• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of aemPermissionsJournals.ASM.orgJournalAEM ArticleJournal InfoAuthorsReviewers
Appl Environ Microbiol. Jun 2011; 77(12): 4230–4233.
PMCID: PMC3131630

Novel (2R,3R)-2,3-Butanediol Dehydrogenase from Potential Industrial Strain Paenibacillus polymyxa ATCC 12321 [down-pointing small open triangle]

Abstract

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 meso- and (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.

TEXT

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)-2,3-butanediol 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 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)-2,3-butanediol 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 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 investigated 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)-2,3-BDL 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 meso- and (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).

Table 1.
Substrate specificities of (2R,3R)- 2,3-butanediol dehydrogenasea
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 ...

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 meso-2,3-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.

Table 2.
Summarized kinetic constants of (2R,3R)- and meso-2,3-butanediol dehydrogenases

As the functionality of BDH99::67 was verified, the amino acid sequence as deduced from the nucleotide sequence was 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 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-E-74023 (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,R-BDH 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).

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 ...

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.

Supplementary Material

[Supplemental material]

Acknowledgments

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).

Footnotes

Supplemental material for this article may be found at http://aem.asm.org/.

[down-pointing small open triangle]Published ahead of print on 29 April 2011.

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 [PMC free article] [PubMed]
2. Celinska E., Grajek W. 2009. Biotechnological production of 2,3-butanediol: current state and prospects. Biotechnol. Adv. 27:715–725 [PubMed]
3. González 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 [PubMed]
4. Jörnvall H., et al. 1995. Short-chain dehydrogenases/reductases (SDR). Biochemistry 34:6003–6013 [PubMed]
5. Jörnvall H., Hedlund J., Bergman T., Oppermann U., Persson B. 2010. Superfamilies SDR and MDR: from early ancestry to present forms. Emergence of three lines, a Zn-metalloenzyme, and distinct variabilities. Biochem. Biophys. Res. Commun. 396:125–130 [PubMed]
6. Kallberg Y., Oppermann U., Jörnvall H., Persson B. 2002. Short-chain dehydrogenases/reductases (SDRs): coenzyme-based functional assignments in completed genomes. Eur. J. Biochem. 269:4409–4417 [PubMed]
7. Kim J. F., et al. 2010. Genome sequence of the polymyxin-producing plant-probiotic rhizobacterium Paenibacillus polymyxa E681. J. Bacteriol. 192:6103–6104 [PMC free article] [PubMed]
8. Ma M., et al. 2011. Complete genome sequence of Paenibacillus polymyxa SC2, a strain of plant growth-promoting rhizobacterium with broad-spectrum antimicrobial activity. J. Bacteriol. 193:311–312 [PMC free article] [PubMed]
9. Nakashimada Y., Marwoto B., Kashiwamura T., Kakizono T., Nishio N. 2000. Enhanced 2,3-butanediol production by addition of acetic acid in Paenibacillus polymyxa. J. Biosci. Bioeng. 90:661–664 [PubMed]
10. Nicholson W. L. 2008. The Bacillus subtilis ydj L (bdh A) gene encodes acetoin reductase/2,3-butanediol dehydrogenase. Appl. Environ. Microbiol. 74:6832–6838 [PMC free article] [PubMed]
11. Oppermann U., et al. 2003. Short-chain dehydrogenases/reductases (SDR): the 2002 update. Chem. Biol. Interact. 143-144:247–253 [PubMed]
12. Otagiri M., et al. 2010. Structural basis for chiral substrate recognition by two 2,3-butanediol dehydrogenases. FEBS Lett. 584:219–223 [PubMed]
13. Peretz M., et al. 1997. Molecular cloning, nucleotide sequencing, and expression of genes encoding alcohol dehydrogenases from the thermophile Thermoanaerobacter brockii and the mesophile Clostridium beijerinckii. Anaerobe 3:259–270 [PubMed]
14. Persson B., Heldlund J., Jörnvall H. 2008. The MDR superfamily. Cell. Mol. Life Sci. 65:3879–3894 [PMC free article] [PubMed]
15. Syu M. J. 2001. Biological production of 2,3-butanediol. Appl. Microbiol. Biotechnol. 55:10–18 [PubMed]
16. Tamura K., Dudley J., Nei M., Kumar S. 2007. MEGA 4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24:1596–1599 [PubMed]
17. Ui S., et al. 1998. Cloning, expression and nucleotide sequence of the L-2,3-butanediol dehydrogenase gene from Brevibacterium saccharolyticum C-1012. J. Ferment. Bioeng. 86:290–295
18. Ui S., Masuda T., Masuda H., Muraki H. 1986. Mechanism for the formation of 2,3-butanediol stereoisomers in Bacillus polymyxa. J. Ferment. Technol. 64:481–486
19. Ui S., et al. 1997. Sequence analysis of the gene and characterization of D-acetoin forming meso-2,3-butanediol dehydrogenase of Klebsiella pneumonia expressed in Escherichia coli. J. Ferment. Bioeng. 83:32–37
20. Xiu Z. L., Zeng A. P. 2008. Present state and perspective of downstream processing of biologically produced 1,3-propanediol and 2,3-butanediol. Appl. Microbiol. Biotechnol. 78:917–926 [PubMed]
21. Yan Y., Lee C., Liao J. C. 2009. Enantioselective synthesis of pure (R,R)-2,3-butanediol in Escherichia coli with stereospecific secondary alcohol dehydrogenases. Org. Biomol. Chem. 7:3914–3917 [PubMed]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...