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Appl. Environ. Microbiol. (full paper) 1
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Efficient PCR-based Amplification of Diverse Alcohol Dehydrogenase Genes from 3
Metagenomes for Improving Biocatalysis: Screening of Gene-specific Amplicons 4
from Metagenomes 5
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Running title: PCR-based Amplification of ADH Genes from Metagenomes 7
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Nobuya Itoh,* Satomi Kariya, Junji Kurokawa 9
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Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural 11
University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan 12
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*To whom correspondence should be addressed: Biotech. Res. Center and Dept. of 15
Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 16
939-0398, Japan, Tel.: +81 766 56 7500, ext. 560; Fax: +81 766 56 2498; E-mail: 17
19
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AEM Accepts, published online ahead of print on 1 August 2014Appl. Environ. Microbiol. doi:10.1128/AEM.01529-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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ABSTRACT 21
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Screening of gene-specific amplicons from metagenomes (S-GAM) has 23
tremendous biotechnological potential. We used this approach to isolate alcohol 24
dehydrogenase (adh) genes from metagenomes based on the Leifsonia sp. adh gene 25
(lsadh), the enzyme product of which can produce various chiral alcohols. A primer 26
combination was synthesized by reference to homologs of lsadh, and PCR was used 27
to amplify nearly full-length adh genes from metagenomic DNAs. All adh 28
preparations were fused with lsadh at the terminal region and used to construct 29
Escherichia coli plasmid libraries. Of the approximately 2000 colonies obtained, 30
1200 clones were identified as adh-positive (~60%). Finally, 40 adh genes, 31
Hladh-001 to -040, were identified from 223 clones with high efficiency, which were 32
randomly sequenced from the 1200 clones. The Hladh genes obtained via this 33
approach encoded a wide variety amino acid sequences (8–99%). After screening, 34
the enzymes obtained (HLADH-012 and -021) were confirmed to be superior to 35
LSADH in some respects for the production of anti-Prelog chiral alcohols. 36
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Keywords: metagenome, gene-specific amplicon, alcohol dehydrogenase, anti-Prelog 38
chiral alcohol, organic solvent tolerance 39
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Introduction 41
Metagenomics is an emerging and powerful tool for the isolation of genes, the 42
enzyme products of which have industrial applications (1–6). Screening of 43
metagenomic libraries to find novel enzymes or enzymes homologous to those 44
described previously, has been used successfully to isolate lipases (7,8) amylases (9), 45
amidases (10), oxidoreductases (11), dehydratases (12), cytochrome P-450 (13), styrene 46
monooxygenase (14), and other enzymes (1–6). However, most previous approaches 47
featured metagenomic DNA extraction and Escherichia coli library construction, 48
followed by sequence- or function/molecule-based screens of the library. Such 49
approaches are very time-consuming and inefficient, especially in terms of detection; 50
much of the DNA sequenced and analyzed is irrelevant, and target genes may be 51
expressed ambiguously in E. coli host cells. PCR amplification of truncated genes from 52
metagenomes would facilitate the identification of genes encoding superior enzymes 53
and yield homologous gene sets that could be used for DNA shuffling (15). Although 54
previous studies based on PCR-mediated methods that utilize primers designed from 55
inner conserved sequences have been conducted for biocatalysts, including lipase (8), 56
cytochrome P-450 (13), 2,5-diketo-D-gluconic acid reductase (16), Pseudomonas 57
alcohol dehydrogenase (ADH) (17), and other biocatalysts (4,6), the methods are not 58
very efficient in many cases and often fail to produce complete functional genes. 59
Enantioselective organic synthesis is useful for producing chiral synthones for the 60
preparation of fine chemicals, including pharmaceuticals and agricultural chemicals. 61
The asymmetric reduction of ketones is one of the most promising approaches because 62
no substrate is lost, in contrast to when racemic separation is performed. Chiral metal 63
complexes such as BINAP-Ru have been used successfully as chemocatalysts in a 64
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number of cases of enantioselective synthesis (18). However, bio-based methods using 65
enzymes or whole-cell systems offer several advantages over the BINAP process for 66
industrial applications, including improved material handling and lower costs for the 67
preparation of catalysts (19–21). 68
Previously, we reported an efficient method for producing both enantiomers of chiral 69
alcohols by asymmetric hydrogen-transfer bioreduction of ketones in a 2-propanol 70
(IPA)-water medium using E. coli biocatalysts expressing a mutated form of 71
phenylacetaldehyde reductase (PAR) (22,23) and Leifsonia ADH (LSADH) (24,25). 72
However, PAR and LSADH do not fully possess the required substrate specificity or 73
stereospecificity; for example, LSADH does not accept methyl benzoylformate, 74
2-acetylpyridine, or 3-quinuclidinone as substrates (26,27). Thus, we sought to clone 75
genes encoding enzymes with properties distinct from those of LSADH. Moreover, 76
dehydrogenases such as LSADH that yield anti-Prelog chiral alcohols (e.g., 77
(R)-1-phenylethanol from acetophenone) are rare in nature (26,27); however, the 78
pharmaceutical industry demands this type of enzyme (28,29). 79
In this paper, we describe the construction of a metagenomic library of enzyme 80
genes that focused on the lsadh gene. Our approach, which involved PCR amplification 81
of nearly full-length genes from metagenomes fused with the terminal region of an 82
lsadh-expressing vector, enabled the isolation of many novel and diverse adh genes and 83
lsadh homologs. This highly efficient approach (S-GAM) shows tremendous 84
biotechnological potential for obtaining gene resources from metagenomes. We also 85
present the potential use of novel enzymes as biocatalysts for converting ketones to 86
various anti-Prelog chiral alcohols at high production levels. 87
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MATERIALS AND METHODS 89
Metagenome preparation. Metagenomic DNA was extracted from 20 environmental 90
samples including various soils collected from farms and paddy fields, gardens at 91
independent sites in Japan, and farm (35–45°C) and bark (50–80°C) composts in 92
Toyama, Japan, using an ISOIL for Beads BeatingTM kit (Nippon Gene, Tokyo, Japan) 93
without further purification. Bark compost samples in fermentation at approximately 94
50–80°C were generously supplied by a compost-producing company (Hokuriku Port 95
Service, Toyama, Japan). Successful extraction of DNA from the soil and compost 96
samples was confirmed using agarose gel electrophoresis; these DNA samples served as 97
templates for PCR. 98
Primers, PCR conditions, and cloning of adh genes. Standard techniques were 99
used for DNA manipulation (30). E. coli JM109 cells were used to host adh genes fused 100
with the pKELA-del plasmid. This vector was derived from pKELA (27), which 101
expresses the lsadh of pKK233-3, by deletion of part of the lsadh gene (100 bp) with 102
XhoI, and then PCR to introduce fusion sites to both 5′ ends using the following 103
primers: F-vec-1, 5′-ACCGCCCAGTGACCGGGCTGCAGGT-3′ and R-vec-1, 104
5′-ACGATCGCGGACCGGTCGGCGACGT-3′ (underlined sequence: fusion sites). 105
PCR was performed using KOD FX Neo DNA polymerase (Toyobo, Osaka, Japan). The 106
reaction mixture contained 10 µL 2× buffer for KOD FX Neo Kit, 2 nmol of each dNTP, 107
8 pmol of each primer, and 0.4 U DNA polymerase in a total volume of 20 µL. PCR 108
commenced at 94°C for 2 min, followed by 30 cycles at 98°C for 10 s, 60°C for 30 s, 109
and 68°C for 3 min, and then the sample was kept at 68°C for 5 min. The linearized 110
vector contained 15 nucleotides at both 5′ ends for fusion with the adh gene amplified 111
directly by PCR using metagenomic DNA. The adh gene of this vector is under the 112
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control of the tac promoter. DNA sequences were determined for both strands using a 113
capillary DNA sequencer (ABI PRISM 310; Applied Biosystems® Life Technologies, 114
Carlsbad, CA). 115
PCR was performed under optimized conditions using KOD FX Neo DNA 116
polymerase or Phusion® Hot Start Flex DNA polymerase (New England BioLabs, 117
Tokyo, Japan) to obtain amplicons from the metagenomes. The reaction conditions were 118
based on those suggested by the manufacturer, except that the reaction mixture 119
contained an approximately 10-fold higher concentration of each primer for KOD FX 120
Neo DNA polymerase and 5% (v/v) dimethyl sulfoxide for Phusion® Hot Start Flex 121
DNA polymerase; both polymerases offer high fidelity and robust performance. 122
Hot-start and step-down PCR protocols were used. Each reaction mixture contained 10 123
µL 2× buffer for KOD FX Neo Kit, 2 nmol of each dNTP, 80 pmol of each primer, 124
10–50 ng metagenomic DNA, and 0.4 U DNA polymerase in a total volume of 20 µL. 125
In the case of Phusion® Hot Start Flex DNA polymerase, the reaction mixture contained 126
4 µL 5× Phusion® GC buffer, 2 nmol of each dNTP, 8 pmol of each primer, 10–50 ng 127
metagenomic DNA, 1 µL dimethyl sulfoxide, and 0.4 U DNA polymerase in a total 128
volume of 20 µL. PCR commenced at 94°C for 2 min, followed by a step-down 129
protocol: 5 cycles at 98°C for 10 s, 74°C for 1 min, 5 cycles at 98°C for 10 s, 72°C for 1 130
min, 5 cycles at 98°C for 10 s, 70°C for 1 min, 5 cycles at 98°C for 10 s, 68°C for 1 min, 131
and finally the sample was kept at 68°C for 7 min. Good amplification of the target 132
genes was obtained from all samples using both sets of primers, F-1/R-1 and F-2/R-2 133
(Table 1). To accelerate the fusion reaction with the plasmid vector, we generally used 134
the F-2/R-2 primer set for the subsequent experiments. 135
Amplified fragments were separated by agarose gel electrophoresis, purified using 136
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the Wizard® Plus SV Miniprep DNA Purification System (Promega, Fitchburg, WI), and 137
fused between the same sites of pKELA-del with an In-Fusion® HD Cloning Kit 138
(Clontech, Mountain View, CA). The reaction mixture consisted of 6 µL pKELA-del (ca. 139
20 ng/µL), 2 µL amplified DNA fragments (5–20 ng/µL), and 2 µL In-Fusion enzyme in 140
a total volume of 10 µL, and the mixture was incubated at 50°C for 15 min. The 141
plasmids obtained were electroporated into E. coli JM109. Clones were grown at 37°C 142
on agar plates containing Luria-Bertani (LB) medium (1% [w/v] tryptone, 0.5% [w/v] 143
yeast extract, and 1.0% [w/v] NaCl; pH 7.0) with 100 µg/mL ampicillin. 144
Screening for ADH activity in E. coli clones. Screening for ADH activity in E. coli 145
was performed for about 2000 E. coli clones in 96-well plates by spectrophotometric 146
measurement of the activity of 1,1-dichloroacetone and phenyl trifluoromethyl ketone 147
(PTK). E. coli cells were cultured at 37°C overnight with shaking in LB liquid medium 148
(0.5 mL) containing 100 µg/mL ampicillin and 0.1 mM 149
isopropyl-β-thiogalactopyranoside (IPTG) in 96-well deep plates. IPTG was added to 150
the culture medium to confirm expression, although the enzyme expression of 151
pKELA-del-HLADH was leaky and barely controlled by IPTG. The cells were collected 152
by centrifugation after cultivation, rinsed in 100 mM potassium phosphate buffer (KPB) 153
(pH 7.0), and disrupted in 0.1 mL BugBusterTM Master Mix (Novagen, Merck 154
Biosciences Japan, Tokyo, Japan) for 20 min at room temperature. Cell debris was 155
removed by centrifugation, and the supernatant was used as a crude enzyme solution. 156
ADH activity was measured using a microplate reader at 340 nm and 25°C; the reaction 157
mixture consisted of 100 mM KPB (pH 7.0), 0.3 µmol NADH, 50 µmol substrate, and 158
10 µL crude enzyme in a total volume of 1.0 mL. 159
Purification of recombinant HLADH-012 and -021. HLADH-012 and -021 were 160
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purified at 0–4°C in 20 mM KPB (pH 7.0), unless otherwise stated. For HLADH-012, 161
washed recombinant E. coli cells from 200 mL culture broth (37°C for 17 h in a shake 162
flask) were suspended in 30 mL buffer and disrupted using an ultrasonic oscillator 163
(Ultra Sonic Disrupter UD-200; Tomy Corp., Tokyo, Japan) for 150 s (5 disruption 164
sequences of 30 s followed by a 60-s interval for cooling). After centrifugation (10 000 165
× g, 10 min), the cell-free extract was mixed with ammonium sulfate up to 40% 166
saturation and maintained overnight under gentle stirring. The precipitate was removed 167
by centrifugation (10 000 × g, 10 min) and the supernatant was recovered. The solution 168
was applied to a Toyopearl Butyl-650 M column (2.5 × 13 cm; Tosoh, Tokyo, Japan) 169
equilibrated with the buffer containing 1.0 M ammonium sulfate. The enzyme was 170
eluted at 1 mL/min with a linear 1.0–0 M ammonium sulfate gradient in the same buffer. 171
Fractions exhibiting high levels of enzyme activity (27–30 min) were collected and 172
dialyzed against the buffer. Next, the enzyme preparation was loaded onto a ResourceTM 173
Q column (0.64 × 3 cm; GE Healthcare Japan, Tokyo, Japan) equilibrated with the 174
above buffer and connected to the AKTApurifier system (GE Healthcare Japan). The 175
enzyme was eluted using a linear 0–0.5 M NaCl gradient in the same buffer at a flow 176
rate of 0.3 mL/min. Fractions exhibiting high levels of enzyme activity (43–46 min) 177
were collected, desalted, and concentrated using a Centriprep YM-30 centrifugal filter 178
unit (EMD Millipore, Billerica, MA). The enzyme solution obtained was the purified 179
preparation used for enzyme characterization. 180
HLADH-021 was purified from 100 mL culture broth in the same way as 181
HLADH-012, and after ammonium sulfate fractionation up to 40%, the supernatant 182
solution was similarly applied to a Toyopearl Butyl-650 M column. Fractions exhibiting 183
high levels of enzyme activity (43–47 min) were collected and desalted using a 184
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Centriprep YM-30 filter unit. The resulting enzyme solution was applied to a 185
DEAE-Toyopearl column (2.5 × 7 cm) equilibrated with the buffer, and the enzyme was 186
eluted using a linear 0–0.5 M NaCl gradient in the same buffer at a flow rate of 1.0 187
mL/min. Fractions exhibiting high levels of enzyme activity (31–33 min) were collected 188
and concentrated using a Centriprep YM-30 filter unit. The enzyme solution obtained 189
was the purified preparation used for enzyme characterization. 190
Analysis of enzymatic properties. General ADH activity was assayed 191
spectrophotometrically at 25°C by measuring the decrease in the absorbance of NADH 192
at 340 nm (ε = 6220 M-1·cm-1). Each reaction mixture contained 10 μmol PTK or other 193
substrates, 0.3 μmol NADH, 1 mmol KPB (pH 7.0), and 10 μL enzyme solution in a 194
total volume of 1.0 mL. The oxidation activity of ADH was also measured at 340 nm in 195
reaction mixtures with a total volume of 1.0 mL containing 10 μmol 2-propanol as the 196
substrate, 1.0 μmol NAD+, 1 mmol KPB (pH 7.0), and 10 μL enzyme solution. One unit 197
of enzyme was defined as the amount that converted 1 μmol NADH or NAD+ in 1 min 198
under these conditions. The kinetic parameters of HLADH-012 and -021 were 199
calculated from a Lineweaver-Burk plot. Protein concentration was estimated by 200
measuring the absorbance of protein-containing solutions at 280 nm or by the method of 201
Bradford (31) using bovine serum albumin as a standard (Bio-Rad Protein Assay Kit; 202
Bio-Rad Lab., Hercules, CA). 203
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 204
performed using 12% (w/v) polyacrylamide slab gels and the Tris-glycine buffer system 205
of Laemmli (32). The molecular mass of the enzyme subunit was determined from the 206
relative mobility of standard proteins. 207
The molecular mass of the native recombinant enzyme was determined using a 208
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Shimadzu LC-10 HPLC system (Shimadzu, Kyoto, Japan) equipped with a TSK-GEL 209
G3000SWXL column (Tosho) as follows: 10 μL of the obtained sample solution was 210
separated by using a mobile phase containing 20 mM KPB and 200 mM NaCl (pH 7.0). 211
The flow rate was 1.0 mL/min and the column temperature was kept at 25°C. The 212
protein was monitored at 280 nm and molecular mass was estimated from the retention 213
times of authentic molecular weight markers (Oriental Yeast Co., Ltd., Tokyo, Japan). 214
Analysis of enzymatic products. For the analysis of enzymatic products, the 215
reaction mixture contained 50 µmol KPB (100 µmol for 1-Boc-pyrrolidinone, 200 µmol 216
for 3-quinuclidinone) (pH 7.0), 50 mg substrate, 1 µmol NAD+, 5% (v/v) 2-propanol, 217
and recombinant E. coli cells collected from a 10-mL culture broth. The resting cell 218
reaction proceeded for 24 h at 25°C with shaking at 2500 rpm using a microtube shaker 219
(M-BR-022UP; Taitec, Saitama, Japan). Next, the mixture was extracted twice with 220
ethyl acetate (1-butanol by raising the pH to 12 with 6 N NaOH for 3-quinuclidinol) 221
(33), and the combined extracts were dried using anhydrous Na2SO4 before analysis. 222
The absolute configuration and enantiomeric purity of the product was evaluated using 223
the peak areas of alcohol products visible on GC or HPLC compared with authentic 224
compounds, following previously reported procedures (23,25,33). 225
Nucleotide sequence accession numbers. The nucleotide sequences of the 226
metagenomic adhs determined, Hladh-001 to -040, homologs of lsadh from L. poae 227
NBRC103069 (lpadh), and L. naganoensis NBRC103131 (lnadh), were submitted to 228
the DNA Data Bank of Japan under the accession numbers AB916600–AB916639, 229
AB917070, and AB917071, respectively. 230
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RESULTS AND DISCUSSION 233
Metagenome preparation, design of primers, and amplification of adh genes. The 234
average weight of DNA obtained from 0.5 g (wet weight) of the soil samples and those 235
of the farm and bark composts used in this study was 1.8 ± 1.3 (standard deviation 236
[SD]) and 2.7 ± 2.3 (SD) µg, respectively; thus, the SDs were very high because yields 237
varied greatly depending on the nature of the soil. We isolated metagenomic DNA from 238
20 soil and compost samples, and these served as templates for the amplification of 239
homologous lsadh genes. 240
Some of the primer sets were designed with reference to the N- and C-terminal region 241
sequences of lsadh and its related genes (Table 1), which belong to the short chain 242
dehydrogenase/reductase (SDR) family. SDRs are proteins of approximately 250–300 243
amino acid residues, have a wide variety of substrate specificities (34), and are useful 244
biocatalysts for producing chiral alcohols from various ketones (35). Alignment analysis 245
of some ADHs belonging to the SDR family at the terminal regions showed that they 246
shared 37–58% identity with LSADH (Fig 1), which clearly indicated the well 247
conserved regions among them. “GXXXGXG” can interact with coenzyme NAD(P)H 248
in the near N-terminus region and “VDGGXTA” in the C-terminus region, which 249
consists of a β-sheet secondary structure in SDRs. Primers of ~30 bp were designed 250
based on these sequences and containing the regions for fusion with the expression 251
vector pKELA-del (Fig. 1). A combination of the primers F-2 and R-2 was used to 252
amplify nearly full-length genes from the metagenomes after they were fine-tuned. 253
Adjustments were made for the regeneration of codon usage, to primer length and Tm 254
for the prevention of primer duplex formation, to primer set combinations, and to 255
optimize other PCR conditions. 256
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We used hot-start and step-down PCR protocols to avoid non-specific amplification 257
and to support the sufficient amplification of DNA using KOD FX Neo DNA 258
polymerase or Phusion® Hot Start Flex DNA polymerase. Both polymerases offer high 259
fidelity and robust performance. It was important to optimize and fine-tune the PCR 260
conditions, including the choice of DNA polymerase, primer concentration, and so on, 261
to obtain successful amplification of the genes from the metagenomes. We observed 262
100% amplification of target genes from 12 general soil metagenomic samples isolated 263
from farms and paddy fields or garden soil samples collected in Japan; in addition, 264
target genes from 3 genomes isolated from genus Leifsonia (L. poae NBRC103069, L. 265
naganoensis NBRC103131, and L. aquatica NBRC15710) were amplified using KOD 266
FX Neo DNA polymerase, and 8 samples of farm and bark composts in fermentation at 267
35–80°C as well as target genes isolated from 4 genomes (L. aurea NBRC104579, L. 268
ginsenji NBRC104580, L. pindaroensis JCM15132, and L. shinshuensis NBRC103132) 269
were amplified using Phusion® Hot Start Flex DNA polymerase. Under the optimized 270
PCR conditions, we successfully amplified genes from all 20 independent 271
environmental metagenomes and 7 Leifsonia sp. genomes. The results showed that once 272
the amplification conditions for PCR were determined, the target genes could be 273
obtained easily from various metagenomes. Thus, this simple PCR-based approach has 274
tremendous biotechnological potential for obtaining useful or novel enzyme genes from 275
metagenomes. 276
Screening and analyses of adh genes from the metagenomic library. 277
PCR-amplified genes were fused with the original lsadh gene at both terminal regions in 278
pKELA-del, an expression vector of the homologous lsadhs in E. coli constructed from 279
pKELA (27), as described in the Materials and Methods. They were then transferred 280
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into E. coli cells and expressed. This simple technique prevented the ambiguous 281
expression of the target genes and enabled high-throughput screening of enzyme activity. 282
Approximately 2000 colonies obtained from 20 soil and compost metagenomes were 283
measured directly using their enzymatic activity for two substrates, 1,1-dichloroacetone 284
and PTK; 1,1-dichloroacetone is a small-sized molecule and a good substrate for many 285
ADHs, whereas PTK is a medium-sized molecule and an original substrate of LSADH. 286
Approximately 1200 (60%) of the 2000 colonies were positive in a spectrophotometric 287
assay using 1,1-dichloroacetone or PTK as the substrate. Thus, a library containing 288
homologous genes of lsadh was constructed efficiently by gene-specific PCR 289
amplification of metagenomes and fusion with a suitable expression vector. 290
To facilitate the analysis of the adh genes from the library, 223 of the 1200 clones 291
(~20%) were selected randomly and sequenced. Genes were given the descriptor Hladh 292
(homologous Leifsonia adh) depending on their similarity to the lsadh gene. After 293
eliminating duplicate genes in terms of amino acid sequence, 40 different genes were 294
isolated from the 223 clones: 29 from bark compost in fermentation (50–80°C), 7 from 295
farm compost (35–45°C), and 4 from general soil samples. Notably, the amino acid 296
sequences encoded by the Hladh genes obtained via this metagenomic approach varied 297
greatly, especially those isolated from the bark compost samples. The results are 298
summarized in Table 2. Amino acid sequence identity was compared with that of lsadh, 299
except at the chimeric regions (1–12 and 249–251 amino acids in LSADH consisted of 300
251 amino acid residues), by BLASTP analysis (36). The isolated genes were divisible 301
into at least 5 groups: Hladh-001 to -011 obtained from the general soil and farm 302
compost (35–45°C) samples, 98–99% amino acid sequence identity with lsadh; 303
Hladh-014, -015, and -016 from bark compost (50–80°C), 73–75%; Hladh-012, -013, 304
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-017 to -025, and -034 to -038 from bark compost (50–80°C), 50–63%; Hladh-026 to 305
-032, from bark compost (50–80°C), 36–44%; and Hladh-033, -039, and -040 from bark 306
compost (50–80°C), <17%. Table 3 shows the results of the BLASTP analyses of the 307
isolated adh gene products with known SDRs. HLADH-012, -013, -017 to -026, and 308
-031 to -040 shared <65% sequence identity with known SDRs including putative ones; 309
therefore, we deduced that they were novel functional ADHs. Interestingly, 45 of the 61 310
LSADH homologs isolated from the general soil and farm compost samples completely 311
matched the amino acid sequence of LSADH (Leifsonia sp. S749, a styrene-tolerant 312
strain isolated from soil in Japan) (24) or LNADH (L. naganoensis isolated from soil in 313
Japan) (37) (Table 2), suggesting that the genes of both strains either fit the primer set 314
very well or they are major Leifsonia habitants in soil environments in Japan. The 315
results also indicated that the origin of the metagenome is the key to obtaining novel 316
ADH genes. We presumed that metagenomic DNA isolated from general soil 317
environments may include DNA from dominant microorganisms, including genus 318
Leifsonia, which could prevent the amplification of diverse adh genes, whereas special 319
or extreme environments (such as bark compost fermented at high temperature) could 320
lead to the amplification of diverse target genes. The fact that HLADH-027 to -030 were 321
very similar to the SDR from Sphaerobacter thermophilus, a thermophilic bacterium, 322
supports this theory. Figure 2 displays the phylogenetic analysis of the gene products 323
with known SDRs. Alignment analysis of the HLADHs and LSADH is shown in Fig. 3. 324
These data showed that our approach could cover a wide range of SDR genes including 325
lsadh and its related homologs. Of course, our library did not necessarily guarantee the 326
isolation of complete adh genes even if they were functional, because the obtained 327
genes were chimeric with lsadh at the terminal regions and thus they were artificial. 328
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However, this gene-specific amplification approach using metagenomes offers an 329
excellent opportunity to identify novel subfamilies of enzyme genes that were 330
previously unknown and uncharacterized. In our approach, the GAM technique attained 331
a high efficiency to obtain target genes; 1200 clones in 2018 colonies (~60%) were 332
adh-positive. On the contrary, previous reports with function-based screening indicate a 333
low hit rate (<1.2%) for the identification of target activity from a metagenomic library 334
(3,4). Thus, the efficiency of our approach to find target activity in an Hladh library is 335
much higher than in previous studies. Recently, some sequence homology-based 336
metagenomic approaches have been used to elucidate the abundance of novel tfdA-like 337
(dioxygenase) genes (38) and aromatic dioxygenase genes (39) in soil and dmdA 338
(dimethylsulfoniopropionate demethylase) genes in marine environments (40) by using 339
suitable primer sets for GAM. Similarly, the high efficiency of our approach is due to 340
the excellent PCR-amplification of target genes from a suitable metagenome-containing 341
sample such as bark compost fermented at a high temperature. The adoption of a fusion 342
technique with a high-level expression vector for the target gene (pKELA-del for lsadh), 343
which can avoid the ambiguous expression of the gene, should also contribute to our 344
high efficiency. Our approach is based on sequence homology, but it is incorporated 345
with the technique of function-based screening. Using the data presented in Table 2, we 346
estimate that 200 or more different adh genes could be discovered if all 1200 positive 347
clones were sequenced. 348
Screening of suitable ADHs for biocatalysis from the isolated adh genes. As 349
confirmed by the primary screening, all isolated HLADHs were active with 350
1,1-dichloroacetone, and most HLADHs including HLADH-001 to -025, -27 to -30, and 351
-34 to -38 were also active with PTK. Therefore, the activities of HLADHs for some 352
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ketones including 1-Boc-pyrrolidinone, 2′-chloroacetophenone, 3-quinuclidinone, and 353
ethyl benzoylformate, which cannot be converted easily to anti-Prelog chiral alcohols 354
with high yield or enantioselectivity by general ADHs including LSADH, were 355
evaluated for their enzymatic function. PTK and acetophenone were used as positive 356
controls, and crude enzymes obtained from the E. coli libraries were used for screening. 357
HLADH-012, -013, and -021 showed relatively high levels of activity toward these 358
ketones compared with LSADH, and HLADH-021 possessed wide substrate specificity 359
and demonstrated activity toward all ketones tested (Fig. 4A). Moreover, the results 360
indicated there were large differences in the enzymatic properties of each enzyme, such 361
as activity and substrate specificity, suggesting that examination of the amplicons from 362
the metagenomic E. coli library can be used to obtain an enzyme catalyst imparting the 363
desired substrate specificity. 364
The high tolerance of enzymes to organic solvents is a desirable function when they 365
are to be applied to organic synthesis as biocatalysts in harsh conditions. From this point 366
of view, the crude enzymes obtained from the libraries were subjected to tolerance 367
testing with 3 organic solvents possessing different logPow ratios (41), in which Pow is 368
defined as the ratio of the equilibrium concentrations of a dissolved substance in an 369
n-octanol and water two-phase system: n-octane (logPow: 4.5), butyl acetate (1.7), and 370
2-methyltetrahydrofuran (1.0). The crude enzyme solution was treated with 50% (v/v) 371
organic solvent and shaking for 30 min at room temperature, and the remaining activity 372
was measured. HLADH-012 and -037 were highly tolerant of all organic solvents tested 373
(Fig. 4B). Conversely, LSADH and LPADH readily lost their activity. Generally, 374
2-methyltetrahydrofuran greatly affects enzymes because of its low logPow, that is, its 375
high polarity; however, HLADH-012 showed relatively high tolerance to this solvent. 376
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The strong inhibitory effect of n-octane on most of the enzymes tested could not be 377
explained despite its medium-range logPow. Purified HLADH-012 and -021 were later 378
subjected to the same test and almost the same results were obtained, which confirmed 379
that these enzymes possess tolerance in the absence of concomitant proteins in a crude 380
solution. 381
We successfully obtained these superior enzymes from our limited library; 382
HLADH-021 possessed wide substrate specificity and HLADH-021/-037 demonstrated 383
organic-solvent tolerance. The results clearly indicated that the S-GAM technique is 384
very efficient and useful for obtaining a target enzyme from a metagenome. 385
Purification and characterization of HLADH-012 and HLADH-021. Preliminary 386
experiments showed that both enzymes were inactive when His × 6 tag was added to 387
their N-termini. Therefore, recombinant HLADH-012 and -021 were purified without 388
the addition of a specific tag. HLADH-012 was purified 11.8-fold, to homogeneity, 389
from the cell-free extract of a 200-mL culture of recombinant E. coli cells (Table 4). 390
Purified HLADH-012 produced 9.0 U/mg of protein when PTK was the substrate. 391
HLADH-021 was purified 13.0-fold, to homogeneity, from the cell-free extract of a 392
100-mL culture by sequential column chromatographic steps. Purified HLADH-021 393
showed relatively high activity, producing 122.3 U/mg of protein when PTK was the 394
substrate. The purity of both enzymes was evaluated by SDS-PAGE (Fig. S1) and 395
analytical HPLC using a TSK Gel 3000SWXL column. Both enzyme preparations 396
appeared to be almost pure. 397
Analytical HPLC yielded molecular masses of 121 kDa for HLADH-012 and 120 398
kDa for HLADH-021. The theoretical subunit molecular mass was 26191 Da for 399
HLADH-012 and 26291 Da for HLADH-021. Thus, like LSADH, both enzymes were 400
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tetramers of identical subunits. 401
The Km values of both enzymes in reductive reactions with PTK and NADH at pH 402
7.0 were calculated by Lineweaver-Burk plots at 0.89 ± 0.14 mM and 0.14 ± 0.002 mM 403
for HLADH-012 and 1.4 ± 0.3 mM and 0.027 ± 0.009 mM for HLADH-021, 404
respectively. Neither enzyme utilized NAPDH as a coenzyme. In oxidative reactions, 405
the Km values of both enzymes with 2-propanol and NAD+ at pH 7.0 were calculated as 406
73.2 ± 7.8 mM and 0.77 ±0.09 mM for HLADH-012 and 1.9 ± 0.3 mM and 0.87 ± 0.09 407
mM for HLADH-021. Notably, the high kcat value (345 ± 49 s-1) of HLADH-021 for 408
PTK indicated the potential of this enzyme as a biocatalyst. 409
The effect of pH on the activity of both enzymes is shown in Fig. S2. HLADH-012 410
showed maximum activity at pH 6.0 in the reductive reaction and at pH 8.0 in the 411
oxidative reaction. The pH profile of HLADH-021 was similar to that of HLADH-012; 412
however, HLADH-012 showed higher thermal stability than HLADH-021. The 413
enzymatic properties of HLADH-012 and -021 are summarized in Table 3. 414
Production of anti-Prelog chiral alcohols using E. coli whole-cell system 415
expressing HLADH-012 or HLADH-021. HLADH-012 and -021 were evaluated as 416
potential biocatalysts. Self-regeneration of NADH with 2-propanol as a hydrogen donor, 417
coenzyme and substrate specificity, activity and stereoselectivity, and robustness during 418
the reaction conditions were assessed (Tables 5 and 6). In addition, an E. coli whole-cell 419
system with 5% (v/v) 2-propanol as a hydrogen donor was used to produce anti-Prelog 420
chiral alcohols from some of the ketones, and the enantiomeric excess (e.e.) of the 421
alcohols produced was determined (Table 6). It was confirmed that the production levels 422
of chiral alcohols were relatively high for HLADH-021, although the reactions were not 423
optimized. Moreover, the e.e. of the chiral alcohols produced was excellent for products 424
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from 2′-chloroacetophenone, 3-quinuclidinone, and ethyl benzoylformate. Interestingly, 425
the stereoselectivity of HLADH-021 toward 1-Boc-3-pyrrolidinone and ethyl 426
benzoylformate was contrary to that of LSADH. As expected, the performance of 427
HLADH-012 in the production of various chiral alcohols was not outstanding because it 428
was selected for its high tolerance to organic solvents as an indicator. 429
These results suggested that the S-GAM technique is quite efficient and useful for 430
identifying suitable biocatalysts with superior functions. It would be easy to obtain 431
additional novel adhs by continuing this approach for the metagenomes of bark compost. 432
Thus, a suitable GAM technique offers the opportunity to make a useful collection of 433
desirable genes that exist in nature. Of course, the S-GAM technique could be applied 434
to other enzyme genes and is a promising approach for enzyme engineering and 435
biocatalysis. 436
437
SUPPLEMENTAL MATERIAL 438
Supplemental materials for this article may be found at ; Figure S1 and 439
Figure S2, PDF file, 375 kB. 440
441
ACKNOWLEDGMENTS 442
This work was supported by a Grant-in-Aid for Scientific Research (B) from the 443
Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by a 444
grant from the Japan Foundation for Applied Enzymology. We thank Sumitomo 445
Chemical Co., Ltd., Osaka, Japan for their financial and technical support. 446
447
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573
574
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FIGURE LEGENDS 575
576
FIG 1. Primer design (A) and schematic procedure of the S-GAM technique (B). Primers were 577
designed based on the alignment of LSADH and putative SDRs from some microorganisms 578
constructed by CLUSTALW. Identical residues at the terminal regions are shown by white 579
letters on a black background. The conserved amino acid sequences surrounded by a dotted 580
square were used as reference points in the design of each primer set. 581
582
FIG 2. Phylogenetic analysis of HLADHs (bold red letters) including the chimeric parts of 583
LSADH with known SDRs from various microorganisms with their accession numbers. The 584
phylogenetic tree was constructed using Kimura’s method with CLUSTALW in the DNA Data 585
Bank of Japan. HBADH-1 and HPADH-24 are gene products isolated from metagenomes (17). 586
The scale bar represents the calculated nucleotide substitution ratio. 587
588
FIG 3. Amino acid sequence alignment of HLADHs with LSADH and LPADH. LSADH 589
(accession no. AB213459); LPADH (AB917070), HLADH-012 (AB916616); HLADH-014 590
(AB916611); HLADH-021 (AB916614); and HLADH-027 (AB916626). Alignment was 591
performed using CLUSTALW software. Identical residues are shown by white letters on a black 592
background. A putative coenzyme-binding region is shown inside a dotted square. The amino 593
acid residues considered important for enzymatic activity are marked with an asterisk. The 594
coenzyme-interacting Asp39 residue for NAD＋/NADH-dependent enzymes is boxed by a solid 595
line. The closed arrows indicate the chimeric regions of LSADH, and the open arrows indicate 596
the binding sites of the primers. 597
598
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FIG 4. Substrate specificity (A) and organic solvent tolerance (B) of HLADHs. Crude enzyme 599
solutions prepared from recombinant E. coli cultures were used for each assay. The bar indicates 600
the SD of 3 measurements. All substrate concentrations were 10 mM, and activity for PKT was 601
defined as 100% (A). Crude enzymes were treated with each organic solvent (50% [v/v]) and 602
were shaken for 30 min at room temperature before the remaining activity was measured (B). 603
604
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TABLE 1. Primers used for gene-specific amplification of lsadh from metagenomes 605 Primer (length) Sequence (5′–3′)
F-1 (25) CGGTCCGCGATCCTVACMGGMGSMG
F-2 (28) GACCGGTCCGCGATCGTVACMGGMGSMG
F-3 (28) GATCGTTCAGCAATCGTVACMGGMGSMG
F-4 (35) CGGTCCGCGATCGTGACCGGMGSCGGSTCSGGSAT
F-5 (35) GCCGACCGGTCCGCGATMGTGACCGGMGSCGGSTC
R-1 (25) TCACTGGGCGGTRTABCCRCCRTCB
R-2 (28) CGGTCACTGGGCGGTRTABCCRCCRTCB
R-3 (29) CGGTCACTGGGCGGTRTABCCRCCRTCBA
R-4 (28) CGGTTATTGAGCAGTRTABCCRCCRTCB
R-5 (29) CGGTTATTGAGCAGTRTABCCRCCRTCBA
R-6 (35) CACTGCTCGGTGTAGCCGCCRTCSACCAGRTGRTA
R-7 (35) CGGTCACTGCGCGGTSTAGCCGCCRTCSACCAGRT
V: G,A,C, M: A,C,S: G,C, R: A,G, B: G,T,C. No amplification was observed for the 606 following primer combinations: F-3/R-3, F-3/R-4, F-3/R-5, F-4/R-6, F-4/R-7, F-5/R-6, and 607 F-5/R-7. Combinations other than F-1/R-1 and F-2/R-2 were not tested. 608 609 610 on January 29, 2018 by guest
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TABLE 2. Numbers of adh amplicons from different metagenomes and their gene analysis data 611 Origin of metagenome
(sample number) Number of genes
sequenced/colonies Number of genes
different from lsadh /duplicates
Soil (12) 27/185 4/23
Farm compost (3) 34/495 7/27
Bark compost (5) 162/1338 29/133
Total (20) 223/2018 40/183
612
613
614
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TABLE 3. BLASTP analysis of isolated Hladh genes from metagenomes 615 Clone No. Description Identity
(amino acid)
(%)
Identity with LSADH
(amino acid) (%)
001–011 Short chain alcohol dehydrogenase (Leifsonia sp. S749) 98–99 98–99
014–016 Short chain alcohol dehydrogenase (Leifsonia sp. S749) 73–75 73–75
012 2,5-Dichloro-2,5-cyclohexadiene-1,4-diol
dehydrogenase (Paenibacillus sp. HGF7)
52 52
013
017–019
021–022
034–038
SDR (Truepera radiovictrix DSM 17093) 55–64 55–63
020 SDR (Pedobacter sp. BAL39) 55 55
023 Oxidoreductase, SDR family protein
(deltaproteobacterium NaphS2)
49 50
024, 025 SDR (Chelativorans sp. BNC1) 51–52 50–52
026 Putative oxidoreductase, SDR family (Variovorax
paradoxus B4)
55 40
027–030 SDR (Sphaerobacter thermophilus DSM 20745) 91–96 44
031 SDR (Desulfatibacillum alkenivorans AK-01) 57 36
032 SDR (Thermobaculum terrenum ATCC BAA-798) 53 43
033, 039, 040 Molybdopterin molybdochelatase (Desulfomonile tiedjei
DSM 6799)
48 8–17
616
617
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TABLE 4. Purification of recombinant HLADH-012 and HLADH-021 618
Step
Total
protein
(mg)
Total
activity
(U)
Specific
activity
(U/mg)
Yield
(%)
Purification
(-fold)
HLADH-012 (200-mL culture)
Cell-free extract 165.2 125.0 0.76 100 1
Butyl-Toyopearl 2.14 7.1 3.3 5.7 4.5
Resource Q 0.20 1.8 9.0 1.4 11.8
HLADH-021 (100-mL culture)
Cell-free extract 67.0 630.1 9.40 100 1
Butyl-Toyopearl 2.05 45.3 22.1 7.2 2.3
DEAE-Toyopearl 0.13 15.9 122.3 2.5 13.0
619
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TABLE 5. Properties of purified HLADH-012 and HLADH-021 compared with those of 620 LSADH 621
Property HLADH-012 HLADH-021 LSADH1)
Molecular mass2) 105 kDa 105 kDa 100 kDa
Subunit structure2) 26.2 kDa
(homotetramer)
26.2 kDa
(homotetramer)
25.0 kDa
(homotetramer)
pI 2) 4.89 4.75 4.58
Coenzyme NAD+/NADH NAD+/NADH NAD+/NADH
Km
PTK (reduction) 0.89 ± 0.14 mM3) 1.4 ± 0.3 mM 13.6 mM
NADH 0.14 ± 0.002 mM 0.027 ± 0.009 mM 0.048 mM
2-propanol (oxidation) 73.2 ± 7.8 mM 1.9 ± 0.3 mM 57.5 mM
NAD+ 0.77 ± 0.09 mM 0.87 ± 0.09 mM 0.12 mM
kcat (s-1)4)
PTK 21 ± 0.3 345 ± 49 127
2-propanol 35 ± 4 47 ± 6 43
Optimum pH,
reduction/oxidation 6.0/8.0 6.0/8.0 6.0/9.5
Thermal stability5) <55°C <40°C -
1) Data from refs. 21, 22, and 24. 622 2) Theoretical calculation derived from study of amino acid sequences. 623 3) The errors indicate the SD of 3 measurements. 624 4) Value for 1 mol enzyme consisting of 4 subunits. 625 5) Temperature indicating more than 70% of the original activity after treatment at each temperature 626 for 30 min at pH 7.0. 627 628
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TABLE 6. Biocatalytic properties of HLADH-012 and HLADH-021 compared to those of 629 LSADH for producing anti-Prelog chiral alcohols 630 Compound Relative activity (%), Production level (mg·mL-1),
Enantiomeric excess (%)/Absolute configuration
HLADH-012 HLADH-021 LSADH1)
CF3
O
PTK
100 (9.0 U·mg-1)
ND2)
>99%/(S)
100 (122.3 U·mg-1)
ND
>99%/(S)
100 (10.3 U·mg-1)
256
>99%/(S)
2′-Chloroacetophenone
9.0 ± 0.43)
26.5 ± 2.1
>99/(R)
1.8 ± 0.2
38.2 ± 3.0
>99/(R)
1.0
1.5
>99/(R)
2-Hydroxyacetophenone
1.3 ± 0.5
ND
ND
2.2 ± 0.06
ND
ND
0
-
-
1-Boc-3-pyrrolidinone
9.6 ± 0.4
19.6 ± 1.5
5.8%/(S)
8.3 ± 0.2
48.1 ± 3.5
51.2%/(S)
2.0
3.5
>99.9/(R)
3-Quinuclidinone
0
-
-
1.3 ± 0.3
19 ± 1.5
>99/(R)
0
-
-
Ethyl benzoylformate
8.3 ± 0.2
40.6 ± 3.2
>99/(R)
8.5 ± 0.4
40.0 ± 3.0
>99/(R)
3
16.3
23%/(S)
1) Data from refs. 23 and 24. 631 2) ND: not determined. 632 3) The errors indicate the SD of 3 measurements. 633 634 635
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Transformation
E.coli JM109
Insert
Vector
In-
Fusion
Hladh genes library
pKELA-del
PCR
Metagenome
Leifsonia sp. S749 1:MAQYDVADRSAIVTGGGSGIGRAVALTLAA 30 232:DAASFITGSYHLVDGGYTAQ 251 Mesorhizobium sp. BNC1 1:-MTGEFKDKVALVTGAGSGIGAAIARELAT 29 232:GRASFITGSYHLVDGGYTTR 251 Paracoccus denitrificans PD1222 1:-MDIRFDNKIALVTGAGSGLGEAIALELAA 29 230:DRASFITGSYHLVDGGYTAL 249 Sphingomonas wittichii RW1 1:-MNRRFEGKVAIVTGAGAGIGRACMARLAS 29 229:DEAPHIHGGAYLIDGGRSAV 248 Clavibacter michiganensis NCPPB 382 1:--MARFDTKTALVTGGGSGIGAAISRALAA 28 231:DDASFISGSYHLVDGGYSAR 250 Stenotrophomonas maltophilia K279a 1:MIDYQLTGKTAIVTGGVSGIGLAVAQTLAA 30 233:DDASFATGAYYAIDGGYLAQ 252 Sinorhizobium meliloti RU11/001 1:-MKLGLEGKIAIVTGAGSGIGAAVSRQLGG 29 232:EQASFITGSYHPVDGAFTAH 251
F-2 primer: GACCGGTCCGCGATCGTVACMGGMGSMG
15 nucleotides for fusion
R-2 primer: CGGTCACTGGGCGGTRTABCCRCCRTCB
15 nucleotides for fusion
(A)
(B)
730 bp
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LSADH, LNADH, HLADH-001～011
LPADH HLADH-014 HLADH-015 HLADH-016
Chelativorans sp. BNC1 (YP_674781.1) Truepera radiovictrix (YP_003704562.1)
HLADH-013, -017～019, -021, -022, -034～037
HLADH-038 HLADH-020
Pedobacter sp. BAL39 (WP_008239543.1) HLADH-023
Gemmatimonadetes bacterium (AHG92594.1) HLADH-012
HLADH-024, -025
Pseudomonas putida (YP_001748299.1)
Pseudomonas fluorescens (YP_005209598.1)
short-chain dehydrogenase (WP_009675721.1)
HPADH-24 (AB823234)
Lactobacillus kefiri (AAP94029.1)
Sphaerobacter thermophilus (YP_003321098.1)
HLADH-029 HLADH-028 HLADH-030
HLADH-026
putative oxidoreductase (YP_008493858.1)
Arthrobacter sp. Rue61a (YP_006661724.1)
Burkholderia multivorans (YP_001585274.1)
Burkholderia cepacia (YP_006619627.1) HBADH-1 (AB823216)
HLADH-031
Desulfatibacillum alkenivorans
(YP_002429702.1)
Bifidobacterium dentium
(YP_003361274.1)
HLADH-032
Thermobaculum terrenum (YP_003324228.1)
Rubrobacter xylanophilus (YP_644628.1)
HLADH-033, -039, -040 Desulfomonile tiedjei (YP_006446649.1)
Mycobacterium gilvum (YP_001135406.1)
Gordonia polyisoprenivorans (YP_005283595.1)
Thermoanaerobacter brockii
(YP_001666148.1)
Burkholderia ambifaria
(YP_774220.1)
Bifidobacterium bifidum (YP_003971063.1)
Riemerella anatipestifer (YP_006017524.1)
Bacillus thuringiensis (WP_003270455.1)
Klebsiella pneumoniae
(YP_003754047.1)
Haloferax mediterranei (YP_006350219.1)
Clostridium stercorarium
(YP_007373341.1)
Bacillus pseudofirmus (YP_003427628.1)
Azoarcus sp. KH32C
(YP_007549497.1)
Amycolatopsis orientalis (YP_008012822.1)
Aromatoleum aromaticum (YP_159700.1)
Parvibaculum lavamentivorans
(YP_001411696.1)
Natrialba magadii (YP_003478984.1)
Desulfobulbus propionicus
(YP_004195929.1)
Paenibacillus sp. Y412MC10
(YP_003244945.1)
Sphingobium japonicum
(YP_003543945.1)
HLADH-027
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LSADH 1 MAQYDVADRSAIVTGGGSGIGRAVALTLPADH 1 MAQYDVADRSAIVTGGGSGIGRAVALT
MAQYDVADRSAIVTGGGSGIGRAVALMAQYDVADRSAIVTGGGSGIGRAVAL
HLADH-012 1 MAQYDVADRSAIVTGGASGIGLATVRLHLADH-014 1 MAQYDVADRSAIVTGGGSGIGRAVALTHLADH-021 1 MAQYDIADRSAIVTGGASGIGAAVVEKHLADH-027 1 MAQYDIADRSAIVTGGGSGIGRAAALA
MAQYDVADRSAIVTGG SGIG AMAQYDVADRSAIVTGGGSGIGRAVALMAQYD ADRSAIVTGG SGIG AVMAQYD ADRSAIVTGGGSGIGRA AL
LSADH 52 IEAAGGKAAALAGDVTDPAFGEASVAGI A GG A DV P A VLPADH 52 IQAAGGTAASLIGDVTDPEFAVASVARHLADH-012 51 LQAQGFRVRFFKVDVSKEEEVIALVQDHLADH-014 52 IQAAGGTAVAHVGDVADPADATAAVEAHLADH-021 52 LTADGKTASFFKVDVAEPDQVEAMVRHHLADH-027 52 ITAVDGEALGIRTDVSRAADVEAMVRT
I A GG A DV P A VA G DV A V
I A GG A DV P A VA G A DV P A V
I A G A DV A V
S 100 G S S G QGLSADH 100 TVGDYSLDSWRTVIEVNLNAVFYGMQPLPADH 100 KVGDYSLDSWRKVIEINLNAVFYSLRAHLADH-012 99 PVGDKTTEEWNRVIGVNLNGVFWCNKYHLADH-014 100 PTGDYPIEAWQKVIDINLSAVMYGMRAHLADH-021 101 PTGEYGIDDWKKVIDINLNGVFYGLRYHLADH-027 103 RVTEIDEAVWDAVLAVNLKGTFLCCKY
VGDY W VI NLN VFYVGDY W VI NLN VFYVGD W VI NLN VFGDY W VI NL V YG Y W VI NLN VFYV W V NL F
* *LSADH 151 FANSSAYVTAKHALLGLTQNAALEYAALPADH 151 FANSSAYVTAKHALLGLTQNAALEYADHLADH-012 150 FNNAPAYCAAKHGIIGMTKTIALDHSKHLADH-014 151 FAGSPAYVAAKHGVVGATKNAALEYAAHLADH-021 152 FANAPAYVAAKHGVVGLTKNAAIEYATHLADH 027 154 EPDLDAYTASKGGVLALTRSIAAGYAR
FAN AYV AKH GLT NAALEYAFAN AYV AKH GLT NAALEYAF N AY AAKHG G T ALFA AYVAAKHG G T NAALEYAFAN AYVAAKHG GLT NAA EYA
AY A K G LT A YA
* *
HLADH-027 154 EPDLDAYTASKGGVLALTRSIAAGYARAY A K G LT A YA
LSADH 202 DALAFLEGKHALGRLGEPEEVASLVAFLPADH 202 DALAFLEGKHALGRLGEPEEVASLVAFHLADH-012 199 DQLDFLTTLHPMGRLGRPEEVAEAVLFHLADH-014 202 DALTFLEGKHALGRLGEPDEVAALVAFHLADH-021 203 ETMKVIAGMHPVQRMGTPDEVANLITY
D L FLEG H LGRLGEPEEVA LV FD L FLEG H LGRLGEPEEVA LV FD L FL H GRLG PEEVA V FD L FLEG H LGRLGEP EVA LV F
G H R G P EVA LHLADH 021 203 ETMKVIAGMHPVQRMGTPDEVANLITYHLADH-027 204 ETLRRFE-QETLLPIGEPEDIGHLVVY
G H R G P EVA LL E L GEPE LV
TLAASGAAVLVTDLNEEHAQAVVAE 51TLAASGASVLVTDLNEKNANAVVAE 51LA GA V V D E A AVV ELA GA V V D E A AVV ELFAQEGAAVVIGDYSD-AGQAVAEE 50TLAANGAKVVVADLKKESADAVVAE 51KFAGNGAKVVVADFDEVGGRAMVDK 51ALAHEGAKVVIADYNEAAAQAVAQE 51
A GA VV D AV ELA GA VVV D A AVV EA GA VVV D E A VLA GA VV D E A AV E
GA-NALAPLKIAVNNAGIG--GEAA 99A L IAVNNAGIG G ARA-NELAPLRIAVNNAGIG--GEAA 99DAVTAYGRLDIMVANAGI---GDSA 98AA-EKLAPLKIAVNNAGIG--GPAA 99HAVDTYGGLHIAVNNAGIG--GASA 100TTVERFGRVDVLFNNAAVALVGRDN 102
A L IAVNNAGIG G AA L I V NAGI G AA L IAVNNAGIG G AA L IAVNNAGIG G A
NNA G
Q GGG S GS G 1 0GGG S G G*
PQLKAMAANGGGAIVNMASILGSVG 150AQLDAIAANGGGSIVNMASILGSVG 150YAIQEFRKVGGGVIVNMASILGHVG 149AQLPAMVKNGGGSIVNIASILGSVG 150YEIPAMLESGGGAIVNVASILGRVE 151YAIPAMTANGGGSIINNASIAALVA 153
AM NGGG IVN ASILG VGA NGGG IVN ASILG VG
GGG IVN ASILG VGAM NGGG IVN ASILG VGAM GGG IVN ASILG VAM NGGG I N ASI V
ADKVRVVAVGPGFIRTPLVEANLSA 201DQKVRVTAVGPGFIRTPLVEANMSA 201KDNIRANCVCPGFILTPLIGS--DK 198AQGVRVNSVGPGFIKTPLVESSLDS 201TQNIRVVSVGPGFIKTPLLDKNLDE 202RDGIRCNAICPGLVRTPMTAS VAN 203
RV VGPGFI TPLRV VGPGFI TPLR V PGFI TPLRV VGPGFI TPLRV VGPGFI TPLR PG TPRDGIRCNAICPGLVRTPMTAS-VAN 203R PG TP
FLASDAASFITGSYHLVDGGYTAQ 251FLASDAASFITGSYHLVDGGYTAQ 251FLASDASSFITGSSLMVDGGYTAQ 248FLASDAASFITGSYHLVDGGYTAQ 251YLVSDEASFLTGGYYLVDGGYTAQ 252
FLASDAASFITGSY LVDGGYTAQFLASDAASFITGSY LVDGGYTAQFLASDA SFITGS VDGGYTAQFLASDAASFITGSY LVDGGYTAQL SD ASF TG Y LVDGGYTAQYLVSDEASFLTGGYYLVDGGYTAQ 252YLASDESRYVTGATFVIDGGYTAQ 252L SD ASF TG Y LVDGGYTAQLASD TG DGGYTAQ
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70
80
90
100
1-boc-3-pyrrolidinoneacetophenone2-chloroacetophenon)
(A)
48.5
35.8
52.6 52.3
40
50
60
703-quinuclidinoneethyl benzoylforma
ive
activ
ity
(%
2 3.80 02
3.5 10.24.4
012.5
04.5
0 00 0 0 0 05
1.25.1
00
10
20
30
Rela
ti
(B)60
n-o(B)(B)
37.8
28 5
51.3
29.330.3 29.9
34.1
30 1
35.540
50
n o
but
2-m
activ
ity
(%)
(B)bu
3 76.0
28.5
19.2 19.0
13.7 14.7
5.3
30.125.3
14.9 14.4
21
10
20
30
Rem
aini
ng
3.7 3.00.2 0.1 0
0
e
ne
56.4
39.135.9
40.1 38.139.6
te
10.1
0
9.29.8
0 0 0
5.4
00 0 0 04.8
00 04.8
0
7.65.4
octane
31.6 35.0
27 5 28 128.1
38.6
30.234.5
45.8
30.330.5
octane
tyl ascetate
methlytetrahydrofuranutyl acetate
17.3
6.6
27.524.9
28.1
7.6
24.619.0
11.8
23.5
16.31.2
25.6
7.210.1 10.7
18.9
6.8
1.7
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Erratum for Itoh et al., Efficient PCR-Based Amplification of DiverseAlcohol Dehydrogenase Genes from Metagenomes for ImprovingBiocatalysis: Screening of Gene-Specific Amplicons from Metagenomes
Nobuya Itoh, Satomi Kariya, Junji Kurokawa
Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, Imizu, Toyama, Japan
Volume 80, no. 20, p. 6280 – 6289, 2014. Page 6281, column 1: Lines 52–54 should read as follows. “. . .20 cycles at 98°C for 10 s, and 68°Cfor 1 min, and finally the sample was kept at 68°C for 7 min.”
Citation Itoh N, Kariya S, Kurokawa J. 2016. Erratum for Itoh et al., Efficient PCR-based amplification of diverse alcohol dehydrogenase genes from metagenomesfor improving biocatalysis: screening of gene-specific amplicons from meta-genomes. Appl Environ Microbiol 82:1976.doi:10.1128/AEM.00317-16.
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
ERRATUM
crossmark
1976 aem.asm.org March 2016 Volume 82 Number 6Applied and Environmental Microbiology
