APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2011, p. 4230–4233 0099-2240/11/$12.00 doi:10.1128/AEM.02998-10 Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 77, No. 12
Novel (2R,3R)-2,3-Butanediol Dehydrogenase from Potential Industrial Strain Paenibacillus polymyxa ATCC 12321䌤† Bo Yu,1§ Jibin Sun,1,2 Rajesh Reddy Bommareddy,1 Lifu Song,1,2 and An-Ping Zeng1* Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, 21071 Hamburg, Germany,1 and Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, People’s Republic of China2 Received 21 December 2010/Accepted 16 April 2011
A (2R,3R)-2,3-butanediol dehydrogenase (BDH99::67) from Paenibacillus polymyxa ATCC 12321 was functionally characterized. The genetic characteristics of BDH99::67 are completely different from those of mesoand (2S,3S)-2,3-butanediol dehydrogenases. The results showed that BDH99::67 belongs to the medium-chain dehydrogenase/reductase superfamily and not to the short-chain dehydrogenase/reductase superfamily, to which meso- and (2S,3S)-2,3-butanediol dehydrogenases belong. report concerning characterization of R,R-BDH from a strain with industrial potential exists. To investigate the (2R,3R)-2,3-BDL formation mechanism, P. polymyxa ATCC 12321 was sequenced (unpublished). From the draft genome sequence, one open reading frame (ORF), scaffold99_orf00067, whose deduced amino acid sequence has a 67% identity with the experimentally verified (2R,3R)-2,3butanediol dehydrogenases from B. subtilis (10), was identified. The gene sequence also has 96% and 95% identities, respectively, with the annotated alcohol dehydrogenase genes from two rhizobacteria, P. polymyxa E681 (7) and P. polymyxa SC2 (8). Therefore, scaffold99_orf00067 was chosen as the candidate R,R-BDH gene for further investigation. The gene is comprised of 1,053 bp, and the GC content is
2,3-Butanediol (2,3-BDL), which has a wide range of important utilizations, was industrially produced in the 1940s by fermentation and again is gaining substantial attention in the new wave of industrial bioprocess development (2, 15, 20). Furthermore, optically active 2,3-BDLs are particular valuable chemicals, which are especially important to provide chiral groups in drugs and high-value pharmaceuticals or for liquid crystals (2). Paenibacillus polymyxa ATCC 12321 has the ability to form almost exclusively the R isomer of 2,3-BDL (over 98%) when grown under anaerobic conditions, and therefore it is considered a promising, nonpathogenic producer with high industrial potential (8, 9). However, the limited knowledge about (2R,3R)-2,3-BDL metabolism and the enzymes responsible for (2R,3R)-2,3-BDL formation, such as (2R,3R)-2,3butanediol dehydrogenase (R,R-BDH), in P. polymyxa has hindered strain improvement. Previously, only one R,R-BDH from Saccharomyces cerevisiae was characterized (3). However, the observed yield in S. cerevisiae is rather low (about 1 mM). S. cerevisiae can be hardly considered an industrial producer for (2R,3R)-2,3-BDL. Nicholson (10) identified a Bacillus subtilis ydjL gene encoding 2,3-butanediol dehydrogenase, and the gene product was later confirmed to be an R,R-BDH (21). However, no more characteristics of the enzyme are available. Besides the above-described enzymes, two alcohol dehydrogenases from the thermophilic organism Thermoanaerobacter brockii and the mesophilic organism Clostridium beijerinckii were reported to have activities toward (3R)-acetoin to produce (2R,3R)-2,3-BDL after gene codon optimization for expression in Escherichia coli (13, 21). To our knowledge, no
TABLE 1. Substrate specificities of (2R,3R)2,3-butanediol dehydrogenasea Reaction and substrate
Activity (%)
Oxidation (2R,3R)-2,3-Butanediol.............................................................100 ⫾ 2 meso-2,3-Butanediol.................................................................. 72 ⫾ 4 (2S,3S)-2,3-Butanediol.............................................................. 0 ⫾ 0 Ethanol ....................................................................................... 5 ⫾ 3 Glycerol ...................................................................................... 25 ⫾ 1 1-Propanol.................................................................................. 5 ⫾ 1 2-Propanol.................................................................................. 15 ⫾ 1 1-Butanol.................................................................................... 6 ⫾ 2 1,2-Propandiol ........................................................................... 51 ⫾ 5 1,3-Propandiol ........................................................................... 5 ⫾ 2 1,2-Pentandiol............................................................................ 65 ⫾ 5 1,5-Pentandiol............................................................................ 7 ⫾ 5 Reduction (3R/3S)-Acetoin.........................................................................100 ⫾ 1 Diacetyl....................................................................................... 91 ⫾ 1 Glyceraldehyde-3-phosphate.................................................... 12 ⫾ 2 Dihydroxyacetone phosphate, dilithium................................. 0 ⫾ 2
* Corresponding author. Mailing address: Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, 21071 Hamburg, Germany. Phone: 49-40-42878 3217. Fax: 49-4042878 2909. E-mail:
[email protected]. § Present address: The Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, People’s Republic of China. † Supplemental material for this article may be found at http://aem .asm.org/. 䌤 Published ahead of print on 29 April 2011.
a Enzyme activities in the diol oxidation reaction were measured with 100 mM substrate and 4 mM NAD⫹ in 100 mM carbonate buffer (pH 11.0). The activity toward (2R,3R)-2,3-butanediol was considered 100%. Ketone reduction activities were measured with 10 mM substrate and 0.2 mM NADH in 100 mM phosphate buffer (pH 8.0). The activity toward (3R/3S)-acetoin was considered 100%. Data are averages ⫾ standard deviations (n ⫽ 3).
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FIG. 1. Chiral-column gas chromatography analysis of the substrates and products of a reaction catalyzed by (2R,3R)-2,3-butanediol dehydrogenase from P. polymyxa ATCC 12321. (a) Profile of mixture of standard chemicals. The retention times in this study were as follows (scale, in minutes, shown at bottom): for (3R)-acetoin, 1.6 min; for (3S)-acetoin, 1.7 min; for (2R,3R)-2,3-butanediol, 6.4 min; and for meso-2,3-butanediol, 7.7 min. (b) Product from (2R,3R)-2,3-butanediol. (c) Product from meso-2,3-butanediol. (d) Product from diacetyl (the peak of diacetyl was in front of the solvent peak and is not shown in the picture).
45.39%. The predicted protein molecular mass is 37.8 kDa, with a slightly acidic pI of 5.37 based on the deduced amino acids. The gene was cloned into pET-22b vector and achieved high expression in E. coli BL21(DE3)/pLysS. After purification, the enzyme, here designated BDH99::67, had clear activities on acetoin, meso-2,3-BDL, and R,R-2,3-BDL with NAD(H) as a coenzyme, which could not be substituted by NADP(H). The
purified enzyme showed a subunit molecular mass of ⬃38.0 kDa from SDS-PAGE analysis, which is consistent with the calculated value. The molecular mass of BDH99::67 was further estimated as about 76.0 kDa by native PAGE, indicating a homodimeric structure of the native enzyme (see Fig. S1 in the supplemental material). The effect of pH on the activity of BDH99::67 was investi-
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gated by using both ketone reduction and diol oxidation reactions over a range of pH 4.0 to 12.0 at room temperature. Maximum activity for ketone reduction was observed at pH 8.0, while that for the diol oxidation reaction was observed at pH 11.0. The heat stability of the enzyme was also investigated. After preincubation of the purified enzyme under different temperatures from 30 to 80°C for 20 min, the remaining enzymatic activities were tested. BDH99::67 was stable under 40°C and began to lose activity above 50°C. Interestingly, the heat stabilities of BDH99::67 enzymatic activities were not consistent between ketone reduction and diol oxidation. After incubation at 80°C for 20 min, BDH99::67 still kept about 25% activity for ketone reduction, while almost no activity remained for diol oxidation (see Fig. S2 in the supplemental material). BDH99::67 could also oxidize several diols (Table 1), not only the secondary alcohols [such as (2R,3R)-2,3-BDL] but also some primary alcohols (such as 1,2-propandiol and 1,2-pentandiol). BDH99::67 could oxidize meso-2,3-BDL, although it showed less activity than with the 2R,3R-isomer. (2S,3S)-2,3BDL was not a substrate for BDH99::67 at all. (3R/3S)-Acetoin was the best substrate in the ketone reduction reaction, followed by diacetyl, which had 91% of the specific activity of (3R/3S)acetoin. Analysis with a gas chromatograph equipped with a chiral column was carried out to further detect the products. With respect to diol oxidation reactions, when (2R,3R)-2,3-BDL served as the substrate with NAD⫹ and BDH99::67, (3R)-acetoin was the only product detected. Accordingly, (3S)-acetoin was, as expected, the only product obtained from meso-2,3-BDL. Furthermore, (3R)-acetoin was the product from diacetyl by BDH99::67 (Fig. 1). When a racemic mixture of (3R/3S)-acetoin was incubated with BDH99::67 and NADH, a product mixture of (2R,3R)-2,3-BDL and meso-2,3-BDL was observed by high-performance liquid chromatography (HPLC) analysis (data not shown). Since BDH99::67 can catalyze both mesoand (2R,3R)-2,3-BDL formation from (3R/3S)-acetoin, the key issue was the configuration of acetoin in P. polymyxa to further improve the optical purity of final (2R,3R)-2,3-BDL production. This study also provides further useful hints for strain improvement, for example, toward diacetyl reductase, which is predicted to be responsible for (3S)-acetoin formation in P. polymyxa under aerobic conditions (18). The diacetyl reductase gene (scaffold80_orf00014) was also annotated from P. polymyxa ATCC 12321 and functionally verified to catalyze diacetyl to (3S)-acetoin (see Fig. S3 in the supplemental material). The comparative data of apparent Km values for R,R-BDHs from P. polymyxa (this study), S. cerevisiae (3), and B. subtilis (21) and one meso-BDH from Klebsiella pneumoniae (19) are summarized in Table 2. The Km values of BDH99::67 were 1.76 mM for (2R,3R)-2,3-butanediol, 5.62 mM for meso-2,3-butanediol, 0.30 mM for (3R/3S)-acetoin, 0.046 mM for NADH, and 0.54 mM for NAD⫹. The much lower Km values of BDH99::67 than of the enzyme from S. cerevisiae also indicated that BDH99::67 is more active for industrial application. The Km value for (2R,3R)-2,3-BDL is much lower than that for meso2,3-BDL, and this result combined with the results from the substrate specificity test show that BDH99::67 could be categorized as an NAD-dependent (2R,3R)-2,3-butanediol dehydrogenase. As the functionality of BDH99::67 was verified, the amino acid sequence as deduced from the nucleotide sequence was
APPL. ENVIRON. MICROBIOL. TABLE 2. Summarized kinetic constants of (2R,3R)- and meso-2,3-butanediol dehydrogenases Km (mM)a
Substrate P. polymyxa
S. cerevisiae
B. subtilis
(2R,3R)-2,31.76 ⫾ 0.29 14 ⫾ 5 Butanediol meso-2,3-Butanediol 5.62 ⫾ 0.81 65 ⫾ 9 (3R/3S)-Acetoin 0.30 ⫾ 0.03 4.5 ⫾ 0.5 0.26 ⫾ 0.02 NADH 0.046 ⫾ 0.009 0.055 ⫾ 0.005 ⫹ 0.54 ⫾ 0.02 0.55 ⫾ 0.003 NAD
K. pneumoniae
5.20 0.72 (R) 0.05 0.06
a All experiments were carried out at room temperature. Km values were extracted by nonlinear regression analysis. Data are averages ⫾ standard deviations (n ⫽ 3). Data for S. cerevisiae are from reference 3. Data for B. subtilis are from reference 21. Data for K. pneumoniae are from reference 19, and no averages ⫾ standard deviations were available; the Km value for acetoin applies only to (3R)-acetoin, and the enzyme was meso-2,3-butanediol dehydrogenase.
compared with those of other reported R,R-BDHs by multiple alignment (see Fig. S4 in the supplemental material). BDH99::67 showed a 69% identity with R,R-BDH from B. subtilis (10), a 35% identity with R,R-BDH from S. cerevisiae (3), and only a 25% identity with the two alcohol dehydrogenases from C. beijerinckii and T. brockii (13, 21). However, there were no significant similarities found with the meso-BDH from K. pneumoniae or S,S-BDH from Brevibacterium saccharolyticum (12). Almost all of the 2,3-butanediol dehydrogenases characterized so far have characteristics of the short-chain dehydrogenase/reductase (SDR) family (4, 6, 11). The meso-BDH from K. pneumoniae and the S,S-BDH from B. saccharolyticum, which exhibit 50% identity in amino acid sequence and belong to the SDR family, have two conserved residues: a coenzyme binding motif (GxxxGxG) at the N terminus and a substrate binding region (YxxxK) at the C terminus. By using the InterProScan web server (http://www.ebi.ac.uk/Tools/InterProScan/) to search the conserved domain of R,R-BDHs, a typical zinc-containing motif (G-H-E-x-{EL}-G-{AP}-x(4)-[GA]-x(2)-[IVSAC]) was identified in the sequence of BDH99::67 from Gly70 to Val84, in which the His71 residue was predicted to be the ligand of the catalytic zinc atom (see Fig. S4 in the supplemental material); this motif was found in all other R,R-BDHs listed above. The possible conserved residues for the coenzyme binding motif (GxxxGxG) of SDRs were found only at the N terminus of R,R-BDHs from P. polymyxa and B. subtilis, while there were no conserved residues for the substrate binding region (YxxxK) of SDR identified (11), implying that BDH99::67 and other R,R-BDHs may differ from the typical SDR superfamily to which meso-BDH and S,S-BDH belong. Additionally, two hydrophobic residues forming the binding site for cofactor NAD(P), Phe138 and Leu141 (numbers refer to R,R-BDH of S. cerevisiae), were found from the listed R,R-BDHs (see Fig. S4); these are the conserved residues in most zinc-containing medium-chain dehydrogenases/reductases (MDR) (7). Furthermore, a GroES-like domain, which is typically a conserved domain in MDR, was found at the N terminus of BDH99::67 (located at amino acid positions 1 to 182), while no GroES-like domains existed in meso-BDH from K. pneumoniae, S,S-BDH from B. saccharolyticum, and other SDRs. The above-described results indicated that R,R-BDH from this study belongs to the MDR but not to the SDR family (5). To investigate if all R,R-BDHs contain the GroES-like domain, R,R-BDH amino
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FIG. 2. Phylogenetic analysis of amino acid sequences of (2R,3R)-, meso-, and (2S,3S)-2,3-butanediol dehydrogenases from different strains in the database. The tree was constructed by the program MEGA 4 using the neighbor-joining method (16). The sequences compared are (2R,3R)-2,3-butanediol dehydrogenases from P. polymyxa (this study), B. subtilis (10), S. cerevisiae (3), and C. beijerinckii and T. brockii (13); meso-2,3-butanediol dehydrogenases from K. pneumoniae IAM1063 (19) and K. terrigena VTT-E-74023 (1); and (2S,3S)-2,3-butanediol dehydrogenase from B. saccharolyticum C-1012 (17). The other sequences without references indicated were selected from the Medium-Chain Dehydrogenase/Reductase Engineering database (MDRED; http://www.mdred.uni-stuttgart.de) and are annotated as (2R,3R)-2,3-butanediol dehydrogenases from genome sequencing.
acid sequences from B. subtilis, S. cerevisiae, C. beijerinckii, and T. brockii were submitted to the InterProScan web server. As expected, all of the R,R-BDHs reported to date contain the GroES-like domains and zinc-containing motifs at the N terminus. One primary conclusion could be made that R,R-BDH has evolved separately from meso-BDH and S,S-BDH. To prove this, the annotated R,R-BDH amino acid sequences in the database and the R,R-BDHs listed above were used to construct a phylogenetic tree, together with the meso- and S,S-BDHs reported to date, including meso-BDH from K. pneumoniae IAM1063 (19) and Klebsiella terrigena VTT-E74023 (1) and S,S-BDH from B. saccharolyticum C-1012 (17), which are known to belong to the SDR family (4). The phylogenetic analysis clearly supported the conclusion that R,RBDH has evolved separately from meso-BDH and S,S-BDH (Fig. 2), and the results also provided an explanation why R,R-BDH has no significant similarities with meso-BDH and S,S-BDH. Therefore, BDH99::67 should be further classified as a zinc-containing, NAD-dependent (2R,3R)-2,3-butanediol dehydrogenase that belongs to the alcohol dehydrogenase family of the MDR superfamily (14). Nucleotide sequence accession number. The DNA sequences of (2R,3R)-2,3-butanediol dehydrogenase and diacetyl reductase from P. polymyxa ATCC 12321 have been deposited in GenBank with accession numbers HQ730089 and JF440646, respectively. B.Y. is the recipient of an Alexander von Humboldt Fellowship for Postdoctoral Researcher, Germany. J.S. is grateful for financial support from the Chinese Academy of Sciences (KSCX2-YW-G030 and 20090461016) and the Chinese Ministry of Science and Technology (973 Program 2007CB707804). REFERENCES 1. Blomqvist, K., et al. 1993. Characterization of the genes of the 2,3-butanediol operons from Klebsiella terrigena and Enterobacter aerogenes. J. Bacteriol. 175:1392–1404. 2. Celinska, E., and W. Grajek. 2009. Biotechnological production of 2,3butanediol: current state and prospects. Biotechnol. Adv. 27:715–725. 3. Gonza ´lez, E., et al. 2000. Characterization of a (2R,3R)-2,3-butanediol dehydrogenase as the Saccharomyces cerevisiae YAL060W gene product: disruption and induction of the gene. J. Biol. Chem. 275:35876–35885.
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