Embed Size (px)
Citation preview
1
Manuscript for submission to Applied and Environmental Microbiology 1
Journal section: Biotechnology 2
3
Synthesis of short-chain diols and unsaturated alcohols from secondary 4
alcoholic substrates by the Rieske non-heme mononuclear iron oxygenase 5
MdpJ 6
7
Running title: MdpJ-catalyzed transformations of secondary alcohols 8
9
10
Franziska Schäfer#, Judith Schuster#, Birgit Würz, Claus Härtig, Hauke Harms, Roland H. 11
Müller, and Thore Rohwerder* 12
13
Helmholtz Centre for Environmental Research - UFZ, Department of Environmental 14
Microbiology, Permoserstr. 15, 04318 Leipzig, Germany 15
16
17
# F. S and J. S. contributed equally to this work. 18
19
* Corresponding author. Mailing address: Helmholtz Centre for Environmental Research - 20
UFZ, Department of Environmental Microbiology, Permoserstr. 15, 04318 Leipzig, Germany, 21
Phone: +49 341 235 1317. Fax: +49 341 235 1351. E-mail: [email protected]. 22
23
24
Copyright © 2012, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.01434-12 AEM Accepts, published online ahead of print on 29 June 2012
on April 12, 2019 by guest
http://aem.asm
.org/D
ownloaded from
2
Abstract 25
26
The Rieske non-heme mononuclear iron oxygenase MdpJ of the fuel oxygenate-degrading 27
bacterial strain Aquincola tertiaricarbonis L108 has been described to attack short-chain 28
tertiary alcohols via hydroxylation and desaturation reactions. Here, we demonstrate that also 29
short-chain secondary alcohols can be transformed by MdpJ. Wild-type cells of strain L108 30
converted 2-propanol and 2-butanol to 1,2-propanediol and 3-buten-2-ol, respectively, 31
whereas an mdpJ knockout mutant did not show such activity. In addition, wild-type cells 32
converted 3-methyl-2-butanol and 3-pentanol to the corresponding desaturation products 3-33
methyl-3-buten-2-ol and 1-penten-3-ol, respectively. The enzymatic hydroxylation of 2-34
propanol resulted in an enantiomeric excess of about 70% for the (R)-enantiomer, indicating 35
that this reaction was favored. Likewise, desaturation of (R)-2-butanol to 3-buten-2-ol was 36
about 2.3-fold faster than conversion of the (S)-enantiomer. The biotechnological potential of 37
MdpJ for the synthesis of enantiopure short-chain alcohols and diols as building block 38
chemicals is discussed. 39
40
41
42
on April 12, 2019 by guest
http://aem.asm
.org/D
ownloaded from
3
Introduction 43
44
Enzymes catalyzing the hydroxylation and desaturation of aliphatic tertiary alcohols seem to 45
be rare and have not been well characterized thus far. In the last years, when studying the 46
biodegradation of the fuel oxygenates methyl tert-butyl ether (MTBE) and tert-amyl methyl 47
ether (TAME), at least one tertiary alcohol-hydroxylating enzymatic reaction has been 48
proposed for the transformation of the central ether metabolites tert-butyl and tert-amyl 49
alcohol (TBA and TAA, respectively) (9, 38). Though this reaction can be expected in several 50
bacterial strains known to completely degrade MTBE and TAME under aerobic conditions 51
(11, 13, 21, 27, 37), only in the fuel oxygenate-degrading bacterial strain Aquincola 52
tertiaricarbonis L108 the identity of the tertiary alcohol-attacking enzyme has recently been 53
revealed by gene knockout experiments (35). In this bacterium, the oxygenase MdpJ catalyzes 54
not only the hydroxylation of TBA to 2-methylpropane-1,2-diol (MPD) but also the 55
desaturation of TAA to 2-methyl-3-buten-2-ol (35). 56
57
MdpJ and its corresponding reductase MdpK have been described for the first time by 58
Hristova and co-workers as MTBE-induced proteins and subunits of the postulated TBA-59
hydroxylating enzyme in Methylibium petroleiphilum PM1 (14). MdpJK belongs to the family 60
of Rieske non-heme iron aromatic ring-hydroxylating oxygenases (RHO), such as the well-61
characterized phthalate and naphthalene dioxygenases (5, 20, 28, 36). These are 62
multicomponent enzymes which use reduced pyridine nucleotide as electron donor. The 63
electrons are transported via a flavin cofactor of the reductase subunit to a [2Fe-2S] iron-64
sulfur cluster, located on the same protein component, as in MdpK (32), or on a separate 65
ferredoxin subunit. The electrons are further passed to a [2Fe-2S] Rieske cluster and a 66
mononuclear iron center of the oxygenase subunit (23, 40). The multifunctionality of MdpJ to 67
catalyze hydroxylation as well as desaturation reactions (35) has likewise been found for 68
on April 12, 2019 by guest
http://aem.asm
.org/D
ownloaded from
4
other RHO enzymes, e. g., naphthalene dioxygenase catalyzes mono- and di-hydroxylations 69
as well as desaturations (22). Practically all known RHO enzymes, however, are described to 70
exclusively attack aromatic compounds (19). Thus, the use of aliphatic substrates underlines 71
the uniqueness of MdpJ among the RHO family. 72
73
The natural substrates of MdpJ-catalyzed hydroxylations and desaturations might be tertiary 74
alcohols, e. g. the fuel oxygenate intermediates TBA and TAA. Accordingly, it has been 75
already shown that MdpJ is also catalyzing the desaturation of the tertiary alcohol 3-methyl-3-76
pentanol to 3-methyl-1-penten-3-ol (35). Here, we demonstrate that MdpJ can also attack the 77
secondary alcohols 2-propanol, 2-butanol, 3-methyl-2-butanol and 3-pentanol. In 1,2-78
propanediol formation from 2-propanol via hydroxylation and 2-butanol desaturation to 3-79
buten-2-ol, a preference for the (R)-enantiomer was found, underpinning the potential of 80
MdpJ for the synthesis of enantiopure short-chain diols and unsaturated alcohols from 81
secondary alcoholic precursors. 82
83
84
Materials and Methods 85
86
Chemicals. (S)-(+)-2-Phenylbutyryl chloride was synthesized as described by Hammarström 87
et al. (12) using (S)-(+)-2-phenylbutyric acid (99% pure, Sigma-Aldrich Chemie GmbH, 88
Steinheim, Germany) and thionylchloride (99% pure, Merck Schuchardt, Hohenbrunn, 89
Germany). Suppliers and purities of other chemicals used in this study are listed in the 90
supplemental material. 91
92
Bacterial strains and growth medium. Aquincola tertiaricarbonis L108, isolated from an 93
MTBE-contaminated aquifer (Leuna, Germany) (21, 31), was cultivated in liquid mineral salt 94
on April 12, 2019 by guest
http://aem.asm
.org/D
ownloaded from
5
medium (MSM, see the supplemental material) containing MTBE at a concentration of 0.3 g 95
liter-1. The previously generated knockout mutant strain A. tertiaricarbonis L108 (ΔmdpJ) 96
K24 (35) was cultivated on 2-methylpropane-1,2-diol (MPD) at 0.5 g liter-1 in MSM 97
supplemented with kanamycin (50 mg liter-1). 98
99
Resting-cell experiments. Cultures were incubated at 30°C on rotary shakers. Bacterial cells 100
were pregrown on MPD (0.5 g liter-1) and then incubated on TBA (0.5 g liter-1) overnight. 101
Afterwards, cells were harvested by centrifugation at 13,000 x g and 4°C for 10 min. After 102
washing twice with MSM, cells were immediately used and biomass was adjusted to values of 103
1.4 g dry weight per liter by dilution with MSM for the 2-butanol experiments and 2.2 g per 104
liter when incubating with 2-propanol, 3-methyl-2-butanol and 3-pentanol. During the 105
experiments, bacteria were incubated at 30°C in 25 ml MSM in glass serum bottles, sealed 106
gas-tight with butyl rubber stoppers. Liquid and gas samples were taken as described before 107
(33), by puncturing the butyl rubber stoppers with syringes equipped with 0.6– by 30-mm 108
Luer Lock needles. For chiral analysis of 1,2-propanediol, the complete culture liquid was 109
harvested by centrifugation at 13,000 x g and 4°C for 10 min in order to obtain a clear 110
supernatant. The shown data represent mean values and standard deviations of four replicate 111
experiments. 112
113
Chiral analysis of 1,2-propanediol. Methods of Powers et al. for chiral analysis of short-114
chain diols and hydroxy carboxylic acids (30) and Jenske et al. for chiral analysis of hydroxy 115
fatty acids (15) were modified for determining the ratio of different 1,2-propanediol 116
enantiomers formed in bacterial cultures. Samples from resting-cell experiments (200 ml) 117
were saturated with NaCl and extracted two times with 100 ml diethylether. The diethylether 118
phase was dried with Na2SO4 and evaporated completely. Standards of the 1,2-propanediol 119
racemate and (S)-enantiomer were applied directly (3 µl). Pyridine (400 µl) and (S)-(+)-2-120
on April 12, 2019 by guest
http://aem.asm
.org/D
ownloaded from
6
phenylbutyryl chloride (100 µl) were added for derivatization. After 2 h incubation on a slow 121
shaker at room temperature, H2O (5 ml) and one spatula tip of K2CO3 were added. Then, the 122
solutions were extracted with MTBE (5 ml) via shaking for 1 h at room temperature. The 123
MTBE phase was dried with Na2SO4 and evaporated to 0.5 ml. Analysis was performed using 124
gas chromatography (GC 7890A; Agilent) coupled to mass spectrometry (MSD 5975C; 125
Agilent) on a RTX-5Sil MS column (30 m, 250 µm, 0.25 µm; Restek) with Integra-Guard 126
column (5 m; Restek). GC conditions: carrier gas helium, constant flow nominal 0.8 ml min-1, 127
injector temperature 230 °C, split injection (20:1), programmed oven temperature (70 °C for 1 128
min, then 1 °C min-1 to 76 °C, then 6 °C min-1 to 350 °C held for 1 min), MSD transfer-line 129
temperature 250 °C. MSD conditions: full scan mode (m/z 40-600), ion source temperature 130
230 °C, quadrupol temperature 150 °C. Compounds were identified by comparison of 131
calibrated retention times and mass spectra of authentic standards. 132
133
Other analytics. Volatile compounds (TBA, isobutene, 2-propanol, acetone, 2-butanol, 3-134
buten-2-ol, 2-butanone, 3-buten-2-one, 3-methyl-2-butanol, 3-methyl-2-butanone, 3-pentanol) 135
were quantified by headspace GC using flame ionization detection (FID) (33). Compounds 136
were assigned according to retention times of pure GC standards. Additionally, ketonic 137
metabolites of 2-butanol as well as metabolites of 3-methyl-2-butanol and 3-pentanol were 138
identified by GC-MS analysis (see Figures S3 to S6 in the supplemental material). 139
Quantitative analysis of 1,2-propanediol was performed using high-performance liquid 140
chromatography (HPLC) with refractive index detection (RID) as described elsewhere (24, 141
25). 142
143
144
Results 145
146
on April 12, 2019 by guest
http://aem.asm
.org/D
ownloaded from
7
Whole-cell assay for analyzing MdpJ substrate specificity. For studying substrate and 147
catalysis specificity of MdpJ, a test system employing the isolated enzyme would be 148
recommendable. However, purification of the active enzyme from cells of strain L108 or after 149
heterologous expression in Escherichia coli was not successful (see the supplemental 150
material). Therefore, we developed a whole-cell assay comparing substrate usage and product 151
formation of wild-type strain L108 with that of the mdpJ knockout strain L108 (ΔmdpJ) K24 152
(35). MPD-grown cells of both strains were incubated in the presence of TBA for 10 to 16 153
hours. This procedure was sufficient to induce MdpJ and MdpK in the wild-type strain 154
(Figure S1 in the supplemental material), whereas the mutant strain K24 did not synthesize 155
these enzymes due to the insertion mutation in the mdpJ gene. In line with this, subsequent 156
resting-cell experiments revealed that only the wild-type cells were able to degrade TBA 157
(Figure S2 in the supplemental material). However, the formation of small amounts of the 158
alkene isobutene from TBA was observed with both strains. This dehydration activity is a side 159
reaction in the MTBE metabolism in strains L108 and PM1 and has previously been ascribed 160
to MdpJ (33). The dehydration activity of the mutant strain clearly shows now that not MdpJ 161
but a different enzyme must be involved. Likely, the same dehydratase forms isobutene from 162
TBA that has recently been considered for the conversion of the tertiary alcohols TAA and 2-163
methyl-3-buten-2-ol to isoamylene and isoprene, respectively (35). Nevertheless, knockout 164
cells formed only less than 30% of the alkene amount of wild-type cells, indicating a higher 165
dehydration activity during complete TBA metabolization. 166
167
Hydroxylation of 2-propanol to 1,2-propanediol. Applying the whole-cell assay, wild-type 168
cells of strain L108 formed 1,2-propanediol from the secondary alcohol 2-propanol (Figure 169
1), by analogy with the hydroxylation of TBA to MPD. Besides, the dehydrogenation product 170
acetone accumulated. By contrast, the mdpJ knockout cells converted 2-propanol exclusively 171
to acetone, providing evidence that MdpJ is catalyzing the hydroxylation of 2-propanol in the 172
on April 12, 2019 by guest
http://aem.asm
.org/D
ownloaded from
8
wild-type strain L108. Acetone formation, on the other hand, is probably caused by an 173
unspecific secondary alcohol dehydrogenase expressed in both strains under the experimental 174
conditions. Formation of 1,2-propanediol was about 7 times slower than TBA conversion (1.5 175
and 10 nmol min-1 mg-1 dry biomass, respectively), indicating that TBA is a much better 176
substrate for MdpJ. As 1,2-propanediol is asymmetric, the stereospecificity of the 177
hydroxylation reaction was investigated. After derivatization to the corresponding (S)-178
phenylbutyryl diester, the (S)- and (R)-enantiomers of 1,2-propanediol could be distinguished 179
by GC analysis. Both enantiomers were formed by MdpJ. However, the hydroxylation to (R)-180
1,2-propanediol was slightly favored resulting in an enantiomeric excess (ee) of about 70% 181
(Figure 2). During resting-cell experiments with the wild-type strain L108, substrate recovery 182
as the conversion products 1,2-propanediol and acetone was below three quarters, as these 183
metabolites only represented less than 25 and 50% of converted substrate, respectively. In 184
contrast, knockout cells, which converted only one third of the 2-propanol amount of wild-185
type cells, accumulated exclusively acetone with close to 100% substrate recovery. This 186
confirms that acetone is not degraded by strain L108 (21), while 1,2-propanediol may be 187
converted slowly to other metabolites not accumulating under the experimental conditions. By 188
analogy to TBA and MPD metabolism, likely degradation products of 1,2-propanediol would 189
be lactaldehyde and lactic acid. 190
191
Desaturation of 2-butanol, 3-methyl-2-butanol and 3-pentanol. As 2-propanol turned out 192
to be a substrate for MdpJ, also the higher homologue 2-butanol was tested. Surprisingly, 193
hydroxylation products were not obtained with wild-type cells, but only the desaturation 194
product 3-buten-2-ol was formed from racemic 2-butanol (Figure 3). Both secondary 195
alcohols, 2-butanol and 3-buten-2-ol, were oxidized to the corresponding ketones, 2-butanone 196
and 3-buten-2-one, respectively (Figure 3 and supplemental Figures S3 and S4). In line with 197
the assumption that MdpJ is responsible for 2-butanol desaturation, neither 3-buten-2-ol nor 198
on April 12, 2019 by guest
http://aem.asm
.org/D
ownloaded from
9
3-buten-2-one was formed by the knockout mutant strain. Likely due to the high 199
dehydrogenase activity, resulting in a nearly complete substrate conversion to 2-butanone, 200
only very small amounts of 2-butanol of maximally 2% and 1% were recovered as the 201
desaturation product 3-buten-2-ol and its corresponding ketone 3-buten-2-one, respectively. 202
In experiments with enantiopure substrates, conversion of (R)-2-butanol to 3-buten-2-ol was 203
about 2.3 times faster than desaturation of the (S)-enantiomer (Figure 4). 204
205
In line with the observed transformation of 2-butanol to 3-buten-2-ol, also 3-methyl-2-butanol 206
and 3-pentanol were converted to the corresponding desaturation products 3-methyl-3-buten-207
2-ol and 1-penten-3-ol, respectively, by wild-type cells of strain L108 (Figures S5 and S6 in 208
the supplemental material). In addition, both wild-type and knockout mutant cells oxidized 209
the secondary alcoholic substrates to the corresponding ketones. In the case of incubation with 210
3-methyl-2-butanol, also the oxidation to the unsaturated ketone 3-methyl-3-buten-2-one by 211
the wild-type cells was observed (Figure S5 in the supplemental material). Generally, 212
desaturation activities were lower than with 2-butanol and prolonged incubation periods of up 213
to 8 hours were necessary to accumulate sufficient amounts of desaturation products for GC-214
MS analysis. 215
216
217
Discussion 218
The Rieske non-heme mononuclear iron oxygenase MdpJ of Aquincola tertiaricarbonis L108 219
has been previously found to convert the tertiary alcohols TBA, TAA and 3-methyl-3-220
pentanol to the corresponding diols and unsaturated alcohols (35). By comparing product 221
formation of wild-type and mdpJ knockout mutant cells, this study now demonstrates that also 222
short-chain secondary alcohols can be attacked by MdpJ. 223
224
on April 12, 2019 by guest
http://aem.asm
.org/D
ownloaded from
10
As already indicated recently by Schuster and co-workers in the context of TAA and 3-225
methyl-3-pentanol metabolism (35), the mode of MdpJ catalysis obviously depends strongly 226
on the molecule structure of the substrate, allowing either hydroxylation or desaturation 227
reactions. An overview of the products obtained from the thus far tested substrates (C3 to C5 228
secondary alcohols and C4 to C6 tertiary alcohols) is given in the supplemental Figure S7. The 229
structures of 2-propanol and TBA do not contain an ethyl group which can be desaturated. 230
Accordingly, only hydroxylation to diols has been observed. On the other hand, 2-butanol, 3-231
methyl-2-butanol, 3-pentanol, TAA and also 3-methyl-3-pentanol are mainly desaturated by 232
MdpJ as they all possess at least one pair of vicinal carbon atoms which can form a double 233
bond. Considering the structural similarity with the MdpJ substrates 2-butanol and TAA, it 234
can also be speculated whether the unsaturated alcohols 3-buten-2-ol and 2-methyl-3-buten-2-235
ol could be used by MdpJ. As these compounds are already desaturated, it appears likely that 236
hydroxylation products would be formed (supplemental Figure S7). However, when testing 237
both unsaturated alcohols in the whole-cell assay, we were not able to detect the formation of 238
1,2-diols, but HPLC analysis indicated the conversion to other products likely representing 239
unsaturated 2-hydroxy carboxylic acids (data not shown). Thus far, these compounds have not 240
been unambiguously assigned by GC-MS measurements and additional identification work is 241
needed. 242
243
The capacity of MdpJ for hydroxylation and desaturation of small aliphatic alcohols as found 244
in strain L108 seems to be quite unique. In the context of MTBE metabolism, a few other 245
strains have been described which should possess enzymes with a similar substrate spectrum. 246
Accordingly, the conversion of TBA to MPD has also been shown for the strains 247
Mycobacterium vaccae JOB5 (37), Hydrogenophaga flava ENV 735 (13), Mycobacterium 248
austroafricanum IFP 2012 (11) and Methylibium petroleiphilum PM1 (27). However, only the 249
MdpJ of strain L108 has been characterized for oxidation of TBA in MTBE metabolism. In 250
on April 12, 2019 by guest
http://aem.asm
.org/D
ownloaded from
11
addition, strain PM1 possesses a nearly identical enzyme, showing 97% sequence identity to 251
MdpJ of strain L108 which has been proposed to be involved in tertiary alcohol metabolism 252
(14). Moreover, MdpJ has been detected in environmental samples. The mdpJ gene is present 253
in MTBE-degrading enrichment cultures from a contaminated groundwater treatment plant in 254
Leuna, Germany (33), and the enzyme has been detected in oxygenate degrading mixed 255
cultures by 13C metagenomic and metaproteomic stable isotope probing (SIP) experiments (2, 256
4). These findings underline the important role of MdpJ in bacterial MTBE degradation. 257
258
Short-chain chiral alcohols and diols are interesting building blocks for the synthesis of 259
pharmaceuticals and other active compounds (3, 18, 39). Enantiopure alcohols, for example, 260
are key intermediates in side chain synthesis of serum cholesterol reducing drugs, i. e. 3-261
hydroxy-3-methylglutaryl-CoA reductase inhibitors (29). In addition, 1,2-propanediol is a 262
chiral building block for the synthesis of antiviral drugs like Tenofovir or Efavirenz (26, 17). 263
Thus far, the most important enzyme-based method to synthesize enantiopure alcohols is 264
lipase-catalyzed kinetic resolution of alcoholic and diolic racemates (6, 18, 34). In addition, 265
enantioselective reduction of ketones (29) or carboxylic acids with specific dehydrogenases 266
can be applied (6). Enantiopure 1,2-propanediol for commercial purposes, on the other hand, 267
is currently synthesized only via chemical routes (1, 6). A more straightforward approach for 268
the synthesis of enantiopure unsaturated alcohols and particularly diols could be the 269
stereospecific desaturation and hydroxylation of the corresponding saturated alcoholic 270
compounds. However, a one-step synthesis employing MdpJ would require a higher 271
stereospecificity. In our preliminary experiments on MdpJ catalysis, only an ee value of about 272
70% of (R)-1,2-propanediol and a likewise 2.3-fold preference of (R)-2-butanol as 273
desaturation substrate was achieved. This indicates at least that for these secondary alcohols 274
tested the same orientation in the reaction center of the enzyme is favored (Figure 5). On the 275
basis of this finding, the poor stereospecificity could be improved by site directed 276
on April 12, 2019 by guest
http://aem.asm
.org/D
ownloaded from
12
mutagenesis or novel approaches like directed enzyme evolution (7, 8). The stereospecificity 277
of the MdpJ-catalyzed desaturation of 3-methyl-2-butanol and 3-pentanol to the hemiterpenic 278
3-methyl-3-buten-2-ol and to 1-penten-3-ol, respectively, has not yet been analyzed. 279
Nevertheless, a similar preference as with (R)-2-butanol could be expected. Hence, an 280
improved MdpJ enzyme showing higher stereospecificity could be employed for the synthesis 281
of chiral allylic alcohols and related compounds currently only available through multi-step 282
chemical synthesis (3, 16). At the present stage of our research, however, we exclude the 283
application of pure MdpJ, because preparation of the cell-free enzyme always resulted in a 284
complete loss of enzymatic activity. Alternatively, application in whole-cell systems is quite 285
promising, but requires either metabolic manipulation of the wild-type strain L108 in order to 286
avoid the ketone-forming side reaction or the transformation of mdpJK genes into a more 287
appropriate host strain lacking such side activities. 288
289
Acknowledgements 290
291
The authors are grateful to DBU (Deutsche Bundesstiftung Umwelt) for financial support of 292
F. S. (AZ: 20008/994). 293
294
295
References 296
297 1 Altaras NE, Cameron D. 1999. Metabolic engineering of 1,2-propanediol 298
pathway in Escherichia coli. Appl. Environ. Microbiol. 65:1180-1185. 299
2 Aslett D, Haas J, Hyman M. 2011. Identification of tertiary butyl alcohol 300
(TBA)-utilizing organisms in BioGAC reactors using 13C-DNA stable isotope 301
probing. Biodegradation 22:961-972. 302
3 Balmer E, Germain A, Jackson WP, Lygo B. 1993. Larger scale preparation of 303
optically-active allylic alcohols. J. Chem. Soc. Perkin Trans. 1:399-400. 304
on April 12, 2019 by guest
http://aem.asm
.org/D
ownloaded from
13
4 Bastida F, Rosell M, Franchini AG, Seifert J, Finsterbusch S, Jehmlich N, 305
Jechalke S, von Bergen M, Richnow HH. 2010. Elucidating MTBE 306
degradation in a mixed consortium using a multidisciplinary approach. FEMS 307
Microbiol. Ecol. 73:370–384. 308
5 Batie C, La Haie JE, Ballou DP. 1987. Purification and characterization of 309
phthalate oxygenase and phthalate oxygenase reductase from Pseudomonas 310
cepacia. J. Biol. Chem. 262:1510-1518. 311
6 Breuer M, Ditrich K, Habicher T, Hauer B, Keßeler M, Stürmer R, Zelinski 312
T. 2004. Industrial methods for the production of optically active intermediates. 313
Angew. Chem. Int. Ed. 43:788-824. 314
7 Chen R. 2001. Enzyme engeneering: rational redesign versus directed evolution. 315
Trends Biotechnol. 19:13-14. 316
8 Dalby PA. 2003. Optimising enzyme function by directed evolution. Curr. Opin. 317
Struct. Biol. 13:500-505. 318
9 Fayolle F, Vandecasteele JP, Monot F. 2001. Microbial degradation and fate in 319
the environment of methyl tert-butyl ether and related fuel oxygenates. Appl. 320
Microbiol. Biotechnol. 56: 339-349. 321
10 FDA, U.S. Food and Drug Administration. 1992. FDA’s statement for the 322
development of new stereoisomeric drugs. Chirality 4:338-340. 323
11 François A, Mathis H, Godefroy D, Piveteau P, Fayolle F, Monot F. 2002. 324
Biodegradation of methyl tert-butyl ether and other fuel oxygenates by a new 325
strain, Mycobacterium austroafricanum IFP 2012. Appl. Environ. Microbiol. 326
68:2754-2762. 327
12 Hammarström S, Hamberg M. 1972. Steric analysis of 3-, ω4-, ω3- and ω2-328
hydroxy acids and various alkanols by gas-liquid chromatography. Anal. 329
Biochem. 52:169-179. 330
on April 12, 2019 by guest
http://aem.asm
.org/D
ownloaded from
14
13 Hatzinger PB, McClay K, Vainberg S, Tugusheva M, Condee CW, Steffan 331
RJ. 2001. Biodegradation of methyl tert-butyl ether by a pure bacterial culture. 332
Appl. Environ. Microbiol. 63:5601-5607 333
14 Hristova KR, Schmidt R, Chakicherla AY, Legler TC, Wu J, Chain PS, 334
Scow KM, Kane SR. 2007. Comparative transcriptome analysis of Methylibium 335
petroleiphilum PM1exposed to the fuel oxygenates methyl tert-butyl ether and 336
ethanol. Appl. Environ. Microbiol. 73:7347–7357. 337
15 Jenske R, Vetter W. 2007. Highly selective gas-chromatography-electron-338
capture negative-ion mass spectrometry method for the indirect enantioselective 339
identification of 2- and 3-hydroxy fatty acids in food and biological samples. J. 340
Chromatogr. A 1146:225-231. 341
16 Jones S, Valette D. 2009. Enantioselective synthesis of allylic alcohols via an 342
oxazaborolidinium ion catalyzed Diels−Alder/Retro-Diels−Alder sequence. Org. 343
Lett. 11:5358-5361. 344
17 Kadyrov R, Koenigs RM, Brinkmann C, Voigtlaender D, Rueping M. 2009. 345
Efficient enantioselective synthesis of optically active diols by asymmetric 346
hydrogenation with modular chiral metal catalysts. Angew. Chem. Int. Ed. 347
48:7556-7559. 348
18 Kourist R, Bornscheuer UT. 2011. Biocatalytic synthesis of optically active 349
tertiary alcohols. Appl. Microbiol. Biotechnol. 91:505-517. 350
19 Kweon O, Kim SJ, Baek S, Chae J, Adjei M, Baek D, Kim JC, Cerniglia C. 351
2008. A new classification system for bacterial Rieske non-heme iron aromatic 352
ring-hydroxylating oxygenases. BMC Biochem. 9:11. 353
20 Larkin MJ, Allen CCR, Kulakov LA, Lipscomb DA. 1999. Purification and 354
characterization of a novel naphthalene dioxygenase from Rhodococcus spec. 355
on April 12, 2019 by guest
http://aem.asm
.org/D
ownloaded from
15
NCIMB12038. J. Bacteriol. 181:6200-6204. 356
21 Lechner U, Brodkorb D, Geyer R, Hause G, Härtig C, Auling G, Fayolle-357
Guichard F, Piveteau P, Müller RH, Rohwerder T. 2007. Aquincola 358
tertiaricarbonis gen. nov., sp. nov., a tertiary butyl moiety-degrading bacterium. 359
Int. J. Syst. Evol. Microbiol. 57:1295–1303. 360
22 Lee K, Gibson DT. 1996. Toluene and ethylbenzene oxidation by purified 361
naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4. Appl. 362
Environ. Microbiol. 62:3101–3106. 363
23 Mason JR, Cammak R. 1992. The electron-transport proteins of hydroxylating 364
bacterial dioxygenases. Annu. Rev. Microbiol. 46:277-305. 365
24 Müller RH, Rohwerder T, Harms H. 2007. Carbon conversion efficiency and 366
limits of productive bacterial degradation of methyl tert-butyl ether and related 367
compounds. Appl. Environ. Microbiol. 73:1783–1791. 368
25 Müller RH, Rohwerder T, Harms H. 2008. Degradation of fuel oxygenates 369
and their main intermediates by Aquincola tertiaricarbonis L108. Microbiology 370
154:1414 –1421. 371
26 Murakami H. 2007. From racemates to single enantiomers – chiral synthetic 372
drugs over the last 20 years. Top. Curr. Chem. 269:273-299. 373
27 Nakatsu CH, Hristova K, Hanada S, Meng XY, Hanson J, Scow KM, 374
Kamagata Y. 2006. Methylibium petroleiphilum PM1T gen. nov., sp. nov., a 375
new methyl tert-butyl ether (MTBE) degrading methylotroph of the beta-376
Proteobacteria. Int. J. Syst. Evol. Microbiol. 56:983-989. 377
28 Nomura Y, Nakagawa M, Ogawa N, Harashima S, Oshima Y. 1992. Genes 378
in PHT plasmid encoding the initial degradation pathway of phthalate in 379
Pseudomonas putida. J. Ferm. Bioeng. 74:333-344. 380
on April 12, 2019 by guest
http://aem.asm
.org/D
ownloaded from
16
29 Panke S, Held M, Wubbolts M. 2004. Trends and innovations in industrial 381
biocatalysis for the production of fine chemicals. Curr. Opin. Biotechnol. 382
15:272- 279 383
30 Powers L, Ciraolo ST, Agarwal KC, Kumar A, Bomont C, Soloviev MV, 384
David F, Desrochers S, Brunengraber H. 1994. Assay of the enantiomers of 385
1,2-propanediol, 1,3-butanediol, 1,3-pentanediol and the corresponding 386
hydroxyacids by gas chromatography-mass spectrometry. Anal. Biochem. 387
221:323-328. 388
31 Rohwerder T, Breuer U, Benndorf D, Lechner U, Müller RH. 2006. The 389
alkyl tert-butyl ether intermediate 2-hydroxyisobutyrate is degraded via a novel 390
cobalamin-dependent mutase pathway. Appl. Environ. Microbiol. 72:4128–391
4135. 392
32 Schäfer F, Breuer U, Benndorf D, von Bergen M, Harms H, Müller RH. 393
2007. Growth of Aquincola tertiaricarbonis L108 on tert-butyl alcohol leads to 394
the induction of a phthalate dioxygenase-related protein and its associated 395
oxidoreductase subunit. Eng. Life Sci. 7:512– 519. 396
33 Schäfer F, Muzica L, Schuster J, Treuter N, Rosell M, Harms H, Müller 397
RH, Rohwerder T. 2011. Alkene formation from tertiary alkyl ether and alcohol 398
degradation by Aquincola tertiaricarbonis L108 and Methylibium spp. Appl. 399
Environ. Microbiol. 77:5981–5987. 400
34 Schmid A, Hollmann F, Byung Park J, Bühler B. 2002. The use of enzymes in 401
the chemical industry in Europe. Curr. Opin. Biotechnol. 13:359-366. 402
35 Schuster J, Schäfer F, Hübler N, Brandt A, Rosell M, Härtig C, Harms H, 403
Müller RH, Rohwerder T. 2012. Bacterial degradation of tert-amyl alcochol 404
proceeds via hemiterpene 2-methyl-3-buten-2-ol by employing the tertiary 405
alcohol desaturase function of the Rieske nonheme mononuclear iron oxygenase 406
on April 12, 2019 by guest
http://aem.asm
.org/D
ownloaded from
17
MdpJ. J. Bacteriol. 194:972-981. 407
36 Simon MJ, Osslunda TD, Saundersa R, Ensley BD, Suggs S, Harcour A, 408
Suen W, Crudenb DL, Gibsonb DT, Zylstrab GJ. 1993. Sequences of genes 409
encoding naphthalene dioxygenase in Pseudomonas putida strains G7 and NCIB 410
98 16-4. Gene 127:31-37. 411
37 Smith CA, O’Reilly T, Hyman MR. 2002. Characterization of the initial 412
reactions during the cometabolic oxidation of methyl tert-butyl ether by propane-413
grown Mycobacterium vaccae JOB5. Appl. Environ. Microbiol. 69:796-804. 414
38 Steffan RJ, McClay K, Vainberg S, Condee CW, Zhang D. 1997. 415
Biodegradation of the gasoline oxygenates methyl tert-butyl ether, ethyl tert-416
butyl ether and tert-amyl methyl ether by propane-oxidizing bacteria. Appl. 417
Environ. Microbiol. 63:4216-4222. 418
39 Vanhessche KPM, Sharpless KB. 1997. Catalytic asymmetric synthesis of new 419
halogenated chiral synthons. Chem. Eur. J. 3:517-522. 420
40 Wackett LP. 2002. Mechanism and applications of Rieske non-heme iron 421
dioxygenases. Enzyme Microbial Technol. 31:577-587. 422
423
on April 12, 2019 by guest
http://aem.asm
.org/D
ownloaded from
18
Figure Captions 424
425
426
Figure 1. Degradation of 2-propanol and accumulation of metabolites in resting-cell 427
experiments of A. tertiaricarbonis wild-type strain L108 (A) and mdpJ knockout strain L108 428
(ΔmdpJ) K24 (B). In the latter case, 1,2-propanediol was below detection limit (10 µM) 429
throughout the whole experiment. The sum of 2-propanol and metabolites represents 430
percentage of all analyzed 2-propanol-derived compounds (2-propanol, acetone and 1,2-431
propanediol) compared to the initial substrate concentration. 432
433
434
Figure 2. GC-MS analysis of the (S)-2-phenylbutyryl derivatives of racemic (RS)-1,2-435
propanediol standard, pure (S)-1,2-propanediol standard and a sample from a resting-cell 436
experiment of A. tertiaricarbonis wild-type strain L108 incubated on 2-propanol. (A) Total 437
ion chromatogram (TIC) signals. (B) Mass spectra of peaks occurring in the TIC of the 438
sample and the racemic standard at 37.9 min, representing the (S)-2-phenylbutyryl derivative 439
of (R)-1,2-propanediol. 440
441
442
Figure 3. Degradation of racemic 2-butanol and accumulation of metabolites in resting-cell 443
experiments of A. tertiaricarbonis wild-type strain L108 (A) and mdpJ knockout strain L108 444
(ΔmdpJ) K24 (B). In the latter case, 3-buten-2-ol and 3-buten-2-one were below detection 445
limit (2 µM) throughout the whole experiment. 446
447
448
449
on April 12, 2019 by guest
http://aem.asm
.org/D
ownloaded from
19
Figure 4. Formation of 3-buten-2-ol from 2-butanol by resting cells of A. tertiaricarbonis 450
wild-type strain L108 applying pure enantiomers as substrate. 451
452
Figure 5. Favored substrate and corresponding product enantiomers of MdpJ-catalyzed 453
hydroxylation of 2-propanol to 1,2-propanediol and desaturation of 2-butanol to 3-buten-2-ol. 454
455
on April 12, 2019 by guest
http://aem.asm
.org/D
ownloaded from
20
Figure 1 456
457
458
459
460
461
462
463
464
465
466
0
1
2
3
4
5
0 1 2 3 4Time (hours)
2-P
ropa
nol,
me
tab
olite
s (m
M)
0
20
40
60
80
100
2-PropanolAcetone1,2-PropanediolSum of 2-propanol
B
+ metabolites
Sum
of
2-p
ropa
nol
+ m
eta
bolit
es
(%)
0
1
2
3
4
5
0 1 2 3 4Time (hours)
2-P
rop
anol
, m
eta
bolit
es
(mM
)
0
20
40
60
80
100
2-PropanolAcetone1,2-PropanediolSum of 2-propanol
A
Sum
of 2
-pro
pano
l + m
eta
bol
ites
(%)
+ metabolites
on April 12, 2019 by guest
http://aem.asm
.org/D
ownloaded from
21
Figure 2 467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
Retention time (min)
Abu
ndan
ce
37.7 37.8 37.9 38.037.7 37.8 37.9 38.0
Sample
Racemicstandard
(S)-enantiomerstandard
Sample (37.9 min)
Abu
ndan
ce
Racemic standard (37.9 min)
50 100 150 200 250
91.1
146.1
205.141.1
119.1
50 100 150 200 250
91.1
146.1
205.141.1
119.1
Abu
ndan
ce
A B m/z
m/z
on April 12, 2019 by guest
http://aem.asm
.org/D
ownloaded from
22
Figure 3 489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
0 1 2 3 4Time (hours)
2-B
utan
ol,
met
abol
ites
(mM
)
2-Butanol 2-Butanone3-Buten-2-ol 3-Buten-2-one
B
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
0 1 2 3 4Time (hours)
2-B
utan
ol,
2-bu
tano
ne (
mM
)
0
10
20
30
40
3-B
ute
n-2-
ol,
3-bu
ten-
2-on
e (
µM
)
2-Butanol 2-Butanone3-Buten-2-ol 3-Buten-2-one
A
on April 12, 2019 by guest
http://aem.asm
.org/D
ownloaded from
23
Figure 4 505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
0
5
10
15
20
25
0 10 20 30 40
Time (min)
3-B
ute
n-2
-ol (
µM
)from (R )-2-butanol at 0.53 ± 0.06 µmol L-1min-1
from (S )-2-butanol at 0.23 ± 0.01 µmol L-1min-1
on April 12, 2019 by guest
http://aem.asm
.org/D
ownloaded from
24
Figure 5 520
521
522
523
524
525
526
527 + O2, NADH
- 2 H2O, NAD
- H2O, NAD
+ O2, NADH
CH3
OHCH3
HCH3
OHCH3
H
2-propanol (R)-1,2-propanediol
(R)-2-butanol
CH2OH
OHCH3
HCH2OH
OHCH3
H
CH2
OHCH3
H
CH3
CH2
OHCH3
H
CH3
(R)-3-buten-2-ol
CH
OHCH3
H
CH2
CH
OHCH3
H
CH2
on April 12, 2019 by guest
http://aem.asm
.org/D
ownloaded from
