investigation of the amycolatopsis sp. atcc 39116...

1

Investigation of the Amycolatopsis sp. ATCC 39116 vanillin 1

dehydrogenase (VDHATCC 39116) and its impact on the biotechnical 2

production of vanillin 3

4

Christian Fleige1 ● Gunda Hansen1 ● Jens Kroll1 ● Alexander Steinbüchel1, 2 5

6

1 Institut für Molekulare Mikrobiologie und Biotechnologie 7

Westfälische Wilhelms-Universität Münster 8

Corrensstrasse 3 9

D-48149 Münster 10

Germany 11

email: [email protected] 12

Phone: +49 (251) 8339821. 13

Fax: +49 (251) 8338388 14

2 Environmental Sciences Department, King Abdulazis University, Jeddah, Saudi Arabia 15

16

Keywords: vanillin, vanillic acid, vanillin dehydrogenase, ferulic acid, 17

Amycolatopsis, vdh, VDH 18

Abbreviations: AmpR, ampicillin resistance cassette; ATCC, American Type Culture 19

Collection; EC no., European Commission number; HPLC, high performance liquid 20

chromatography; KmR, kanamycin resistance cassette 21

Copyright © 2012, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.02358-12 AEM Accepts, published online ahead of print on 12 October 2012

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ABSTRACT 22

The actinomycete Amycolatopsis sp. ATCC 39116 is capable of synthesizing large 23

amounts of vanillin from ferulic acid, which is a natural cell wall component of higher plants. 24

The desired intermediate vanillin is subject to undesired catabolism caused by the 25

metabolic activity of a hitherto unknown vanillin dehydrogenase (VDHATCC 39116). In order to 26

prevent the oxidation of vanillin to vanillic acid and thereby to obtain higher yields and 27

concentrations of vanillin, the responsible vanillin dehydrogenase in Amycolatopsis sp. 28

ATCC 39116 was investigated for the first time using the data of our genome sequence 29

analysis and further bioinformatic approaches. The vdh gene was heterologously 30

expressed in E. coli, and the encoded vanillin dehydrogenase was characterized in detail. 31

VDHATCC 39116 was purified to apparent electrophoretic homogeneity and exhibited NAD+-32

dependent activity towards vanillin, coniferylaldehyde, cinnamaldehyde and benzaldehyde. 33

The enzyme showed its highest activity towards vanillin at a pH of 8.0 and a temperature 34

of 44°C. In a next step, a precise vdh-deletion mutant of Amycolatopsis sp. ATCC 39116 35

was generated. The mutant lost its ability to grow on vanillin and did not show vanillin 36

dehydrogenase activity. A 2.3 times higher vanillin concentration and a substantially 37

reduced amount of vanillic acid occurred in the mutant Amycolatopsis sp. ATCC 39116 38

∆vdh::KmR when ferulic acid was provided for biotransformation in a cultivation 39

experiment at the 2-L bioreactor scale. Based on these results and taking further metabolic 40

engineering into account, Amycolatopsis sp. ATCC 39116 ∆vdh::KmR represents an 41

optimized and industrial applicable platform for biotechnological production of natural 42

vanillin. 43

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INTRODUCTION 45

Vanillin is one of the most important and widely used flavour compounds in the food and 46

fragrance industry. The extraction of natural vanillin from cured seed pods of the orchid 47

Vanilla planifolia is highly cost and labor intensive and does not satisfy the global demand 48

of the continuously increasing market. Therefore, new sources for the synthesis of vanillin 49

are of great interest for the industry. More than 99 % of the industrially produced vanillin is 50

chemically synthesized from petrochemicals or lignin derivatives (8). Due to the increasing 51

interest in ’natural‘ and ’healthy‘ ingredients, especially in food industry, alternative sources 52

for natural vanillin originating from biotechnical processes were intensively investigated 53

(37). Vanillin can be labeled as ’natural’ if it is extracted from natural sources or “obtained 54

by appropriate physical, enzymatic or microbiological processes from material of 55

vegetable, animal or microbiological origin” (11). 56

The most attractive approaches for microbial vanillin synthesis are the biotransformation 57

of cheap natural aromatic compounds, such as ferulic acid (3-(4-hydroxy-3-methoxy-58

phenyl)prop-2-enoic acid), or the de novo biosynthesis from primary metabolites like 59

glucose (17). Bioconversion of ferulic acid into vanillin was demonstrated for various 60

microorganisms, including bacteria like Pseudomonas and Rhodococcus (27, 31, 33, 34) 61

or fungi such as the basidiomycete Pycnoporus cinnabarinus (44). Additionally, several 62

genes encoding the responsible enzymes in some of these organisms were heterologously 63

expressed in E. coli and enabled the corresponding strains to convert ferulic acid into 64

vanillin (5, 32). However, high concentrations of vanillin are attended by serious problems 65

for the cells due to the toxicity of this highly reactive aromatic aldehyde, which is detoxified 66

in the course of further catabolism or leads to cell death. Therefore, most processes did 67

not achieve industrial applicability because of insufficient vanillin yields. 68


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In order to overcome this drawback, screenings for vanillin tolerant bacterial strains 69

were performed, which are able to convert ferulic acid and thereby accumulate high 70

amounts of the biotransformation product vanillin (26, 38). Rabenhorst et al. (38) reached 71

a vanillin concentration of 11.5 g/L after biotransformation of 19.9 g/L ferulic acid using 72

Amycolatopsis sp. HR167, corresponding to a molar yield of 77.8%. With Streptomyces 73

setonii ATCC 39116, which meanwhile has been reclassified to Amycolatopsis sp. ATCC 74

39116, Muheim et al. (26) achieved a final vanillin concentration of 13.9 g/L with a molar 75

yield of 75%. In contrast to most other used strains, both approaches are applicable in 76

industrial processes, although they also face the problem of further vanillin degradation to 77

vanillic acid and other unwanted degradation products (1, 43). 78

For an improved vanillin production process using the actinomycete Amycolatopsis sp. 79

ATCC 39116, whose 16S-rDNA-sequence is identical to that of Amycolatopsis sp. HR167, 80

the whole genome was sequenced to identify enzymes involved in the vanillin catabolism, 81

as previously done for other vanillin-producing strains (9, 10, 23, 31, 34). In order to 82

promote a more efficient accumulation of vanillin in by preventing its oxidation to vanillic 83

acid, enzymes with homology to characterized vanillin dehydrogenases of other bacteria 84

were screened. After identifying a set of enzymes with homologies to vanillin and other 85

aldehyde dehydrogenases, the related genes were heterologously expressed in E. coli, 86

and the encoded enzymes were tested for their activity on vanillin. The enzyme exhibiting 87

VDH activity showed highest homology to an aldehyde dehydrogenase of 88

Nocardioidaceae bacterium Broad-1 (ZP_08197264) and was therefore designated 89

VDHATCC 39116. In a next step, a precise deletion mutant was generated and the impact of 90

VDHATCC 39116 on the catabolism of vanillin was examined. First attempts were made to 91

employ the deletion mutant in fermentation processes as novel production platform for 92

natural vanillin. 93


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MATERIALS AND METHODS 94

95

Bacterial strains, plasmids and cultivation conditions. All bacterial strains and 96

plasmids used in this study are listed in Table 1. Cells of Escherichia coli were grown in 97

Lysogeny Broth- (LB-) medium at 30 °C (40). Cells of Amycolatopsis sp. ATCC 39116 were 98

grown at 42 °C in Caso-medium (Merck, Darmstadt, Germany) or in a modified mineral 99

salts medium (19). Carbon sources were provided from stock solutions, which were 100

sterilized by filtration before usage, as indicated in the text. For selection of plasmid 101

harboring strains, antibiotics were added to the medium at the following concentrations: 102

Kanamycin: 50 µg/ml for E. coli, 100 µg/ml for Amycolatopsis sp. ATCC 39116; Ampicillin: 103

100 µg/ml for E. coli. Cell growth was measured photometrically using a Klett-Summerson 104

photometer equipped with filter no. 54 (520-580 nm) or by measuring the optical density at 105

400 nm (Amycolatopsis sp. ATCC 39116) or 600 nm (E. coli). 106

107

Biotransformation experiments. For biotransformation experiments of E. coli or 108

Amycolatopsis sp. ATCC 39116, cells were harvested within the stationary growth phase, 109

washed twice with phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM 110

Na2HPO4, 1.8 mM KH2HPO4, pH 7.5) and resuspended in an appropriate volume of the 111

same buffer or mineral salts medium (19). The experiments were carried out with 50 ml 112

mineral salts medium in 250 ml Erlenmeyer flasks (E. coli) or in a 2-L bioreactor with a 113

total volume of 1,000 ml PBS (Amycolatopsis sp. ATCC 39116). Ferulic acid or vanillin 114

were added from sterile stock solutions as indicated in the text. 115

116

Determination of metabolic intermediates. Excreted intermediates of the ferulic acid 117

metabolism were analyzed by high performance liquid chromatography using a “UltiMate 118


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3000 HPLC System“ (Dionex, Idstein, Germany) without prior extraction. Culture 119

supernatants were obtained after centrifugation (5 min, 16,000 x g) and catalytic reactions 120

were stopped by acidification of the withdrawn samples to pH 2 by the addition of 121

trichloroacetic acid to a concentration of 6.1 mM. Intermediates were separated by 122

reverse-phase chromatography using an Acclaim® 120 C8 column (particle size: 5 µm; 123

column: 250 x 2.1 mm) with a gradient of 0.1% (vol/vol) formic acid (eluant A) and 124

acetonitrile (eluant B) in a range from 26 to 100% (vol/vol) eluant B. The run started with a 125

flow rate of 0.1 ml/min, which was raised to 0.3 ml/min after 16 min when eluent B reached 126

100 %. For quantification, all intermediates were calibrated with external standards. The 127

compounds were identified by their retention times and their absorption at a specific 128

wavelength determined using a multiple wavelength detector “MWD – 3000” (259 nm, 129

280 nm, 285 nm, 340 nm; Dionex, Idstein, Germany). Data analyses and quantifications 130

were performed with the associated software “Chromeleon 6.8 Chromatography Data 131

Systems“ (Dionex, Idstein, Germany). 132

133

DNA isolation and manipulation. Genomic DNA of Amycolatopsis sp. ATCC 39116 134

was isolated with “DNeasy Blood & Tissue Kit” (Qiagen GmbH, Hilden, Germany). Plasmid 135

DNA was isolated from E. coli using the “peqGOLD Plasmid MiniPrep Kit I” (PEQLAB 136

GmbH, Erlangen, Germany). Plasmid isolation from Amycolatopsis sp. ATCC 39116 was 137

performed using the “Qiagen Plasmid Purification Kit (Mini)“ (Qiagen GmbH, Hilden, 138

Germany). DNA was digested with restriction endonucleases (Fermentas GmbH, St. Leon-139

Rot, Germany) under the conditions described by the manufacturer. Phusion high-fidelity 140

DNA polymerase and other DNA manipulating enzymes (Fermentas GmbH, St. Leon-Rot, 141

Germany) were used according to the instructions of the manufacturer. Oligonucleotides 142

were purchased from Eurofins MWG Synthesis GmbH (Ebersberg, Germany). DNA 143


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fragments were isolated from agarose gels or reaction mixtures using the “peqGOLD Gel 144

Extraction Kit” (PEQLAB GmbH, Erlangen, Germany). 145

146

Genome sequencing, gene prediction and annotation. Sequencing of the whole 147

genome of Amycolatopsis sp. ATCC 39116, prediction of open reading frames and 148

automatic annotation was performed as described by Hiessl et al. (18). Homologues of 149

putative VDHs were identified using BLAST analyses, and the corresponding genes were 150

ranked based on their homology to already characterized VDHs. 151

152

Transfer of DNA. Plasmids were transferred into E. coli by employing the CaCl2-153

method (40), whereas the transfer of plasmids and linear DNA fragments into 154

Amycolatopsis sp. ATCC 39116 was performed by direct mycelia transformation as 155

described by Priefert et al. (35). 156

157

Plasmid and mutant construction. The vdh gene of Amycolatopsis sp. ATCC 39116 158

was amplified with its own ribosomal binding site (RBS) via PCR from total chromosomal 159

DNA using the Phusion high-fidelity polymerase and oligonucleotides vdh_for_RBS_NdeI 160

and vdh_rev_XhoI (Tab. S1). For an additional C-terminal His-tag, vdh was also amplified 161

without its native stop codon using vdh_for_RBS_NdeI and vdh_rev_nostop_XhoI 162

(Tab. S1) as reverse primer. Both fragments were digested with NdeI/XhoI and ligated with 163

the NdeI/XhoI-linearized vector pET23a (+). After transformation of E. coli Mach-1 T1, the 164

resulting plasmids pet23::vdh and pet23::vdhHis were sequenced and subsequently 165

transferred to the expression host E. coli BL21(DE3). 166

A precise deletion of vdh was accomplished via homologous recombination by replacing 167

the native gene with a kanamycin resistance cassette. For this purpose, the flanking 168


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regions upstream and downstream of vdh in the genome were amplified using the 169

following oligonucleotides (Tab. S1): upstream region: vdhLF_for3 and vdhLF_rev_KmR; 170

downstream region: vdhRF_for_PromKmR and vdhRF_rev3. Additionally, the kanamycin 171

resistance cassette of pRLE6 was amplified using KmR_rev_vdhLF and 172

PromKmR_for_vdhRF. The resulting fragments were purified and combined in a 173

subsequent fusion-PCR using primers vdhLF_for3 and vdhRF_rev3. The resulting 174

fragment vdhLF_KmR_vdhRF combined the upstream and downstream region of vdh with 175

the kanamycin resistance cassette and was transferred into Amycolatopsis sp. ATCC 176

39116 as described above. Transformants of Amycolatopsis sp. ATCC 39116 were 177

selected on solid Caso-medium containing 100 µg/ml kanamycin. Mutants were screened 178

by colony-PCR and gene replacement was finally analyzed via diagnostic PCR with 179

different primer combinations showing a specific integration of the kanamycin resistance 180

cassette into the vdh-locus. Furthermore, the resulting PCR-fragments were verified via 181

digestion and sequencing. 182

183

Preparation of crude cell extracts and soluble fractions. Cell pellets of E. coli were 184

resuspended in 3-4 ml of 100 mM potassium phosphate buffer (pH 7.1). Crude extracts 185

were obtained by three passages through a French pressure cell (120 MPa). For the 186

preparation of soluble fractions, cell debris was removed by centrifugation for 60 min at 187

20,000 x g at 4 °C. For subsequent enzyme assays, samples were stored on ice, while 188

samples for polyacrylamide gel electrophoresis were stored at -20 °C. His6-tagged proteins 189

were purified using “His SpinTrap“-columns (GE Healthcare Europe GmbH, Freiburg, 190

Germany) according to the instructions of the manufacturer. 191

192


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Polyacrylamide gel electrophoresis and Western blot analysis. Samples from crude 193

cell extracts and soluble fractions were analyzed for their protein contents according to the 194

method of Bradford (7). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-195

PAGE) was performed in 12.5% (wt/vol) gels as described by Laemmli (20). Amounts of 196

proteins were added as indicated in the text. Staining of proteins was performed with 197

Serva Blue G (47). Molecular weight reference proteins were purchased from GE 198

Healthcare Europe GmbH (Freiburg, Germany), “LMW SDS Markers“: Phosphorylase b, 199

rabbit muscle, 97,000 kDa; bovine serum albumin, 66,000 kDa; Ovalbumin, chicken egg 200

white, 45,000 kDa; Carbonic anhydrase, bovine erythrocyte, 30,000 kDa; Trypsin inhibitor, 201

soybean, 20,100 kDa; α-Lactalbumin, bovine milk, 14,400 kDa. 202

Proteins were blotted from SDS-polyacrylamide gels onto PVDF membrane “PVDF 203

Transfer Membrane Hybond-P“ (GE Healthcare Europe GmbH, Freiburg, Germany) 204

according to the method of Towbin et al. (45), using a “PeqLab Western Blotting Semi Dry 205

Sedec M“ apparatus (PEQLAB GmbH, Erlangen, Germany). Transfer of proteins was 206

accomplished for 120 min at a voltage of 14 V and a constant current of 5 mA/cm2. 207

Immuno detection of His6-tagged proteins was performed with a primary antibody 208

“Mouse anti histidine tag“ (AbD Serotec, MorphoSys AbD GmbH, Düsseldorf, Germany) 209

and a secondary antibody “AP-goat anti-mouse IgG“ (Zymed, Invitrogen GmbH, Karlsruhe, 210

Germany). Membranes with blotted proteins were incubated in skim milk (2.5 %, wt/vol, 211

Tris-buffered saline (TBS; total volume 1L: TRIS/HCl (pH 7.6), 2.42 g; NaCl, 8 g; Tween 20, 212

0.05 % (wt/vol)) at room temperature to block nonspecific binding. After washing with TBS, 213

the membrane was incubated with the primary antibody (1:2,000 in TBS-Tween (0.1 %), 214

200 µL/cm² membrane) overnight at room temperature. After threefold washing with TBS 215

for 10 min, the membrane was incubated with the secondary antibody (1:10,000 in TBS-216

Tween (0.1 %), 200 µL/cm² membrane) for 2 h at room temperature. After finally washing 217


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threefold with TBS as described above, the antigen-antibody complexes were visualized 218

using “Western Blue stabilized substrate for AP“ (Promega GmbH, Mannheim, Germany). 219

220

VDH Enzyme assay. VDH enzyme activity was assayed according to Gasson et al. 221

(13). The reaction mixture contained in a total volume of 1 ml: 0.1 mM potassium 222

phosphate buffer (pH 7.1), 0.5 mM NAD+, 1.2 mM pyruvate (sodium salt), lactate 223

dehydrogenase (rabbit muscle, 1 U), 500 µg of the enzyme preparation of interest and 224

0.125 mM vanillin, benzaldehyde, coniferylaldehyde or cinnamaldehyde, respectively. 225

Oxidation of the substrates was measured at 30 °C and a suitable wavelength: vanillin 226

340 nm (ε= 11.492 cm2/µmol), cinnamaldehyde 340 nm (ε= 0.588 cm2/µmol), 227

benzaldehyde 293 nm (ε= 0.339 cm2/µmol) and coniferylaldehyde 410 nm 228

(ε= 3.452 cm2/µmol). Enzyme activity is stated in units (U). One unit is defined as the 229

amount of enzyme which converts 1 µmol vanillin per minute. 230

The temperature and pH optimum of VDH ATCC 39116 was determined in a discontinuous 231

assay by measuring the vanillic acid formation using HPLC, since the spectrophotometric 232

assay is additionally dependent on the activity of the lactate dehydrogenase. The reaction 233

mixture contained in a total volume of 1 ml: 0.1 mM potassium phosphate buffer (pH 7.1), 234

2 mM NAD+, 500 µg of the enzyme preparation of interest and 1.25 mM vanillin. The 235

reactions were started by the addition of NAD+, and the mixture was incubated for 15 min 236

before the reaction was stopped by heating to 85°C for 5 min. To test the temperature 237

preference, the assay was carried out at 15-65°C in steps of 5°C. After allocating the 238

optimum between 40 and 45°C, the temperature steps were refined to 1°C. The effect of 239

different pH values (5.0 – 9.5 in 0.5 steps) was observed at 30°C in Britton-Robinson-240

buffer consisting of 40 mM boric acid, 40 mM acetic acid and 40 mM phosphoric acid and 241

titrated to the desired pH with 200 mM NaOH. 242


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243

Nucleotide sequence accession numbers. The genome sequence of Amycolatopsis 244

sp. ATCC 39116 has recently been deposited in GenBank under the accession no. 245

AFWY00000000 (9). Additionally, the nucleotide sequence of vdh is provided within the 246

patent PCT/EP2012/061600 (12). Moreover all five tested gene candidates are available in 247

GenBank under the accession numbers: JX292129 (vdh), JX292130, JX292131, 248

JX292132, JX292133 249

250

251

RESULTS 252

In silico analyses of the genome and identification of the Amycolatopsis sp. ATCC 253

39116 vanillin dehydrogenase (VDHATCC 39116). Based on the genome sequence of 254

Amycolatopsis sp. ATCC 39116 and an automatic annotation, five genes, coding for 255

putative aldehyde dehydrogenases were identified. After heterologous expression of these 256

gene candidates in E. coli, only one of them lead to vanillin dehydrogenase activity and 257

might therefore be involved in the catabolism of vanillin. The gene, designated vdh, 258

consists of 1461 bp and encodes a protein of 486 amino acids with a theoretical molecular 259

weight of 50.6 kDa. A comparison of the deduced amino acid sequence of VDHATCC 39116 260

with internet-based sequence data using “BLASTP 2.2.25” (2, 3) showed homology to 261

different (benz)aldehyde dehydrogenases of various sources. The highest homology (73%) 262

was determined for an aldehyde dehydrogenase of Nocardioidaceae bacterium Broad-1 263

(ZP_08197264). By using “ClustalX 1.83” (16), a multiple sequence alignment (MSA) with 264

various representative aldehyde dehydrogenases was done for assessing their 265

relationship to VDHATCC 39116 in a dendrogramm (Fig. 1). Additionally, a MSA using “BioEdit 266


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Sequence Alignment Editor 7.0.5.3“(16) was performed to demonstrate the similarity on 267

the amino acid level of VDHATCC 39116 to different enzymes with already verified vanillin 268

dehydrogenase activity (Fig. S1). Further in silico analysis using “ScanProsite” (Expert 269

Protein Analysis System, ExPASy; (14)) revealed a characteristic pattern of aldehyde 270

dehydrogenases within the amino acids 251 to 258 including glutamic acid in the catalytic 271

center. 272

273

Characterization of VDHATCC 39116. Beside the in silico analyses, the specific vanillin 274

dehydrogenase activity in cells of Amycolatopsis sp. ATCC 39116 was investigated after 275

cultivating the strain with or without vanillin and structurally related compounds like ferulic 276

acid as carbon source, since former investigations with Pseudomonas showed an inducing 277

effect (4, 13). Amycolatopsis sp. ATCC 39116 was cultivated in mineral salts medium 278

containing 0.5 % (wt/vol) gluconate for 32 h at 42 °C. In addition, 0.1 % (wt/vol) of vanillin 279

or ferulic acid was added to one of the cultures. The cells were harvested after cultivation, 280

soluble fractions were prepared, and the specific activity of the vanillin dehydrogenase was 281

measured photometrically as described in materials and methods. The activity assays 282

revealed specific vanillin dehydrogenase activities of 25 ± 2 U/g protein if cells were 283

previously cultivated with vanillin as carbon source. In cells, which were cultivated only 284

with gluconate as sole carbon source, no VDHATCC 39116 activity was detectable. The 285

enhancing effect was also obvious during SDS-PAGE analysis of the soluble fractions 286

which revealed a prominent protein band at a size of approximately 54 kDa in case of the 287

vanillin-induced cells. A corresponding band was missing in samples of cells which were 288

not incubated with vanillin. Comparable results could be observed, if cells were cultivated 289

with ferulic acid as inducer (data not shown). 290


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After demonstrating the activity of a vanillin dehydrogenase in cells of Amycolatopsis sp. 291

ATCC 39116 grown with vanillin and after identifying a putatively responsible gene, vdh 292

was cloned and heterologously expressed in E. coli. For this, the gene was inserted into 293

the vector pET23a (+) and expressed in E. coli BL21 (DE3) under the control of the strong 294

T7 promoter. In order to purify the heterologous enzyme for further investigations, vdh was 295

also expressed without its native stop-codon to append a C-terminal His6-tag provided by 296

the vector pET23a (+). SDS-PAGE and subsequent Western blot analysis were carried out 297

to verify the correct gene expression and purification of VDHATCC 39116 with the His6-tag 298

(Fig. 2). 299

Protein analyses by SDS-PAGE and Western blot revealed the presence of an 300

appropriate protein with an apparent molecular weight of about 54 kDa. The purification of 301

VDHHis using “His SpinTrap“-columns yielded an almost homogenous protein (lane 7). 302

Immuno detection of the C-terminal His6-tag additionally confirmed the assumption that the 303

occurring protein signal represents the heterologously expressed VDHHis. Further analysis 304

of vanillin dehydrogenase activity within the eluate showed a specific activity of 72 ± 2 U/g 305

protein, which was remarkably low due to activity loss during the purification, which we 306

could not overcome, although we changed the composition of all used buffers. No enzyme 307

activity was observed in the absence of the cofactor NAD+. Furthermore, no oxidation of 308

vanillin was detectable, if NADP+ was used as cofactor (data not shown). 309

The optimal condition for the oxidation of vanillin was determined by measuring the 310

formed vanillic acid using cell free extracts of the recombinant vdh-expressing E.coli strain. 311

Maximum activities were found at a pH of 8.0 and a temperature of 44°C. The enzyme was 312

active at a broad pH range from 5.0 to 9.0 and was able to catalyze the reaction at 313

temperatures between 15°C and 65°C. After storage at 4°C for 24h VDHATCC 39116 exhibited 314

86% of its former activity. 315


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The most specific substrate was found to be vanillin, but VDHATCC 39116 also catalyzed 316

the oxidation of coniferylaldehyde, benzaldehyde and cinnamalaldehyde with specific 317

activities up to 66% (benzaldehyde) in comparison to vanillin. 318

In a next step, vanillin biotransformation experiments using the recombinant E. coli 319

strain expressing VDHATCC 39116 were performed. The cells were cultivated until they 320

reached the stationary growth phase, were then harvested and washed twice with 321

potassium phosphate buffer. After suspending the cells in 50 ml mineral salts medium, 14 322

mM vanillin was added, and the formation of vanillic acid was observed using HPLC 323

analysis of culture supernatants (Fig. 3). As a control experiment, E. coli BL21(DE3), 324

harboring the plain vector pET23a, was incubated under identical conditions. 325

The biotransformation experiments demonstrated that the VDHATCC 39116 expressing 326

strain is able to metabolize vanillin to vanillic acid (Fig. 3). Vanillic acid was synthesized in 327

equimolar amounts immediately after the addition of vanillin, while a transformation could 328

not be observed in the control experiment (data not shown). 329

After identifying the responsible gene and allocating vanillin dehydrogenase activity to 330

VDHATCC 39116, vdh was additionally overexpressed episomally in Amycolatopsis sp. 331

ATCC 39116 using a derivative of the Amycolatopsis – E. coli shuttle vector pRLE6 (35): 332

The homologous expression led to a 3.7-fold increase of VDHATCC 39116 activity in 333

comparison to the wild type. 334

335

Construction of a vdh-deletion mutant of Amycolatopsis sp. ATCC 39116. In order 336

to investigate the impact of VDHATCC 39116 on the catabolism of vanillin in Amycolatopsis sp. 337

ATCC 39116, a precise deletion mutant was generated by replacing the native gene with a 338

kanamycin resistance cassette. For this, the flanking regions located upstream and 339

downstream of vdh in the genome were amplified and combined with the resistance gene 340


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in a fusion-PCR. Amycolatopsis sp. ATCC 39116 was transformed with the resulting linear 341

fragment, and the gene replacement in the resulting mutant Amycolatopsis sp. 342

ATCC 39116 ∆vdh::KmR was confirmed by a diagnostic PCR. In a first step, the integration 343

of the deletion construct into the genome was demonstrated by amplifying a 3.36 kb-344

fragment (Fig. 4, lane 2). This fragment distinguishes in size from the genomic 345

organization of the wild type as a consequence of the smaller size of the resistance gene 346

in comparison to that of vdh. A second PCR with two different primer combinations verified 347

the specific integration of the deletion fragment into the vdh-locus by amplifying segments 348

which started upstream (Fig. 4, lane 3) or downstream (Fig. 4, lane 4), respectively, of the 349

used flanking regions. Finally, the amplified fragments were sequenced to ensure the 350

correct replacement of vdh with the kanamycin resistance gene. 351

352

Characterization of Amycolatopsis sp. ATCC 39116 ∆vdh::KmR. The vdh-deletion 353

mutant was characterized to determine the impact of the enzyme on the catabolism of 354

vanillin in Amycolatopsis sp. ATCC 39116. For this purpose, biotransformation experiments 355

in a 2-L bioreactor scale were carried out to analyze the utilization of ferulic acid. Ferulic 356

acid is catabolized via vanillin and subsequently to vanillic acid and is therefore a potential 357

substrate for the biotechnical production of vanillin. The difference between the vdh-358

deletion mutant and the wild type Amycolatopsis sp. ATCC 39116 in terms of ferulic acid 359

utilization was demonstrated for ’resting cells‘, independently from growth. The cells were 360

grown in Caso-medium at 42 °C until they reached the stationary growth phase. 361

Afterwards, they were harvested, washed twice with PBS and resuspended in the same 362

buffer (1,000 ml, pH 7.5, 0.5 g/L cell dry weight). Ferulic acid was added at an initial 363

concentration of 2.0 mM (stock concentration: 210 mM, pH 8, NaOH) to the cell 364

suspension, and the occurring intermediates were analyzed by HPLC. During the 365


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transformation process 0.75 mM/h ferulic acid were fed continuously. In order to promote 366

continuing vanillin synthesis, the feeding rate was readjusted to 3.1 mM/h for 3 hours, after 367

the cells had adapted to the conversion conditions and the substrate concentration was 368

decreasing (7h from start). Accordingly, feeding was stopped after 10 hours, and the 369

disappearance of ferulic acid and the occurrence of the products were observed for 370

additional 20 hours. 371

The biotransformation experiments revealed a significant difference between the wild 372

type Amycolatopsis sp. ATCC 39116 and the vdh-deletion mutant with regard to the 373

occurrence of catabolism products (Fig. 5). While Amycolatopsis sp. ATCC 39116 started 374

to accumulate vanillic acid at first, Amycolatopsis sp. ATCC 39116 ∆vdh::KmR 375

accumulated vanillin as the first over-flow product. Concerning the wild type experiment, 376

vanillin was not detectable in the supernatants until vanillic acid was formed at a 377

concentration of about 1 mM. Additional vanillic acid was further synthesized from vanillin, 378

which was meanwhile formed. The latter was completely catabolized after ferulic acid was 379

no longer available for the cells. In contrast, only a very low concentration of vanillic acid 380

was formed by the cells of the mutant strain, while cultures of these cells accumulated 381

vanillin continuously up to 6.8 mM (wild type 2.9 mM) as long as ferulic acid was present in 382

the experiment. Furthermore, in contrast to the wild type, degradation of the synthesized 383

vanillin occurred only at a very low rate, even during the last 10 hours, when ferulic acid 384

was completely degraded. Further catabolism intermediates like guaiacol were only 385

detected in trace amounts in cultures of the wild type. 386

Growth analysis on vanillin and ferulic acid in mineral salts medium with 0.1 % (wt/vol) 387

of the substrates as sole source of carbon were also carried out. While the wild type 388

exhibited slow growth on both substrates after a long lag-phase of about 30-40h, the 389

mutant did not grow at all on vanillin. During the experiment no utilization of vanillin was 390


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detectable for the mutant strain. On ferulic acid Amycolatopsis sp. ATCC 39116 391

∆vdh::KmR showed a significantly reduced growth rate when compared to the wild type, 392

indicating that VDHATCC 39116 has a substantially impact on the metabolism of ferulic acid. 393

However, the experiment suggests that ferulic acid can serve as source of carbon and 394

energy despite of the knockout. VDH activity of both, the wild type strain and the mutant 395

was observed subsequently. While the wild type exhibited dehydrogenase activity after 396

growth with ferulic or vanillin as previously observed, no VDH activity was detected in the 397

mutant strain, even despite of induction. 398

399

DISCUSSION 400

401

The actinomycete Amycolatopsis sp. ATCC 39116 is capable of synthesizing large 402

amounts of vanillin from the natural substrate ferulic acid (26). This bacterium therefore 403

represents the most promising platform for the biotechnical production of natural vanillin. 404

However, significant amounts of the desired product are lost because of the inherent 405

vanillin catabolism via vanillic acid (43). The availability of the genome sequence of 406

Amycolatopsis sp. ATCC 39116 contributed to our understanding of the vanillin catabolism 407

in this strain. Furthermore, it offered the possibility to identify responsible genes as a target 408

for metabolic engineering of this production strain. Systematic gene deletions have been 409

intensively used for strain development of various production processes. In order to 410

improve industrial applications that approach the theoretical product yields, catabolic or 411

competing pathways of the desired product were disrupted. Prominent examples are the 412

production of amino acids, ethanol and succinate by Corynebacterium glutamicum, 413

S. cerevisiae and E. coli, respectively (6, 48). 414


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In several vanillin-degrading bacteria, a vanillin dehydrogenase was identified to 415

catalyze the oxidation of vanillin to vanillic acid (13, 23, 34, 36, 46). The activity of a vanillin 416

dehydrogenase could now be demonstrated in Amycolatopsis sp. ATCC 39116, too. 417

Moreover, the expression of the corresponding gene seems to be inducible by the 418

enzyme’s substrate and structurally related compounds, such as ferulic acid. As already 419

shown for P. fluorescens AN103 (13, 21) enzyme activity could not be observed in cells 420

cultivated in the absence of these substance. 421

Based on the genome sequence of Amycolatopsis sp. ATCC 39116 and its annotation, a 422

set of putatively responsible genes was identified showing homology to previously 423

characterized enzymes with aldehyde dehydrogenase activity of other organisms. 424

Selected gene candidates were heterologously expressed in E. coli and VDH activity was 425

monitored. One of the encoded enzymes was capable of catalyzing the oxidation of vanillin 426

to vanillic acid and thus was designated as VDHATCC 39116. The enzyme exhibited homology 427

to several aldehyde dehydrogenases including also a characteristic motif with glutamic 428

acid as a central amino acid of the catalytic center (Fig. S1). Interestingly the protein does 429

not show a remarkably high homology to the group of enzymes with already verified 430

vanillin dehydrogenase activity (Fig. 1). However, all up to now characterized proteins 431

derive from a phylogenetically closely related group of bacteria. VDHATCC 39116 represents 432

the first vanillin dehydrogenase that was characterized in detail in actinobacteria and might 433

therefore represent an independently evolved protein. This is confirmed by the observation 434

that the encoding gene vdh seems not to be part of cluster as previously described for 435

other studied strains (9, 29, 34, 36, 46). 436

Upon the heterologous expression of the encoding gene vdh in E. coli, a protein signal 437

with an apparent molecular weight of 54.0 ± 2.0 kDa was obtained in SDS-PAGE analysis. 438

This is in agreement with both, the calculated molecular weight of the vdh translational 439


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product and the sizes of other enzymes exhibiting VDH activity (4, 23, 36, 46). The 440

recombinant protein was purified to apparent homogeneity carrying a 6xHis6-tag, which 441

was confirmed by immuno detection. VDHATCC 39116 activity was detected in the eluated 442

protein fraction verifying the identity of the vanillin dehydrogenase of Amycolatopsis sp. 443

ATCC 39116. In line with this, VDH activity was only detectable in presence of the cofactor 444

NAD+, indicating an NAD+-dependent aldehyde dehydrogenase as already supposed 445

based on the theoretical analysis of the amino acid sequence. In contrast, former studies 446

stated that the catabolism of vanillin in Amycolatopsis sp. ATCC 39116 is only catalyzed by 447

an oxidase (43). NADP+ could not be used as cofactor. A preference towards NAD+ was 448

also reported for other studied vanillin dehydrogenases (13, 23). Moreover, VDH activity of 449

Amycolatopsis sp. ATCC 39116 could be enhanced by supplementary episomal 450

expression of vdh, which thereby validates the enzymes function. VDHATCC 39116 was 451

shown to catalyze the oxidation of different aromatic aldehydes, but has a preference 452

towards vanillin as previously shown for Sphingomonas paucimobilis SYK-6 (23). The 453

enzyme was active in a broad range of conditions including different pH and temperatures 454

and showed its maximum activity at a pH of 8.0 and at a temperature of 44°C, respectively. 455

It exhibited a considerably storage stability since 86 % of its former activity could be 456

determined after 24h at 4°C. 457

Based on the results of former studies (1, 25, 43) and in correspondence with the 458

results obtained in this study, we propose for Amycolatopsis sp. ATCC 39116 the following 459

CoA-dependent pathway for the degradation of ferulic acid via vanillin and vanillic acid to 460

protocatechuic acid or guaiacol with VDHATCC 39116 as a key enzyme (Fig. 6). 461

Following the identification of the VDHATCC 39116 encoding gene, the physiological impact 462

of VDHATCC 39116 for the catabolism of vanillin and related compounds, such as ferulic acid, 463

was determined. Therefore, a precise vdh-deletion mutant was generated, and the 464


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catabolism of ferulic acid of the mutant was investigated and compared to the wild type of 465

Amycolatopsis sp. ATCC 39116. A ‘resting cell’ approach was employed, and the occurring 466

catabolism intermediates, including vanillin and vanillic, were observed by HPLC. 467

Both, the wild type and Amycolatopsis sp. ATCC 39116 ∆vdh::KmR, degrade ferulic acid 468

in a comparable manner (Fig. 5A). In contrast to former studies no negative effect, 469

regarding the catabolism of ferulic acid was observed (21, 33, 34). The authors assumed 470

polar effects, as a consequence of the vdh deletion or disruption, respectively, to be 471

responsible for the resulting phenotype. Since the vdh gene of Amycolatopsis sp. 472

ATCC 39116, unlike in other studied strains, is not part of a cluster with other ferulic acid 473

catabolism genes, a similar effect was not to be expected for the present strain (9, 29, 34, 474

36, 46). A remarkable difference was, however, observed for the synthesis of vanillin and 475

vanillic acid (Fig. 5B, 5C). The vdh-deletion mutant showed a 2.3 times enhanced vanillin 476

accumulation, which started immediately after exposure of the cells to the substrate. A final 477

concentration of 6.8 mM was reached, and the excreted vanillin in the culture supernatants 478

was degraded only very slowly, even after ferulic acid was completely metabolized. The 479

wild type synthesized and excreted vanillin only up to 2.9 mM but only after an initial 480

accumulation of vanillic acid up to 1 mM had occurred. This observation has previously 481

also been made for the wild type Streptomyces sp. ATCC 39116 by Muheim and Lerch 482

(25). Since the accumulation of vanillin by the deletion mutant started independently from 483

vanillic acid, a regulatory effect, which influences the expression of vdh or the activity of 484

the enzyme in the wild type depending on the vanillic acid concentration, could be a 485

possible explanation. Moreover, as a result of the decreasing ferulic acid concentration, 486

vanillin was subsequently degraded by the wild type cells. In line with this, further 487

catabolism intermediates like guaiacol or protocatechuic acid were formed in detectable 488

amounts only in cultures of the wild type. 489


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Growth analysis of the mutant strain revealed, as previously described for other 490

knockout mutants (21, 23), that the bacteria are no longer able to use vanillin as sole 491

source of carbon and energy. The subsequent enzyme assay showed in coincidence with 492

this that in contrast to the wild type, no oxidation of vanillin was detectable under the used 493

conditions. This observation is in accordance with the fact, that no other tested homologue 494

of VDH ATCC 39116 showed the desired enzyme activity. However, an alternative minor 495

pathway of vanillin oxidation might be present, since a slow degradation of vanillin was 496

observable in the biotransformation experiments, even despite of the knockout. 497

These experiments clearly showed that VDHATCC 39116 plays a significant role in the 498

course of ferulic acid degradation in Amycolatopsis sp. ATCC 39116. Therefore, the 499

deletion mutant represents a promising production strain to compete with the already 500

established vanillin production processes using wild type strains (26, 39). 501

Further metabolic engineering will improve the already obtained outcome since a slow 502

degradation of vanillin, probably due to unspecific aldehyde dehydrogenases or oxidases 503

activities (43), occurred even for Amycolatopsis sp. ATCC 39116 ∆vdh::KmR. Similar 504

observations were also made for different other vanillin production strains, in which VDH-505

encoding genes were deleted or disrupted (21, 31, 33, 34). In addition, ferulic acid could 506

be completely degraded to serve as source of energy through competing pathways, such 507

as decarboxylation to 4-vinylguaiacol (37). This could be the reason why vanillin or vanillic 508

acid, respectively, were not produced in equimolar amounts in the biotransformation 509

experiments (42% molar yield for the mutant, 19% for the wild type). Optimization of the 510

fermentation process is therefore aiming at an improved vanillin synthesis rate and at a 511

higher molar yield of vanillin with regard to ferulic acid. 512

Based on the present study, a highly efficient microbial production strain for natural 513

vanillin from ferulic acid can now be developed. Complete prevention of the catabolism of 514


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vanillin during the biotransformation and optimizing the fermentation process will 515

significantly improve the production of natural vanillin using Amycolatopsis sp. 516

ATCC 39116 ∆vdh::KmR. 517

518

ACKNOWLEDGEMENTS 519

The project was financially supported by SYMRISE AG, Holzminden, Germany, which is 520

gratefully acknowledged. We thank Isabelle van Hamme for technical assistance. 521

522

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684

685 686


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LEGENDS TO FIGURES 687

FIG. 1. Phylogenetic tree of the putative VDH of Amycolatopsis sp. ATCC 39116 and 688

related enzymes with ALDH or VDH activity. Multiple sequence alignment using 689

“ClustalX 1.83“ based on the amino acid sequences of the following enzymes: putative 690

VDHATCC 39116 of Amycolatopsis sp. ATCC 39116; VDH of Pseudomonas sp. HR199 (36); 691

VDH of Pseudomonas fluorescens AN103 (12); VDH of P. putida WCS358 (46); VDH of 692

P. putida KT2440 (28); VDH of Sphingomonas paucimobilis SYK-6 (22); Benzaldehyde 693

dehydrogenase (NAD+) of Nocardioidaceae bacterium Broad-1 (ZP_08197264); 694

Benzaldehyde dehydrogenase of Rhodococcus erythropolis PR4 (41); Benzaldehyde 695

dehydrogenase (NAD+) of Saccharopolyspora erythraea NRRL2338 (29); Benzaldehyde 696

dehydrogenase (NAD+) of Rhodococcus jostii RHA1 (24); Aldehyde dehydrogenase of 697

Streptomyces lividans TK24 (ZP_06526522); Benzaldehyde dehydrogenase (NAD+) of 698

Burkholderia cenocepacia AU 1054 (YP_621190); Benzaldehyde dehydrogenase (NAD+) 699

of Pseudomonas fluorescens Pf0-1 (42); Benzaldehyde dehydrogenase of Pseudomonas 700

putida (15); Aldehyde dehydrogenase of Pseudonocardia sp. P1 (ZP_08121488); NAD+-701

dependent Aldehyde/ benzaldehyde dehydrogenase of Amycolatopsis mediterranei U32 702

(49). Calculations were performed using the neighbour joining method (39). Enzymes with 703

verified vanillin dehydrogenase activity are shown in bold. 704

705

FIG. 2. Documentation of the purification of His6-tagged VDHATCC 39116 by SDS-706

PAGE (A) and immuno detection of the His6-tag (B). The samples were separated in 707

two identically prepared 12.5 % (wt/vol) SDS-polyacrylamide gels. The first gel was stained 708

with Serva Blue G (A), while the proteins of the second gel were transferred onto a PVDF-709

membrane, and the His6-tag was immuno detected as described in materials and methods 710


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(B). Lanes 1 and 8: 8 µl molecular weight marker; 2: 25 µg protein of crude extract of 711

E. coli BL21(DE3) pET23a; 3: 25 µg protein of crude extract of E. coli BL21(DE3) 712

pET23a::vdhHis; 4: 25 µg protein of soluble fraction of E. coli BL21(DE3) pET23a::vdhHis; 713

5: 25 µg protein of flowthrough of the purification; 6: 10 µg protein of the first washing step; 714

7: 10 µg protein of the eluate. 715

716

FIG. 3. Biotransformation of vanillin by E. coli BL21(DE3) pET23a::vdh. Cells were 717

grown in LB with 100 µg/ml ampicillin at 30 °C and harvested after 16 h in the stationary 718

growth phase. After two washing steps with 100 mM potassium phosphate buffer (pH 7.5), 719

cells were resuspended in 50 ml mineral salts medium containing 100 µg/ml ampicillin and 720

14 mM vanillin. The cultures were incubated at 30 °C and 100 rpm for 8 h and 721

biotransformation of vanillin to vanillic acid was observed at the given times using HPLC 722

analyses as described in materials and methods. () vanillin; () vanillic acid. 723

724

FIG. 4. Verification of the gene replacement in Amycolatopsis sp. ATCC 39116 725

∆vdh::KmR. In order to verify a specific gene replacement of the vdh gene by the 726

kanamycin resistance cassette different diagnostic PCRs were carried out using 727

chromosomal DNA as template. The resulting fragments were analyzed in a 1 % agarose 728

gel. Molecular weight marker: “GeneRuler™ 1 kb DNA Ladder” (Fermentas GmbH, St. 729

Leon-Rot, Germany). 730

Lanes 1 and 2 represent the amplificate using oligonucleotides vdhLF_for_diag and 731

vdhRF_rev2: Showing the difference between the wild type Amycolatopsis sp. 732

ATCC 39116 (1) and the mutant Amycolatopsis sp. ATCC 39116 ∆vdh::KmR (2). Lanes 3 733

and 4 present the results using primer vdhLF_for_diag and pRLE6for2 (3) as well as 734

KmR_rev_vdhLF and vdhRF_rev2 (4): Shows the proof of the specific integration of the 735


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kanamycin resistance cassette into the vdh locus for the mutant Amycolatopsis sp. 736

ATCC 39116 ∆vdh::KmR 737

738

FIG. 5. Biotransformation of ferulic acid by Amycolatopsis sp. ATCC 39116 and 739

the mutant Amycolatopsis sp. ATCC 39116 ∆vdh::KmR. Cells were grown in 250 ml 740

Caso-Medium at 42 °C and harvested within the stationary growth phase. After two 741

washing steps with phosphate buffered saline (pH 7.5), cells were resuspended in 742

1,000 of the same buffer containing 2 mM ferulic acid. The cultures were incubated in a 743

2L-bioreactor at 42 °C for 30 h. Biotransformation of ferulic acid (A) to vanillin (B) and 744

vanillic acid (C) was observed at the given times using HPLC analyses as described in 745

materials and methods. Initially, ferulic acid was fed at a rate of 0.75 mM/h during the first 746

7 hours. Afterwards, substrate was fed for 3 hours at a rate of 3.1 mM/h, before feeding 747

was stopped. () Amycolatopsis sp. ATCC 39116; () Amycolatopsis sp. ATCC 39116 748

∆vdh::KmR 749

750

FIG. 6. Proposed pathway of the CoA-dependent catabolism of ferulic acid via 751

vanillin in Amycolatopsis sp. ATCC 39116 752

753

754

755

756

757

758

759

760


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TABLES 761

TAB. 1: Bacterial strains and plasmids used in the present study 762

Bacterial strains

and plasmids

Description, sequence Reference

Strains

Amycolatopsis sp.

ATCC 39116

wild-type, kanamycin-sensitive American Type

Culture Collection,

No. 39116

Amycolatopsis sp.

ATCC 39116

∆vdh::KmR

vdh-deletion-mutant, kanamycin-resistant This study

E. coli Mach-1 T1 F - φ80(lacZ)ΔM15 ΔlacX74 hsdR (rK– mK

+)

ΔrecA1398 endA1 tonA

Invitrogen GmbH

(Karlsruhe,

Germany)

E. coli BL21(DE3) F – ompT hsdSB(rB– mB

–) gal dcm (DE3) Novagen Inc.

(Madison, WI,

USA)

Plasmids

pET23a T7-promotor, His6-tag, T7-terminator, ColE1 pBR322

ori, AmpR,f1 ori

Novagen Inc.

(Madison, WI,

USA)

pET23a::vdh pET23a harboring vdh (NdeI/XhoI) coding for

VDHATCC 39116 colinear to T7-promotor

This study


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pET23a::vdhHis pET23a harboring vdh (NdeI/XhoI) without native

stop codon coding for VDHATCC 39116 with C-terminal

His6-tag, colinear to T7-Promotor

This study

763

764

765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799


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FIGURES 800

Fig. 1 801

802 803 804 805 806 807 808


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809

Fig. 2 810

811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833


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834

Fig. 3 835

836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853


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854

Fig. 4 855

856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871


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872

Fig. 5 873

874

875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894

895

896


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897

Fig. 6 898

899

900

901

902

903

904

905 906


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C C

H

OH

O

CH3

H

C

O

OH

C C

H

OH

O

CH3

H

C

O

S CoAC C

OH

O

CH3

H

C

O

S CoA

H

OH

H

OHO

OH

O

CH3

OHO

OH

OH

VanillinVanillic acidProtocatechuic acid

Ferulic acid Feruloyl-CoA 4-Hydroxy-3-methoxyphenyl-

β-hydroxypropionyl-CoA

O2

NADH+H+NAD+

H2OHCHOH2O

NAD+NADH+H+

CoA-SH

ATP AMP + PPi

H2O

C

H

C

O

S CoA

H

H

HO

OH

O

CH3Vanillin

dehydrogenase

(VDH)

Vanillate

demethylase

(VanA und VanB)

Feruloyl-CoA

synthetase

(FCS)

Enoyl-CoA

hydratase/aldolase

(ECH)

Enoyl-CoA

hydratase/aldolase

(ECH)

Guaiacol

OH

O

CH3

CO2


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investigation of the amycolatopsis sp. atcc 39116...

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