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Applied and Environmental Microbiology, December 2006, p. 7510-7517, Vol. 72, No. 12
0099-2240/06/$08.00+0     doi:10.1128/AEM.01541-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Identification of Novel Benzoylformate Decarboxylases by Growth Selection^,

Helge Henning,^1, Christian Leggewie,^1, Martina Pohl,¹ Michael Müller,² Thorsten Eggert,¹ and Karl-Erich Jaeger^1*

Institute of Molecular Enzyme Technology, Heinrich Heine University Duesseldorf, Research Centre Juelich, D-52426 Juelich, Germany,¹ Institute of Pharmaceutical Sciences, Albert Ludwigs University, D-79104 Freiburg, Germany²

Received 4 July 2006/ Accepted 23 September 2006

	ABSTRACT

Top Abstract Introduction Materials and Methods Results and Discussion References

 
A growth selection system was established using Pseudomonas putida, which can grow on benzaldehyde as the sole carbon source. These bacteria presumably metabolize benzaldehyde via the ß-ketoadipate pathway and were unable to grow in benzoylformate-containing selective medium, but the growth deficiency could be restored by expression in trans of genes encoding benzoylformate decarboxylases. The selection system was used to identify three novel benzoylformate decarboxylases, two of them originating from a chromosomal library of P. putida ATCC 12633 and the third from an environmental-DNA library. The novel P. putida enzymes BfdB and BfdC exhibited 83% homology to the benzoylformate decarboxylase from P. aeruginosa and 63% to the enzyme MdlC from P. putida ATCC 12633, whereas the metagenomic BfdM exhibited 72% homology to a putative benzoylformate decarboxylase from Polaromonas naphthalenivorans. BfdC was overexpressed in Escherichia coli, and the enzymatic activity was determined to be 22 U/ml using benzoylformate as the substrate. Our results clearly demonstrate that P. putida KT2440 is an appropriate selection host strain suitable to identify novel benzoylformate decarboxylase-encoding genes. In principle, this system is also applicable to identify a broad range of different industrially important enzymes, such as benzaldehyde lyases, benzoylformate decarboxylases, and hydroxynitrile lyases, which all catalyze the formation of benzaldehyde.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion References

 
White biotechnology uses microorganisms and (microbial) enzymes to manufacture a wide variety of different chemicals, including polymers, bulk chemicals, agrochemicals, and pharmaceuticals (18). The biocatalytic production of these chemicals offers a number of advantages, including usually mild reaction conditions, the avoidance of toxic wastes, and, in particular, access to a variety of enantiopure compounds due to the high substrate specificity and enantioselectivity of many enzymes. However, the identification of novel biocatalysts that possess desired properties is still a challenge. Classical strain-screening approaches that are used for the isolation of microorganisms are laborious and often unsuccessful. Recently, the metagenome approach was developed to access useful genetic information encoded by environmental DNA, which is directly isolated from various habitats, cloned, and expressed in appropriate microbial host strains (37). However, the identification of a clone producing a biocatalyst of interest within a large metagenomic library requires high-throughput screening technologies, which are difficult to set up and usually expensive to run. Selection provides an elegant solution to the problem. Here, the desired enzyme activity is linked to the survival of the respective clone. Novel selection strategies that use natural or modified transcriptional regulators that bind to the product of an enzymatic reaction have recently been developed (22). Transcriptional control can also be accomplished by a modified eukaryotic nuclear receptor (39) or by a "riboswitch" RNA (3). Enzymes producing compounds such as prephenate (7, 28), pyruvate (8), ammonia (36), and 2-hydroxybenzaldehyde (48) and 1,2,4-trichlorobenzene (29) were identified. A similar approach, termed chemical selection or complementation (1), is based on the detection of small molecules in an in vivo assay of lyase- and ligase-type reactions with a modified yeast three-hybrid system.

In the present work, we report on the construction of a selection system useful to identify novel enzymes producing benzaldehyde as the reaction product, which can be used by the bacterial selection host as the sole carbon source. The respective strain must possess the enzyme benzaldehyde dehydrogenase and the ß-ketoadipate pathway to convert benzoate into the tricarboxylic acid cycle intermediates succinyl-coenzyme A (CoA) and acetyl-CoA (Fig. 1). The genes encoding enzymes of the ß-ketoadipate central pathway were detected in many bacteria, mainly belonging to the genera Acinetobacter and Pseudomonas (10). An example is Pseudomonas putida ATCC 12633, which is able to grow on aromatic compounds as the sole carbon source (13-15). In this strain, mandelate is converted to acetyl-CoA via the mandelate/ß-ketoadipate pathway (Fig. 1), and the enzyme benzoylformate decarboxylase MdlC (synonym, BFD) (E.C. 4.1.1.7) catalyzes the formation of benzaldehyde from benzoylformate by decarboxylation. The structure of BFD was solved in the absence (11) and the presence (34) of mandelic acid as an inhibitor, confirming that the enzyme acts as a tetramer, and active-site residues have been elucidated by site-directed mutants (34, 40) and directed-evolution studies (26, 27).

View larger version (40K): [in this window] [in a new window]   FIG. 1. Mandelate and ß-ketoadipate pathways in P. putida. The mandelate pathway in Pseudomonas putida ATCC 12633 is shown in dark gray, and the conversion of benzoylformate to benzaldehyde by a benzoylformate decarboxylase (MdlC) is highlighted. The ß-ketoadipate pathway (gray) also exists in the majority of all pseudomonads. Additionally, P. putida KT2440 and P. putida DSM50198 possess a benzaldehyde dehydrogenase of unknown function. The metabolization of succinyl-CoA and acetyl-CoA via the tricarboxylic acid cycle is shown in light gray. MdlA, mandelate racemase; MdlB, S-mandelate dehydrogenase; MdlDE, NAD+- and NADP+-benzaldehyde dehydrogenases; BenABC, benzoate dioxygenase; BenD, 2-hydro-1,2-dihydroxybenzoate dehydrogenase; CatA, catechol-1,2-dioxygenase; CatB, cis,cis-muconate lactonizing enzyme (cycloisomerase); CatC, muconolactone isomerase; PcaD, ß-ketoadipate enolactone hydrolase I; PcaIJ, ß-ketoadipate succinyl-CoA transferase subunit; PcaF, ß-ketoadipyl CoA thiolase; TCA, tricarboxylic acid.

The functionality of the selection system was proven by the isolation of three novel benzoylformate decarboxylases, one of them originating from a metagenomic library. These novel enzymes showed only low sequence similarity to presently known BFDs.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Bacterial strains, plasmids, and growth conditions.
The bacterial strains and plasmids used in this study are listed in Table 1. The Escherichia coli strain DH5 was used as a host for cloning and construction of recombinant plasmids. The strain E. coli DH5 carrying the plasmid pRK2013 with the tra genes was used for triparental conjugation. E. coli strains were cultured at 37°C and Pseudomonas strains at 30°C in solid or liquid Luria-Bertani medium. Pseudomonas strains displaying benzoylformate decarboxylase activity were selected by growth in liquid or solid minimal medium M9 (38) without glucose supplemented with 10 mM benzoylformate (purchased from Sigma-Aldrich) as the sole carbon source. The bacteria were incubated for at least 2 days at 30°C. When necessary, antibiotics were added at the following concentrations: chloramphenicol, 600 µg/ml; kanamycin, 50 µg/ml; and gentamicin, 30 µg/ml for Pseudomonas strains, and chloramphenicol, 50 µg/ml; kanamycin, 25 µg/ml; gentamicin, 10 µg/ml; and ampicillin, 100 µg/ml for E. coli strains.

Construction of the deletion mutant P. putida mdlC.

A gentamicin resistance cassette from plasmid pWKR202 was inserted between these two 500-bp fragments of mdlC by using the restriction site HindIII. The cloning steps were carried out in E. coli DH5 by using the plasmid pBluescript SK(+). The mutagenesis cassette consisted of three different fragments in the order mdlC', gentamicin resistance cassette, and 'mdlC. Subsequently, the mutagenesis cassette was cloned into the suicide vector pSUP202, and the resulting plasmid, pSUPMDLC, was transferred via triparental conjugation (6) into P. putida ATCC 12633. The benzoylformate decarboxylase gene, mdlC, was interrupted by homologous recombination, and the correct insertion of the mutagenesis cassette was confirmed by PCR analysis.

Construction of a genomic-DNA library from P. putida ATCC 12633 and P. putida ATCC 12633 mdlC.
The genomic DNAs from P. putida ATCC 12633 and P. putida ATCC 12633 mdlC were isolated by a standard procedure (38) and partially digested with the restriction endonuclease Sau3AI. The broad-host-range vector pBBR1MCS was restricted with BamHI and used for cloning of genomic-DNA fragments of 2 to 8 kb. Finally, the genomic-DNA library was transferred into P. putida KT2440 by conjugation. The average insert size and the frequency of clones harboring only vector DNA within each library were determined by restriction analysis of 24 clones.

Cloning and expression of the mdlC genes from P. putida ATCC 12633 and P. aeruginosa PAO1.
Both mdlC genes were amplified using genomic DNA from P. putida ATCC 12633 and P. aeruginosa, respectively. The following primers were used: BFDPpup (5'-ATA TCCATG GCT TCG GTA CAC GGC ACC ACA TAC-3'), BFDPpdw (5'-ATA TCT CGA GTC ACT TCA CCG GGC TTA CGG TGC TTA C-3'), BFDPaup (5'-ATA TCA TAT GAA AAC CGT CCA TTC CGC G-3'), and BFDPadw (5'-ATA TAA GCT TTC AGG GTT CGA TGG TTT GCG-3'). The primers were designed for cloning into plasmid pBBR1MCS, which was used for subsequent screening of genomic or metagenomic libraries. Both PCR products were ligated into SmaI-restricted pBBR1MCS-2, conferring kanamycin resistance. The plasmids were designated pBBRBFDPp for the mdlC gene from P. putida ATCC 12633 and pBBRBFDPa for the mdlC gene from P. aeruginosa. The correct integration of the genes was confirmed by DNA sequencing. Subsequently, the recombinant plasmids were transferred to P. putida KT2440 and analyzed for growth on agar plates containing 10 mM benzoylformate as the sole carbon source.

Cloning and expression of novel bfd genes from P. putida ATCC 12633.
Putative bfd genes were amplified using genomic DNA from P. putida ATCC 12633. The following primers for cloning into expression plasmids pET16b and pET22b (Novagen) were used: BFD3NdepET (5'-ATA TCA TAT GAA AAC TGT TCA CGG CGC CAC-3'), BFD3BampET (5'-ATA TGG ATC CGG GCT CGA TGG TCT GGG TCG-3'), and BFD2BamHIoSTC (5'-TGG CCT TGA GGA TCC GCG GCT GCT-3'). The PCR products were cloned into the NdeI/BamHI-digested expression vectors pET16b and pET22b (Novagen). The correct integration of bfdB and bfdC was confirmed by DNA sequencing. The heterologous expression of both genes was accomplished in E. coli BL21(DE3), with coexpression of chaperones from plasmid pG-TF2 (31), as recommended by the manufacturer (Takara Bio Inc.).

Isolation and cloning of DNA from soil.
A soil sample was collected from a meadow near Juelich, Germany, and the metagenomic DNA was isolated from 5 g soil based on the direct-lysis method of Zhou et al. (49). Additionally, the isolated metagenomic DNA was purified using the DNeasy Tissue Kit (QIAGEN), starting the protocol with the washing step. The purified metagenomic DNA was partially digested with the restriction endonuclease Sau3AI. Fragments of 2 to 9 kb were isolated and ligated into the BamHI-restricted broad-host-range vector pBBR1MCS. The resulting metagenomic-DNA library was transferred to P. putida KT2440 by conjugation. The average insert size and the frequency of clones harboring only vector DNA within each library were determined by restriction analysis of 24 plasmids isolated from randomly chosen clones.

Cloning and expression of the novel BFD gene bfdM from a metagenomic library.
The plasmid DNA from a clone exhibiting decarboxylase activity was sequenced by genome walking, and the following primers were used for the amplification of the novel bfdM gene and subsequent cloning into the expression vector pET22b (Novagen): BFDM1Nde (5'-ATA TCA TAT GCA AGA GAC AAC CCC CCA GAA T-3') and BFDM1BamH (5'-ATA GGA TCC GGC CAC TTC GAC GAG CAC GGG C-3'). The PCR product was cloned into the NdeI/BamHI-digested expression vector pET22b (Novagen), and the correct integration of bfdM was confirmed by DNA sequencing. The heterologous expression was accomplished as described for bfdB and bfdC.

Coupled decarboxylase assay.
The decarboxylase activity toward benzoylformate was determined as described previously (17). In brief, the assay mixture was prepared from the following stock solutions in standard buffer: benzoylformate solution (100 µl, 50 mM, adjusted to pH 6.0), NADH (100 µl, 3.5 mM), horse liver alcohol dehydrogenase (50 µl, 10 U; Sigma), and potassium phosphate buffer (700 µl, 50 mM, pH 6.0). The components were mixed in a 1.7-ml cuvette and incubated for 3 min at 30°C, and the reaction was started by the addition of sonicated cell extracts from overexpression cultures. These extracts were prepared as follows: 100 µl (equal to an optical density of 20 at 580 nm) supernatant of extracts centrifuged at 13,000 rpm for 20 min was sonicated twice for 3 min each time (50 cycles; 80% power; Sonoplus HD2070; Bandelin, Berlin, Germany). The slope was calculated from the linear part of the descending absorption curve determined at 340 nm and within a reaction time of 15 to 90 s.

Sequence analysis.
The similarity of newly identified genes to known DNA or amino acid sequences was determined by Blastn or Blastp (http://www.ncbi.nlm.nih.gov/BLAST/). The sequence alignment was performed with ClustalW (http://www.ebi.ac.uk/clustalw/).

Nucleotide sequence accession numbers.
The sequences of the three novel benzoylformate decarboxylase genes described here have been deposited in the EMBL database. They are AM284966 for bfdB, AM284967 for bfdC, and AM284968 for bfdM.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Construction of a Pseudomonas putida selection strain.
The elimination of a functional mdlC gene in P. putida ATCC 12633 interrupts the mandelate pathway and prevents the corresponding knockout mutant, P. putida ATCC 12633 mdlC, from growing on mandelate as the sole carbon source. Consequently, this strain should be usable as a selection host to identify novel benzoylformate decarboxylase genes that can be introduced in trans and that may originate from a genomic, a metagenomic, or a directed-evolution library. The deletion mutant P. putida ATCC 12633 mdlC was constructed by the insertion of a gentamicin resistance cassette into the functional gene mdlC and was cultivated on selective medium containing benzoylformate as the sole carbon source, enabling growth of the recombinant bacteria only upon expression of a functional plasmid-encoded BFD. Surprisingly, the mutant P. putida ATCC 12633 mdlC was still able to grow on benzoylformate-containing medium. Therefore, this strain could not be used as a selection host; however, this result clearly indicated either the presence of so far unknown genes encoding enzymes with BFD activity or an unknown pathway for the degradation of benzoylformate.

Thus, we searched for bacterial strains having the ability to grow with benzaldehyde as the sole carbon source but lacking the enzymes catalyzing the reactions from (R)-mandelate to benzoylformate (Fig. 1). These bacteria are expected to possess a benzaldehyde dehydrogenase and to metabolize benzaldehyde via the ß-ketoadipate pathway to yield the products acetyl-CoA and succinyl-CoA. A database analysis revealed that the strains P. putida KT2440 and P. putida DSM50198 should exhibit these features (21, 30). Furthermore, Tsou et al. described the presence of a ß-ketoadipate pathway in P. putida DSM50198, which lacks genes of the mandelate pathway (47). First, we confirmed that both strains were unable to grow in selective medium containing benzoylformate as the sole carbon source. As a functional test, we then cloned into the broad-host-range vector pBBR1MCS the mdlC genes encoding BFDs from both P. aeruginosa PAO1 and P. putida ATCC 12633. The corresponding plasmids were transferred into the putative selection host strains, P. putida KT2440 and P. putida DSM50198, and both recombinant strains were able to grow on benzoylformate-containing medium, whereas the control strains, which lack the BFD genes, did not grow at all (data not shown). These results clearly indicated that both P. putida strains were useful as selection hosts.

Identification of two novel BFD-encoding genes in the genome of P. putida ATCC 12633.
Our results suggested that P. putida ATCC 12633 may possess at least one so far unknown gene encoding a BFD activity. We therefore constructed DNA libraries from chromosomal DNAs of P. putida ATCC 12633 and the deletion mutant P. putida ATCC 12633 mdlC using the plasmid pBBR1MCS. The resulting genomic-DNA libraries consisted of 4,500 and 4,000 clones for the P. putida wild type and the mdlC mutant, respectively, both with an average insert size of 5 kb, thereby reaching a fourfold coverage of the P. putida genome. Initially, these libraries were constructed in E. coli and then transferred to P. putida KT2440 by triparental mating. We identified several clones that grew on benzoylformate selective media. The corresponding plasmids were isolated, and restriction analysis revealed two different fragment patterns. Subsequent DNA sequencing identified two novel genes, which were designated bfdB and bfdC. The two genes share identical DNA sequences, except for those nucleotides encoding the C-terminal nine amino acids. The clones expressing these genes were able to grow on benzoylformate selective media, whereas the respective control strain showed no growth at all (Fig. 2). The two BFD-encoding genes are located in different regions of the P. putida ATCC 12633 genome, as deduced from the sequences of the respective flanking regions (Table 2). The genes up- and downstream of bfdC resemble the genomic region surrounding mdlC in P. aeruginosa, where a putative benzoate transporter of the major facilitator superfamily and a putative transcriptional regulator are encoded in the same orientation. Thus, the bfdC gene seems to be part of a gene cluster encoding proteins for the degradation of aromatic compounds, as suggested for MdlC of P. aeruginosa (44). On the other hand, bfdB seems more likely to have originated from a gene transfer. To our knowledge, the region surrounding this gene does not show any homology to known regions adjacent to other BFD genes identified in published bacterial genomes. The enzymes BfdB and BfdC exhibit 83% identity to the benzoylformate decarboxylase from P. aeruginosa and 63% to the previously described enzyme MdlC from P. putida ATCC 12633. These results demonstrate that (i) P. putida KT2440 is an appropriate selection host strain and (ii) P. putida ATCC 12633 possesses two additional hitherto-unknown BFD-encoding genes, bfdB and bfdC, one of which was most likely duplicated during evolution.

View larger version (46K): [in this window] [in a new window]   FIG. 2. Growth of recombinant P. putida KT2440 displaying benzoylformate decarboxylase activity on an agar plate containing benzoylformate selective medium. P. putida KT2440 possessing the plasmids pBFDB (coding for BfdB from the genomic-DNA library of P. putida ATCC 12633), pBFDC (coding for BfdC from the genomic-DNA library of P. putida ATCC 12633 mdlC), and pBFDM (coding for BfdM from a metagenomic library) expressed active BFD enzymes enabling growth by converting benzoylformate. P. putida strains harboring the corresponding plasmids, pBBRCmR, pBBRGmR, and pBBRKmR, served as controls.

Overexpression and characterization of the novel BFDs.
The primary structures of the three novel BFDs we have identified were analyzed with respect to conserved regions, including active and cofactor binding sites. Figure 3 shows a sequence alignment of the novel enzymes with BFDs from P. putida KT244 and P. aeruginosa (MdlC), which shows the highest identity to BfdB and BfdC. All amino acid side chains forming the active site, the substrate binding site, and the thiamine diphosphate binding site are highly conserved in the new enzymes. This also holds for the metagenome-derived enzyme BfdM, indicating that the reaction mechanism of benzoylformate decarboxylases is well conserved.

View larger version (82K): [in this window] [in a new window]   FIG. 3. Sequence alignment of MdlC (BfdA), BfdB, and BfdC from P. putida ATCC 12633 (Pp), BFD from Pseudomonas aeruginosa (Pa), and BfdM isolated from the metagenome. Highly conserved regions are highlighted in black, and similar amino acid residues are shown in gray. Amino acids forming the catalytic site are indicated by an asterisk. Residues tagged with a dot are involved in substrate binding (34, 40), and the boxed residues are involved in thiamine diphosphate cofactor and metal binding.

Conclusions.
We have described here an efficient bacterial selection system which is based on a P. putida strain growing with benzaldehyde as the sole carbon and energy source. The selective capacity of this system was demonstrated by the successful identification of three novel BFDs, using benzoylformate as a model substrate. Apart from identifying BFDs, this system is more generally applicable, because benzaldehyde is not only part of the mandelate pathway but is also formed during phenylalanine degradation (25), in the veratryl alcohol pathway (19), from toluene degradation (32) and by cyanogenesis in plants (5). Therefore, the system we have constructed can be used to identify a broad range of different enzymes, including the industrially important enzymes benzaldehyde lyase, benzoylformate decarboxylase (33), hydroxynitrile lyase (5), and alcohol dehydrogenase, which all produce benzaldehyde by conversion of benzoin, benzoylformate, mandelonitrile, or benzyl alcohol, respectively (Fig. 4).

View larger version (11K): [in this window] [in a new window]   FIG. 4. Growth selection on benzaldehyde to identify industrially important biocatalysts from genomic- or metagenomic-DNA libraries. Accessible enzymes include (i) alcohol dehydrogenase, (ii) hydroxynitrile lyase, (iii) benzoylformate decarboxylase, and (iv) benzaldehyde lyase.

	ACKNOWLEDGMENTS

 
This work was supported by the Federal Ministry of Education and Research, Berlin, Germany, in the framework of the Competence Network Göttingen entitled "Genome Research on Bacteria for the Analysis of Biodiversity and Its Further Use for the Development of New Production Processes."

We thank Gerhard Gottschalk, Göttingen Genomics Laboratory, Göttingen, Germany, for continuous support.

	FOOTNOTES

 
* Corresponding author. Mailing address: Institute of Molecular Enzyme Technology, Heinrich Heine University Duesseldorf, Research Centre Juelich, D-52426 Jülich, Germany. Phone: (49)-2461-613716. Fax: (49)-2461-612490. E-mail: karl-erich.jaeger{at}fz-juelich.de.

Published ahead of print on 29 September 2006.

This paper is dedicated to Maria-Regina Kula on the occasion of her 70th birthday.

H.H. and C.L. contributed equally to this work.

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