Identification and functional characterization of phenylglycine biosynthetic genes involved in pristinamycin biosynthesis in Streptomyces pristinaespiralis

Journal of Biotechnology 155 (2011) 63–67

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Identification and functional characterization of phenylglycine biosynthetic genes involved in pristinamycin biosynthesis in Streptomyces pristinaespiralis Yvonne J. Mast ∗ , Wolfgang Wohlleben, Eva Schinko 1 Mikrobiologie/Biotechnologie, Interfakultäres Institut für Mikrobiologie und Infektionsmedizin, Fakultät für Biologie, Eberhard Karls Universität Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany

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Article history: Received 10 June 2010 Received in revised form 3 November 2010 Accepted 1 December 2010 Available online 10 December 2010 Keywords: Antibiotics Nonribosomal polypeptide Streptogramin Aproteinogenic amino acid Precursor supply Phenylglycine

a b s t r a c t Pristinamycin I (PI), a streptogramin type B antibiotic produced by Streptomyces pristinaespiralis, contains the aproteinogenic amino acid l-phenylglycine. Recent sequence analysis led to the identification of a set of putative phenylglycine biosynthetic genes. Successive inactivation of the individual genes resulted in a loss of PI production. Production was restored by supplementation with externally added l-phenylglycine, which demonstrates that these genes are involved in phenylglycine biosynthesis and thus probably disclosing the last essential pristinamycin biosynthetic genes. Finally, a putative pathway for phenylglycine synthesis is proposed. © 2010 Elsevier B.V. All rights reserved.

1. Introduction The cyclohexadepsipeptide antibiotic pristinamycin I (PI) is a member of the B group of streptogramin antibiotics (Fig. 1). In Streptomyces pristinaespiralis PI is coproduced with the polyunsaturated cyclopeptidic macrolactone antibiotic, pristinamycin II (PII), which belongs to the A group of streptogramins (Cocito, 1979). PI and PII are produced in a 30:70 ratio, and their semisynthetic derivatives, quinupristin and dalfopristin, are used in combination as a therapeutic drug (Synercid® ) against multiresistant Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VREF) strains (Eliopoulos, 2003). PI consists of two proteinogenic and five aproteinogenic amino acids and is synthesized by nonribosomal peptide synthetases (NRPSs):

Abbreviations: Dpg, 3,5-dihydroxyphenylglycine; Hpg, 4hydroxyphenylglycine; HPLC, high performance liquid chromatography; HPLC-DAD, HPLC-diode array detection; MRSA, methicillin-resistant Staphylococcus aureus; NRPS, nonribosomal peptide synthetase; ORF, open reading frame; PI, pristinamycin I; PII, pristinamycin II; PCR, polymerase chain reaction; Phg, phenylglycine; pgl, designation of the phenylglycine genes; VREF, vancomycin-resistant Enterococcus faecium. ∗ Corresponding author. Tel.: +49 07071 2978865; fax: +49 07071 295979. E-mail addresses: [email protected] (Y.J. Mast), [email protected] (W. Wohlleben), [email protected] (E. Schinko). 1 née Eva Heinzelmann. 0168-1656/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2010.12.001

SnbA activates the starter unit, 3-hydroxypicolinic acid. SnbC is responsible for the incorporation of the two amino acids, lthreonine and l-␣-aminobutyric acid, and SnbDE is involved in the activation of the last four amino acids of the PI macrocycle: lproline, 4-N,N-dimethylamino-l-phenylalanine, 4-oxo-l-pipecolic acid and l-phenylglycine (l-Phg) (de Crécy-Lagard et al., 1997). So far, almost all PI biosynthetic genes have been identified and characterized (Blanc et al., 1994), except those that are responsible for the biosynthesis of l-Phg, which is the seventh and hence the last amino acid that is incorporated into PI. Aproteinogenic amino acids, such as 4-hydroxyphenylglycine (Hpg) and 3,5-dihydroxyphenylglycine (Dpg), are well known components of glycopeptide antibiotics like vancomycin and teicoplanin. These amino acids are specifically provided by secondary metabolite biosynthesis as shown, for example, by balhimycin of Amycolatopsis balhimycina (Pfeifer et al., 2001) and complestatin from Streptomyces lavendulae (Chiu et al., 2001). Phg, as an aproteinogenic amino acid, is only found in streptogramin antibiotics such as virginiamycin. The enantiomeric isomer d-Phg plays an important role in the fine chemicals industry, where it is used as a precursor for the production of semisynthetic ␤-lactam antibiotics, such as cephalosporins and penicillins (Müller et al., 2006). In this study, we describe the cloning, sequence analysis, and functional verification of the genes involved in Phg biosynthesis. Furthermore we propose a biosynthetic pathway for the synthesis of the aproteinogenic amino acid Phg.

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Fig. 2. Genetic organization of the Phg biosynthetic genes pglA–E (dashed arrows). Insertion sites used for inactivation of the pgl genes by the aprP cassette are shown. Noncoding regions are marked by brackets.

Fig. 1. Chemical structure of PI from S. pristinaespiralis. R = CH3 (PIA ), R = H (PIB ).

2. Results and discussion 2.1. In silico analysis of Phg biosynthetic genes in S. pristinaespiralis In the course of recent sequence analysis of the pristinamycin biosynthetic gene region, the genes pglA, pglB, pglC, pglD, and pglE were identified (Mast et al., 2010). The predicted products of these genes showed similarity to some of the enzymes involved in the biosynthesis of the aproteinogenic amino acids Hpg and Dpg. These amino acids are utilized in the biosynthesis of glycopeptides such as balhimycin from Amycolatopsis balhimycina (Table 1). This suggests that the pgl gene products may have a function in synthesizing the aromatic amino acid Phg in S. pristinaespiralis. The putative Phg biosynthetic genes pglA–E are arranged in an operon-like structure between the genes snbDE and snaD. This gene region has a size of around 6 kb and includes six orfs, designated as pglA, pglB, pglC, pglD, mbtY, and pglE (Fig. 2). All genes have overlapping start and stop codons, which suggests that they are translationally coupled. The gene snbDE encodes the PI peptide synthetase SnbDE, of which SnbE has already been shown to be responsible for the incorporation of the Phg residue into the peptide chain of PI (Thibaut et al., 1997; Blanc et al., 1994) and snaD codes for the PII peptide synthetase SnaD. Noncoding regions with promoter elements (imperfect −10 and −35 motifs) are present between snbDE and pglA (17 nucleotides), as well as between pglE

and snaD (334 nucleotides), which indicates that snbDE and snaD are probably not part of the “Phg operon”. To predict the function of the deduced pgl gene products, sequence comparisons were carried out using the Blast data base (Altschul et al., 1990): the deduced gene product of pglA is similar (47% identity, 62% similarity) to the hydroxyacyldehydrogenase (dioxygenase) DpgC of A. balhimycina, which is a key enzyme for Dpg synthesis, a component of balhimycin (Pfeifer et al., 2001). DpgC converts 3,5-dihydroxyphenylacetyl-CoA to 3,5dihydroxyphenylglyoxylate (Widboom et al., 2007). Due to the similarity of PglA to the DpgC dioxygenases of glycopeptide biosyntheses, we suggest that PglA catalyzes a similar oxygenase reaction during the formation of Phg. The predicted pglB and pglC gene products show similarity to the ␣- and ␤subunit of the pyruvate dehydrogenase from Mycobacterium avium, respectively (PdhA, 46% identity, 57% similarity; PdhB, 58% identity, 70% similarity) (Li et al., 2005). In primary metabolism, pyruvate dehydrogenases catalyze the conversion of pyruvate to acetylCoA during glycolysis, which suggests that PglB/C might convert a pyruvate-related substance in secondary metabolism (see below). The deduced pglD gene product shows similarity (39% identity, 53% similarity) to the type II thioesterase RifR of Amycolatopsis mediterranei, which is proposed to have a corrective function during rifamycin biosynthesis (Claxton et al., 2009). This similarity indicates that PglD may act as a thioesterase during Phg biosynthesis. The predicted gene product of the small (215 nucleotides) gene mbtY shows similarity to the MbtH-like protein (59% identity, 78% similarity) of A. balhimycina (Stegmann et al., 2006). mbtH-like genes are well conserved and were found in numerous gene clusters for the biosyntheses of nonribosomally synthesized peptides and siderophores (Quadri et al., 1998). It was suggested that MbtHlike proteins are involved in the stabilization of NRPS complexes (Stegmann et al., 2006), or in the mediation of protein–protein interactions that are important for the assembly of metabolites (Lautru et al., 2007). MbtH-like proteins may also be directly involved in the catalysis of biosynthetic reactions (Wolpert et al.,

Table 1 Genes located in the Phg biosynthetic operon of S. pristinaespiralis and their deduced functions. Gene

Size (bp)

Size (aa)

MW (kDa)

Predicted function

ID/SMa (%)

Match

Origin

Accession number

pglA pglB pglC pglD mbtY pglE

1407 1059 1041 855 216 1314

468 352 346 284 72 437

51 37 37 30 8 49

Hydroxyacyl-dehydrogenase Pyruvate dehydrogenase E1 component ␣-subunit Pyruvate dehydrogenase E1 component ␤-subunit Thioesterase type II MbtH-like protein Phenylglycine aminotransferase

47/62 46/57 58/70 39/53 59/78 54/67

DpgC PdhA PdhB RifR Orf1 Pgat

A. balhimycina M. avium M. avium A. mediterranei A. balhimycina A. balhimycina

CAC48380 AAS04626 AAS04625 AAG52991 CAC48363 CAC48367

MW: molecular weight. a % similarity/identity of amino acid sequences.

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Table 2 Bacterial strains, plasmids, and cosmids used in this study. Strains/plasmids/cosmids S. pristinaespiralis Pr11 pglA::aprP pglB::aprP pglC::aprP pglD::aprP pglE::aprP E. coli XL1 Blue Cosmid pYJM1 Plasmids pEH13 pSLE39 pK18 pYJM6 pYJM7 pYJM8 pYJM9 pYJM10

Genotype/phenotype

Source plasmids/reference

Pristinamycin producing strain/wildtype, natural isolate of S. pristinaespiralis ATCC 25486 Gene interruption of pglA, aprP, PI non-producing Gene interruption of pglB, aprP, PI non-producing Gene interruption of pglC, aprP, PI non-producing Gene interruption of pglD, aprP, PI and PII non-producing Gene interruption of pglE, aprP, PI non-producing

Sanofi-Aventis This study This study This study This study This study

recA1 end A1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F’, proAB lac1q Z M15 Tn10(tetr )]

Bullock et al. (1987)

Cosmid carrying the pglA–E gene, aprar

This study

pUC21 derivative carrying the apramycin-PermE resistance cassette (aprP) pUC21 derivative carrying aphII of transposon Tn5 pUC derivative, aphII, lacZ -complementation system pGEM-TEasy (Promega) derivative carrying a 1.4-kb EcoRV/SmaI fragment of pSLE39 (aphII cassette) and a 2.5-kb fragment, which contains pglA interrupted by aprP pGEM-TEasy (Promega) derivative carrying a 1.4-kb EcoRV/SmaI fragment of pSLE39 (aphII cassette) and a 2.0-kb fragment, which contains pglB interrupted by aprP pk18 Derivative carrying a 2.3-kb fragment, which contains pglC interrupted by aprP pk18 Derivative carrying a 1.5-kb fragment, which contains pglD interrupted by aprP pk18 Derivative carrying a 2.8-kb fragment, which contains pglE interrupted by aprP

Heinzelmann et al. (2001) Muth unpubl. Pridmore (1987) This study

2007). Recently, it has been shown that MbtH-like proteins are integral components of NRPSs, where they stimulate amino acid activation (Felnagle et al., 2010; Zhang et al., 2010). Due to the localization of mbtY within the “Phg operon”, which itself is enclosed by the two NRPS genes snbDE and snaD, MbtY may interact with one of the NRPS. The likely candidate is SnbDE because this peptide synthetase incorporates Phg into PI. The predicted pglE gene product shows similarity (54% identity, 67% similarity) to the phenylglycine aminotransferase Pgat of A. balhimycina (Pelzer et al., 1999), which is involved in the transamination processes leading to the aproteinogenic amino acids Hpg and Dpg (Pfeifer et al., 2001). Thus, PglE may catalyze an analogous reaction during Phg biosynthesis.

2.2. Gene insertion mutagenesis of the pgl genes To confirm the involvement of the pgl genes in pristinamycin biosynthesis, the individual genes were inactivated by gene insertion mutagenesis. Inactivation of pglA, pglB, pglC, pglD, and pglE was carried out with the Streptomyces nonreplicative plasmids, pYJM6, pYJM7, pYJM8, pYJM9, and pYJM10, respectively. These plasmids carried large, by PCR amplified, fragments, containing the target genes plus surrounding regions, disrupted by insertion of the apramycin resistance cassette, which was equipped with the constitutive ermE promoter (aprP) (Heinzelmann et al., 2001) (Table 2). The presence of the ermE promoter, downstream from the apr gene, should prevent polar effects on downstream lying genes (Heinzelmann et al., 2001). The mutants pglA::aprP, pglB::aprP, pglC::aprP, pglD::aprP, and pglE::aprP were generated by polyethylene glycol-mediated transformation (Kieser et al., 2000) of S. pristinaespiralis wildtype protoplasts with plasmids pYJM6, pYJM7, pYJM8, pYJM9, and pYJM10, respectively (Table 2). Apramycinresistant and kanamycin-sensitive transformants were analyzed by PCR and/or Southern hybridization experiments (data not shown) to identify those clones in which a double-crossover event between the chromosomal copy of pglA, pglB, pglC, pglD, and pglE and the mutated fragment located on pYJM6, pYJM7, pYJM8, pYJM9, and pYJM10, respectively, had occurred. For the analysis of pristinamycin production, strains were cultivated in 100 ml inoculum medium (Blanc et al., 1994). After 48–72 h, 17 ml of the precultures were inoculated into 200 ml of production medium and cultivated for 3–4 days (Blanc et al., 1994). Aliquots (5 ml) of S. pristinaespiralis culture were extracted with 5 ml ethyl acetate for 20 min, and

This study This study This study This study

then concentrated completely in vacuo. The extract was redissolved in propan-2-ol (0.75 ml) and samples were analyzed by an HPLC equipped with diode-array detection (HPLC-DAD) (Fiedler, 1993). Pristinamycin was detected at 210 nm and its spectrum was compared to that of purified substance (provided by Sanofi-Aventis) and to an HPLC-UV/Vis spectral library (Fiedler, 1993) (Fig. 3). Due to the capability of S. pristinaespiralis to produce several pristinamycin congeners, different pristinamycin-specific peaks were detected by HPLC. PIA was detected by its specific retention time (7.5 min), which was verified by UV–Vis spectrometry, whereas the PIB concentration was too low to be measured. In comparison to the wildtype, the mutants pglA::aprP, pglB::aprP, pglC::aprP, and pglE::aprP lost the ability to produce PI, since the PIA specific peak was absent their HPLC chromatograms (Fig. 3). In each case PII production was unimpaired. These results prove that the genes pglA, pglB, pglC, and pglE are involved in PI biosynthesis. Surprisingly, pglD::aprP produced neither PI nor PII, which suggests that the thioesterase gene pglD is essential for both, PI and PII biosynthesis. During PII biosynthesis PglD may have an editing function as this has been shown for several other type II thioesterases (Claxton et al., 2009; Heathcote et al., 2001; Schwarzer et al., 2002), whereas its role in PI biosynthesis is described below.

2.3. Feeding of l-Phg to S. pristinaespiralis pgl mutants Feeding studies were performed to examine whether the deficiency of PI production in pglA::aprP, pglB::aprP, pglC::aprP, pglD::aprP, and pglE::aprP was due to the lack of the ability to synthesize Phg. The S. pristinaespiralis mutants pglA::aprP, pglB::aprP, pglC::aprP, pglD::aprP, and pglE::aprP were inoculated in 10 ml inoculum medium supplemented with 25 mg l-Phg (solubilized in 0.7 ml 1 M NaOH and neutralized with 0.5 ml 1 M HCl). After 48 h, aliquots (10 ml) of the precultures were inoculated into 100 ml of production medium that was supplemented with l-Phg (0.25 g solubilized in 7 ml 1 M NaOH and neutralized with 5 ml 1 M HCl), and cultivated for 3–4 days. Samples (5 ml) were harvested and treated as mentioned above. Antibiotic production was monitored by HPLC analysis. Feeding l-Phg to S. pristinaespiralis pglA::aprP, pglB::aprP, pglC::aprP, and pglE::aprP restored PI production, which demonstrated that the mutants had taken up l-Phg and had incorporated it into PI (Fig. 3). In contrast, feeding l-Phg to pglD::aprP could not restore PI production. Besides that pglD::aprP did not pro-

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Fig. 3. Production profiles of S. pristinaespiralis Pr11 wildtype (left), mutant pglA::aprP (middle) and mutant pglA::aprP fed with l-Phg (right). Samples were harvested at time point 93.5 h. Pristinamycin PIA (Rt = 7.5 min) and PIIA (Rt = 8.3 min) were detected by UV–Vis spectrometry. The corresponding UV–Vis spectra of retention region 7.5–7.7 min are listed below. Wavelength monitoring was performed at 210 nm.

Fig. 4. Putative pathway for the synthesis of the aproteinogenic amino acid l-Phg.

duce PII. Altogether these results indicate that the genes pglA, pglB, pglC, and pglE are responsible for the biosynthesis of the PI precursor Phg. Furthermore, these results reveal that the mutations have no polar effect on the synthesis of the pristinamycin compounds. However, downstream effects cannot be excluded for the mutant pglD::aprP, because this strain does not produce any pristinamycin, and cannot be supplemented with externally added Phg or be complemented with the native pglD gene (data not shown). In future experiments, further genetic and biochemical analyses will be performed to investigate the function of PglD. However, because of the genetic localization of pglD within the “Phg operon” and its expected function during Phg biosynthesis (see below), we suggest that pglD is involved in Phg biosynthesis.

from benzoylformate by the action of the thioesterase PglD, leading to benzoylformate (phenylglyoxylate). For Dpg biosynthesis during vancomycin production, it was suggested that the cleavage of the CoA-thioester bond of 3,5-dihydroxyphenylacetyl-CoA is catalyzed by DpgC. However, DpgC was reported to lack a standard hydrolytic thioesterase activity (Tseng et al., 2004). Because there is a thioesterase gene present in the Phg operon, we predict that the encoded PglD catalyzes this reaction step during Phg synthesis. Finally, an amino group may be transferred by the action of the aminotransferase PglE, which would result in the formation of l-Phg. In conclusion we propose that, together with the reported Phg genes, all essential PI biosynthetic genes have been identified.

2.4. Proposed pathway for Phg biosynthesis

Nucleotide sequence accession numbers

As a result of the findings from the sequence analysis of the pgl genes and the comparisons of their deduced gene products (see above), a pathway for Phg biosynthesis is proposed (Fig. 4). We propose that Phg is derived from phenylpyruvate, which is an intermediate of the primary metabolic shikimate pathway as has been similarly suggested for Hpg synthesis during complestatin biosynthesis in S. lavendulae (Chiu et al., 2001). Phenylpyruvate may then be converted to phenylacetyl-CoA by the action of the pyruvate dehydrogenase-like complex PglB/C, which is similar to the conversion of pyruvate to acetyl-CoA in primary metabolism. In the next step the dihydroxyphenylglycine dehydrogenase-like enzyme PglA catalyzes the conversion of phenylacetyl-CoA to benzoylformyl-CoA, which is probably the pivotal reaction step during Phg biosynthesis. CoA may be released

A cosmid containing the pglA–E gene was sent to GATC Biotech for sequencing. The DNA sequences were determined by standard techniques (Sanger et al., 1977) and were examined for ORFs with the codon usage program of Staden and McLachlan (Bibb et al., 1984; Staden and McLachlan, 1982). The nucleotide sequences reported here have been assigned GenBank accession nos. FN563137, FN563138, FN563139, FN563140, FN563141, FN563142, FN563143, and FN563144 in the EMBL data library. Acknowledgements We wish to thank Anne Gondran from Sanofi-Aventis Pharma for helpful discussions and for providing us with strain S. pristinaespiralis Pr11. We thank R. Ort-Winklbauer for technical assistance

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