Borneol Dehydrogenase from Pseudomonas sp. Strain TCU-HL1Catalyzes the Oxidation of (�)-Borneol and Its Isomers to Camphor

Hoi-Lung Tsang,a Jui-Lin Huang,a Yu-Hsuan Lin,a Kai-Fa Huang,b Pei-Luen Lu,c Guang-Huey Lin,d Aye Aye Khine,a Anren Hu,e

Hao-Ping Chena

Department of Biochemistry,a Department of Microbiology and Immunology,d and Department of Medical Technology,e School of Medicine, Tzu Chi University, Hualien,Taiwan; Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei, Taiwanb; Department of Bioresources, Da-Yeh University, Changhua, Taiwanc

ABSTRACT

Most plant-produced monoterpenes can be degraded by soil microorganisms. Borneol is a plant terpene that is widely used intraditional Chinese medicine. Neither microbial borneol dehydrogenase (BDH) nor a microbial borneol degradation pathwayhas been reported previously. One borneol-degrading strain, Pseudomonas sp. strain TCU-HL1, was isolated by our group. Itsgenome was sequenced and annotated. The genome of TCU-HL1 consists of a 6.2-Mbp circular chromosome and one circularplasmid, pTHL1 (12.6 kbp). Our results suggest that borneol is first converted into camphor by BDH in TCU-HL1 and is furtherdecomposed through a camphor degradation pathway. The recombinant BDH was produced in the form of inclusion bodies.The apparent Km values of refolded recombinant BDH for (�)-borneol and (�)-borneol were 0.20 � 0.01 and 0.16 � 0.01 mM,respectively, and the kcat values for (�)-borneol and (�)-borneol were 0.75 � 0.01 and 0.53 � 0.01 s�1, respectively. Two plantBDH genes have been reported previously. The kcat and kcat/Km values of lavender BDH are about 1,800-fold and 500-fold lower,respectively, than those of TCU-HL1 BDH.

IMPORTANCE

The degradation of borneol in a soil microorganism through a camphor degradation pathway is reported in this study. We alsoreport a microbial borneol dehydrogenase. The kcat and kcat/Km values of lavender BDH are about 1,800-fold and 500-fold lower,respectively, than those of TCU-HL1 BDH. The indigenous borneol- and camphor-degrading strain isolated, Pseudomonas sp.strain TCU-HL1, reminds us of the time 100 years ago when Taiwan was the major producer of natural camphor in the world.

One of the bicyclic plant secondary metabolites in the terpenefamily is (�)-borneol. It can be isolated from the resin and

essential oil of plants of the families Dipterocarpaceae, Lamiaceae,Valerianaceae, and Asteraceae (1). Its optical isomer counterpart,(�)-borneol, can be obtained from the plant Blumea balsamifera(2). Both (�)-borneol and (�)-borneol are used in traditionalChinese medicine. Currently, commercially available borneol ismainly synthesized through Meerwein-Ponndorf-Verley reduc-tion of camphor. Four different stereoisomers, i.e., (�)-isobor-neol, (�)-isoborneol, (�)-borneol, and (�)-borneol, are presentin chemically synthesized borneol (Fig. 1). Camphor, a bicyclicsaturated terpene ketone, is structurally similar to borneol andalso is widely used in traditional Chinese medicine. Camphor andborneol are included together in many dermal herbal recipes forexternal use, to subdue itching and pain.

Natural borneol is one of the intermediates present in plantcamphor biosynthesis pathways (3). The interactions betweenplant secondary metabolites and soil microorganisms are compli-cated (4). As far as we know, with respect to the biodiversity of soilmicroorganisms, the microbial degradation pathway of borneolhas not yet been reported. One borneol-degrading strain, Pseu-domonas sp. strain TCU-HL1, was isolated from the mountainarea in Hualien, Taiwan, by our group. The camphor degradationpathways and the camphor degradation genes in Pseudomonasputida were reported previously (5–7). Our results show that both(�)-borneol and (�)-borneol are decomposed to camphor in thisstrain. However, the gene encoding borneol dehydrogenase(BDH) (i.e., bdh) had not been identified in the previously de-scribed camphor degradation gene cluster. Here, we report thecloning, sequencing, overexpression, purification, and character-

ization of borneol dehydrogenase, as well as the degradation path-way for borneol in Pseudomonas sp. TCU-HL1.

MATERIALS AND METHODSMaterials. (�)-Borneol, (�)-camphor, and (�)-camphor were obtainedfrom Sigma-Aldrich (St. Louis, MO, USA). (�)-Borneol, (�)-isoborneol,(�)-2-butanol, cyclohexanol, and cyclopentanol were purchased fromAlfa Aesar (Lancashire, United Kingdom). Racemic borneol and isobor-neol were bought from Cheng Yi Chemical Co., Ltd. (Taipei, Taiwan).Phenyl-Sepharose High Performance hydrophobic interaction andQ-Sepharose Fast Flow anion-exchange gel media were obtained from GEHealthcare Life Sciences (Pittsburgh, PA, USA). All chemicals used wereof ACS grade or higher.

Bacterial isolation and identification. Pseudomonas sp. TCU-HL1was isolated from soil samples collected in Hualien County, Taiwan. It wasselectively cultured at 30°C with 0.4 mg/ml (�)-borneol as the sole carbonsource, in liquid M9 minimal medium supplemented with Goodies solu-tion (8) (3 g/liter KH2PO4, 6 g/liter Na2HPO4, 0.5 g/liter NaCl, 1 g/literNH4Cl, 2.625 mM MgSO4, and 0.1 mM CaCl2, supplemented with 1 �M

Received 15 June 2016 Accepted 12 August 2016

Accepted manuscript posted online 19 August 2016

Citation Tsang H-L, Huang J-L, Lin Y-H, Huang K-F, Lu P-L, Lin G-H, Khine AA, Hu A,Chen H-P. 2016. Borneol dehydrogenase from Pseudomonas sp. strain TCU-HL1catalyzes the oxidation of (�)-borneol and its isomers to camphor. Appl EnvironMicrobiol 82:6378 –6385. doi:10.1128/AEM.01789-16.

Editor: V. Müller, Goethe University Frankfurt am Main

Address correspondence to Hao-Ping Chen, hpchen@mail.tcu.edu.tw.

H.-L.T., J.-L.H., and Y.-H.L. contributed equally to this work.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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thiamine, 140 �M MgCl2, 25 �M CaCO3, 42.5 �M FeSO4, 6.25 �MZnSO4, 6.25 �M MnSO4, 1.25 �M CuSO4, 1.25 �M CoCl2, and 1.25 �MH3BO3). The isolate was identified on the basis of 16S rRNA gene se-quence analysis. The genomic DNA was purified by phenol-chloroformextraction methods (9). The PCR conditions for amplification of the 16SrRNA gene were 5 min at 95°C to denature the DNA, followed by 3 cyclesof denaturation at 95°C for 1 min, primer annealing at 55°C for 2 min 15s, and strand extension at 72°C for 1 min 15 s and then 25 cycles ofdenaturation at 95°C for 35 s, primer annealing at 55°C for 1 min 15 s, andstrand extension at 72°C for 7 min, on a thermal cycler (Major Science,Saratoga, CA, USA). The following pair of oligonucleotides was used toamplify the 16S rRNA gene of TCU-HL1: 5=-AGAGTTTGATCATGGCTTAG-3= and 5=-GGTTACCTTGTTACGACTT-3=.

GC-MS analysis of borneol degradation metabolites. TCU-HL1 wasgrown at 30°C with 100 mg chemically synthesized borneol as the solecarbon source in 100 ml M9 minimal medium supplemented as describedabove, with shaking at 150 rpm. The bacterial cells were harvested bycentrifugation when they entered the stationary phase. The bacterial su-pernatant was then immediately extracted with 10 ml ethyl ether. The

ether extracts were directly subjected to gas chromatography-massspectrometry (GC-MS). Analysis was performed using a Thermo TSQQuantum gas chromatograph (Thermo Fisher Scientific, Waltham, MA)equipped with a split/splitless injector, a silanized quartz liner with aninner diameter of 2 mm (effective volume, 3.75 ml), and a TriPlus au-tosampler for 150 vials. An HP-5ms capillary GC column (12 m by 0.2mm i.d.; film thickness, 0.33 �m) was obtained from Agilent Technolo-gies (Santa Clara, CA, USA). Chromatographic conditions were as fol-lows: gas carrier, helium; constant flow rate, 0.7 ml/min; injection vol-ume, 2 �l (splitless mode); and injector temperature, 290°C. The GCtemperature program was as follows: 40°C for 3 min, 40°C to 190°C in 150min, 190°C for 3 min, 190°C to 290°C in 5 min, and 290°C for 10 min. TheThermo TSQ Quantum mass spectrometer was operated in selective ionmonitoring (SIM) mode with ionization by electron impact at 70 eV, withthe transfer line at 290°C, the ion source at 290°C, and the quadrupole at150°C.

Partial purification of borneol dehydrogenase from TCU-HL1crude extract. All purification steps were performed on ice or at 4°C. In atypical purification, 15 g of cells (wet weight) was resuspended in 30 ml of

FIG 1 Degradation pathway of (�)-borneol, (�)-borneol, (�)-isoborneol, and (�)-isoborneol in Pseudomonas sp. TCU-HL1. Bdh, borneol dehydrogenase;CamC, camphor-5-monooxygenase; CamD, 5-exo-hydroxycamphor dehydrogenase.

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10 mM potassium phosphate buffer (pH 7.0) and ruptured by sonication.Cell debris was removed by centrifugation at 25,000 � g for 15 min at 4°C,and the supernatant was brought to 20% ammonium sulfate saturationwith the slow addition of 3.2 g solid into 30 ml supernatant. The proteinsolution was cleared by centrifugation at 25,000 � g for 30 min at 4°C andpurified with a Phenyl-Sepharose High Performance hydrophobic inter-action column (2.6 by 70 cm) and a Q-Sepharose Fast Flow anion-ex-change column (1.6 by 15 cm), using a published purification scheme(10). The following profile was used for separation with the anion-ex-change column: 30 ml of isocratic elution with 100% buffer A (10 mMpotassium phosphate buffer [pH 7.0]), 240 ml of a linear gradient from100% buffer A to 100% buffer B (10 mM potassium phosphate buffercontaining 0.5 M KCl [pH 7.0]), and 50 ml of isocratic elution with 100%buffer B. The protein homogeneity and molecular masses of fractionscontaining activity were examined by SDS-PAGE.

Protein identification by mass spectrometry. Two protein bands,with relative molecular masses of 40 and 30 kDa, were cut from the SDS-PAGE gel and subjected to trypsin digestion. The resultant peptide libraryfrom both bands was analyzed by nano-liquid chromatography-tandemmass spectrometry (LC-MS/MS) analysis (11). For identification of pro-tein bands, a monoisotopic peak list file (in MASCOT generic format[MGF]) of the raw files obtained from an LTQ XL mass spectrometer wasgenerated by MASCOT Daemon 2.2.2, and the MGF files were searchedagainst an in-house server of MASCOT v2.3.2 (Matrix Science, UnitedKingdom) and the Swiss-Prot protein database (released on 21 January2015, containing 547,357 sequences). A total of 331,549 bacterial se-quences in the Swiss-Prot protein database were chosen for taxonomiccategorization. Precursor and production mass tolerances were set as �2and �0.5 Da, respectively.

Gene synthesis. The synthetic gene coding for short-chain dehydro-genase/reductase (SDR) from Pseudomonas putida (GenBank accessionno. WP_032492645.1) was obtained from MDBio, Inc. (Taipei, Taiwan).Its genetic codons were optimized for Escherichia coli class II codon usage.The synthetic sdr gene was delivered in a pET28a expression vector togenerate the plasmid psdrX.

Genome sequencing of strain TCU-HL1. For PacBio library con-struction and sequencing, genomic DNA was sheared using a Covarisg-TUBE, followed by purification via binding to prewashed AMPure PBbeads (PacBio part no. PB100-265-900). After end repair, the blunt adapt-ers were ligated, followed by exonuclease incubation to remove all nonli-gated adapters and DNA. The final SMRTbell templates were annealedwith primers and bound to the proprietary polymerase using PacBioDNA/polymerase binding kit P6 v2 (PacBio part no. PB100-372-700) toform the binding complex. After dilution, the library was loaded onto aPacBio RS II sequencer with a DNA sequencing kit 4.0 (PacBio part no.PB100-356-200) and a SMRT Cell 8Pac for sequencing. The primary fil-tering analysis was performed by the RS II instrument, and the secondaryanalysis was performed using SMRT Analysis software. After assembly,only one contig was obtained for both chromosomal and plasmid DNA.

Open reading frames (ORFs) were identified using the Prodigal pro-gram (http://prodigal.ornl.gov). The bacterial RefSeq database wassearched for homologs using BLASTp, with an E cutoff value of 10. Au-tomatic annotation results were manually verified. The observation thatTCU-HL1 was able to grow in M9-based medium suggested that all genesrequired for de novo biosynthesis of all amino acids, nucleotides, coen-zymes, and carbohydrates were present in its genome. Those biosyntheticpathways were also examined manually using the Pathway Tools program(http://bioinformatics.ai.sri.com/ptools).

Construction of expression vector for recombinant BDH. A pair ofoligonucleotides, i.e., 5=-ATTTAGACATATGAAACTGCTAGAAGGTAAAAGAATCATC-3= and 5=-TAAGCGGATCCTAGCGTACAGCGTACAGCGATCAGGCCAT-3=, was designed using the nucleotide sequence ofthe bdh gene, in order to enable amplification by PCR. NdeI and BamHIsites were introduced at the start and end of the bdh gene. The codingregions for BDH were then amplified by PCR using TCU-HL1 genomic

DNA as the template. Amplification was achieved using 28 cycles at thefollowing temperatures: 95°C for 30 s, 52°C for 45 s, and 72°C for 1 min;finally, the reaction was maintained at 72°C for 10 min. The PCR productswere gel purified, digested with NdeI and BamHI, and ligated with NdeI/BamHI-digested pET-30 vector. The ligation mixture was used to trans-form E. coli DH5�. The plasmid that carried the bdh gene in the correctorientation was designated pbdhX.

Production and purification of recombinant SDR from P. putidaand BDH from Pseudomonas sp. TCU-HL1. The induction and produc-tion of the two recombinant enzymes were carried out by following aprocedure reported previously (12). Both enzymes were produced in aninsoluble form at 20°C. All purification steps were performed on ice or at4°C. In a typical purification, 15 g of cells (wet weight) was resuspended in30 ml of 0.1 M potassium phosphate buffer (pH 7.0) containing 100 mMNaCl, 1 mM dithiothreitol (DTT), and 1 mM EDTA. The cells were rup-tured using a low-temperature ultra-high-pressure continuous-flow celldisrupter (JN-02C; JNBIO, Guangzhou, China). The insoluble fractionwas collected by centrifugation at 25,000 � g for 15 min, and the super-natant was discarded. The pellet was washed twice with 25 ml of the samebuffer supplemented with 0.5% Triton X-100, to remove lipophilic con-taminants. Each wash was followed by centrifugation at 25,000 � g for 15min. The pellet was solubilized in 25 ml of 0.1 M potassium phosphatebuffer (pH 7.0) containing 6 M urea, 10 mM DTT, and 1 mM EDTA. Thesolution was stirred at room temperature for 2 h and cleared by centrifu-gation at 25,000 � g for 15 min. At that point, the purity of the recombi-nant enzymes was �90%, as judged by SDS-PAGE. For SDR, no stepswere taken to purify the enzyme further before refolding. The supernatantwas used as the SDR stock solution for refolding experiments and wasstored at �80°C.

Recombinant denatured BDH was further purified with a Q-Sephar-ose Fast Flow anion-exchange column (1.6 by 5.0 cm). About 100 mg ofproteins was loaded onto the anion-exchange column, which was equili-brated in buffer A (10 mM potassium phosphate buffer containing 10%glycerol and 2 M urea [pH 7.0]). The proteins were eluted with the fol-lowing program: from 0 to 25 ml, 100% buffer A; from 25 to 75 ml, 100%buffer A to 100% buffer B (buffer A containing 0.5 M KCl); and from 75 to125 ml, 100% buffer B. The fractions containing BDH were pooled. Thedenatured BDH solution was used as the stock solution for refoldingexperiments and was stored at �80°C. The protein concentrations ofrecombinant SDR and BDH in the stock solutions were estimated from aCoomassie blue-stained 12% SDS-polyacrylamide gel.

Refolding of recombinant SDR from P. putida and BDH from Pseu-domonas sp. TCU-HL1. All steps were performed at 4°C or on ice. About10 mg of recombinant enzymes in 3 ml of stock solution was added drop-wise, with gentle stirring, to 100 ml of 0.1 M potassium phosphate buffer(pH 7.0) containing 1 M urea, 10% glycerol, and 1 mM DTT. The solutionwas dialyzed overnight against 3 times 2 liters of 0.1 M potassium phos-phate buffer (pH 7.0) containing 10% glycerol and 1 mM DTT. The dia-lysate was then cleared by centrifugation at 25,000 � g for 15 min. Furtherconcentration or purification inevitably led to protein aggregation. Theclarified protein solution was stored on ice.

Enzyme assay. A spectrophotometric method was used to assay theactivity of borneol dehydrogenase. The reaction was initiated by the ad-dition of NAD�, and the reaction mixture was incubated at room tem-perature for 5 min. As BDH oxidized borneol to camphor, the rate ofNADH formation was monitored by the increase in absorbance at 340nm. In a typical reaction, 7 �g of BDH proteins was used in a total volumeof 600 �l of 100 mM Tris buffer (pH 8.5). The Km and kcat of NAD� weredetermined in the presence of 2 mM (�)-borneol and 0.1, 0.2, 0.4, 0.8, 2.0,and 5.0 mM cofactor, whereas the Km and kcat of (�)- and (�)-borneolwere determined in the presence of 2 mM NAD� and 0.1, 0.2, 0.5, 1.0, 2.0,and 3.0 mM substrate. The Km and kcat of (�)-isoborneol were deter-mined in the presence of 2 mM NAD� and 0.08, 0.1, 0.2, 0.5, 1.0, and 2.0mM substrate.

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Molecular modeling and docking. The three-dimensional (3D)structural models of Pseudomonas sp. TCU-HL1 BDH and Pseudomonasputida SDR were generated with the program Discovery Studio (AccelrysSoftware, Inc.), using the crystal structure of meso-2,3-butanediol dehy-drogenase (PDB code no. 1GEG) as the template (13). To generate theinitial binding poses of the cofactor NAD and the substrate borneol, theNAD and borneol models were manually docked into the models ofthe enzymes with the program Coot (14), based on the binding mode ofNAD in meso-2,3-butanediol dehydrogenase and on the position of thecatalytic tyrosine residue, respectively. The initial models were optimizedby energy minimization with Discovery Studio. The stereochemical qual-ity of the resulting models was further checked with the programs 3D-profile and PROCHECK (15, 16). The structure figures were generatedwith the program PyMOL (Schrödinger, New York, NY, USA).

Accession number(s). The Pseudomonas sp. strain TCU-HL1 genomeis available in GenBank under accession numbers CP015992 (chromo-somal; TCU-HL1) and CP015993 (plasmid; pTHL1). Data are also avail-able under BioSample number SAMN05195958 and BioProject numberPRJNA324156.

RESULTSCharacterization of strain TCU-HL1. Pseudomonas sp. TCU-HL1 (BioSample accession number SAMN05195958) was isolatedfrom soil samples. As shown by the BLAST search results for its

16S rRNA gene sequence, it is closely related to the members ofPseudomonas resinovorans. Strain TCU-HL1 is a Gram-negative,nonsporulating, straight rod that is 1.5 to 2.5 �m in length. Itsbacterial morphologies were observed by scanning electron mi-croscopy (SEM) (Fig. 2). It can grow in M9 medium by usingvanillin as the sole carbon source in our laboratory, which is ingood agreement with the previous report (17).

TCU-HL1 is able to use borneol as the sole carbon source. Theborneol degradation metabolites were extracted with ethyl etherand characterized by GC-MS analysis. The results revealed thepresence of two metabolites, i.e., camphor and 2,5-dibornane-2,5-dione, with retention times of 43.72 min and 59.74 min, respec-tively. The degradation of camphor by Pseudomonas putida hasbeen studied extensively (5–7, 18). Perhaps not surprisingly, ourresults suggest that borneol is first converted to camphor by BDHin TCU-HL1. The resultant camphor metabolite is then furtherdegraded by another camphor catabolism enzyme (Fig. 1).

Partial purification of native BDH from TCU-HL1 crude ex-tract. Native BDH in the crude TCU-HL1 cell extracts was firstpurified by phenyl-Sepharose hydrophobic interaction columnchromatography. BDH activity eluted toward the end of the gra-dient, in a broad peak that was not resolved from many othercontaminating proteins. The active fractions were pooled, dia-lyzed overnight against 10 mM potassium phosphate buffer (pH7.0), and loaded onto a Q-Sepharose Fast Flow anion-exchangecolumn. Fractions containing BDH activity were analyzed bySDS-PAGE. The results revealed 30-kDa and 40-kDa proteinbands, the intensities of which correlated with enzyme activity inthe respective fractions. Both bands were cut out and analyzed bynano-LC-MS/MS.

Protein identification by mass spectrometry and implica-tions for genomic analysis. Protein identification by nano-LC-MS/MS analysis and Mascot search was carried out. The four best-matched proteins of the aforementioned 30-kDa and 40-kDa proteinbands are listed in Table 1. Because BDH is a member of the NAD�-dependent dehydrogenases, both putative short-chain dehydroge-nase/reductase (SDR) (GenBank accession no. WP_032492645.1)from Pseudomonas putida and aldehyde dehydrogenase (GenBankaccession no. WP_028626884.1) from Pseudomonas resinovoransappear to be plausible candidates. However, it is worth noting thatthe gene encoding putative SDR is located in the camphor degra-dation gene cluster in Pseudomonas putida (Fig. 3A). This result is

FIG 2 Scanning electron micrograph of Pseudomonas sp. TCU-HL1.

TABLE 1 Identification by nano-LC-MS/MS analysis of protein bands in fractions containing BDH activity

Protein band and protein name

Theoreticalmolecularweight Scorea

No. ofpeptidesmatched

Sequencecoverage(%)

GenBankaccession no. Species

30-kDa bandPantoate--alanine ligase 31,064 475 25 44 WP_028630029.1 Pseudomonas resinovoransSerine/threonine protein kinase 73,742 452 17 18 WP_028631084.1 Pseudomonas resinovoransPutative short-chain dehydrogenase/reductase 30,418 434 42 18 WP_032492645.1 Pseudomonas putidaPrkA-family serine protein kinase 73,660 375 14 12 WP_012074172.1 Pseudomonas aeruginosa

40-kDa bandABC transporter substrate-binding protein 38,209 1,327 196 50 WP_028628212.1 Pseudomonas resinovoransABC-type Fe3� transport system, periplasmic component 38,140 1,037 170 52 WP_003453559.1 Pseudomonas pseudoalcaligenesAldehyde dehydrogenase 53,296 922 64 35 WP_028626884.1 Pseudomonas resinovoransPutative ABC transporter substrate-binding protein 38,138 882 142 42 WP_016491213.1 Pseudomonas resinovorans

a Mascot database search score.

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in accord with our finding that camphor is the first degradationproduct of borneol.

Production and refolding of SDR from P. putida. The sdrgene encoding putative SDR was chemically synthesized. The plas-mid psdrX was used to transform E. coli BL21 and to enable theoverexpression of a recombinant short-chain dehydrogenase, pre-dicted to be borneol dehydrogenase, from P. putida. The produc-tion of recombinant proteins through isopropyl--D-thiogalacto-pyranoside (IPTG) induction was carried out as describedpreviously (12). The proteins were produced at high levels in theform of inclusion bodies (data not shown). The insoluble recom-binant proteins were refolded by dissolution in 8 M urea and weredialyzed against 10 mM potassium phosphate buffer in the pres-ence of 10% glycerol. However, no BDH activity could be detectedby spectrophotometric methods after refolding.

Genome sequencing and camphor degradation gene clusterof strain TCU-HL1. To determine the genes that are involved inthe degradation of borneol, the bacterial genome of TCU-HL1was sequenced and assembled by a single-molecule sequencer, i.e.,a PacBio RS II sequencing instrument from Pacific Biosciences.The genome of TCU-HL1 consists of a 6.2-Mbp circular chromo-some and one circular plasmid, pTHL1 (12.6 kbp). The annotatedgenome includes 70 tRNA genes, representing all 20 amino acids,and 16 rRNA genes (all of which occur in the chromosome). Met-abolic reconstruction of 5,770 annotated ORFs using PathwayTools v19.5 resulted in the prediction of 231 metabolic pathwaysin TCU-HL1, including 167 biosynthesis pathways, 97 degrada-tion and assimilation pathways, 5 detoxification pathways, and 10pathways involved in precursor metabolite production and energygeneration. The taxonomic identification of TCU-HL1 basedon whole-genome information was also determined by usingANItools (19) (http://ani.mypathogen.cn). The species closest to

TCU-HL1 in the ANItools database is Pseudomonas resinovoransNBRC 106553. This result is in accord with the results from 16SrRNA sequence comparisons.

The protein sequence of the aforementioned putative SDRfrom Pseudomonas putida was used for a BLAST search against allprotein sequences in the annotated TCU-HL1 genome with E val-

FIG 3 Comparison of the camphor degradation gene clusters between Pseudomonas putida (A) and Pseudomonas sp. TCU-HL1 (B). The genes involved inborneol or camphor degradation are shown in gray. The genes shown in dark gray were expressed and the properties of their product proteins were investigatedin this study. bdh, borneol dehydrogenase; C, camphor-5-monooxygenase; D, hydroxycamphor dehydrogenase; E25, 2,5-diketocamphane-1,2-monooxygenase;F, 2-oxo-3-4,5,5-trimethylcyclopentenyl-acetyl-coenzyme A (CoA) synthase; G, 2-oxo-3-4,5,5-trimethylcyclopentenyl-acetyl-CoA 1,2-monooxygenase; E36,3,6-diketocamphane-1,6-monooxygenase.

TABLE 2 Sequences of trypsin-digested fragments present in both BDHfrom Pseudomonas sp. TCU-HL1 and SDR from P. putida

Sequence Mass (Da)

Position (residues)

In BDH In SDR

INAVLPYMVTPMYVDFR 2,029.03 182–198 182–198IIVTGGAQGIGASVVR 1,497.87 9–24 9–24AAVHTWTR 941.49 162–169 162–169QWGPDGIR 928.46 174–181 174–181

FIG 4 SDS-PAGE (12% gel) of samples taken after each step in the purifica-tion of the recombinant BDH. Lane 1, size markers; lane 2, crude cell extractbefore IPTG induction; lane 3, crude cell extract after IPTG induction; lane 4,insoluble protein isolated from induced cells; lane 5, denatured proteins puri-fied by Q-Sepharose Fast Flow anion-exchange column chromatography.

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ues of 0.001. Only one sequence exactly matched our query. Asshown in Fig. 3B, the predicted borneol dehydrogenase is alsolocated in a camphor degradation gene cluster. However, the or-ganization of the camphor degradation gene cluster in Pseudomo-nas sp. TCU-HL1 is quite different from that in Pseudomonasputida.

The bdh gene (THL1_4180) in the TCU-HL1 genome encodesa polypeptide chain of 260 amino acid residues, with a predictedmolecular mass of 27.6 kDa and a pI of 5.66. An N-terminal con-served Gly13-X-X-X-Gly17-X-Gly19 sequence motif is present inthe NAD(H)-binding region (20). More importantly, a catalyti-cally essential Tyr157-X-X-X-Lys161 motif and a Ser144 residue,which are conserved in many short-chain dehydrogenases, can befound (21). As shown in Table 2, four trypsin-digested peptidefragments of BDH and SDR have the same amino acid sequencesand molecular masses. These results are also in good agreement

with the 18% coverage shown in the nano-LC-MS/MS analysisand Mascot search.

Production and refolding of putative recombinant BDHfrom strain TCU-HL1. The gene encoding BDH in TCU-HL1 wasamplified by PCR and subcloned into the expression vector pET-30. The resulting plasmid, pbdhX, was used to transform E. coliBL21. As shown in Fig. 4, recombinant BDH was produced in theform of an inclusion body in E. coli. The refolding conditions forrecombinant BDH were the same as those for the aforementionedSDR from P. putida. The refolded recombinant BDH from TCU-HL1 was enzymatically active toward (�)-borneol, (�)-borneol,and (�)-isoborneol. Further attempts to concentrate the proteinsolution, either by ultrafiltration or with spin columns, resulted inthe precipitation of refolded proteins. This result implies that itwould be technically difficult to obtain BDH protein crystals tosolve the 3D structures. The very dilute protein solution is still

FIG 5 (A) Surface representations showing the tertiary structures of BDH (left), SDR (center), and meso-2,3-butanediol dehydrogenase (right). Red dashedcircles, entrances of the substrate access tunnels. (B) Representation of the active sites of BDH (pale cyan) and SDR (light orange). The hydroxyl group of catalyticTyr is located between the hydroxyl group of borneol and the nicotinamide group of NAD�. Amino acid residue Gln91 in SDR blocks the substrate access tunnel.

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active for at least 10 days, without significant loss of activity, whenit is kept on ice.

Kinetic analysis. To investigate the optimal pH value for theenzyme, the pH of the assay solution was varied between 6.5 and9.0. A pH optimum was observed in the alkaline range, and themaximal rate was achieved at about pH 8.5. Therefore, all furtherkinetic and substrate specificity experiments were conducted atpH 8.5. Computer analysis gave apparent Km values for (�)-bor-neol, (�)-borneol, (�)-isoborneol, and NAD� of 0.20 � 0.01,0.16 � 0.01, 0.10 � 0.01, and 0.28 � 0.01 mM, respectively. Thekcat values for (�)-borneol, (�)-borneol, and (�)-isoborneolwere 0.75 � 0.01, 0.53 � 0.01, and 0.39 � 0.01 s�1, respectively. Astrong preference for NAD� over NADP� was observed. Whencofactor NAD� was replaced by another cofactor, NADP�, thecatalytic rate of refolded BDH was reduced to �5% (data notshown). The recombinant BDH also did not use isopropanol, cy-clohexanol, cyclopentanol, or (�)-2-butanol as a substrate (datanot shown).

Molecular modeling and docking. The amino acid sequenceof TCU-HL1 BDH shows 34% identity and 51% similarity to thepreviously crystallized meso-2,3-butanediol dehydrogenase fromKlebsiella pneumoniae (13). Because of its sequence similarity, thisstructure was used as the template to build the TCU-HL1 BDHhomology model and the P. putida SDR homology model.

The results of the kinetic assay showed that the cofactorNADP� was barely taken up by BDH from TCU-HL1. To eluci-date these catalytic properties, NAD� was docked into the activesite of the BDH homology model to examine the interactions in-side the conserved NAD�-binding domain. Perhaps not surpris-ingly, the model suggests that the negatively charged phosphategroup of bound NADP� is repulsed by the negatively chargedresidue Asp37 and also is sterically hindered by the bulky residuesGln16, Met38, and Asn39.

Comparative structure analysis results show that a tunnel forthe substrate to access the active site is located at the center of BDHfrom TCU-HL1, as shown in Fig. 5A. In contrast, in SDR from P.putida, the bulky amino acid residue Gln91 blocks the tunnel tothe active site so as to inactivate the enzyme’s activities towardborneol (Fig. 5B).

DISCUSSION

One hundred years ago, Taiwan was the major producer of naturalcamphor, accounting for more than 70% of the worldwide naturalcamphor production (22). In addition to the major terpene cam-phor, many other terpenes, including borneol, are present in theessential oil of the camphor tree. This is probably one of the rea-sons why so many camphor- or borneol-degrading strains of mi-croorganisms have been isolated by our group on this camphortree-covered evergreen island.

As far as we know, neither the microbial BDH gene nor theborneol degradation pathway has been reported. Herein we de-scribe the degradation of borneol in microorganisms. The role ofSDR in the P. putida camphor degradation gene cluster, the TCU-HL1 BDH homolog, remains obscure. No enzymatic activitycould be detected when methanol, ethanol, isopropanol, or (�)-2-butanol was used as the SDR substrate (data not shown).

Two plant genes encoding borneol dehydrogenase were previ-ously identified, cloned, and sequenced (3, 23). The sequencecomparison shows that the deduced amino acid sequence of theBDH gene from TCU-HL1 shares 48% similarity and 32% identity

with the BDH gene from Artemisia annua L. (GenBank accessionno. ADK56099.1) (23) and 44% similarity and 31% identity withthe BDH gene from Lavandula � intermedia (GenBank accessionno. AFV30207.1) (3). However, the Km and kcat values of lavenderBDH for (�)-borneol are 53.6 �M and 0.0004 s�1, respectively.The kcat and kcat/Km values of lavender BDH are about 1,800-foldand 500-fold lower, respectively, than those of TCU-HL1. Thekinetic properties of plant and microbial BDHs might reflect theirdifferent physiological roles. The physiological role of soil micro-bial BDH is to degrade borneol in order to provide an energysupply to keep cells alive. Microbial BDHs with weak activitywould indicate that microbial cells cannot obtain energy fromborneol very efficiently. However, plant BDH is responsible forthe biosynthesis of the secondary metabolite camphor. Generallyspeaking, secondary metabolites are not essential for plant sur-vival. Plant BDHs with low catalytic activities could still synthesizeand accumulate camphor throughout the life of the plant.

ACKNOWLEDGMENTS

We declare no conflicts of interest.Author contributions were as follows: H.-L.T., overexpression, puri-

fication, and characterization of recombinant BDH and SDR; J.-L.H. andY.-H.L., isolation of strain TCU-HL1, preparation of TCU-HL1 genomicDNA, partial purification of native BDH, and protein identification bymass spectrometry; K.-F.H., homology modeling and molecular dockingof BDH; P.-L.L., borneol degradation gene cluster analysis; G.-H.L., elec-tron microscopy of strain TCU-HL1; A.A.K., borneol degradation samplepreparation; A.H., GC-MS analysis; and H.-P.C., TCU-HL1 genome an-notation, design of experiments, and manuscript preparation.

FUNDING INFORMATIONThis work, including the efforts of Hao-Ping Chen, was funded byMinistry of Science and Technology, Taiwan (MOST) (MOST103-2311-B-320-002).

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Borneol Degradation in Pseudomonas spp.

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Erratum for Tsang et al., “Borneol Dehydrogenase fromPseudomonas sp. Strain TCU-HL1 Catalyzes the Oxidation of(�)-Borneol and Its Isomers to Camphor”

Hoi-Lung Tsang,a Jui-Lin Huang,a Yu-Hsuan Lin,a Kai-Fa Huang,b Pei-Luen Lu,c Guang-Huey Lin,d Aye Aye Khine,a Anren Hu,e

Hao-Ping Chena

Department of Biochemistry,a Department of Microbiology and Immunology,d and Department of MedicalTechnology,e School of Medicine, Tzu Chi University, Hualien, Taiwan; Institute of Biological Chemistry,Academia Sinica, Nankang, Taipei, Taiwanb; Department of Bioresources, Da-Yeh University, Changhua,Taiwanc

Volume 82, no. 21, p. 6378 – 6385, 2016, https://doi.org/10.1128/AEM.01789-16. Page6379: In Fig. 1, the labels for (�)-isoborneol and (�)-isoborneol should be transposed.

Citation Tsang H-L, Huang J-L, Lin Y-H, HuangK-F, Lu P-L, Lin G-H, Khine AA, Hu A, Chen H-P.2018. Erratum for Tsang et al., “Borneoldehydrogenase from Pseudomonas sp. strainTCU-HL1 catalyzes the oxidation of (+)-borneoland its isomers to camphor.” Appl EnvironMicrobiol 84:e02230-18. https://doi.org/10.1128/AEM.02230-18.

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