JBC Papers in Press. Published on October 20, 2010 as Manuscript M110.

139659
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M110.139659

PROSPECTING METAGENOMIC ENZYME SUBFAMILY GENES FOR DNA

FAMILY SHUFFLING BY A NOVEL PCR-BASED APPROACH
Qiuyan Wang 1, Huili Wu 1, Anming Wang 1, Pengfei Du 1, Xiaolin Pei 1, Haifeng Li 1, Xiaopu
Yin 1, Lifeng Huang 1, Xiaolong Xiong 2
Center for Biomedicine and Health 1, College of Life Sciences 2,
Hangzhou Normal University; Hangzhou 310012, P. R. China
Running head: TMGS-PCR based DNA family shuffling
Address correspondence to: Qiuyan Wang, Center for Biomedicine and Health, Hangzhou
Normal University, No. 222 Wenyi Road, Hangzhou 310012, Zhejiang, China. Fax:
(86)571-28865630, E-mail: qywanghznu@gmail.com.

DNA family shuffling is a powerful Directed evolution mimics the processes of

method for enzyme engineering, which Darwinian evolution in a test tube, combining
utilizes recombination of naturally occurring random mutagenesis and/or recombination with
functional diversity to accelerate screening or selection for enzyme variants that
laboratory-directed evolution. However, the have the desired properties (1). Directed

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use of this technique has been hindered by the
scarcity of family genes with the required
level of sequence identity in the genome
database. We describe here a strategy for
collecting metagenomic homologous genes for
DNA shuffling from environmental samples
by truncated metagenomic gene specific PCR
(TMGS-PCR). Using identified metagenomic
gene specific primers, twenty-three 921 bp
truncated lipase gene fragments, which
shared 64 to 99% identity with each other and
formed a distinct subfamily of lipases, were
retrieved from 60 metagenomic samples.
These lipase genes were shuffled, and selected
active clones were characterized. The
chimeric clones show extensive functional and
genetic diversity, as demonstrated by
functional characterization and sequence
analysis. Our results indicate that homologous
sequences of genes captured by TMGS-PCR
can be used as suitable genetic material for
DNA family shuffling with broad applications
in enzyme engineering.
Many excellent examples of the application
of directed evolution to a wide range of enzymes
have clearly demonstrated its critical role in
tailoring enzymes for industrial applications(1-4).
evolution through random mutagenesis of a
single starting sequence can result in significant
improvement toward the required function.
However, this strategy has the disadvantage of
having to screen an extremely high number of
candidates to find the rare positive mutants that
are needed as the genetic material for DNA
shuffling (5). A more potent directed evolution
strategy is known as DNA family shuffling; it
refers to the recombination of equivalent genes
from natural homologous families rather than
random mutagenesis of a single gene (6). This
approach takes advantage of the fact that most of
the deleterious mutations have long ago been
removed by natural selection, while diverse
function has been created by the positive
mutation interchange. The reassortment of
proven mutations yields a higher frequency of
functional progeny sequences, and because
multi-gene shuffling starts with more than one
parental sequence, it accesses a broader range of
progenitor combinations. These attributes make
the approach more efficient, reducing
loss-of-function mutations dramatically so that
fewer progeny molecules need to be screened to
discover superior performers (7-9).
Although DNA family shuffling has the
above-mentioned advantages over random

Copyright 2010 by The American Society for Biochemistry and Molecular Biology, Inc.
mutagenesis, there are only dozens of published
reports using it, unlike the thousands published
that use random mutagenesis. The modest
number of published cases of DNA family
shuffling is indicative of its limitation: reliance
on natural homologous gene materials. It is
always frustrating searching for qualified
homologous sequences in the genome database.
Recently, several techniques for the
combinatorial generation of protein chimeras in
regions of low homology have been developed
that can circumvent homology dependence for a
DNA shuffling process. These techniques
include non-homologous random recombination
(10), sequence homology-independent protein
recombination (11) and incremental truncation
for the creation of hybrid enzymes (ITCHY)
(12-14). However, random non-homologous
recombination results in the creation of libraries
containing a large number of out-of-frame or
otherwise inactive clones. The theoretical size of
such libraries renders them unsuitable for
manual screening because a vast number of
clones must be interrogated to identify highly
active enzymes (14). However, Castle et al. have
provided an alternative and ultimately
complementary solution to this problem:
building synthetic libraries using a subset of
diversity from an alignment (15). This method
has been proven to be successful for providing
sufficient genetic diversity for directed enzyme
evolution, even based on information from
alignments with few genes of low homology.
However, the need to develop more alternatives
to collecting natural homologous genes
efficiently for DNA family shuffling remains
urgent.
In the past decade, advances in the field of
‘metagenomics’ have dramatically revised our
view of biodiversity (16). Considering the
estimation that >99% of microorganisms in most
environments are not amenable to culturing, very
little is known about their genomes. The
isolation, archiving and analysis of
2
environmental DNA (or so called
‘metagenomes’) have enabled us to mine
microbial diversity, allowing us to access their
genomes (17-20). Two strategies are generally
used to screen and identify novel genes from
metagenomic libraries: function-based and
sequence-based screening. In function-based
screening, clones expressing desired traits are
selected from libraries, and aspects of the
molecular biology and biochemistry of the active
clones are analyzed. Conversely, sequence-based
screening is not dependent on the expression of
cloned genes in heterologous hosts. Generally, it
is based on the conserved DNA sequences of
target genes. Hybridizations or PCRs are
performed based on the deduced DNA consensus
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(21).
We report here the development of a
homologous gene collection method termed
“truncated metagenomic gene specific PCR”
(TMGS-PCR). This approach led to success in
tapping into homologous sequences of enzymes
from environmental DNA with PCR with a
designated metagenomic starting gene and
experimentally validated truncated gene specific
primers (Fig. 1). Sequences analysis and
functional characterization of the resulting
shuffled library demonstrated that TMGS-PCR
can provide homologous genes with great
genetic diversity for DNA family shuffling.
EXPERIMENTAL PROCEDURES
Bacterial strains, plasmids and growth
conditions - E. coli DH5α and pBluescript SK+
(Stratagene, La Jolla, CA, USA) were used as
host and vector, respectively, for cloning of the
lipase. E. coli strain BL21-CodonPlus
(DE3)-RIPL (Stratagene, La Jolla, CA, USA)
and pET-24a(+) (Novagen, Inc., Madison, WI)
were used as host and vector, respectively, for
heterologous expression of the lipase. Plasmid
pMD18-T (TaKaRa Ltd., Otsu, Japan) was used
for gene cloning and sequencing. E. coli cells
were routinely grown in Luria–Bertani (LB)
medium at 37°C, supplemented with 100 µg/ml
ampicillin, 50 µg/ml streptomycin sulfate, 10
µg/ml tetracycline HCl and 12.5 µg/ml
chloramphenicol, if needed.
Library construction- A total of 60
environmental samples were collected from
terrestrial microenvironments in China and were
named as S01-60. DNA extraction from samples
was carried out using the PowerSoil® DNA
Isolation Kit (MOBIO, Carlsbad, CA, USA)
according to the manufacturer's instructions.
Electrophoresis for DNA from envireomental
samples was performed with a Bio-Rad CHEF
DR II system (Bio-Rad, Richmond, CA, USA).
A metagenomic library of the S05 sample was
constructed using the approximately 40 kb
end-repaired DNA fragments and
TM
CopyControl Fosmid Library Production Kit
(Epicentre Technologies, Madison, WI, USA)
using protocols provided by the manufacturer.
Screening and subcloning- For detection of
lipolytic activity, transformed cells were spread
immediately after electroporation on LB agar
containing 1% (w/v) tributyrin (Merck,
Darmstadt, Germany) and incubated overnight at
37°C. After 3–5 days at room temperature or
4°C , colonies were monitored for the presence
of clear halos (22). Sau3A I partially digested
fosmid DNA from the positive clone was ligated
to BamHI-linearized and dephosphorylated
pBluescript SK+ vector, and the ligation mixture
was used to transform E. coli DH5α. The new
clones were examined on the same type of
indicator plates for lipase activity. A
tributyrin-positive clone was selected and
sequenced. The gene encoding LipS05 was
amplified by PCR using Lip24aF and Lip24aR
primers,
5′-CGCCATATGATGGAATTTATTCCCCCT
G-3′ and 5′-
TAGCTCGAGTTTGGCACGCAGCG-3′,
which resulted in Nde I and Xho I sites at the 5′
and 3′ ends, respectively. The digested PCR
fragment was inserted between the Nde I and
3
Xho I sites of pET-24a(+), creating
pET-24a-LipS05. The recombined plasmid was
transformed into competent cells of E. coli strain
BL21-CodonPlus (DE3)-RIPL using the CaCl2
method following standard protocols.
Homologous gene amplification - Sets of
primers, which were assigned designations
comprising the first three letters Lip referring to
lipase and the sequence length (bp) of the
product, were used to amplify related lipase
fragments from enviromental DNA samples. The
product was amplified using a BIO-RAD DNA
Engine Peltier Thermal Cycler. The conditions
were as follows. Thirty cycles of reactions were
performed under the conditions of denaturation
at 94°C for 1 min, annealing at 58°C for 1 min,
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and an extension step at 72°C for 1 min. This
sequence was repeated 35 times followed by a
10-min final extension step at 72°C. All PCR
products were cloned into the TA vector and
confirmed by DNA sequencing.
Sequences analysis - The Lipase
Engineering Database
(http://www.led.uni-stuttgart.de/) was
searched using the BLASTP program (23).
Multiple sequence alignments were constructed
using the CLUSTAL-X program (24). To
determine the evolutionary relationship of
enviromental Lipases with other lipases,
phylogenetic analysis was performed with the
amino acid sequences of environmental lipases
and 50 lipases obtained from Lipase Engineering
Database using neighbor-joining methods with
1000 bootstrap replications performed by MEGA
version 4.0 (25).
DNA shuffling - The chimeric library was
constructed using a modified DNA shuffling
method (26). Twenty-four ~1.3 kb DNA
chimeric parent genes containing the LipS01-50
truncated fragments were amplified using
Lip24aF/R primers. The reaction mixture (50 µl)
contained 1 µl of Pfu polymerase (2.5 units/µl)
(Stratagene, La Jolla, CA), 1 µl of 20 µM each
primer, 30 ng of each plasmid DNA, 5 µl of
dNTP (2.5 mM each) and 5 µl of 10x Pfu buffer.
The resulting parent DNA fragments were then
digested with bovine pancreas DNase I (TaKaRa
Ltd., Otsu, Japan) as follows. A 25 µl volume of
solution containing 0.05 µg of each parent DNA
was mixed with 6.25 µl of 0.2 M Tris-HCl (pH
7.5), 0.25 µl of 10 mg/ml BSA and 0.25 µl of 0.1
M MnCl2. Digestion was initiated with the
addition of 2.5 µl of DNase I (0.5 unit/ml) into
the mixture at 37°C. Following incubation for 30
min, the digestion was stopped by adding 0.5 M
EDTA to a final concentration of 50 mM and
heat inactivating at 95°C for 10 min. The
digested fragments were separated by gel
electrophoresis. The desired 100-200 bp DNA
fragments were isolated and purified using the
QIAquick gel extraction kit (Qiagen, Valencia,
CA).
Reassembly of the DNase I digested
fragments was conducted in a 50 µl reaction
mixture containing 37 µl of fragment DNA(~3
µg), 5 µl of 10x Pfu buffer, 8 µl of dNTP (2.5
mM each) and 1 µl (2.5 units) of Pfu polymerase.
A progressive hybridization PCR cycling
program was used for reassembly. The
reassembled reaction mixture (1 µl) along with
primers Lip24aF/R was used to amplify the
full-length genes using PCR. Then the Nde
I-Xho I restriction fragment was inserted into
pET-24a(+) at the corresponding sites, and the
resulting plasmid was used to transform E. coli
BL21-CodonPlus (DE3)RIPL by
electroporation.
Expression, refolding and purification - A single
colony of selected mutants was used to inoculate
5 mL LB, containing 50 µg/ml kanamycin, 50
µg/ml streptomycin sulfate, 10 µg/ml
tetracycline HCl and 12.5 µg/ml
chloramphenicol, and the culture was agitated at
225 rpm overnight at 37°C. The cells were
grown in 1 L of LB media at 37°C until
OD600 nm=0.8, then protein expression was
induced using 0.5 mM
isopropyl-β-D-thiogalactopyranoside (IPTG),
4
and the incubation temperature was lowered to
20°C for 20 h.
Inclusion bodies were solubilized using the
protein refolding kit (Novagen), according to the
manufacturer's protocol, with slight
modifications. E. coli BL21 cells expressing
lipases were harvested, resuspended in 0.1
culture volume of wash buffer (20 mM Tris-HCl
pH 7.5/10 mM EDTA/1% Triton X-100), and
broken by sonication. After centrifugation, the
pellet was washed twice with 0.1 culture
volumes of wash buffer and then solubilized at
room temperature in solubilization buffer (0.1 M
glycine, pH 11/0.3% N-lauroylsarcosine) at a
concentration of 5 to 10 mg/ml wet weight of the
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pelleted cell debris, including the inclusion
bodies. For refolding, the denatured protein was
dialyzed twice (for 3 h and 12 h) against 50
sample volumes of dialysis buffer (20 mM
Tris-HCl, pH 8.5) supplemented with 0.1 mM
DTT, twice again (3 h each) against dialysis
buffer, and then once again (12 h) against 25
sample volumes of dialysis buffer supplemented
with 1.0 mM reduced glutathione/0.2 mM
oxidized glutathione. All dialysis steps were
performed at 4°C.
Soluble protein was incubated with
Ni-NTA agarose (Qiagen, Hilden, Germany) for
1 h at 4°C, and the mixture was loaded onto a
chromatography column. The column was
washed with a buffer containing 100 mM
sodium phosphate (pH 8.0), 200 mM sodium
chloride, and 10 mM imidazole. The N-terminal
His-tagged lipase was eluted from the column
with the same buffer containing 500 mM
imidazole. The protein was dialyzed overnight
against 20 mM sodium phosphate (pH 8.0) and
concentrated with polyethylene glycol (PEG)
20000. Then the purfied enzyme was stored at
4°C and used within three days for enzyme
assays. The purified enzymes were assayed by
SDS-PAGE followed by Coomassie brilliant
blue G-250 staining and the protein
concentration was determined by the Bradford
method.
Activity assay - The time course of the
esterase-catalyzed hydrolysis of p-nitrophenyl
alkanoate esters was followed by monitoring the
production of p-nitrophenyl at 405 nm in 1 cm
pathlength cells with UV-Visible
spectrophotometer (Shimadzu Co., Kyoto,
Japan). In the standard assay, 20µl of 10 mM
p-nitrophenyl butyrate (pNPC4), p-nitrophenyl
caprylate (pNPC8) or p-nitrophenyl laurate
(pNPC12) solution was added to the reaction
system for a final concentration of 0.2 mM in 50
mM phosphate buffer (pH 8.0) and incubated at
25°C. Then, the reaction was started with the
addition of 20 µl of the enzymatic solution. The
background hydrolysis of the substrate was
deducted with a reference sample of identical
composition to the incubation mixture, except
that lipase was omitted. One unit of enzymatic
activity was defined as the amount of protein
releasing 1 µmol of p-nitrophenyl from
p-nitrophenyl alkanoate esters per minute (27).
Nucleotide sequence accession numbers -
Nucleotide sequences described in this paper
have been submitted to GenBank under the
following accession numbers: GU942683 to
GU942706.
RESULTS
Isolation of starting lipase gene - To isolate
the starting lipase gene from a metagenomic
sample, S05 environmental DNA library was
constructed using a CopyControl fosmid library
production kit (Epicentre) according to the
manufacturer’s instructions. This process was
followed by the screening of the lipolytic
activity on a tributyrin agar plate. A positive
clone showing the lipolytic activity among
approximately 30,000 colonies was selected (Fig.
2A). Sequence analysis of the short insert DNA
obtained by subsequent subcloning experiments
revealed the presence of one open reading frame
consisting of 1,311 nucleotides, encoding a
5
protein (LipS05) with a molecular mass of 48
kDa. A BLAST search in the Lipase Engineering
Database (http://www.led.uni-stuttgart.de/)
revealed that the LipS05 can be aligned to
several lipases around the catalytic site (E value
<0.01). Those sequences showing the highest
homology were from Rhodococcus sp.
(gi|111021394|; identity, 25/76 32%; similarity,
39/76 51%), Coprinopsis cinerea (gi|169844727|;
identity, 27/82 32%; similarity, 45/89 50%),
Streptomyces griseus (gi|182439565|; identity,
19/58, 32%; similarity, 41/82 50%) and
Cryptococcus neoformans (gi|134109957|;
identity, 25/89 28%; similarity, 48/89 53%).
LipS05 and the homologous uncharacterized
lipases have a conserved active-site motif
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consisting of the pentapeptide GXSXG (Fig. 2B)
that is characteristic of lipases (28).
The LipS05 gene was subcloned into the
pET-24a(+) vector and overexpressed in E. coli
BL21-CodonPlus (DE3)-RIPL. The supernatant
of cell extract from E. coli harboring
pET-24a-LipS05 showed a significantly elevated
activity compared with that of E. coli harboring
pET-24a without the subcloned lipase sequence.
Therefore, high-throughput functional screening
can be applied to detect the phenotypic
variability of mutants of LipS05 using this
expression system (29).
Recovery of homologous sequences - Since
there are no conserved domains in the LipS05
equence, only sequences that correspond to tens
of amino acids that allow alignment, it is not
possible to design degenerate primers to isolate
related family genes from other environmental
samples as in traditional PCR-based
metagenomic screening. Sets of primers specific
to arbitrary locations of the LipS05 sequence
were designed (Table 1). Initially, overlapping
primers targeted terminal to the LipS05
sequence were designed to amplify full length
homologous genes from environmental DNA
samples. For full length Lip1311 primer sets, no
more than 3 specific amplification products of
the correct size (1311 bp) were generated from
reactions for 60 templates. For the other two
overlapping terminal primers, Lip1275 and
Lip1247, none of the products were observed by
gel analysis. Despite repeated attempts to
optimize PCR conditions, only nontarget
amplification products were obtained for the
above-mentioned primers. Therefore, sets of
primers with a series of progressively longer
deletions were designed to collect more
homologous genes. For the Lip1095 primer set,
four specific amplification products with a size
of 1095 bp were found under similar PCR
conditions. While for Lip923 primer set, 20
specific amplification products was generated
under the same PCR condition. A total of 60
inserts were sequenced from those 20 clone
libraries with inserts from at least 3 randomly
selected clones sequenced from each library.
Twenty-three novel lipase gene fragments, with
conserved pentapeptide GXSXG and between 68
to 97% nucleotide identity to LipS05, were
retrieved. Corresponding to soil sample name,
these lipase genes were named LipS01-1,
LipS01-2, LipS03, LipS05-2, LipS07, LipS08-1,
LipS08-2, LipS09, LipS10, LipS11, LipS12,
LipS13, LipS16, LipS17, LipS19, LipS30,
LipS33, LipS48-1, LipS48-2, LipS49, LipS50-1,
LipS50-2 and LipS51, respectively. To confirm
the correlation between the number of specific
PCR products and the length of the truncated
fragments, the Lip481 primer set, which had
further truncation of sequence length, was
applied in PCR screening of those environmental
DNA samples, which resulted in 16 specific
amplification products under similar reaction
conditions. Optimized amplification reaction
conditions and an increase in the number of
sequenced clones may result in more novel
sequences retrieved. As the isolated sequences
were of the appropriate number and length for
following DNA family shuffling, no attempts
were made to isolate more sequences.
Twenty-three 923 bp retrieved lipase genes and
6
the starting gene LipS05 were chosen for use as
genetic materials for DNA family shuffling.
Sequence analysis of LipS05 and subfamily
sequences - A multiple alignment was
constructed to illustrate conserved and variable
sites within the retrieved lipase subfamily. The
classical signature of lipases, the catalytic site
(GXSXG), was also conserved in the subfamily.
The pairwise identities of the protein sequences
ranged from 64% to 99% (68–99% DNA
sequence identity). This high sequence identity
showed the close relationship between these
proteins and indicated they can be considered to
belong to the same subfamily. In spite of this
high pairwise identity between sequences, 141
variable sites with different homology levels
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were also observed in multiple alignments (Fig.
4). Therefore, a combination of 141 variable
sites with a minimum of two amino acids change
in one site would comprise at least 2141, or
2.8×1042, different sequences. Although the vast
majority of the sequence space that can be
explored will be contingent on the recombining
capacity of DNA family shuffling, it can be
assumed that the DNA family shuffling of those
homologous genes will result in extensive
genetic diversity.
The phylogenetic tree constructed by the NJ
method for the lipase family proteins is shown in
Fig. 5. All of the lipases from environmental
samples cluster closely together on the left,
while all of the known lipases cluster loosely
together on the right. The lipases map together
on a branch of the tree separate from those
known lipases and form a clearly distinct group.
The phylogenetic tree clearly indicates that
novel lipases form a well-supported clade (NJ
bootstrap, 100%) that is loosely related to the
other known lipases (bootstrap, <50%).
Analysis of shuffled clones - To explore
whether those homologous genes can be used as
suitable genetic materials for DNA family
shuffling, we created a shuffled library with 24
homologues and characterized a small number of
chimeric clones. The shuffled library of
chimeras was transformed into E. coli and
screened for lipolytic activity on a tributyrin agar
plate (Fig. 6A). About 10% of the transformants
formed clearing halos, indicating expression of
active enzyme. The size of the resulting clear
halos in active clones was uniform, indicating
that phenotypic diversity was generated from the
chimeric library. To gain a deep insight into the
functional diversity of the chimeric mutants, the
three active clones with the largest halos were
overexpressed and purified to apparent
homogeneity. The clones’ specific activity on
pNPC4, pNPC8 and pNPC12 were measured to
demonstrate varied activity on short-, medium-,
or long-chain p-nitrophenyl alkanoate esters
substrates of the mutant enzymes. The results
demonstrated that the mutants exhibited a
distinct substrate specificity, with significantly
varied specific activity on different substrates
(Fig. 6B). The mutants C3 and E5 showed a
preference for the short-chain pNPC4, while
mutant D5 had its maximal activity on the
medium-chain pNPC8. Although both C3 and
E5 exhibited maximal activity on pNPC4, an
obvious difference in activity on pNPC4 and
pNPC8 was observed between them: for mutant
C3, the activity on pNPC8 was 54% relative to
that of pNPC4, while mutant E5 demonstrated a
comparable activity on pNPC4 and pNPC8. All
of the mutants indicated similar substrate
specificity for the long-chain substrate pNPC12,
with no more than 20% of maximal activity on
their preferred substrates. In addition to this
distinct substrate specificity, the mutants also
had a definite difference in their specific activity
on those substrates. For the short-chain substrate
pNPC4, the mutant C3 showed the highest
specific activity (87.4 U mg-1) among the
mutants, which was 1.5 and 3.5 fold higher than
that of D5 and E5, respectively. For the
medium-chain substrate pNPC8, although the
mutant D5 showed the maximal activity relative
to its activity on the short- and long-chain
7
substrates, the value of its specific activity (56.2
U mg-1) was slightly higher (about 1.3 fold) than
that of the other two mutants. For the long-chain
substrate pNPC12, the mutant C3 showed the
highest specific activity with 17.4 U mg-1, which
was more than 2 fold more than that of the other
two mutants. To investigate the correlation
between structure and observed activity, these
three clones were sequenced. The sequence of
C3 is a chimera of LipS33, LipS11, LipS30,
LipS11, LipS01-2, LipS08-2 and LipS11. The
E5 gene encodes a chimeric enzyme with
sequence elements originating from LipS01-2,
LipS08-2, LipS30, LipS33, LipS11, LipS30,
LipS08-2, LipS33, LipS13, LipS12, LipS13,
LipS30 and LipS16. D5 contained sequence
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elements from LipS09, LipS48-2, LipS30,
LipS13 and LipS30 (Fig. 6C). As such strikingly
high crossover frequency was observed in the
selected clones, another 7 unselected chimeric
clones in small library were sequenced to
determine the average crossover frequency in the
library. A per-gene average of 8 crossovers and
3 spontaneous mutations were observed in
sequenced genes.
DISCUSSION
In the present study, a novel metagenomic
gene-specific strategy was chosen to retrieve
related genes, in contrast to traditional
PCR-based metagenomic screening, which uses
known gene information to design degenerate
primers corresponding to conserved regions of
an enzyme family (21). Total DNA extracted
directly from environmental samples does not
typically contain an even representation of the
population's genomes within the sample. (30).
Therefore, the targeted homologous genes of
known sequences might be overshadowed by
sequences from unknown dominant microbial
populations in environmental DNA, which would
severely decrease the efficiency and specificity of
gene amplification and affect the product yield
(18). In fact, LeCleir G et al. failed to retrieve
any chitinase sequences from environmental
samples using known sequences based PCR
approach, despite evidence suggesting high
levels of chitinolytic activity in the sample (31).
A previous study retrieved only one lipase
sequence from the environmental DNA of olive
oil percolation using known sequences based
PCR approach, even with extensive analysis of
conserved regions and careful primer design (32).
In contrast, metagenomic gene-specific PCR
uses an gene identified by functional screening
of soil DNA library, instead of a known gene in
database as the startpoint for retrieving family
genes, which has a inherent property of biasing
for genomes of dominant microorganisms of soil
DNA. This distinguishing feature make
metagenomic gene a good start point for
retrieving metagenomic homolgous family genes
from soil DNA samples by PCR. It is worth
noting that 20 bands of correct size and without
any non-specific bands was observed in the gel
analysis of the products amplified from DNA of
60 soil samples with metagenomic gene specific
Lip923 primer set (Fig. 3). That was achieved by
only one experiment without any attempts at
optimizing reaction conditions. Nevertheless,
neither our method nor traditional metagenomic
sequence-based PCR guarantees acquisition of
full-length family genes because identical
sequences are conserved in the interior regions
of subfamily genes rather than at terminal
regions (6).
Phylogenetic analysis indicated that the
metagenomic gene specific amplification
strategy offers a chance to access to a novel
subfamily that was previously unknown and
completely uncharacterized. Traditional
degenerate primer-based PCR for metagenomic
screening is based on the conserved DNA
sequences of known family genes. Generally, the
amino acid identities of newly discovered
sequences from a metagenome to those in the
protein databases have been above 50% and
usually around 80–98%, which is rarely possible
8
to shed light on a novel gene family (33-37).
The functional diversity of chimeric
phenotypes generated by DNA-family-shuffling
was evident in experiments assessing both
activity on indicative plates and substrate
specificity of purified mutants. The varied size
of the clear halos around the mutant clones
indicated an apparently varied lipolytic activity.
In addition, characterization of the specific
activity on substrates with various chain lengths
provides definite evidence for the functional
diversity of chimeric clones. Such a property of
diversity is expected and is indeed a hallmark of
successful libraries created by DNA family
shuffling (6,7). The crossover frequency of
chimeric clones in this study is higher than that
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reported for normal enzyme shuffling and lower
that that of a shuffled library created with
methods for generating highly recombined genes,
such as RICHT (38). Given that we used the
same shuffling procedure as in normal enzyme
shuffling reports to create our chimeric library,
the high crossover frequency can be attributed to
the diversity of the genetic material we used. In
fact, there are usually no more than 4
homologous genes of enzymes from
microorganisms used to shuffle in reports using
DNA family shuffling (38). Additionally, in the
13,310 bases sequenced, 3 spontaneous
mutations were found. This rate of 1 mutation
per 4,436 bases is well within the number of
spontaneous mutations expected from the rounds
of PCR used to generate the fragment and
template DNA and to amplify the chimeric
products. Further, no unexpected insertions,
deletions, or rearrangements were detected by
DNA sequencing. These data suggest that the
DNA shuffling procedure using genetic material
collected by TMGS-PCR can creates a fair
qualified chimerical library.
CONCLUSION
In conclusion, we have presented a novel
TMGS-PCR approach with unique features that
distinguish it from the traditional PCR-based
metagenomic screening method. Functional
analysis of the shuffled library, coupled with the
sequence information from the selected chimeric
progeny, indicated that this method can provide
high-quality genetic materials for DNA family
shuffling. We expect that TMGS-PCR could
provide a new alternative to collecting
metagenomic homologous genes of microbial
enzymes for DNA family shuffling, which is an
excellent tool for the irrational design of
biocatalysts with tailored properties. Meanwhile,
one can also use TMSG-PCR to generate
breeding stock and then use “synthetic
shuffling” to breed divergent sequences for
efficient directed enzyme evolution.

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FOOTNOTES

Acknowledgement: The work was supported by National Natural Science Foundation of

China (30900253, 20906016), Natural Science Foundation of Zhejiang Province (Y4080317).
The abbreviations used are: TMGS-PCR, truncated metagenomic gene specific
polymerase chain reaction; p-nitrophenyl butyrate (pNPC4); p-nitrophenyl caprylate (pNPC8);
p-nitrophenyl laurate (pNPC12).

10
 FIGURE LEGENDS

Fig. 1. Schematic overview of homologous genes collected by truncated metagenomic

gene specific amplification. Initially, the metagenomic starting gene was isolated from
environmental samples by functional screening. Based on the sequence of this identified gene,
truncated gene specific primers were used to amplify homologous genes from different
environmental samples. The retrieved homologous family genes were subjected to shuffling
to generate the chimeric libraries for isolated clones with desired properties.
Fig. 2. Isolation of starting lipase from metagenomic library. (A) Detection of lipase on
the plates containing tributyrin. (B) Multiple alignment of the partial amino acid sequences
containing the conserved GxSxG motifs of LipS05 and other lipases. GI number and
organisms: gi|111021394|, Rhodococcus sp; gi|169844727|, Coprinopsis cinerea;
gi|182439565|, Streptomyces griseus; gi|134109957|, Cryptococcus neoformans. The putative
catalytic triad residues are shaded and denoted by arrowheads.
Fig. 3. Agarose gel electrophoresis of TMGS-PCR products amplified from DNA of 60
soil samples using Lip923F/R primer set. M indicates the 5.0 kb ladder lane.

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Fig. 4. Amino acid sequence alignment of lipases. The alignment quality curve is
displayed below the alignment. Asterisks, colons and dots indicate completely, strongly and
weakly conserved positions, respectively.
Fig. 5. Phylogenetic analysis of lipase sequences (partial sequence, 307 aa). The data are
illustrated as a neighbor-joining tree based on 24 new lipases and 50 previously known
sequences from the Lipase Engineering Database. The name at the nodes is assigned
according to those in the Lipase Engineering Database and is shown for the major clades
only. Each geographic origin is represented by a grey dot.
Figure 6. Characterization of chimerical clones. (A) lipase activity screens. The chimeric
lipase library was plated to rich medium containing 1% tributyrin, grown one day, and
incubated for 5-7 additional days at 4°C. Transformants exhibiting diverse activity produce
clear variably-sized halos. (B) Specific activity on varied substrates for selected chimeric
mutants obtained by family shuffling. Data reported are the mean of at least two independent
experiments. (C) Sequence of selected chimaeric mutants obtained by family shuffling. The
segments derived from different enviromental lipases are shown as white bars and noted as
their name above the bars. Because of sequence identity in crossover, shown as black bars, the
exact derivation of the sequence cannot be determined exactly. The aminoacid point
mutations is indicated by carets (v) above the bars.

11
 TABLE LEGENDS

Table 1 Primers used in environmental sample amplification.

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12
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13
Figure 1
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14
Figure 2
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15
Figure 3
Figure 4
. ***** ******* : : . ***: . *. **** ** : : * *: : : ** **: **: *: **. *: * ***: *******: *: **: : : * : : : *. *: . **: . *************: **. : * . * ********. ** **** : . . . * : * * **
LipS08-1 P YKE I I QE L R YNL C T S E DQP VP VF P F AYDWR L P L E I I E T QF AE F VE E VI DR T KL MAHYVNAGYVAHP T VNL I GHS MGGL I I T GYL DKKGR S AP VS KVAT L GT P YKGS F E AVI KI AT GT ANL GS DAP NS R E R E AAR L T S S L YHL L P AI QDAL E L DDP S L P T 160
LipS33 P YKE I I QE L R YNL C T S E DQP VP VF P F AYDWR L P L E I I E T QF AE F VE E VI DR T KL MAHYVNAGYVDHP T VNL I GHS MGGL I I T GYL DKKGR S AP VS KVAT L GT P YKGS F E AVI KI AT GT ANL GS DAP NS R E R E AAR L T S S L YHL L P AI QDAL E L DDP S L P T 160
LipS19 P YKE I I QE L R YNL C T S E DQP VP VF P F AYDWR L P L E I I E T QF AE F VE E VI DR T KL MAHYVS AGYVDHP T VNL I GHS MGGL I I T GYL DKKGR S AP VS KVAT L GT P YKGS F E AVI KI AT GT ANL GS DAP NS R E R E AAR L T S S L NHL L P AI QDAL GL DDP S L P T 160
LipS16 P YKE I I QE L R YNL C T S E DQP VP VF P F AYDWR L P L E I I E AQF AE F VE E VI DR T KL MAHYVS AGYVDHP T VNL I GHS MGGL I I T GYL DKKGR S AP VS KVAT L GT P YKGS F E AVI KI AT GT ANL GS DAP NS R E R E AAR L T S S L YHL L P AI QDAL E L DDP S L P T 160
LipS11 P YKE I I QE L R YNL C P S E DQP VP VYP F AYDWR L P L E I I E R QF AE F VE E VI DR T R L MAHYVQAGYI KR P T VNL VGHS MGGL I I T GYL DKKGR S AP VS KVVT L GT P YKGS F E AVI KI AT GT ANL GS DAP NS R E R E AAR L T S S L YHL L P E I QDAL E L DDP S L P N 160
LipS13 P YKE I I QE L R YNL C P S E DQP VP VF P F AYDWR L P L E I I E E QF AE F VDE VI DR T KL MAHYVQAGYT E HP T VNL I GHS MGGL I I T GYL DKKGKS AP VS KVVT MGT P YKGS F E AVI KI AT GT ANL GS DAP NS R E R E AAR L T S S L YHL L P AI QDAL E L DDP S L P T 160
LipS30 P YKE I I QE L R YNL C S S E DQP VP VF P F AYDWR L P L E I I E KQF AE F VDE VI DR T KL MVHYVQAGYAE HP T VNL I GHS MGGL I I T GYL DKKGKS AP VS KVVT MGT P YKGS F E AVI KI AT GT ANL GS DAP NS R E R E AAR L T S S L YHL L P AL E NAL E L DDP S L P T 160
LipS48-1 P YKE I I QE L R YNL C ANE E QP VP VF P F AYDWR L P L AI L E KQL ADF VE E VI AR T QL MS HYVE AGYR E NP R VNL I GHS MGGL L I T GYL DR KGKS AR VAKVVT L GT P YKGS F E AVI KI AT GT ANL GADS P NS R E R E AAR L T S S L YHL L P AI KGAL E L DP P E L P D 160
LipS08-2 P YKE I I QE L R YNL C P S E E QP VP VF AF AYDWR L P L E I I E R QF S DF VE E VI AR T KL I AHYVE T GYVE NP R VNL I GHS MGGL I I AGYL DKKGKS AP VS R VVT L AT P YKGS F E AVI KI AT GT ANL GS DT P NS R E R E AAR L T S S L YHL L P S I E DAL E VDDP AL P T 160
LipS17 P YKE I I QE L R YNL C P S E DQP VP VF P F AYDWR L P L E I I E KQF S DF VDE VI AR T R L VGHYVE S GF L E NP KVNL I GHS MGGL I I T GYL DKKGKT AP VS KVVT L AT P YKGS F E AVI KI AT GT ANL GS DAP NS R E R E AAR L T S S L YHL L P T I S DAL E VDDP E L P T 160
LipS50-1 P YKE I I QE L R YNL C P S NDQP VP VF P F AYDWR L P L E I I E R QF S DF VAE VI DR T KL I NHYVE KGYVE NP KVNL I GHS MGGL I I T GYL DKKGKS AP VS KVVT L AT P F HGS F E AVI KI AT GT ANL GS DP P NS R E R E AAR L T S S L YHL L P AI KNAL E I DDP KL P A 160
LipS09 P YKE I I QE L R YNL C P S E DHL VP VF P F AYDWR L P L GI I E KQF S NF VE E VI DR T KL I GHYVE AGYVE NP T VNL I GHS MGGL I I T GYL DKKGKAAP VS KVVT L AT P YE GS F E AVI KI AT GT ANL GS DQP NS R E R E AAR L T S S L YHL L P AI KDAL E VDDP T L P A 160
LipS05 P YKE I I QE L R YNL C HDE DQQVP VF P F AYDWR L P L E I I E KQF S DF VDE VI DR T KL I R HYVE AGYVNNP KVNL I GHS MGGL I I T GYL DKKGT S AP VS KVVT L AT P YKGS F E AVI KI AT GT S NL GS DT S NS R E R E AAR L T S S L YHL L P AI QDAL E VDDP KL P T 160
LipS05-2 P YKE I I QE L R YNL C HDE DQQVP VF P F AYDWR L P L E I I E KQF S DF VDE VI DR T KL I R HYVE AGYVDNP KVNL I GHS MGGL I I T GYL DKKGT S AP VS KVVT L AT P YKGS F E AVI KI AT GT S NL GS DT S NS R E R E AAR L T S S L YHL L P AI QDAL E VDDP KL P T 160
LipS07 S YKE I I QE L R YNL C HDE DQQVP VF P F AYDWR L P L E I I E R QF S DF VDE VI DR T KL I AHYVE AGYVDHP T VNL I GHS MGGL I I T GYL DKKGT S AP VAKVAT L AT P YKGS F E AVI KI AT GT S NL GS DT S NS R E R E AAR L T S S L YHL L P AI QDAL E VDDP NL P T 160
LipS01-1 P YKE I I QE L R YNL C HDE DQQVP VF P F AYDWR L P L E I I E R QF S DF VDE VI DR T R L I S HYVE AGYVQNP KVNL I GHS MGGL I I AGYL DT KKDS AR VAKVAT L AT P YKGS F E AVI KI AT GT S NL GS DT S NS R E R E AAR L T S S L YHL L P AI QDAL E I DDP NL P T 160
LipS01-2 P YKE I I QE L R YNL C T NE DQQVP VF P F AYDWR L P L DI I E KQF S DF VDE VI DR T KL I S HYVE AGF VQNP KVNL I GHS MGGL I I AGYL DT KKDS AR VAKVAT L AT P YKGS F E AVI KI AT GT S NL GS DT S NS R E R E AAR L T S S L YHL L P AI QDAL E I DDP NL P T 160
LipS10 P YKE I I QE L R YNL C T YE DQQVP VF P F AYDWR L P L DI I E KQF S DF VDE VI DR T KL I AHYVE AGYVE NP KVNL I GHS MGGL I I AGYL DT KKE S AQVAKVAT L AT P YKGS F E AVI KI AT GT S NL S S DT S NS R E R E AAR L T S S L YHL L P AI QDAL E VDDP NL P T 160
LipS51 P YKE I I QE L R YNL C T NE DQL VP VF P F AYDWR L P L E I I E KQF S DF VDE VI DR T KL I T HYVE AGYVE NP KVNL I GHS MGGL I I AGYL DT KKDS AR VAKVAT L AT P YKGS F E AVI KI AT GT S NL GS DT S NS R E R E AAR L T S S L YHL L P AI QDAL E VDDP NL P R 160
LipS49 P YKE I I QE L R YNL C T NE DE QVP VF P F AYDWR L P L DI I E KQF S DF VDE VI NR T R L I S HYVE AGYVE NP KVNL I GHS MGGL I I AGYL DT KKDS AR VAKVAT L AT P YKGS F E AVI KI AT GT S NL GS DT S NS R E R E AAR L T S S L YHL L P AI QDAL E VDDP S L P K 160
LipS03 P YKE I I QE L R YNL C HDE DKQVP VF P F AYDWR L P L E I I E R QF S DF VDE VI DR T KL I S HYVE AGYVE NP KVNL I GHS MGGL I I AGYL DT KKDS AR VAKVAT L AT P YKGS F E AVI KI AT GT S NL GS DT S NS R E R E AAR L T S S L YHL L P AI KGAL E I DDP KL P N 160
LipS12 P YKE I I QE L R YNL C T DE NHQVP VYP F AYDWR L P L DMI E KQF S DF VDE VVE R T KL I S HYVE DGYVDHP T VNL I GHS MGGL I I AGYL DT KGKS AP I S KVAT L AT P YKGS F E AVI KI AT GT S NL GADAS NS S E R E AAR L T S S L YHL L P AI QDAL E VDDP KL P T 160
LipS48-2 P YKE I I QE L R YNL C P S E DE P VP VF P F AYDWR L P L E I I E KQF S DF VE E VI DR T KL I R HYAL AGYAANP KVNL I GHS MGGL I I T GYVDKKGKS AP VS KVAT L AT P YHGS F E AVI KI AT GT ANL GS DT S QS R E R E AAR L T AS L YHL L P DI KNAL E VDDP KL P L 160
LipS50-2 P YKE I I QE L R YNL R P KE DKP VP VF P F GYDWR QP L DL I E AQF ADF VDE VI AR T KL L R HYAE AGYADDP KVNL VGHS MGGL I I AGYL E R I GKS AP VAKVAT L AT P YR GS F E AVI KI AT GT ANL GT DE P T S R E R E AAR L T P S L YHL L P DI E GGL E I DDP KL P K 160
ruler 1. . . . . . . 10. . . . . . . . 20. . . . . . . . 30. . . . . . . . 40. . . . . . . . 50. . . . . . . . 60. . . . . . . . 70. . . . . . . . 80. . . . . . . . 90. . . . . . . 100. . . . . . . 110. . . . . . . 120. . . . . . . 130. . . . . . . 140. . . . . . . 150. . . . . . . 160

. : *. . . *** . : : *: : *: * . : ** ** : *: . . : * *: : * : . : * . ***** **: * ***: : : : *. **: ***. *. : * * *: . . * ******* : : * * * ***: **********

LipS08-1 S F F NP GL WQL S I I AS VL E YVR T QMT F I T E QE - - - QKAQAL F L R F L E AAQAYR T R I DNF R L S R T R L KAADWL C VVGVNS E T R VR MR I T KT E R GP MF DL S S KYR QNL WR KNAE NQAE WR L T GDGT VP F E AAL P NF L AL E NI VC VT P E DYGYW 307
LipS33 S F F NP GL WQL S I I AS VL E YVR T QMT F I T E QE - - - QKAQAL F L R F L E AAQAYR T R I DNF R L S R T R L KAADWL C VVGVNS E T R VR MR I T KT E R GP MF DL S S KYR QNL WR KNAE NQAE WR L T GDGT VP F E AAL P NF L AL E NI VC VT P E DYGYW 307
LipS19 S F F NP GL WQL S I I AS VL E YVR T QMT F I T E QE - - - QKAQAL F L R F L E AAQAYR T R I DNF R F S R T R L KAADWL C VVGVNS E T R VR MR I T KT E R GP MF DL S S KYR QNL WR KNAE NQAE WR L T GDGT VP F E AAL P NF L AL E NI VC VT P E DYGYW 307
LipS16 S F F NP GL WQL S I I AS VL E YVR T QMT F I T E QE - - - QKAQAL F L R F L E AAQAYR T R I DNF R F S R T R L KAADWL C VVGVNS E T R VR MR I T KT E R GP MF DL S S KYR QNL WR KNAE NQAE WR L T GDGT VP F E AAL P NF L AL E NI VC VT P E DYGYW 307
LipS11 S F F NP GL WQL S I VAS VME YVR T QMAF I T E KE - - - QKAQAL F L R F L E AAE AYR S R I DNF R L S T T R L KAADWL C VVGVNS E T R VR MR VT KT E R GP MF DL GS KYR L NL WR KNP E NQAE WR L T GDGT VP F E AAL P NF L AP E NI VC VT P E DYGYW 307
LipS13 S F F NP GL WQL S VVAS VL E YVR T QMT F I I E QE - - - QKAQAL F L R F L E AAQAYR T R I DNF R L S KT KL R AE DWL C VVGVNS E T R VR MR I AKT E R GP L F DL S S KYR L NL WR KNP E HE ME WR L T GDGT VP F E AAL P NF L KL E NI VC VT P E DYGYW 307
LipS30 S F F S P GL WQL S VVAS VL E YVR T QMT F I T E QE - - - QKAQAL F L QF L E AAQVYR T R I DNF R L S KT NL KAGDWL C VVGVNS E T R VR MR I AKT E R GP L F DL S S KYR L NL WR KNP E NE ME WR L T GDGT VP F E AAL P DF L KL E NI VC VT P E DYGYW 307
LipS48-1 NF F DP AL WQL S I VAS VL AYVQKQMS F I S E QD- - - QKAQE L F I R F L QAGE AYR NR I DR F R L T KT NL T AADWL C VVGVDS E T R VR MR I AKT QR GP MF DL S S KYR L NL WR KNL E NP AE WR L T GDGT VP F E AAL P NF L DL E NI L C VT P E DYGYW 307
LipS08-2 S L F DP AL WQL S VVAS VL AYVQR QMAF L T DQN- - - QKAQE L F AR F L E AAR AYR T R I DKF R L T KT NL KP E DWL C VVGVDS E T R VR MR I AMT E R GP MF DL S S KYR L NL WR QDP QNQAE WR WT GDGT VP F E AAL P NF L KL E NI VC VT P E DYGYW 307
LipS17 NL F DP AL WQL S VVAS VL AYVQR QMAF I T NQN- - - QKAQE L F T R F L E AAQAYR NR I DKF R L T R T AL QP E DWL C VAGVNS E T R VR MR I AKT E R GP L F DL S S KYR L NL WR KNP GNP AE WR L T GDGT VP F E AAVP NF L KP E NI VC VT P E DYGYW 307
LipS50-1 NL F DP AL WQL S VL AS VL AYVQAQMAF VS E KE - - - QKAQE L F VR F L E AAQAYR NR I DKF R L T KT GL E P E DWL C VVGVNS E T R VR MR I T S T E R GP MF DL S S KYR L NR WR S DL ANP AE WR L T GDGT VP F E AAL P NS L KL E NI VC VT P E DYGYW 307
LipS09 NL F DP AL WQL S VVAS VL AYVQR QMAF L T DHD- - - QKAQE L F AR F L KAAQAYR NR L DKF R L S KT NL KP E DWL C VVGVNS E T R VR MR VQR T E R GP L F DL S S KYR L NR WKS DL ANP ME WR L T GDGT VP F E AAL P NF L E L E NI I C VT P E DYGYW 307
LipS05 S L F DP AL WQS S VVAS VL AYVQKQMAF I AE QN- - - QKAQAL F AR F L E AAQAYR AR I DQF QL S KT NL KP E DWL C VAGVNS E T R VR MR I T S T E R GP L F DL S S KYR L NL WR KNKQDQS KWGL T GDGT VP F E AAL P NF L KP E NI VC VT P E DYGYW 307
LipS05-2 S L F DP AL WQS S VVAS VL AYVQKQMAF I AE QN- - - QKAQAL F AR F L E AAQAYR AR I DQF QL S KT NL KP E DWL C VAGVNS E T R VR MR I T S T E R GP L F DL S S KYR L NL WR KNKQDQS KWGL T GDGT VP F E AAL P NF L KP E NI VC VT P E DYGYW 307
LipS07 S L F DP AL WQS S VVAS VL AYVQKQMAF I T E QD- - - QKAQE L F AR F L E AAQAYR AR I DQF QL S KT NL KP E DWL C VAGVNS E T R VR MR I T R T E R GP L F DL S S KYR L NL WR KNKKNP T E WQL T GDGT VP F E AAL P NF L KP E NI VC VT P E DYGYW 307
LipS01-1 S L F DP AL WQS S I L AS VL AYVQKQMAF I KE QDE QT QKAQAL F AKF L E AAQAYR AR I DQF QL S T T NL KP E DWL C VAGVNS E T R VR MR I S R T E R GP L F DL S S KYR L NL WR KNKKNP T E WGL T GDGT VP F E AAL P NF L KP E NI VC VT P E DYGYW 310
LipS01-2 S L F DP AL WQS S I L AS VL AYVQKQMAYI KE QDE QT QKAQAL F AKF L E AAQAYR AR I DQF QL S T T NL KP ADWL C VAGVNS E T R VR MR I S R T E R GP L F DL S S KYR L NL WR KNKKNP T AWGL T GDGT VP F E AAL P NF L KP E NI VC VT P E DYGYW 310
LipS10 S L F DP AL WQS S I L AS VL AYVQKQMAS I KE QDE QT QKAQAL F T KF L E AAR AYR AR I DQF QL S P T NL KP E DWL C VAGVNS E T R VR MR I T R T E R GP L F DL S S KYR L NL WR KNKANP S DWGL T GDGT VP F E AAL P NF L KP E NI VC VT P E DYGYW 310
LipS51 S L F DP AL WQS S I VAS VL AYVQKQMAF I KE QDQQT QKAQT L F AQF L E AAQAYR T R I DQF QL T E T NL QP E DWL C VAGVNS E T R VR MR I S R T E R GP L F DL S S KHR L NL WR KNNVNP T E WKL T GDGT VP F E AAQP T F L KP E NI VC VT P E DYGYW 310
LipS49 S L F DP AL WQS S I L AS VL AYVQKQMAF I KE QDQQT QKAQAL F AKF L E AAQAYR T R I DQF QL P E T NL QP KDWL C VAGVNS DT R VR MR I T R T E R GP L F DL S S KYR L NL WR KNKQNP T E WGL T GDGT VP F E AAL P NF L QP E NI VC VT P E DYGYW 310
LipS03 S L F DP GL WQT S VI AS VL AYVQKQMAF I T KQE - - - QKAQE L F T KF L E VAQAYR AR I DKF KL S KT NL NS DDWL C VVGVNS E T R VR MR I T S T DHGP L F DL S S KYR L NL WR KNAGNQT E WGL T GDGT VP F E AAL P NF L KP E NI VC VT P E DYGYW 307
LipS12 S L F NP AL WQS S I I AS VL AYVQQQMAF I KGQDE KT QKAQAL F AQF L E R AQMYR S R I DQF QL S KT NL T ADDWL C VVGVNS QT R VR MR I S S T DS GP L F DL S S KYR L NL WR NKDYS P E QWR WT GDGT VP YE AAL P NF L GP E NI VC VT P E DYGYW 310
LipS48-2 S L F DP AL WQAS VI AS VL AYVQKQMAVI E QE S - - - - KAQAL F AR F L E AAQAYR T R I DNF DL S T T NL QS GDWL C VMGVNS E T R VR MR I T S T DR GP L F DL GS KYR L NL WR KKS E AE ANWR L T GDGT VP F E AAL P NF L GL E NI I C VT P E DYGYW 306
LipS50-2 S L F DAGL WQR GI I DS I T E F VR L QAVS KI DR P - - - QQAQDL F NS L L QDAQAHR KR VE S F R L S KAGL R P E DWL C VAGVDS VT R VKL KI T KT S R GP MF DL GS DYR QNL WR KDP R DQT DWHL T GDGT VP L DS AVP T F L KP E NI VC VT P E DYGYW 307
ruler . . . . . . . 170. . . . . . . 180. . . . . . . 190. . . . . . . 200. . . . . . . 210. . . . . . . 220. . . . . . . 230. . . . . . . 240. . . . . . . 250. . . . . . . 260. . . . . . . 270. . . . . . . 280. . . . . . . 290. . . . . . . 300. . . . . . . 310

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Figure 5
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Figure 6
 Table 1
Location Size of No. of
Tm
Name Sequence(5’→3) within Product amplification
(°C)
gene (bp) products
Lip1311F ATGGAATTTATTCCCCCTGTCATCTTTGTG 62.03 1-30 1311 0
Lip1311R TTATTTGGCACGCAGCGGCTTGAC 63.69 1287-1311 1311 0
Lip1275F TCATCTTTGTGCCGGGCATCACC 63.73 20-42 1275 0
Lip1275R GCTTGACCGCCGGATCCCATTT 63.8 1273-1294 1275 0
Lip1247F GCATCACCGGGACGTATTTGCGA 63.73 35-57 1247 0
Lip1247R ATCCCATTTTTCAGCCGTGACGC 61.96 1259-1280 1247 0
Lip1095F CGTGTGGCGCTTCACCCAGATG 65.66 115-136 1095 4
Lip1095R GAAGTGACGGACAATGAGTCGATGC 63.62 1185-1209 1095 4
Lip923F CCCTACAAGGAAATCATTCAGGAACTGC 63.48 196-223 923 20
Lip923R TCCCAGTATCCATAATCCTCAGGTGTCAC 64.85 1090-1118 923 20
Lip481F CATTCGATGGGCGGATTGATCATCACC 65.04 415-441 481 16
Lip481R TAACGCCTGCTACGCACAGCCAGTCTTCT 67.68 867-896 481 16

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