttacctgg
tcttgacatc cacggaagtt ttcagagatg agaatgtgcc ttcgggaacc
3180gtgagacagg tgctgcatgg ctgtcgtcag ctcgtgttgt gaaatgttgg
gttaagtccc 3240gcaacgagcg caacccttat cctttgttgc cagcggtccg
gccgggaact caaaggagac 3300tgccagtgat aaactggagg aaggtgggga
tgacgtcaag tcatcatggc ccttacgacc 3360agggctacac acgtgctaca
atggcgcata caaagagaag cgacctcgcg agagcaagcg 3420gacctcataa
agtgcgtcgt agtccggatt ggagtctgca actcgactcc atgaagtcgg
3480aatcgctagt aatcgtggat cagaatgcca cggtgaatac gttcccgggc
cttgtacaca 3540ccgcccgtca caccatggga gtgggttgca aaagaagtag
gtagcttaac cttcgggagg 3600gcgcttacca ctttgtgatt catgactggg
gtgaagtcgt aacaaggtaa ccgtagggga 3660acctgcggtt ggatcacctc
cttaccttaa agaagcgtac tttgcagtgc tcacacagat 3720tgtctgatag
aaagtgaaaa gcaaggcgtc ttgcgaagca gactgatacg tccccttcgt
3780ctagaggccc aggacaccgc cctttcacgg cggtaacagg ggttcgaatc
ccctagggga 3840cgccacttgc tggtttgtga gtgaaagtcg ccgaccttaa
tatctcaaaa ctcatcttcg 3900ggtgatgttt gagatatttg ctctttaaaa
atctggatca agctgaaaat tgaaacactg 3960aacaatgaaa gttgttcgtg
agtctctcaa attttcgcaa ctctgaagtg aaacatcttc 4020gggttgtgag
gttaagcgac taagcgtaca cggtggatgc cctggcagtc agaggcgatg
4080aaggacgtgc taatctgcga taagcgtcgg taaggtgata tgaaccgtta
taaccggcga 4140tttccgaatg gggaaaccca gtgtgtttcg acacactatc
attaactgaa tccataggtt 4200aatgaggcga accgggggaa ctgaaacatc
taagtacccc gaggaaaaga aatcaaccga 4260gattccccca gtagcggcga
gcgaaaattg aagagtttga tcatggctca gattgaacgc 4320tggcggcagg
cctaacacat gcaagtcgaa cggtaacagg aagcagcttg ctgcttcgct
4380gacgagtggc ggacgggtga gtaatgtctg ggaaactgcc tgatggaggg
ggataactac 4440tggaaacggt agctaatacc gcataacgtc gcaagaccaa
agagggggac cttcgggcct 4500cttgccatcg gatgtgccca gatgggatta
gctagtaggt ggggtaacgg ctcacctagg 4560cgacgatccc tagctggtct
gagaggatga ccagccacac tggaactgag acacggtcca 4620gactcctacg
ggaggcagca gtggggaata ttgcacaatg ggcgcaagcc tgatgcagcc
4680atgccgcgtg tatgaagaag gccttcgggt tgtaaagtac tttcagcggg
gaggaaggga 4740gtaaagttaa tacctttgct cattgacgtt acccgcagaa
gaagcaccgg ctaactccgt 4800gccagcagcc gcggtaatac ggagggtgca
agcgttaatc ggaattactg ggcgtaaagc 4860gcacgcaggc ggtttgttaa
gtcagatgtg aaatccccgg gctcaacctg ggaactgcat 4920ctgatactgg
caagcttgag tctcgtagag gggggtagaa ttccaggtgt agcggtgaaa
4980tgcgtagaga tctggaggaa taccggtggc gaaggcggcc ccctggacga
agactgacgc 5040tcaggtgcga aagcgtgggg agcaaacagg attagatacc
ctggtagtcc acgccgtaaa 5100cgatgtcgac ttggaggttg tgcccttgag
gcgtggcttc cggagctaac gcgttaagtc 5160gaccgcctgg ggagtacggc
cgcaaggtta aaactcaaat gaattgacgg gggcccgcac 5220aagcggtgga
gcatgtggtt taattcgatg caacgcgaag aaccttacct ggtcttgaca
5280tccacagaac tttccagaga tggattggtg ccttcgggaa ctgtgagaca
ggtgctgcat 5340ggctgtcgtc agctcgtgtt gtgaaatgtt gggttaagtc
ccgcaacgag cgcaaccctt 5400atcctttgtt gccagcggtc cggccgggaa
ctcaaaggag actgccagtg ataaactgga 5460ggaaggtggg gatgacgtca
agtcatcatg gcccttacga ccagggctac acacgtgcta 5520caatggcgca
tacaaagaga agcgacctcg cgagagcaag cggacctcat aaagtgcgtc
5580gtagtccgga ttggagtctg caactcgact ccatgaagtc ggaatcgcta
gtaatcgtgg 5640atcagaatgc cacggtgaat acgttcccgg gccttgtaca
caccgcccgt cacaccatgg 5700gagtgggttg caaaagaagt aggtagctta
accttcggga gggcgcttac cactttgtga 5760ttcatgactg gggtgaagtc
gtaacaaggt aaccgtaggg gaacctgcgg ttggatcacc 5820tccttacctt
aaagaagcgt actttgcagt gctcacacag attgtctgat gaaaatgagc
5880agtaaaacct ctacaggctt gtagctcagg tggttagagc gcacccctga
taagggtgag 5940gtcggtggtt caagtccact caggcctacc aaatttgcac
ggcaaatttg aagaggtttt 6000aactacatgt tatggggcta tagctcagct
gggagagcgc ctgctttgca cgcaggaggt 6060ctgcggttcg atcccgcata
gctccaccat ctctgtagtg attaaataaa aaatacttca 6120gagtgtacct
gcaaaggttc actgcgaagt tttgctcttt aaaaatctgg atcaagctga
6180aaattgaaac actgaacaac gaaagttgtt cgtgagtctc tcaaattttc
gcaacacgat 6240gatgaatcgc aagaaacatc ttcgggttgt gaggttaagc
gactaagcgt acacggtgga 6300tgccctggca gtcagaggcg atgaaggacg
tgctaatctg cgataagcgt cggtgaggtg 6360atatgaaccg ttataaccgg
cgatttccga atggggaaac ccagtgtgat tcgtcacact 6420atcattaaat
tgaagagttt gatcatggct cagattgaac gctggcggca ggcctaacac
6480atgcaagtcg aacggtaaca ggaagcagct tgctgcttcg ctgacgagtg
gcggacgggt 6540gagtaatgtc tgggaaactg cctgatggag ggggataact
actggaaacg gtagctaata 6600ccgcataacg tcgcaagacc aaagaggggg
accttagggc ctcttgccat cggatgtgcc 6660cagatgggat tagctagtag
gtggggtaac ggctcaccta ggcgacgatc cctagctggt 6720ctgagaggat
gaccagccac actggaactg agacacggtc cagactccta cgggaggcag
6780cagtggggaa tattgcacaa tgggcgcaag cctgatgcag ccatgccgcg
tgtatgaaga 6840aggccttcgg gttgtaaagt actttcagcg gggaggaagg
gagtaaagtt aatacctttg 6900ctcattgacg ttacccgcag aagaagcacc
ggctaactcc gtgccagcag ccgcggtaat 6960acggagggtg caagcgttaa
tcggaattac tgggcgtaaa gcgcacgcag gcggtttgtt 7020aagtcagatg
tgaaatcccc gggctcaacc tgggaactgc atctgatact ggcaagcttg
7080agtctcgtag aggggggtag aattccaggt gtagcggtga aatgcgtaga
gatctggagg 7140aataccggtg gcgaaggcgg ccccctggac gaagactgac
gctcaggtgc gaaagcgtgg 7200ggagcaaaca ggattagata ccctggtagt
ccacgccgta aacgatgtcg acttggaggt 7260tgtgcccttg aggcgtggct
tccggagcta acgcgttaag tcgaccgcct ggggagtacg 7320gccgcaaggt
taaaactcaa atgaattgac gggggcccgc acaagcggtg gagcatgtgg
7380tttaattcga tgcaacgcga agaaccttac ctggtcttga catccacgga
agttttcaga 7440gatgagaatg tgccttcggg aaccgtgaga caggtgctgc
atggctgtcg tcagctcgtg 7500ttgtgaaatg ttgggttaag tcccgcaacg
agcgcaaccc ttatcctttg ttgccagcgg 7560tccggccggg aactcaaagg
agactgccag tgataaactg gaggaaggtg gggatgacgt 7620caagtcatca
tggcccttac gaccagggct acacacgtgc tacaatggcg catacaaaga
7680gaagcgacct cgcgagagca agcggacctc ataaagtgcg tcgtagtccg
gattggagtc 7740tgcaactcga ctccatgaag tcggaatcgc tagtaatcgt
ggatcagaat gccacggtga 7800atacgttccc gggccttgta cacaccgccc
gtcacaccat gggagtgggt tgcaaaagaa 7860gtaggtagct taaccttcgg
gagggcgctt accactttgt gattcatgac tggggtgaag 7920tcgtaacaag
gtaaccgtag gggaacctgc ggttggatca cctccttacc ttaaagaagc
7980gtactttgca gtgctcacac agattgtctg atagaaagtg aaaagcaagg
cgtcttgcga 8040agcagactga tacgtcccct tcgtctagag gcccaggaca
ccgccctttc acggcggtaa 8100caggggttcg aatcccctag gggacgccac
ttgctggttt gtgagtgaaa gtcgccgacc 8160ttaatatctc aaaactcatc
ttcgggtgat gtttgagata tttgctcttt aaaaatctgg 8220atcaagctga
aaattgaaac actgaacaat gaaagttgtt cgtgagtctc tcaaattttc
8280gcaactctga agtgaaacat cttcgggttg tgaggttaag cgactaagcg
tacacggtgg 8340atgccctggc agtcagaggc gatgaaggac gtgctaatct
gcgataagcg tcggtaaggt 8400gatatgaacc gttataaccg gcgatttccg
aatggggaaa cccagtgtgt ttcgacacac 8460tatcattaac tgaatccata
ggttaatgag gcgaaccggg ggaactgaaa catctaagta 8520ccccgaggaa
aagaaatcaa ccgagattcc cccagtagcg gcgagcgaaa attgaagagt
8580ttgatcatgg ctcagattga acgctggcgg caggcctaac acatgcaagt
cgaacggtaa 8640caggaaacag cttgctgttt cgctgacgag tggcggacgg
gtgagtaatg tctgggaaac 8700tgcctgatgg agggggataa ctactggaaa
cggtagctaa taccgcataa cgtcgcaaga 8760ccaaagaggg ggaccttcgg
gcctcttgcc atcggatgtg cccagatggg attagctagt 8820aggtggggta
acggctcacc taggcgacga tccctagctg gtctgagagg atgaccagcc
8880acactggaac tgagacacgg tccagactcc tacgggaggc agcagtgggg
aatattgcac 8940aatgggcgca agcctgatgc agccatgccg cgtgtatgaa
gaaggccttc gggttgtaaa 9000gtactttcag cggggaggaa gggagtaaag
ttaatacctt tgctcattga cgttacccgc 9060agaagaagca ccggctaact
ccgtgccagc agccgcggta atacggaggg tgcaagcgtt 9120aatcggaatt
actgggcgta aagcgcacgc aggcggtttg ttaagtcaga tgtgaaatcc
9180ccgggctcaa cctgggaact gcatctgata ctggcaagct tgagtctcgt
agaggggggt 9240agaattccag gtgtagcggt gaaatgcgta gagatctgga
ggaataccgg tggcgaaggc 9300ggccccctgg acgaagactg acgctcaggt
gcgaaagcgt ggggagcaaa caggattaga 9360taccctggta gtccacgccg
taaacgatgt cgacttggag gttgtgccct tgaggcgtgg 9420cttccggagc
taacgcgtta agtcgaccgc ctggggagta cggccgcaag gttaaaactc
9480aaatgaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc
gatgcaacgc 9540gaagaacctt acctggtctt gacatccaca gaactttcca
gagatggatt ggtgccttcg 9600ggaactgtga gacaggtgct gcatggctgt
cgtcagctcg tgttgtgaaa tgttgggtta 9660agtcccgcaa cgagcgcaac
ccttatcctt tgttgccagc ggtccggccg ggaactcaaa 9720ggagactgcc
agtgataaac tggaggaagg tggggatgac gtcaagtcat catggccctt
9780acgaccaggg ctacacacgt gctacaatgg cgcatacaaa gagaagcgac
ctcgcgagag 9840caagcggacc tcataaagtg cgtcgtagtc cggattggag
tctgcaactc gactccatga 9900agtcggaatc gctagtaatc gtggatcaga
atgccacggt gaatacgttc ccgggccttg 9960tacacaccgc ccgtcacacc
atgggagtgg gttgcaaaag aagtaggtag cttaaccttc 10020gggagggcgc
ttaccacttt gtgattcatg actggggtga agtcgtaaca aggtaaccgt
10080aggggaacct gcggttggat cacctcctta ccttaaagaa gcgtactttg
cagtgctcac 10140acagattgtc tgatgaaaat gagcagtaaa acctctacag
gcttgtagct caggtggtta 10200gagcgcaccc ctgataaggg tgaggtcggt
ggttcaagtc cactcaggcc taccaaattt 10260gcaccgcaaa tttgaagagg
ttttaactac atgttatggg gctatagctc agctgggaga 10320gcgcctgctt
tgcacgcagg aggtctgcgg ttcgatcccg catagctcca ccatctctgt
10380agtgattaaa taaaaaatac ttcagagtgt acctgcaaag gttcactgcg
aagttttgct 10440ctttaaaaat ctggatcaag ctgaaaattg aaacactgaa
caacgaaagt tgttcgtgag 10500tctctcaaat tttcgcaaca cgatgatgaa
tcgcaagaaa catcttcggg ttgtgaggtt 10560aagcgactaa gcgtacacgg
tggatgccct ggcagtcaga ggcgatgaag gacgtgctaa 10620tctgcgataa
gcgtcggtga ggtgatatga accgttataa ccggcgattt ccgaatgggg
10680aaacccagtg tgattcgtca cactatcatt aaattgaaga gtttgatcat
ggctcagatt 10740gaacgctggc ggcaggccta acacatgcaa gtcgaacggt
aacaggaaac agcttgctgt 10800ttcgctgacg agtggcggac gggtgagtaa
tgtctgggaa actgcctgat ggagggggat 10860aactactgga aacggtagct
aataccgcat aacgtcgcaa gaccaaagag ggggaccttc 10920gggcctcttg
ccatcggatg tgcccagatg ggattagcta gtaggtgggg taacggctca
10980cctaggcgac gatccctagc tggtctgaga ggatgaccag ccacactgga
actgagacac 11040ggtccagact cctacgggag gcagcagtgg ggaatattgc
acaatgggcg caagcctgat 11100gcagccatgc cgcgtgtatg aagaaggcct
tcgggttgta aagtactttc agcggggagg 11160aagggagtaa agttaatacc
tttgctcatt gacgttaccc gcagaagaag caccggctaa 11220ctccgtgcca
gcagccgcgg taatacggag ggtgcaagcg ttaatcggaa ttactgggcg
11280taaagcgcac gcaggcggtt tgttaagtca gatgtgaaat ccccgggctc
aacctgggaa 11340ctgcatctga tactggcaag cttgagtctc gtagaggggg
gtagaattcc aggtgtagcg 11400gtgaaatgcg tagagatctg gaggaatacc
ggtggcgaag gcggccccct ggacgaagac 11460tgacgctcag gtgcgaaagc
gtggggagca aacaggatta gataccctgg tagtccacgc 11520cgtaaacgat
gtcgacttgg aggttgtgcc cttgaggcgt ggcttccgga gctaacgcgt
11580taagtcgacc gcctggggag tacggccgca aggttaaaac tcaaatgaat
tgacgggggc 11640ccgcacaagc ggtggagcat gtggtttaat tcgatgcaac
gcgaagaacc ttacctggtc 11700ttgacatcca cagaactttc cagagatgga
ttggtgcctt cgggaactgt gagacaggtg 11760ctgcatggct gtcgtcagct
cgtgttgtga aatgttgggt taagtcccgc aacgagcgca 11820acccttatcc
tttgttgcca gcggtccggc cgggaactca aaggagactg ccagtgataa
11880actggaggaa ggtggggatg acgtcaagtc atcatggccc ttacgaccag
ggctacacac 11940gtgctacaat ggcgcataca aagagaagcg acctcgcgag
agcaagcgga cctcataaag 12000tgcgtcgtag tccggattgg agtctgcaac
tcgactccat gaagtcggaa tcgctagtaa 12060tcgtggatca gaatgccacg
gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca 12120ccatgggagt
gggttgcaaa agaagtaggt agcttaacct tcgggagggc gcttaccact
12180ttgtgattca tgactggggt gaagtcgtaa caaggtaacc gtaggggaac
ctgcggttgg 12240atcacctcct taccttaaag aagcgtactt tgcagtgctc
acacagattg tctgataaaa 12300agtgaaaagc aaggcgtctt gcgaagcaga
ctgatacgtc cccttcgtct agaggcccag 12360gacaccgccc tttcacggcg
gtaacagggg ttcgaatccc ctaggggacg ccacttgctg 12420gtttgtgagt
gaaagtcacc tgccttaata tctcaaaact catcttcggg tgatgtttga
12480gatatttgct ctttaaaaat ctggatcaag ctgaaaattg aaacactgaa
caacgagagt 12540tgttcgtgag tctctcaaat tttcgcaaca cgatgatgaa
tcgaaagaaa catcttcggg 12600ttgtgaggtt aagcgactaa gcgtacacgg
tggatgccct ggcagtcaga ggcgatgaag 12660gacgtgctaa tctgcgataa
gcgtcggtaa ggtgatatga accgttataa ccggcgattt 12720ccgaatgggg
aaacccagtg tgtttcgaca cactatcatt aactgaatcc ataggttaat
12780gaggcgaacc gggggaactg aaacatctaa gtaccccgag gaaaagaaat
caaccgagat 12840tcccccagta gcaaattgaa gagtttgatc atggctcaga
ttgaacgctg gcggcaggcc 12900taacacatgc aagtcgaacg gtaacaggaa
gcagcttgct gcttcgctga cgagtggcgg 12960acgggtgagt aatgtctggg
aaactgcctg atggaggggg ataactactg gaaacggtag 13020ctaataccgc
ataacgtcgc aagaccaaag agggggacct tagggcctct tgccatcgga
13080tgtgcccaga tgggattagc tagtaggtgg ggtaacggct cacctaggcg
acgatcccta 13140gctggtctga gaggatgacc agccacactg gaactgagac
acggtccaga ctcctacggg 13200aggcagcagt ggggaatatt gcacaatggg
cgcaagcctg atgcagccat gccgcgtgta 13260tgaagaaggc cttcgggttg
taaagtactt tcagcgggga ggaagggagt aaagttaata 13320cctttgctca
ttgacgttac ccgcagaaga agcaccggct aactccgtgc cagcagccgc
13380ggtaatacgg agggtgcaag cgttaatcgg aattactggg cgtaaagcgc
acgcaggcgg 13440tttgttaagt cagatgtgaa atccccgggc tcaacctggg
aactgcatct gatactggca 13500agcttgagtc tcgtagaggg gggtagaatt
ccaggtgtag cggtgaaatg cgtagagatc 13560tggaggaata ccggtggcga
aggcggcccc ctggacgaag actgacgctc aggtgcgaaa 13620gcgtggggag
caaacaggat tagataccct ggtagtccac gccgtaaacg atgtcgactt
13680ggaggttgtg cccttgaggc gtggcttccg gagctaacgc gttaagtcga
ccgcctgggg 13740agtacggccg caaggttaaa actcaaatga attgacgggg
gcccgcacaa gcggtggagc 13800atgtggttta attcgatgca acgcgaagaa
ccttacctgg tcttgacatc cacggaagtt 13860ttcagagatg agaatgtgcc
ttcgggaacc gtgagacagg tgctgcatgg ctgtcgtcag 13920ctcgtgttgt
gaaatgttgg gttaagtccc gcaacgagcg caacccttat cctttgttgc
13980cagcggtccg gccgggaact caaaggagac tgccagtgat aaactggagg
aaggtgggga 14040tgacgtcaag tcatcatggc ccttacgacc agggctacac
acgtgctaca atggcgcata 14100caaagagaag cgacctcgcg agagcaagcg
gacctcataa agtgcgtcgt agtccggatt 14160ggagtctgca actcgactcc
atgaagtcgg aatcgctagt aatcgtggat cagaatgcca 14220cggtgaatac
gttcccgggc cttgtacaca ccgcccgtca caccatggga gtgggttgca
14280aaagaagtag gtagcttaac cttcgggagg gcgcttacca ctttgtgatt
catgactggg 14340gtgaagtcgt aacaaggtaa ccgtagggga acctgcggtt
ggatcacctc cttaccttaa 14400agaagcgtac tttgcagtgc tcacacagat
tgtctgatag aaagtgaaaa gcaaggcgtc 14460ttgcgaagca gactgatacg
tccccttcgt ctagaggccc aggacaccgc cctttcacgg 14520cggtaacagg
ggttcgaatc ccctagggga cgccacttgc tggtttgtga gtgaaagtcg
14580ccgaccttaa tatctcaaaa ctcatcttcg ggtgatgttt gagatatttg
ctctttaaaa 14640atctggatca agctgaaaat tgaaacactg aacaatgaaa
gttgttcgtg agtctctcaa 14700attttcgcaa ctctgaagtg aaacatcttc
gggttgtgag gttaagcgac taagcgtaca 14760cggtggatgc cctggcagtc
agaggcgatg aaggacgtgc taatctgcga taagcgtcgg 14820taaggtgata
tgaaccgtta taaccggcga tttccgaatg gggaaaccca gtgtgtttcg
14880acacactatc attaactgaa tccataggtt aatgaggcga accgggggaa
ctgaaacatc 14940taagtacccc gaggaaaaga aatcaaccga gattccccca
gtagcggcga gcga 14994171755DNAPhotobacterium profundum 17tcatgatgcc
tttccctctt tttgcgccca tacagccagt ttttctctaa aaatcttggc 60gttatggcgt
atatcaacgg gaaaattaga gtgaattaga aaatcgacaa tacccacagt
120ttgttgatgt ttagcgccaa tcgttttaag ctcggcaaaa aacgtattac
gggcagtatc 180atccgtcagg cttgtgcctt tattaagctc aacacacagc
agtggcactg tttcaccatg 240acgttcaatg ccgaccaatg cggtacgctt
gaccgaagag tgagtattaa atatacgttc 300acaaggaata gaaaaatacg
gcagcttgtc tgatgcaacc gcaacgcgat tggcttcaac 360acggtgagct
ttacgaccac acatccacag ttgcccgctg tcgtccaagt agcctaaatc
420ccccatgcgg tgacgtacag tattcgcccc atcggtaatt ttagcctgaa
cggttgcatg 480atcacggtga taatacgcac ggctcaccat gggacctttt
acaacgattt cgccaatttg 540gtttacaggt aaacgtaacg tttcgtccca
cgtagtaatt ggatcgtcgg tgatcgcaat 600gatcgcaata tccacaccat
caacagcttg accgacacaa ataccaccgc cattatcagt 660gacatccgtg
gttttcatta actgatcact accaatcatg gtaagaggca atgattctgt
720cgcaccataa gaattcagta cttcaacccc atcctttagc attttactaa
agcgtgaaat 780agacgaaata gtagcaggtg cgccagcgga aataacacgt
ttgatactgg gtaatgtatg 840aggtttatga tgctgtgtac ccgctttccc
taaacgctct atcaacgcag gattcacaaa 900catattggta cattggtatt
gttcaatcgc tgcaaacaaa ctgtcgggat tagccgttat 960tggtttactg
gcatccatat caggcacaat cgatgccatg cctaaagctg gaccaaaaag
1020agagaataac gggaaggtcg caagatcacg ctcgccgtgg gcgataccat
aatcattttt 1080caatacactg atttgtgctt caaacattgc atgggtatac
acaacacctt tcggcgtacc 1140cgtactgcca ctggtaaata gaatggctgc
catttcatca tttttaagct tggcaatatc 1200ataatcaatc gcttgatgca
ttttactgcg cggcttaata acagtattac gtttaagtaa 1260tgcttttagt
gttgtaccac caaaaatatt ggcaaataca tcatttccac caacggtgag
1320taaacgtcga acactcggtt taccccaacc aaataaacaa cgggcaatat
gcgctttagg 1380gataccaata aacgcatcgg gcttagcttc atcaaagcac
tgtttgaggt ttttaacccc 1440catgccagga tcaaccaaaa taggaacaat
acccgcttta aataatgcaa atgttagagc 1500aaaaaaatca agactaggcg
ttaccatcag taccgctttc atgcctcgtt taataccgtg 1560atcattcaac
gcttgtgcaa ttgcgttact gtcagtgttt aactgcccaa acgttaattc
1620ttcataccgt agtttgccga ataacgatcg tttttgaaca gcaacagcaa
gcgaagcagg 1680tgtttctttt gccgcgcgtg ttaagtggcg acagatattg
gctttagagg catcaatctc 1740atgcccttta tccat 1755
* * * * *