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Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 319-326 |
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doi:10.1111/j.1745-7270.2008.00408.x |
Alternative method for
site-directed mutagenesis of complex polyketide synthase in Streptomyces
albus JA3453
Danfeng Song1, Jane
Coughlin2,
Jianhua Ju2,
Xiufen Zhou1,
Ben Shen2,3,4,
Chunhua Zhao1*,
and Zixin Deng1*
1
Laboratory of Microbial
Metabolism and College of Life Sciences and Biotechnology, Shanghai Jiaotong
University, Shanghai 200030, China
2
Division of
Pharmaceutical Sciences, 3 National Cooperative Drug Discovery Group of University
of Wisconsin, and 4 Department of Chemistry, University of
Wisconsin, Madison, Wisconsin 53705, USA
Received: December 22, 2007�������
Accepted: February
14, 2008
This study was supported
by the grants from the �973 Program� of the Ministry of Science and Technology
(No. 2003CB114205), the National Natural Science Foundation of China (No.
30470941), and the Shanghai Municipal Council of Science and Technology (No.
04JC14058)
*Corresponding
authors:
Zinxin Deng: Tel,
86-21-62933404; E-mail, [email protected]
Chunhua
Zhao: Tel, 86-21-62933765; E-mail, [email protected]
Sequence
analysis of oxazolomycin (OZM) biosynthetic gene cluster from Streptomyces
albus JA3453 revealed a gene, ozmH, encoding a hybrid polyketide and
non-ribosomal peptide enzyme. Tandem ketosynthase (KS) domains (KS10-1 and KS10-2) were
characterized and they show significant homology with known KSs. Using an alternative
method that involves RecA-mediated homologous recombination, the negative
selection marker sacB gene, and temperature-sensitive replications,
site-directed mutagenesis of the catalytic triad amino acid cysteines were
carried out in each of the tandem KS domains to test the function they play in
OZM biosynthesis. HPLC-mass spectrometry analysis of the resulting mutant
strains showed that KS10-2 is essential
for OZM biosynthesis but KS10-1 is not
indispensable and might serve as a "redundant domain". These results
confirmed the existence of an "extra domain" in complex polyketide
synthase.
Keywords������� oxazolomycin; polyketide synthase; RecA-mediated homologous
recombination; Streptomyces; tandem ketosynthase
Polyketide and non-ribosomal peptide produced by bacteria, fungi, or plants comprise two families of natural products including many clinically important drugs exemplified by erythromycin (polyketide), vancomycin (non-ribosomal peptide), and bleomycin (hybrid polyketide and non-ribosomal peptide) [1]. These compounds are biosynthesized by polyketide synthase (PKS) or non-ribosomal peptide synthase (NRPS) that are recruited as "co-linearity" assembly lines by sequential condensation of short carboxylic acids or amino acids. Most type I PKSs are multifunctional, non-iteratively acting modular enzymes, each minimally consisting of a b-ketoacyl synthase (KS), an acyltransferase, and an acyl carrier protein (ACP), represented by 6-deoxyerythromycin B synthase involved in erythromycin biosynthesis [2]. Optional domains are available in a specific module such as ketoreductase, dehydratase (DH), enoyl reductase, and methyltransferase [3]. Surprisingly, additional domains were identified from certain polyketide synthases in recent studies. For example, two ACPs (ACP6-1 and ACP6-2) flanking one methyltransferase domain were characterized in LnmJ PKS module 6 of the leinamycin biosynthetic gene cluster [4]. Genetic investigations suggest that either ACP is sufficient for leinamycin biosynthesis. In vitro studies also show that discrete acyltransferase LnmG can load malonyl-CoA onto both ACPs, only with a difference of 5-fold loading efficiency. These results have eventually led to a polyketide chain biosynthesis mechanism established by Tang et al that the elongation polyketide chain can skip onto either ACP to load the malonyl-CoA extender unit [5,6]. Multiple ACP domains were also identified in other biosynthetic gene clusters, such as mupirocin PKS [7]. Tandem KS domains were discovered in several polyketide biosynthetic gene clusters, such as LnmI module 3 involved in leinamycin biosynthesis [4]. In this paradigm, Tang et al proposed that the first KS domain catalyzes the transfer of the growing peptide intermediate of peptidyl-S-PCP from the upstream NRPS module to its Cys residue, and the second KS catalyzes the condensation between peptidyl-S-KS and the cognate malonyl-S-ACP to complete the elongation step.
Oxazolomycin (OZM) is a hybrid NRPS-PKS natural product with antitumor, antivirus, and limited antibacterial activity [8]. We localized and sequenced the OZM biosynthetic gene cluster from Streptomyces albus JA3453 in our previous study. Analysis of the complete sequence revealed that OZM is biosynthesized by PKSs, NRPSs, and hybrid NRPS/PKS metasynthases. One of the genes, ozmH, was characterized with five modules, of which module No. 10 has an unusual domain organization of tandem KSs (KS10-1 and KS10-2). Both share significant identity to known type I KSs. In this study, we report site-directed mutagenesis of individual KS domains to test their functions in OZM biosynthesis using a procedure that involves RecA-mediated homologous recombination, the negative selection marker sacB gene, and temperature-sensitive replication. HPLC-mass spectrometry (MS) analysis of the resulting mutants suggests that the first KS domain is not necessary for OZM biosynthesis and could be assigned as an "extra domain", but that the second KS is essential for OZM biosynthesis. These results are consistent with the fact that the decarboxylation amino acid of the first KS is an Asn rather than a normal His in the catalytic triad. Also, previous investigations for site-directed mutagenesis of PKS were carried out using conventional methods involving massive DNA recombination [5]. But in this study, a two-step procedure was used to mutate the amino acid on the Streptomyces chromosome. First, we introduced a spectinomycin resistance gene into the chromosome at a locus adjacent to the mutation site as a selection marker by the REDIRECT system. Second, using the negative selection marker sacB gene, we constructed a cosmid carrying the desired mutation residues to mediate homologous recombination on the Streptomyces chromosome [9].
Materials and Methods
Bacterial strains and plasmids
The Escherichia coli and Streptomyces strains, vectors, and plasmids used in this study are summarized in Table 1.
Chemicals, biochemicals, and
media
Commonly used chemicals and biochemicals were from commercial sources. E. coli strains carrying plasmids were grown in Luria-Bertani (LB) medium and selected with appropriate antibiotics [10]. All media for Streptomyces growth were prepared according to standard protocols. YEME (10.3% sucrose) and tryptic soy broth were from Difco Laboratories (Detroit, USA). Modified GS agar medium (soluble starch, 20 g/L; KNO3, 1 g/L; K2HPO4, 0.5 g/L; MgSO4, 0.5 g/L; NaCl, 0.5 g/L; FeSO4, 0.01 g/L; and agar, 20 g/L) supplemented with 0.5% yeast extract was used for conjugation. Spore suspensions were prepared on Murashige and Skoog medium [soy bean meal (degreased), 20 g/L; mannitol, 20 g/L; and agar, 20 g/L (pH 7.0)]. Tryptic soy broth medium was used for the vegetative growth of the S. albus JA3453 wild-type and recombinant strains [11].
Conjugation between E. coli
ET12567 (pUZ8002) and S. albus
Introduction of plasmids into S. albus JA3453 wild-type or mutant strains was carried out by conjugation, following a standard procedure with minor modifications [11]. S. albus spores were heat-shocked in LB medium at 42 �C for 10 min, followed by incubation at 30 �C for 2.5 h. Spore germination was monitored microscopically every 30 min, after 1 h of incubation at 30 �C. Germinated S. albus spores were pelleted and resuspended in LB broth as the recipient strain. E. coli ET12567 (pUZ8002) carrying the donor plasmid was grown in LB broth with appropriate antibiotics for selection to an OD600 of 0.3 to 0.4. Cells from 2 ml culture were pelleted, washed twice with LB broth, and resuspended in 100 ml LB broth as the E. coli donors. For conjugation, the donor (100 ml) and recipient (100 ml, 108 spores) were mixed and spread onto modified GS plates freshly supplemented with 10 mM MgCl2. The plates were incubated at 28 �C for 16-22 h. After removal of most of the E. coli ET12567 donors from the plates by washing the surface with sterile water, each plate was overlaid with 1 ml sterilized water containing apramycin (Apr) and nalidixic acid at a final concentration of 50 mg/ml. Incubation continued at 28 �C until exconjugants appeared (in approximately 3 d).
Construction of plasmids
mediating insertion of spc resistance gene
Insertion of the spc resistance gene in ozmH adjacent
to the KS10-1 mutation site was carried out with REDIRECT protocols provided by
the John Innes Institute (Norwich, UK) [12,13]. The primers were designed as
follows: 5'-ACGTCGAGGCGGCCGTGG�TCGCCGGCGTCAC�CC�T�G�CTGCattccggggatccgtcgacc-3'
(KS10-1-FP1) and 5'-GT�CCCC�GCCGGTCGAAGAGGC�GGTGCCC�TCCCGCGT�C�GGtgtaggctggagctgcttc-3'
(KS10-1-RP1). The FLP targeting sequences (lowercase letters) were attached
to the 39 nt corresponding to the internal coding sequences of ozmH (capital
letters) and these primers used to amplify the spectinomycin resistance cassette
from pIJ778. The resulting polymerase chain reaction (PCR) products were
introduced into E. coli BW25113/pIJ790 carrying cosmid pJTU1060 by
electroporation. Recombination between the PCR-amplified spectinomycin
resistance cassette and the cosmid pJTU1060 yielded the mutated plasmid
pJTU1067, in which 1378 bp spectinomycin resistance gene fragments were
inserted into ozmH at the expected locus.
Similar approaches were used to insert the spectinomycin resistance
gene near to the KS10-2 mutation locus, except that different
primers were used, 5'-AGTGC�GAG�GTC�G�C�CGTCGCGGGCG�GCGTCAACCTCTCGC�attccg�ggg�atccgtcgacc-3'
(KS10-2-FP1) and 5'-TGCCC�AC�G�G�C�T�CC�G�TAGGTG�CGGTACTTGCCC�GGGTGCAtgt�aggct�gga�gc�tg�cttc-3'
(KS10-2-RP1). PCR targeting between the amplified spectinomycin resistance
cassette and pJTU1060 afforded the mutated construct pJTU1068, in which the
spectinomycin resistance gene was inserted into ozmH adjacent to the
expected mutagenesis locus of KS10-2.
Construction of subclones with
site-directed mutation of ozmH
To clone the KS10-1 coding region, PCR was carried out with pfu polymerase and primers 5'-TCCACT�GCG�T�TT�C�TGCGTGCTGTTC-3' (KS10-1-FP2) and 5'-GCCG�TGG�A�AGTCGCAGACGAA GGAG-3' (KS10-1-RP2). Using pJTU1060 as the template, PCR product with the predicted size of 3.0 kb was cloned into the EcoRV site of pBluescript SK (Stratagene, La Jolla, USA) to verify PCR fidelity by sequencing to yield pJTU1141. The exogenous fragment was then moved again as a 3.0 kb EcoRI-HindIII fragment from pJTU1141 and ligated into the same site of pIJ2925 [11], resulting in the plasmid pJTU1143.
Similar approaches were adapted to clone the KS10-2 coding region using primers 5'-GGTTACGCCCCCGACG�AG�CTGAAGG-3' (KS10-2-FP2) and 5'-GACGGCCTTGACC�A�G�T�CGCAGCAG-3' (KS10-2-RP2) on the pJTU1060 template. A distinctive product with the predicted size of 2.4 kb was cloned into the EcoRV site of pBluescript SK to verify PCR fidelity affording pJTU1142. The insert was excised from pJTU1142 with EcoRI-HindIII and ligated into the same site of pIJ2925 to produce pJTU1146.
To mutate the KS10-1 and KS10-2 active site Cys into Gly, the primers were designed as follows (the mutated codons are underlined): for KS10-1, 5'-GGTCGTGGACACCGCC������G�G�ATCCTCCGCGCTCGTGGCCCT-3' (KS10-1-FP3) and 5'-AGGGCCACGAGCGCGGAGG�ATCCGG�CGGT�GT�C�C�ACGACC-3' (KS10-1-RP3); and for KS10-2, 5'-GAC�G�G�T�G�GACACCCTG�GGATCCTCCTCGCTCACCGCGC-3' (KS10-2-FP3) and 5'-GCGCGGTGAGCGAGGAGGA����T�C�C�C�AGGGTGTCCACCGTC-3' (KS10-2-RP3). Site-directed mutagenesis was carried out by a QuickChange kit (Stratagene) to afford mutated plasmids pJTU1145 and pJTU1147. Both Cys active sites that had been mutated into Gly were confirmed by DNA sequencing. Finally, the 3.0/2.5 kb BglII fragment containing the point mutation was subcloned from pJTU1145/1147 and moved into the BamHI site of pKOV-Kan (Kan) to afford pJTU2299/pJTU2300 [9].
Transformation of E. coli
and selection of recombinants
The plasmids pDF25 (cml) and pJTU2299/pJTU2300 (kan) were cotransformed into DH10B highly efficient competent cells containing the cosmid pJTU1060 (apr), with Cml, Kan, and Apr for selection at 30 �C. Colonies were picked separately and plated at 43 �C to select co-integrant clones. DH10B cells harboring the vector pDF25 were made competent again using CaCl2 at 30 �C and the co-integrant clones were transfected into the 50 ml cell suspensions followed by incubation at 30 �C with Cml, Kan, and Apr for selection. Then transformants were subjected to Apr plates at 43 �C for 24 h to allow double cross-over homologous recombination. Several colonies were selected and streaked on Apr/sucrose and incubated at 30 �C for 24 h. The larger colonies were picked and restreaked on Apr/sucrose at 30 �C to grow overnight. The colonies that grew only on Apr/sucrose were analyzed by restriction enzyme digestion followed by DNA sequencing.
Production, isolation, and
analysis of OZM
Streptomyces albus JA3453 wild-type and recombinant strains were cultivated on Murashige and Skoog medium at 30 �C for 7 d. Spore and aerial mycelia suspensions were prepared under sterile conditions by adding 5 ml deionized water containing 100 ml of Triton X-100. The fermentation broth was centrifuged (4000 g for 20 min) and the supernatant was harvested and extracted twice with an equal volume of EtOAc. HPLC was carried out using a Prodigy ODS-2 column (150 mm4.6 mm; Phenomenex, Torrance, USA) developed with a linear gradient from 60% to 90% CH3OH in H2O over 20 min at a flow rate of 1 ml/min with ultraviolet detection at 278 nm. ESI-MS was carried out on an Agilent 1000 HPLC-MDS SL instrument (Agilent Technologies, Palo Alto, USA) to compare with the published data for OZM [8].
Results
Characterization of tandem KSs
in gene ozmH and proposed biosynthetic mechanism
In our previous study, we localized the OZM biosynthetic gene cluster as a 135 kb contiguous DNA represented by five overlapping cosmids [14]. Complete sequencing revealed 21 genes putatively responsible for OZM biosynthesis, and designated as ozmA to ozmU. Of these, the gene ozmH encodes a giant protein of 7737 amino acids characteristic of a complex PKS and NRPS hybrid megasynthase composed of five PKS or NRPS modules [Fig. 1(A)]. Of these modules, module 10 has an unusual domain organization of KS-KS-ketoreductase-ACP. KS10-1 shows highly homology with the type I KSs in known PKS modules, such as BaeJ involved in bacillaene biosynthesis from Bacillus amyloliquefaciens (GenBank accession No. CAG23957; 53% similarity and 67% identity). KS10-2 shows significant homology with KS-like BaeM (GenBank accession No. CAG23959; 63% similarity and 75% identity). The catalytic triad decarboxylation active site of the first KS (KS10-1) is asparagine instead of histidine but that of the second KS (KS10-2) is a normal amino acid histidine [Fig. 1(B)]. Similar domain organization of tandem KSs in a single module has been discovered in several other polyketide gene clusters, exemplified by LnmI module 3 of the leinamycin biosynthetic gene cluster [4]. Because upstream and downstream of ozmH module 10 presents only PKS modules, we presume that in the ozm biosynthetic model, KS10-1 might be assigned as an "extra domain" due to the changed decarboxylation active site.
To unambiguously confirm our hypothesis that KS10-1 is an "extra domain" for OZM biosynthesis, we mutated its catalytic triad residue Cys responsible for ketoacyl condensation into Gly on the chromosome of S. albus JA3453, together with that of KS10-2 as a control.
Introduction of spectinomycin
resistance gene
To mutate the KS10-1 and KS10-2 active-site cysteines into glycines, we inserted a spectinomycin resistance gene into the chromosome adjacent to the target mutation locus to facilitate subsequent screening of the recombinant strains. According to protocols provided by Gust et al [12], PCR targeting and l-RED-mediated recombination through a double cross-over between the PCR amplified spectinomycin resistance cassette on the template pIJ778 and pJTU1060 yielded the mutant plasmids pJTU1067 and pJTU1068, in which a 1378 bp spectinomycin resistance gene fragment was inserted into ozmH at the locus 50 bp downstream of the target mutagenesis sites of KS10-1 and KS10-2 (see "Materials and Methods" section). Then the above plasmids were introduced into S. albus JA3453 by ET12567 (pUZ8002)-mediated E. coli-Streptomyces bi-parental conjugation [11] stepwise by first screening the mutants with spectinomycetin and apramycin resistance phenotype, followed by serial rounds of propagation on non-selective plates to isolate colonies with spectinomycetin resistance and apramycin-sensitive phenotype to afford mutant strains SDF1 and SDF2, respectively. The genotypes of SDF1 and SDF2, in which spectinomycetin resistance markers were introduced into the desired position of the S. albus genome, were confirmed by PCR and Southern blot analysis (data not shown).
Construction of cosmids
containing the expected site-directed mutation in ozmH
The genomic fragments of S. albus containing KS10-1 and KS10-2 were amplified from the cosmid harboring the OZM biosynthesis genes. The conserved catalytic triad amino acid cysteines were engineered into glycines to produce pJTU2299/pJTU2300 separately (see "Materials and Methods"). Also, a new BamHI restriction site was introduced in order to distinguish the recombinant plasmids. To transfer modification of these plasmids into a cosmid target, we used two vectors that had been well described in other studies, pKOV-Kan [9] and pDF25 [15] (Fig. 2). Both vectors possess a temperature-sensitive DNA replication (pSC101-ts) and propagate at a permissive temperature below 33 �C, but replication is deficient at 43 �C. The pKOV-Kan plasmid is also characteristic of positive selection markers cml and kan resistance gene and negative selection gene sacB origin from B. amyloliquefaciens that permits the host to grow very slowly on media supplemented with 5% sucrose. The vector pDF25 harbors Cm resistance marker and the recA gene mediates high-frequency homologous recombination in E. coli cells.
The inserts containing point mutations were excised from subclones and ligated with the vector pKOV-Kan (Kan). The nucleotide changes were located in the center of exogenous fragments which would facilitate homologous recombination. The pKOV-Kan derived plasmids were co-transformed into E. coli cells carrying cosmid target together with plasmid pDF25 with appropriate antibiotic selection at a permissive temperature of 30 �C. the transformants were subsequently transferred to 43 �C to allow first homologous recombination to yield co-integrants. E. coli cells harboring the vector pDF25, which carries gene recA, were made competent cells again and the co-integrants were transfected into the cells to induce double cross-over homologous recombination with a negative selection marker sacB to isolate colonies that can not grow on sucrose-containing media. The expected genotypes of resulting recombinants were confirmed by restriction enzyme digestion followed by DNA sequencing, that is, cosmid containing the desired site-directed mutation of ozmH could be digested by BamHI. The modified cosmids originating from subclones pJTU2299 and pJTU2300 afforded pJTU2301 and pJTU2302, respectively (Fig. 3).
Generation of KS10-1 and
KS10-2
single inactive Streptomyces mutant strains
Introduction of mutated cosmids pJTU2301/pJTU2302 into S. albus SDF1/SDF2, respectively, was carried out by E. coli-Streptomyces conjugation [11]. The desired double crossover homologous recombinants were selected with spectinomycetin sensitive and apramycin resistance phenotype, leading to the isolation of mutant strains SDF7/SDF8, whose expected genotype was confirmed by PCR analysis of the mutated locus followed by DNA sequencing.
HPLC analysis of mutants SDF7
and SDF8
HPLC analysis showed that OZM production in the mutant strain SDF7 is comparable with that of the wild-type JA3453 strain as a positive control under identical fermentation conditions, but the mutant SDF8 lost its ability to produce OZM. The identity of OZM was further confirmed by ESI-MS analysis, yielding the characteristic (M+H)+ ion at m/z=656.1, consistent with the molecular formula C36H49N3O9 [8] (Fig. 4).
Discussion
The natural compound OZM produced by S. albus JA3453 is a hybrid PKS-NRPS antibiotic showing important bioactivity. Complete sequencing of the biosynthetic gene cluster has revealed multiple PKS, NRPS, and hybrid PKS-NRPS megasynthase. Of these, the ozmH gene encodes a giant protein identified with five modules. ozmH module 9 is characteristic of tandem KS domains, which seemed to be unusual for PKS organization. To test the role the domains played in OZM biosynthesis, site-directed mutagenesis was used to change their conserved catalytic triad Cys residues into Gly, to afford mutant strains SDF7 and SDF8. SDF7 still produces OZM comparable with wild-type strain JA3453, but SDF8 loses its ability to produce OZM. This suggests that the first KS is redundant for OZM biosynthesis and might serve as an "extra domain", but the second KS is indispensable for OZM biosynthesis. These results are consistent with fact that this domain has an Asn instead of a His codon in this particular location, which seems to be of no function.
Gust et al developed a rapid and efficient protocol that was used to inactivate a large amount of Streptomyces genes by insertional mutagenesis [12,16]. In polyketide and non-ribosomal peptide biosynthetic gene clusters, multiple domains are always organized into a single protein to assemble fatty acids or amino acids into natural compounds, so disruption of a domain by the insertion of a resistance gene always results in malfunction of the intact protein. So site-directed mutation, rather than insertional inactivation, is needed for engineering of PKS. A previous study of site-directed mutagenesis of PKS exemplified by tandem ACPs for LNM biosynthesis was carried out by conventional DNA recombination requiring multi-step clone construction [5]. The strategy in our study for mutation, which involves sacB-mediated negative selection, has been successfully used in E. coli to generate point mutations for bacterial artificial chromosomes by homologous recombination [9]. As oriT-mediated conjugation allowed convenient intergeneric transfer of cosmid DNA from E. coli into Streptomyces [11], we extended the above strategy in Streptomyces to mutagenesis genes, especially those responsible for natural compound biosynthesis, which not only decreased our efforts to construct mutant plasmids, but also improve screening of the mutants due to the long double cross-over recombination DNA arms. Interestingly, the frequency of double cross-overs in Streptomyces was nearly 50% when mutagenized cosmids were used for conjugation, as >15 kb homologous DNA sequences were present on both sides of the mutated locus.
With the proliferating genes characterized or predicted in genomic databases [18], there is a growing requirement for high throughput techniques to determine their functions. To the best of our knowledge, this study represents the only application of recA- and sacB-mediated strategies to introduce point mutations into Streptomyces chromosomes or complex PKS.
Acknowledgements
We thank the Analytical Instrumentation
Center of the School of Pharmacy, University of Wisconsin (Madison, usa) for support in obtaining MS and
HPLC data, the John Innes Centre (Norwich, UK) for providing the REDIRECT
Technology kit, Werner F. Fleck (Hans Knoell Institute for Natural Product
Research, Jena, Germany) for providing the S. albus JA3453
strain, and John K. Heath, University of Birmingham (Birmingham, UK) for
providing plasmids pKOV-Kan and pDF25.
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