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Applied and Environmental Microbiology, July 2000, p. 3113-3116, Vol. 66, No. 7
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Screening of Environmental DNA Libraries for the
Presence of Genes Conferring Lipolytic Activity on
Escherichia coli
Anke
Henne,1
Ruth
A.
Schmitz,1
Mechthild
Bömeke,2
Gerhard
Gottschalk,1,2 and
Rolf
Daniel1,*
Abteilung Allgemeine
Mikrobiologie1 and Göttingen
Genomics Laboratory,2 Institut für
Mikrobiologie und Genetik der Georg-August-Universität, 37077 Göttingen, Germany
Received 24 January 2000/Accepted 5 April 2000
 |
ABSTRACT |
Environmental DNA libraries prepared from three different soil
samples were screened for genes conferring lipolytic activity on
Escherichia coli clones. Screening on triolein agar
revealed 1 positive clone out of 730,000 clones, and screening on
tributyrin agar revealed 3 positive clones out of 286,000 E. coli clones. Substrate specificity analysis revealed that one
recombinant strain harbored a lipase and the other three contained
esterases. The genes responsible for the lipolytic activity were
identified and characterized.
| |
TEXT |
Lipases catalyze both the hydrolysis
and the synthesis of esters formed from glycerol and long-chain fatty
acids. These enzymes usually exhibit broad substrate specificity and
degrade acyl p-nitrophenyl esters, Tweens, and
phospholipids, often with positional selectivity, stereoselectivity,
and chain length selectivity (16). Lipases resemble
esterases but differ markedly from them in their ability to act on
water-insoluble esters (7). Both types of enzymes have been
recognized as very useful biocatalysts because of their wide-ranging
versatility in industrial applications. The term "lipolytic
enzymes" used throughout this report comprises lipases (EC 3.1.1.3)
and esterases (EC 3.1.1.1).
The classical and cumbersome approach to isolate new lipolytic proteins
is to screen a wide variety of microorganisms for the desired lipolytic
activity. The enzymes and the corresponding genes are then recovered
from the identified organisms. In this method, a large fraction of the
microbial diversity in an environment is lost due to difficulties in
enriching and isolating microorganisms in pure culture. It has been
estimated that >99% of microorganisms observable in nature typically
cannot be cultivated using standard techniques (3). An
alternative approach is to use the genetic diversity of the
microorganisms in a certain environment as a whole to encounter new or
improved genes and gene products for biotechnological purposes. One way
to exploit the genetic diversity of various environments is to isolate
DNA without culturing the organisms present. Subsequently, the desired
genes are amplified by PCR and cloned. The sequences of the primers
used are derived from conserved regions of already known genes or
protein families (22). Thus, the identification of entirely
new genes or gene products by PCR-based methods is limited. Another way
is to use the DNA for the construction of DNA libraries and to clone
directly functional genes from environmental samples. The knowledge of sequence information prior to cloning is not required. Another advantage is that the already prepared environmental libraries can be
employed for the screening of various targets. This approach shows an
alternative way to access and exploit the immense pool of genes from
microorganisms that have not been cultivated so far. We and other
authors applied this method successfully, i.e., for the direct cloning
of genes encoding 4-hydroxybutyrate dehydrogenases (14) and
chitinases (9).
In this study, three different environmental DNA libraries were
screened for the presence of genes conferring lipolytic activity. The
genes encoding lipolytic activity were recovered from the obtained
positive Escherichia coli strains and then sequenced. Subsequently, the corresponding gene products were analyzed.
Screening for genes conferring lipolytic activity.
The
environmental DNA libraries were constructed from soil samples using
E. coli DH5
as a host and pBluescript SK(+) [pSK(+)] as
a vector (14). The samples were collected in Germany from a
meadow near Northeim (library I), a sugar beet field near
Göttingen (library II), and the valley of the Nieme River
(library III) as described previously (14). The DNA was
isolated from the samples by direct lysis of the microorganisms in the
soil. The three libraries revealed average insert sizes of 5 to 8 kb.
The percentage of plasmids containing inserts was approximately 80% (14).
For the detection of E. coli clones exhibiting lipolytic
activity, two types of indicator plates were employed. Lipase activity was detected on Luria-Bertani agar containing triolein (1%, vol/vol) and the fluorescent dye rhodamine B (0.001%, wt/vol). Orange
fluorescent halos around lipase-positive E. coli strains
could be seen when these plates were exposed to UV light of 254 nm
(18). Lipolytic activity was detected on Luria-Bertani agar
containing tributyrin (1%, vol/vol). For determination of substrate
specificity tributyrin was replaced by other triglycerides (see below).
Positive E. coli clones were detected by zones of clearance
around the colonies.
One out of approximately 730,000 and 3 out of approximately 286,000
E. coli clones were positive during the initial screens
on
triolein- and tributyrin-containing agar plates, respectively
(Table
1). In order to confirm that the
lipolytic phenotype of
the clones is plasmid encoded, the recombinant
plasmids were isolated
and retransformed into
E. coli, and
the resulting
E. coli strains
were screened again on
indicator plates (see above). All four
different recombinant plasmids,
designated pAH110 to pAH113, conferred
a stable lipolytic phenotype on
the resulting recombinant
E. coli strains. One was obtained
from library I, one was from library
II, and two were from library III
(Table
1). The insert sizes
of pAH110 to pAH113 were in the range of
2,185 to 6,661 bp (Fig.
1).
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FIG. 1.
Restriction maps of the inserts of pAH110 to pAH113 and
of the corresponding subcloned inserts of pAH114 to pAH117. The arrows
represent length, location, and orientation of the genes
lip, orf111, orf112, and
orf113.
|
|
Characterization of the lipolytic E. coli clones.
We investigated the substrate specificity of the lipolytic enzymes of
E. coli/pAH110 to E. coli/pAH113 by using various
substrates. The following triglycerides were tested in the plate assay:
tributyrin, tricaproin, tricaprylin, tricaprin, trilaurin, and
triolein. Since lipases are, by definition, carboxylesterases that have
the ability to hydrolyze long-chain acylglycerols (
C10),
whereas esterases hydrolyze ester substrates with short-chain fatty
acids (
C10) (25), this assay allowed us to
differentiate between both types of lipolytic enzymes. E. coli/pAH110 behaved like the lipase-producing control strain
Acinetobacter sp. strain BD413 (17) with all substrates. Lipolytic activity of E. coli/pAH111 to E. coli/pAH113 was recorded only with tributyrin as a substrate. This
is typical for esterases (18, 19).
A direct quantification of hydrolytic activity in crude extracts of
E. coli/pAH110 to
E. coli/pAH113 was done
spectrophotometrically
at 410 nm and 37°C with emulsified
p-nitrophenyl esters of different
fatty acids as substrates
(
26). One enzyme unit corresponded
to the hydrolysis of 1 µmol of substrate per min. The preparation
of crude extracts was
performed by sonication as described previously
(
14).
Protein concentrations were measured by the method of
Bradford
(
6) with bovine serum albumin as a standard. With
p-nitrophenyl esters of fatty acids as substrates, activity
was
observed only in crude extracts of
E. coli/pAH110. The
rates of
hydrolysis for the following fatty acids were as indicated (in
units per milligram): butyrate, 68.8; caprylate, 38.7; laurate,
16.5;
and palmitate, 16.4. The enzyme showed higher activity towards
esters
of short- to medium-chain (C
4 and C
8) fatty
acids. A similar
substrate specificity was found for the crude
preparation of lipase
2 from
Moraxella sp. strain TA144
(
13) and the cold-adapted
lipase (LipP) from
Pseudomonas sp. strain B11-1 (
8). The lack
of
activity with
p-nitrophenyl esters of fatty acids as found
in crude extracts of
E. coli/pAH111 to
E. coli/pAH113 was also
reported for the lipolytic enzyme of
Streptomyces cinnamoneus (
23). Thus, the results
of both assays indicated that the enzyme
produced by
E. coli/pAH110 is a lipase, whereas the other three
enzymes are
esterases.
Because of its broad substrate specificity, the lipase was studied
further. In order to determine the molecular mass of the
enzyme, the
crude extract of
E. coli/pAH110 was subjected to sodium
dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis
(
21)
and activity staining. For activity staining the sample
applied
to the gel was not boiled, and SDS was removed after
electrophoresis
by gentle shaking of the gel for 20 min in 20%
(vol/vol) isopropanol
and for 10 min in distilled water. Subsequently,
the gel was transferred
for detection of lipase activity to an agar
plate containing 1%
tributyrin or tricaprylin, 25 mM Tris-HCl (pH
8.0), 5 mM CaCl
2,
and 1.3% agar (
23).
Afterward, the gel was incubated for 12
h at room temperature. As
shown in Fig.
2, the crude extract of
E. coli/pAH110 revealed a band with lipolytic activity at
29,000
Da. No reaction was observed with the crude extract of the
negative
control
E. coli/pSK(+), which contained only the
cloning vector.
Identical results were obtained when tributyrin agar
was used
instead of tricaprylin agar (data not shown). In addition, the
temperature dependence of the lipase with
p-nitrophenyl
palmitate
as a substrate was determined. The enzyme was active at
temperatures
from 12 to 60°C. The maximal activity was recorded at
42°C (data
not shown).
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FIG. 2.
Lipase activity staining after SDS-polyacrylamide gel
electrophoresis of E. coli/pAH110 crude extract. Lanes: 1, marker proteins; 2, Coomassie blue-stained crude extract of E. coli/pAH110; 3, activity-stained crude extract of E. coli/pAH110; 4, activity-stained crude extract of E. coli/pSK(+). In lanes 3 and 4, tricaprylin was used as a
substrate.
|
|
Molecular analysis of pAH110 to pAH113.
The inserts of all
recombinant plasmids recovered from E. coli/pAH110 to
E. coli/pAH113 were sequenced by the Göttingen
Genomics Laboratory (Göttingen, Germany). The sequences were
analyzed with the Genetics Computer Group program package
(11) and compared to the sequences in the National Center
for Biotechnology Information databases (2). In order to
identify the genes on pAH110 to pAH113, which are responsible for the
lipolytic activity of the corresponding recombinant E. coli
strains, the inserts were partially subcloned by restriction digestion
with various enzymes and subsequent ligation into pSK(+). The resulting
constructs were transformed into E. coli. The recombinant
E. coli strains were screened again on indicator plates
containing tributyrin or triolein as a substrate. This strategy was
successful for all four plasmids. The four corresponding positive
E. coli strains (E. coli/pAH114 to E. coli/pAH117) were indistinguishable from the original clones with
respect to substrate specificity. The restriction maps, the designation
of the recombinant plasmids derived from pAH110 to pAH113, and the
localization of the identified genes are given in Fig. 1. All four of
the below-mentioned genes encoding lipolytic enzymes were preceded by a
potential ribosome binding site, appropriately spaced from the start codon.
The 1,232-bp insert of pAH114 revealed an open reading frame (846 bp),
which is similar to the
lip gene of
Streptomyces
albus (
10). Therefore, the open reading frame was
designated
lip accordingly.
The predicted molecular mass of
28,683 Da for the gene product
is in good accordance with that obtained
during activity staining
of crude extract of
E. coli/pAH110
(see above). The deduced gene
product of
lip (281 amino
acids) is 30% identical (35.9% similar)
to the lipase (Lip) of
S. albus. Similar amino acid identities
were obtained for
the lipases of
Streptomyces coelicolor (
24)
and a
Moraxella sp. (
12). The enzymes of the latter two
organisms
revealed the same broad substrate spectrum as Lip from
E. coli/pAH110
(
12,
13,
24). A comparison of the
lipase from
E. coli/pAH110
with other known lipases showed
high similarity with regard to
the conserved motif
[LIV]-X-[LIVFY]-[LIVMST]-G-[HYWV]-S-X-G-[GSTAC],
commonly
found within the sequences of lipases and esterases (
5).
This motif carries the active-site serine of hydrolytic enzymes.
This
signature pattern was also more or less conserved in the
deduced amino
acid sequences of the other three genes identified
in this study, which
encode putative esterases (Fig.
3).
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FIG. 3.
Amino acid alignment of the protein regions of the
deduced lip, orf111, orf112, and
orf113 gene products containing the conserved motif around
the proposed active-site serine of lipolytic enzymes. Matching amino
acids of the consensus sequence for this region
([LIV]-X-[LIVFY]-[LIVMST]-G-[HYWV]-S-X-G-[GSTAC])
(5) are shaded.
|
|
The sequence of the 2,136-bp insert of pAH115 harbored a single large
open reading frame (1,248 bp), designated
orf111 (Fig.
1).
The deduced gene product (415 amino acids), with a predicted
molecular
mass of 44,628 Da, is 34.3% identical (40.0% similar)
to one
hypothetical lipase/esterase of
S. coelicolor (SCH10.22c)
(
20).
DNA sequence analysis of the insert of pAH116 (1,472 bp) revealed one
potential gene, designated
orf112, within the sequence.
The
orf112 gene (927 bp) codes for 308 amino acids with a
predicted
molecular mass of 34,161 Da. Database searches showed
similarities
(47.8 and 34.6% identity and 53.5 and 47.2% similarity)
to the
hypothetical esterase from
S. coelicolor (SCE9.22;
27,211 Da)
(
20) and the esterase from
Acinetobacter
lwoffii (Est; 33,974
Da) (
1,
19). Both esterases belong
to the G-D-X-G family
of lipolytic enzymes. For the detection of
members of this protein
family two specific fingerprint patterns are
available in the
PROSITE database (
15); the first one,
including the putative
active-site histidine, is
[LIVMF] - X(2) - [LIVMF] - H - G - G
- [SAG] - [FY] - X(3) - [STDN] -X(2)-[ST]-H,
and the second one
harboring the putative active-site serine, is
[LIVM]-X-[LIVMF]-[SA]-G-D-S-[CA]-G-[GA]-X-L-[CA].
The
orf112 gene product showed both fingerprint
patterns.
The plasmid pAH117 (1,474 bp) contained a single open reading frame of
927 bp designated
orf113 (Fig.
1). The deduced gene
product
(307 amino acids), with a predicted molecular mass of
32,484 Da,
exhibited 43.8% identity (53.6% similarity) to the
above-mentioned
esterase of
A. lwoffii, but in contrast to the
orf112 gene product, the signature pattern for members of
the
G-D-X-G family of proteins was not present in the amino acid
sequence.
In addition, the
orf113 gene product revealed
28.6% identity (36.3%
similarity) to the lipolytic enzyme of
Sulfolobus acidocaldarius,
which, like the
orf113
gene product, hydrolyzes only short-chain
triacylglycerols, such as
tributyrin (
4).
In summary, four novel lipolytic enzymes (one putative lipase and three
esterases) were found during screening of environmental
DNA libraries.
The deduced amino acid sequences of the four proteins
revealed only
moderate identity (<50%) to any other sequence available
in the
various databases. This result indicates that the constructed
DNA
libraries harbor genes from a wide variety of microorganisms
of which
many have not been investigated or even cultivated. Thus
far,
characterized genes encoding lipolytic proteins from cultivated
microorganisms are very different (
16). Therefore, sequence
information collected solely with cultivated microorganisms will
not be
sufficient to design universal PCR primers to retrieve
the variety of
genes encoding lipolytic enzymes from natural microbial
communities.
Molecular methods that do not rely on isolation of
microorganisms into
pure culture are needed to recover novel genes.
The presented
recombinant approach showed the feasibility of direct
cloning of
functional genes from environmental DNA. This method
of accessing and
exploiting the natural biodiversity, together
with high-throughput
screening systems, will have a great impact
on microbial biotechnology
in the
future.
Nucleotide sequence accession numbers.
The nucleotide
sequences of the inserts of pAH114, pAH115, pAH116, and pAH117 have
been deposited in the GenBank database under accession numbers AF223645
to AF223648, respectively.
| |
ACKNOWLEDGMENTS |
We thank the Deutsche Bundesstiftung Umwelt for support.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institut
für Mikrobiologie und Genetik der Georg-August-Universität,
Grisebachstr. 8, 37077 Göttingen, Germany. Phone: 49-551-393827. Fax: 49-551-393808. E-mail: rdaniel{at}gwdg.de.
| |
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Applied and Environmental Microbiology, July 2000, p. 3113-3116, Vol. 66, No. 7
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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