Departments of Chemical Engineering1, Chemistry and Biochemistry2, Stanford University, Stanford, CA 94305-5025, USA
School of Pharmacy3 and Department of Bacteriology4, University of Wisconsin, Madison, WI 53706, USA
Author for correspondence: Chaitan Khosla. Tel: +1 650 723 6538. Fax: +1 650 723 6538. e-mail: ck{at}chemeng.stanford.edu
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ABSTRACT |
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Keywords: rifamycin, polyketide synthase, Amycolatopsis
Abbreviations: AHBA, 3-amino-5-hydroxybenzoic acid; PKS(s), polyketide synthase(s)
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INTRODUCTION |
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The mechanism of action of rifamycin against bacteria and viruses (Lowder & Johnson, 1987 ; Riva et al., 1972
) as well as its structureactivity relationships (Arora & Main, 1984
; Arora, 1985
; Bacchi et al., 1998
; Bartolucci et al., 1995
) have been extensively studied. More recently, the biosynthesis of rifamycin has attracted attention. The polyketide chain starts with a 3-amino-5-hydroxybenzoic acid (AHBA) primer unit (Ghisalba & Nuesch, 1981
; Ghisalba et al., 1981
; Hatano et al., 1982
). Initially, the gene encoding AHBA synthase, which catalyses the last step in AHBA biosynthesis, was cloned (Kim et al., 1998
); in turn, this led to cloning and sequencing of the whole gene cluster (Schupp et al., 1998
; Tang et al., 1998
). Encoded within the gene cluster is a 10-module polyketide synthase (PKS) presumably responsible for the biosynthesis of proansamycin X, the polyketide precursor of rifamycin B (Fig. 1
). This multifunctional enzyme complex presents an attractive opportunity for engineering novel rifamycin analogues, as has been repeatedly demonstrated for the better-studied erythromycin PKS (Cane et al., 1998
). However, a central prerequisite for such genetic engineering is the availability of suitable genetic tools and methods.
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In Amycolatopsis, unlike Streptomyces, genetic tools and methods, such as transformation and transfection, and cloning vectors, are relatively undeveloped. Plasmids that replicate in Streptomyces cannot be maintained in Amycolatopsis mediterranei (Schupp & Diver, 1986 ; Pelzer et al., 1997
), although plasmids such as pIJ702 and pKC505 from Streptomyces could replicate in Amycolatopsis orientalis (Matsushima et al., 1987
). A few indigenous plasmids such as pMEA100 (Moretti et al., 1985
; Madon & Hutter, 1991
), pMEA300 (Vrijbloed et al., 1994
) and pA387 (Lal et al., 1991
) have been found in Amycolatopsis species, but have not yet been fully developed into cloning and expression vectors. Consequently, genetic manipulation of the rifamycin PKS genes in A. mediterranei is very difficult, and primarily relies on the use of suicide delivery systems.
Here we describe the construction of specific deletion mutants of A. mediterranei S699 as hosts for PKS gene expression. Engineered forms of the rifamycin PKS can be introduced into these hosts either on plasmid vectors or through single crossovers into the chromosome. We demonstrate the utility of this approach by reinserting the rifA gene into the chromosome to produce the expected tetraketide P8/1-OG.
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METHODS |
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Media, chemicals and growth conditions.
E. coli strains were grown in LuriaBertani (LB) medium at 37 °C. When necessary, 50 µg carbenicillin ml-1 or 60 µg apramycin ml-1 was present in the medium. A. mediterranei strains were grown in YMG (also called YM) (Lal et al., 1998 ) medium at 30 °C, supplemented with 60 µg apramycin ml-1 and/or 50 µg hygromycin ml-1 when necessary. All antibiotics were purchased from Sigma; 3-hydroxybenzoic acid was obtained from Aldrich, whereas 3-amino-5-hydroxybenzoic acid (AHBA) was synthesized in this laboratory and verified by NMR.
DNA manipulation.
DNA fragments for subcloning were purified with Qiaex I (for fragments smaller than 10 kb) or Qiaex II (for fragments larger than 10 kb) gel extraction kits (Qiagen). DNA blotting and Southern hybridization made use of protocols recommended for the DIG labelling and detection kit (Boehringer). All other DNA manipulations utilized standard methods (Sambrook et al., 1989 ).
Construction of a genomic DNA library.
Genomic DNA was isolated from A. mediterranei S699 and partially digested with Sau3A. The fraction containing 3542 kb fragments was purified from a 0·3% agarose gel and ligated with BamHI/XbaI-linearized SuperCos 1 (Stratagene). The ligation mixture was packaged using Gigapack III Gold packaging extract (Stratagene), and used to infect E. coli XL-1 Blue. Recombinants were selected on LB agar plates containing carbenicillin.
Purification of P8/1-OG.
Spores of A. mediterranei were plated on Petri dishes containing 40 ml YMG medium to give lawns. Following growth at 30 °C for 3 d, the mycelium was overlaid with a solution of the starter unit AHBA in 10% DMSO diluted with water (pH adjusted to 7·2) to give 1·5 ml per plate. The agar in plates was allowed to dry for 45 min before use. After addition of the starter unit, incubation was continued for another 10 d. The medium was then homogenized and extracted three times with an equal amount of ethyl acetate containing 1% acetic acid. The crude extract was pre-purified on a short (5 cm) silica gel column using CHCl3/CH3OH 4:1 (containing 1% acetic acid) as an eluant. All fractions containing product were combined and evaporated. The residue was rechromatographed on a 10 cm column using the same solvent system; the chromatographic purification removed most of the contaminants except AHBA. P8/1-OG was further purified by a second chromatography (5% CH3OH in ethyl acetate, 1% acetic acid overall content) after application to the column in a small amount of CHCl3/CH3OH 4:1. Fractions containing pure product were combined and evaporated to give a yellowish residue that was identified as P8/1-OG by: RF 0·41 (5% CH3OH in ethyl acetate, 1% acetic acid), 0·37 (CHCl3/CH3OH 4:1, 1% acetic acid); HPLC (see below for conditions) tr=10·28 min; 1H-NMR (400 MHz, CD3OD) (refer to Ghisalba et al., 1981 for 1H-NMR data previously reported) 6·24 (t, br, 1 H, J=1·60 Hz), 6·16 (t, br, 1 H, J=1·46 Hz), 6·13 (t, 1 H, J=2·10 Hz), 6·04 (s, 1 H), 4·53 (d, 1 H, J=8·85 Hz), 2·77 (qd, 1 H, J=8·92, 7·09 Hz), 1·86 (s, 3 H), 0·97 (d, 3 H, J=7·02 Hz) (br, broad; qd, quartet of doublets).
Quantitative HPLC analysis of production levels.
3-Hydroxybenzoic acid (10 mg per plate) was fed to both A. mediterranei HGF003 and A. mediterranei HZ149 as described above. Following incubation, culture media were extracted with ethyl acetate containing 1% acetic acid. The extracts were evaporated in vacuo and the residue was redissolved in CH3OH (2 ml per plate extracted). The resulting solution was analysed by HPLC [Beckman ULTRASPHERE C18, 5 µ, 0·46x25 cm; flow rate, 1·0 ml min-1; injection volume, 20 µl; UV detection at 254 and 306 nm; solvent gradient, 01 min: 100% A, 120 min: 100% A100% B, 2025 min: 100% B (solvent A was 1% acetic acid in water and solvent B was 1% acetic acid in CH3CN)]. The expected tetraketide eluted at tr=13·06 min.
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RESULTS |
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Construction of a deletion host
DNA fragments A, B and C were subcloned from cosmids cos2, cos42 and cos6, respectively (Fig. 2). Fragment A starts at nt 651 and continues to nt 1438 (where nucleotides are numbered from the first base of the rifamycin PKS genes). Fragment B extends from nt 28464 to nt 29417, and includes the end of module 6 as well as the start of module 7. Fragment C contains the end of module 10 starting from nt 47393 and continues to nt 50159; it includes the 3' end of the rifF amide synthase gene. Two suicide plasmids were constructed in pSET152 (NheIEcoRI) with the hygromycin-resistance gene (hyg) (Malpartida et al., 1983
) flanked by either fragments A and B or A and C in their natural orientations. These constructs, designated pHU130 and pHU131, respectively, were introduced into A. mediterranei S699 by electroporation. Approximately 10 hyg+ apr+ colonies were obtained from each transformation, presumably as a result of a single crossover in one of the homologous regions. One such transformant was randomly selected in each case, and propagated through two rounds of subsequent growth on non-selective YMG agar. More than 200 single colonies were tested for resistance to apramycin and hygromycin on selective YMG plates. Two derivatives of pHU130::S699 and three derivatives of pHU131::S699 had lost their resistance to apramycin but were still resistant to hygromycin. Southern analysis of XmnI + EcoRI-digested genomic DNA from two of the three pHU131::S699 derivatives, and of HindIII + EcoRI-digested genomic DNA from one of the two pHU130::S699 derivatives, using the hyg gene as a probe, confirmed that all three colonies were derived from double-crossover events, as expected (Fig. 3
). Randomly chosen derivatives of pHU130::S699 and pHU131::S699 were designated A. mediterranei strains HZ14 and HZ17, respectively (see Fig. 2
).
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Expression of a truncated form of the rifamycin PKS in a deletion host
The XmnI (position -651)RcaI (position 14757) fragment from cos2 was end-filled using T4 DNA polymerase and cloned into the SmaI site of pUC119 to give pHU42. The XbaIEcoRI fragment from pHU42 was then ligated with NheI+EcoRI-digested pSET152 to give the plasmid pHU149. Plasmid pHU149 was introduced into the deletion host A. mediterranei strain HZ17 by electroporation. Two apramycin-resistant transformants, HZ149-1 and HZ149-3, were obtained (Fig. 4). Extraction with ethyl acetate containing 1% acetic acid and TLC comparison with a comparable extract from the deletion host HZ17 revealed no major metabolite in the fermentation medium. Moreover, no AHBA was produced by either transformant. Analysis by reverse-phase HPLC yielded the same result, leading us to suspect that HZ149-1 or HZ149-3 might be capable of producing tetraketides, but that tetraketide production was down-regulated due to attenuated biosynthesis of AHBA. Therefore, A. mediterranei strains HZ149-3, HZ17 and HGF003 were subjected to more detailed metabolite analysis.
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Earlier studies had shown that A. mediterranei HGF003 is capable of producing polyketides in high yields when fed a variety of exogenous starter units (Hunziker et al., 1998 ). However, whereas AHBA is converted into rifamycin B by this strain, alternative starter units such as 3-hydroxybenzoic acid and 3,5-dihydroxybenzoic acid are processed only into tetraketide analogues of P8/1-OG. Therefore, the production levels of A. mediterranei strains HZ149-3 and HGF003 were compared by adding 3-hydroxybenzoic acid to growing cultures of both strains. Following incubation, culture extracts were analysed by HPLC for tetraketide (desamino P8/1-OG) levels. Desamino P8/1-OG production by HZ149 was approximately 35% relative to HGF003. Purification of desamino P8/1-OG from the HZ149-3 extract gave 7·7 mg pure compound, which corresponded to a titre of approximately 50 mg l-1. HGF003 has been shown previously to produce desamino P8/1-OG at 135 mg l-1 (Hunziker et al., 1998
).
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DISCUSSION |
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Although A. mediterranei is taxonomically related to Streptomyces, methods for gene cloning in A. mediterranei are relatively underdeveloped. In particular, a suitable low-copy plasmid capable of holding large inserts is not yet available. Moreover, the absence of endogenous promoters suitable for polyketide gene expression is a disadvantage. Because our hostvector strategy uses a suicide vector to insert the desired gene(s) into the chromosome of A. mediterranei through a selectable single crossover, the expressed genes can use the same promoter that is presumably responsible for transcription of the rifamycin PKS genes. Although we were able to generate HZ149 in a single-step process, we were unable to deliver very large fragments (approx. 50 kb, carrying the first six modules of the rifamycin PKS gene cluster) into the chromosomes of the deletion hosts (data not shown). This may be due to either poor transfer of extremely large fragments into A. mediterranei, or the greater likelihood of their destruction by endogenous nucleases. As improved plasmids and promoters are developed for A. mediterranei, we expect that the utility of our deletion hosts will increase further.
Finally, it is intriguing that production of P8/1-OG in HZ149 depends on the addition of exogenous AHBA. Since both wild-type A. mediterranei and its deletion derivatives can produce large amounts of AHBA, our results may suggest that one or more genes downstream of the rifamycin PKS operon are required for AHBA biosynthesis, and that integration of a suicide vector disrupts their transcription. Further experiments may help resolve this interesting conundrum.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 23 February 1999;
revised 18 May 1999;
accepted 25 May 1999.
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