1Department of Medicinal Chemistry and 2Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, VA 23219 and 3Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109-1065, USA
4 To whom correspondence should be addressed. e-mail: kareynol{at}hsc.vcu.edu
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Abstract |
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Keywords: acyl-carrier protein/acyltransferase/erythromycin/hybrid PKS/pikromycin/typeI polyketide synthase
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Introduction |
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One such example has been replacement of the six methylmalonyl-CoA specific AT domains in DEBS with malonyl-CoA specific AT domains from heterologous systems. The corresponding hybrid PKS thus generates a desmethyl analog of 6-dEB at the predicted location (Ruan et al., 1997). This work was first successfully reported for modules 1 and 2 by workers at Abbott Laboratories (Ruan et al., 1997
). Independent work at Kosan Biosciences led to extension of this work to include modules 3, 5 and 6 (McDaniel et al., 1999
), but not module 4 (Reeves et al., 2001
). A successful switch at module 4 has been accomplished recently by a third and independent group of investigators from Biotica Technology (Petkovic et al., 2003
). A different approach in which site-specific mutations have been made in module 4 AT has provided a mixture of the normal product and the desired 6-desmethyl analog (Reeves et al., 2001
). Despite these and other successes (Stassi et al., 1998
), many of the AT switches yielded hybrid systems that produce little or no product (Reeves et al., 2001
). Similar observations have been made with changes to the AT domain in module 6 of the pikromycin PKS system (PikPKS), where only some AT switches have been marginally successful (Chen et al., 2000
; Yoon et al., 2002
).
Changes to loading domains, including AT switches have also been reported. The first successful example engineered broader substrate specificity by replacement of the AT0 of DEBS1 (which loads propionyl-CoA primers preferentially) (Figure 2B) with the corresponding AT0 of the avermectin PKS (which loads a wide range of branched-chain acyl-CoA primers) (Marsden et al., 1998). Subsequently, the loading domain of DEBS1 was replaced with that from the monensin PKS (Bisang et al., 1999
). The loading domain of monensin, pikromycin and many other type I PKSs contains an N-terminal domain termed KSQ, which resembles the KS domain but contains a glutamine residue in place of the active site cysteine (Figure 2A) (Xue et al., 1998
; Bisang et al., 1999
). In such systems, the AT0 domain selectively loads a dicarboxylated acyl-CoA primer (malonyl-CoA and methylmalonyl-CoA for the monensin and pikromycin PKS, respectively). The KSQ domain subsequently catalyzes the decarboxylation of these units (Figure 2A) to provide the corresponding acyl ACP substrate for KS1 (Bisang et al., 1999
; Witkowski et al., 1999
). Attempts to use site-specific mutations to switch AT0 specificity between mono- and dicarboxylated acyl-CoA primers have not been successful (Long et al., 2002
).
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Numerous genetic techniques, including family shuffling, SCRATCHY, ITCHY and SHIPREC, have been successfully used for generating highly diversified mutant libraries (Stemmer, 1994; Ostermeier et al., 1999
; Lutz et al., 2001a
,b; Sieber et al., 2001
; Kawarasaki et al., 2003
). When combined with an appropriate screen these methods allow for directed evolution of an enzyme or hybrid enzyme with specific desired properties. Application of these approaches to the creation of functional and efficient hybrid type I PKSs has not yet been described, primarily due to a number of technical issues. Using the PikPKS we describe herein a method for generating and screening a library of hybrid PikAI molecules containing changes in AT0. The methodology is applicable to both homologous and homologous-independent combinatorial engineering techniques, can be extended to other domains and modules of the PikPKS, and is potentially applicable to other type I PKS systems.
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Materials and methods |
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DNase I was purchased from Promega (Madison, WI). PCRs were performed using the GC-Rich PCR system from Roche (Mannheim, Germany) with only minor modifications to the manufacturers instructions. All other chemicals were purchased from Sigma and were of the highest available grade. Standard molecular cloning techniques were employed (Sambrook et al., 1989). The Escherichia coli TG2 strain was used as the standard cloning host for the plasmid constructions. Escherichia coli transformants were selected and cultured following standard procedures.
Streptomyces venezuelae BB138 (a pikAI deletion mutant) (Yoon et al., 2002) was used for expression of hybrid PikAI proteins and was transformed by the standard procedures (Kieser et al., 2000
).
Generation of mutant (pBK77) and hybrid PikAI (pBK79) complementation plasmids
The plasmid pDHS722 is a pikAI complementation plasmid (Yoon et al., 2002), which fully restores pikromycin production in the pikAI deletion S.venezuelae mutant BB138. A silent BamHI site was introduced in the beginning of region encoding AT0 in this pDHS722 to give pBK76. The engineered BamHI site was created by two-step cloning procedures. An EcoRI (shown in italics)BamHI (introduced site, underlined sequence) fragment was amplified using pDHS722 as template and the following primer set: 5'-GTGTCCAAGAGTGAGTCCGAGGAATTCGTGTCC-3' and 5'-CGCCACTCGCCCGGATCCGGAAGCCACGCC. The amplified fragment replaced the wild-type EcoRIBamHI (natural site) fragment in pDHS722, then the omitted AT0-ACP0 region was refilled by a PCR-amplified fragment. This PCR fragment was generated using pDHS722 as a template and the following primer set: 5'-ggcgtggcttccggatccgggcgagtggcg-3' (with the engineered BamHI site) and 5'-ggggtgctccggatccgggtggtacagccc-3' (with the natural BamHI site). Macrolide antibiotic production by strain BB138 transformed with pBK76 and pDHS722 was indistinguishable, demonstrating that the engineered restriction site did not have a deleterious effect.
A pikAI complementation plasmid (pBK77), which encodes a mutant PikAI in which the conserved glutamine residue of the KSQ domain was replaced with alanine, was subsequently constructed. Briefly, an EcoRIBamHI fragment from pBK76 was subcloned and the QuikChange kit and a primer set (5'-gactccggcgctagctcgtcgctcgtc-3' and 5'-gacgagcgacgagctagcgccggagtc-3', bold sequence indicates mutation) were then used to introduce the desired mutation. The resulting fragment was cloned back into pBK76 to give pBK77.
A hybrid pikAI complementation plasmid (pBK79) containing eryAI (encoding DEBS1 AT0-ACP0) was generated from pBK77. Briefly, the DNA region encoding the AT0-ACP0 of DEBS1 was amplified using a primer set (5'-GGCGTGGCCCGGGGATCCGCGCCG-3' and 5'-CGAGCGCGTGGGATCCGGGTGGAACAGCG-3') and pBK3 (Kim et al., 2002a). The resulting fragment was cloned into pBK77 using BamHI sites to give pBK79. This hybrid complementation plasmid and pBK77 were transformed into S.venezuelae BB138 to assess pikromycin production (Xue et al., 1998
).
Generation of pBK84, a recipient plasmid bearing a counter selectable marker
The sacB gene and its promoter, flanked by BamHI sites, were obtained by PCR amplification from pEX100T (ATCC 87436) (Schweizer and Hoang, 1995) using the following primer pair: 5'-ctagctagaggatccatcctttttaaccc-3' and 5'-tttggatcccgtttttatttgttaactg-3'. The PCR product was cloned into pBK77 using the BamHI sites, providing the recipient plasmid pBK84.
Preparation of shuffled DNA library
The AT0-ACP0 region of pikAI was obtained as a BamHI PCR fragment using pDHS722 as a template and the following primer set: 5'-ggcgtggcttccggatccgggcgagtggcg-3' and 5'-ggggtgctccggatccgggtggtacagccc-3'. The corresponding loading didomain region of eryAI was amplified with the same primers used to make pBK79 (see above). The PCR products were ligated into pCR2.1 vector to give pBK82 and pBK83, respectively, which were maintained in E.coli TG2. When the didomain DNA fragments were needed for DNA shuffling, the plasmids were isolated using the Qiagen Mini Prep Kit (Qiagen GmbH, Germany) and excised using BamHI.
The DNA fragments encoding the AT0-ACP0 didomain of PikAI and DEBS1 (5 µg) were digested with 0.0015 U of DNase I to produce 50300 bp fragments. The digested DNA fragments were separated on 2% agarose gel and purified using DE81 ion-exchange filter paper (Whatman). The fragmented DNA was subjected to a gradient PCR (50 µl of reaction volume) which uses a low initial annealing temperature (the first 21 cycles were 95°C for 60 s, 20°C for 60 s with 0.5°C increments each cycle, and 72°C for 60 s; the second 21 cycles were 95°C for 60 s, 30°C for 60 s with 0.5°C increments and 72°C for 60 s with 2 s increments each cycle; the last cycle was 95°C for 30 s, 42°C for 30 s and 72°C for 100 s). Two microliters of the PCR were subjected to an enrichment PCR which was initiated at 96°C for 8 min followed by 15 cycles of 95°C for 45 s, 60°C for 30 s and 72°C for 90 s. To prevent amplification of the parental gene sequences, the shuffled PCR constructs were enriched using hybrid primer sets. These primers were from the appropriate regions of genes encoding the loading domain of DEBS1 and PikAI. One set consisted of a forward primer for pikAI sequence (5'-gcgcctgagggtctggtccggggcgtggcttccggatccgggcg-3') and a reverse primer for eryAI (5'-gcgcctgagggtctggtccggggcgtggcTTCCggatccgcg ccg-3'). The second set comprised a eryA1 forward (5'-gccctggcggacgtacgacgtgccggggtgctccggatccgggtggaacagcg-3') and pikAI reverse primer (5'-gccctggcggacgtacgacgtgccggggtgctccggatccgggtggtacagccc-3'). Each set of hybrid primers contained a 39 nt region at the 5' end which was essential for the subsequent in vivo recombination step (Gust et al., 2003). The enriched DNA fragments were cloned to pCR2.1. The resulting constructs were used to transform E.coli. Plasmids were purified from 18 randomly chosen clones and the corresponding inserts were sequenced (depicted as P8P25 in Figure 5).
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The shuffled didomain fragments were cloned back into the appropriate region of the pikAI gene using the recipient plasmid pBK84. This plasmid was introduced to E.coli BW25113/pIJ790 (Gust et al., 2003), which expresses the
-Red recombinase. Escherichia coli BW25113/pIJ790/pBK84 was cultured in 40 ml of SOB media [containing 20 mM MgSO4, 10 mM arabinose, ampicillin (100 µg/l) and chloramphenicol (25 µg/l)] at 30°C to an OD600 of 0.4. The cells were harvested and washed with 10 ml of ice-cold 10% glycerol, and resuspended with 50 µl of 10% glycerol. The shuffled DNA fragments (46 µg) were introduced to the cells by electroporation (0.2 cm BioRad GenePulser II cuvette, 2.5 kV). The shocked cells were transferred to 2 ml of ice-cold LB media and incubated for 1 h at 37°C and dispensed into LB containing ampicillin. After a 57 h incubation at 37°C, the E.coli cells were harvested to isolate plasmid and re-transferred to TG2 strain by electroporation, as described above. The transformed cells were cultured onto non-salt LB agar containing ampicillin and 5% sucrose for 1620 h at 30°C. A BamHI digest of plasmid isolated from six separate E.coli colonies all revealed only a 1.3 kb fragment for the DNA encoding the hybrid AT0-ACP0 didomain, and no detectable 1.8 kb sacB fragment. All of the colonies that grew on the sucrose-LB medium were then pooled and subjected to a plasmid purification procedure. The pooled purified plasmids were then used for transformation of the BB138 pikAI deletion mutant using standard protocols.
Screening for functional hybrid PikAI
The pikAI deletion mutant BB138 was transformed with the library of complementation plasmids encoding PikAI with chimeric AT0-ACP0 loading domains. The transformants were grown on R2YE agar plates overnight at room temperature and overlaid with thiostrepton (1.5 ml of 14 µg/ml thiostrepton per 150 mm diameter plate). Functional complementation was screened for after 23 days incubation at 30°C by overlay with thiostrepton-resistant Bacillus subtilis (this strain was specifically generated for this project by a standard UV mutagenesis approach). Two milliliters of molten SCM agar (Kim et al., 2002b) seeded with 108109 c.f.u. of B.subtilis were overlaid onto the transformants. After 12 days incubation at 30°C, clear zones of inhibition were produced. Three transformants giving rise to inhibition zones were isolated and their production of pikromycin confirmed again by the same bioassay. Quantitation of production levels by HPLC analyses was carried out as described previously (Kim et al., 2002b
). Five transformants that did not produce any detectable levels of pikromycin were also taken. The AT0-ACP0 DNA region of the pikAI complementation plasmids (Figure 5, P1P8, P27) from these nine transformants was PCR amplified (Van Dessel et al., 2003
) for sequence analysis.
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Results and discussion |
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The product of pikromycin biosynthesis in S.venezuelae is glycosylated and has significant bioactivity. Thus, working in the natural host permits rapid screening for production of pikromycin or related bioactive compounds. Previous work has demonstrated that a number of novel compounds generated in this strain from hybrid PKS systems, based primarily around the pikromycin PKS, are also glycosylated and biologically active (Yoon et al., 2002). The strain has rapid growth in both solid and liquid media, and is genetically well characterized (it is easily transformed). Finally, numerous mutants in which specific genes encoding the Pik PKS modules have been generated and pikromycin production shown to be readily restored by a complementation plasmid (Kim et al., 2002a
; Yoon et al., 2002
). In other well studied PKS systems such as DEBS, genetic manipulation of the host organism presents many technical difficulties (Katz, 1997
) leading to most analyses being carried out in a heterologous host (Cortes et al., 1995
; Kao et al., 1995
; Katz, 1997
; Kinshota et al., 2001
; Pfeifer et al., 2001
; Reeves et al., 2001
; Wu et al., 2002
). In this case, the work is often carried out with just the PKS component (or portion thereof) and does not result in production of a bioactive compound.
Replacement of the AT0-ACP0 didomain of pikromycin with the corresponding region of DEBS1 generates an inactive PikAI
A two-step approach to changing the loading domain of the PikA1 was taken. In the first step we generated a pikAI complementation plasmid pBK77, encoding a PikAI mutant in which the conserved glutamine residue was replaced by alanine. Evidence has shown that glutamine residues are important for the decarboxylase activity of KSQ domains (Bisang et al., 1999; Witkowski et al., 1999
; Long et al., 2002
). It was expected that in this PikAI mutant the AT0 would prime with methylmalonyl-CoA but would be unable to generate efficiently the propionyl ACP required to initiate pikromycin biosynthesis (Figure 2A). Consistent with this expectation, the strain BB138/pBK77 produced <1% of the pikromycin levels made by BB138/pDHS722 and BB138/pBK76 (plasmids expressing the wild-type PikAI). Consistent and detectable low levels of pikromycin made by BB138/pBK77 (observed by bioassay and HPLC analyses of solid and liquid cultures, respectively) demonstrated that the plasmid-derived protein product was both expressed and soluble (no product was generated by BB138). Initiation of the low levels of pikromycin biosynthesis may result from a slower decarboxylation of methylmalonyl-CoA, or direct loading of propionyl-CoA. In the former case it is likely that the KSA mutant may retain some modest decarboxylase activity, particularly as it has been demonstrated that mutation of an active site cysteine to alanine of a KS component of a type II fatty acid synthase leads to a mutant with greater in vitro decarboxylase activity than the corresponding cysteine to glutamine mutant (Smirnova and Reynolds, 2001
). Production of low levels of pikromycin by PikAI containing a KSA loading domain differs from observations of an analogous mutation in a hybrid DEBS system containing a KSQ initiation mechanism, which resulted in a complete loss of detectable product formation (Long et al., 2002
). The reasons for these different observations are unknown.
In the second step of this process we attempted to recover the pikromycin production by the KSA-PikAI by constructing hybrid PKS in which the PikAI AT0-ACP0 didomain was replaced with the corresponding DEBS1 AT0-ACP0. As this DEBS1 loading didomain can load propionyl-CoA directly (Figure 2B), we anticipated that this switch could compensate for the glutamine to alanine mutation. Furthermore, as the DEBS1 loading didomain has a capacity to utilize acyl-CoA primers other than propionyl-CoA (Pieper et al., 1995; Pacey et al., 1998
; Lau et al., 2000
; Kim et al., 2002b
), we predicted that this change might provide an avenue for production of novel pikromycin-type compounds. However, BB138/pBK79 (expressing the hybrid PikAI) produced no detectable pikromycin. The sensitivity of our bioassay permits detection of pikromycin levels significantly <0.1% of that observed from our control (BB138/pDHS722). Thus, this hybrid PKS generates at least 10-fold less product than the PKS initiated by KSA-PikAI, is essentially not functional and represents a further example of an unsuccessful AT switch. It has previously been demonstrated that DEBS1 containing the entire loading domain and first two extension modules is functional in S.venezuelae under these growth conditions (triketide lactone products are observed), demonstrating that the appropriate precursors for AT0 of DEBS are available (Kim et al., 2002a
). Direct loading by propionyl-CoA would result in pikromycin production, ruling out concerns regarding specificity of subsequent steps. Thus, it is likely that in the context of the hybrid PikAI the DEBS1 AT0-ACP0 does not function.
Development of a method for efficient generation of a library of hybrid PikAI complementation plasmids
We reasoned that use of different fusion points or use of site-specific mutations may ultimately provide a functional hybrid PikAI. The standard approach of selecting the fusion point, generating the appropriate DNA fragment and introducing this into the appropriate expression host is a time-consuming process and has no predictable outcome. In fact several additional attempts to make functional hybrid PikAI molecules using different loading domains and fusion points proved unsuccessful (T.A.Cropp and K.A.Reynolds, unpublished results). We considered that recent methods (Stemmer, 1994; Ostermeier et al., 1999
; Lutz et al., 2001a
,b; Sieber et al., 2001
; Kawarasaki et al., 2003
) for directed evolution of chimeric proteins using two or more parents could be applied for directed evolution of a functional hybrid PikAI system. Thus, a library of hybrid pikAI complementation plasmids containing a wide range of different fusion points could be generated simultaneously. Screening of this library for restoration of pikromycin biosynthesis to BB138 would rapidly identify fusion points and chimeric proteins which are functional, offering a clear advantage over the current methods of generating hybrid PKS systems. BB138/pBK77 is an excellent model system for application of these techniques as the pBK77 complementation plasmid is readily transformed into the host strain and generates low levels of pikromycin. Most hybrid systems were expected to lose production while a small number were anticipated to make pikromycin. The bioassay using B.subtilis overlay as a reporter was very sensitive and the KSA mutation ensured wild-type levels of pikromycin (detected as an 1116 cm diameter zone of inhibition on a 2-day-old culture) could only be achieved if loading domain specificity changed from loading methylmalonyl-CoA to propionyl-CoA.
Nonetheless, application of molecular breeding technologies to type I PKS systems presents several technical challenges, including low overall homology of DNA encoding comparable domains from heterologous PKS, and the tremendous size of a polypeptide such as PikAI which harbors multiple catalytic domains. The DNA encoding the AT0-ACP0 region of DEBS1 and PikAI has only 53% sequence identity, relatively low compared to the genes used for other family shuffling studies in which 6090% identical genes were used (Crameri et al., 1998; Chang et al., 1999
; Ness et al., 1999
). This low sequence homology makes it difficult to reassemble the original length of the DNA fragment. Moreover, there is a high frequency of unshuffled parental sequences in the progeny (Arnold, 1998
; Kikuchi et al., 1999
; Moore et al., 2001
). To overcome these restrictions we designed hybrid or skewed primer sets for enrichment of the shuffled DNA from a temperature-gradient PCR method starting from a very low annealing temperature (Figures 3A and 4). The reassembled DNA was enriched with a skewed primer set which paired a forward primer of PikAI and DEBS1 AT0 sequence with reverse primer for the DEBS1 and PikAI ACP0 sequence, respectively. As predicted, PCRs using these skewed primer sets produced significantly less of the correct-sized DNA fragment (Figure 4, lanes 2 and 3) than a matched primer set (Figure 4, lane 1). Thus, the shuffled DNA fragments should exclude the parental sequences and contain a minimum of one crossover between the sequences encoding AT0-ACP0 of PikAI and DEBS1. To examine the diversity of this library of shuffled DNA fragments, the PCR product was cloned using a TA cloning kit and 18 randomly selected clones were sequenced. This analysis revealed 12 single crossovers, five triple crossovers, and one of quintuple crossover (P8P25 in Figure 5). In all 18 clones there was only one mutation, a consequence of the use of a high-fidelity polymerase. One of the crossovers occurred at a site with 5 bp of contiguous identity, while the rest occurred in regions containing at least 6 bp of contiguous sequence. As predicted, this family shuffling resulted in crossovers in conserved regions of the DNA encoding the PikAI and DEBS AT-ACP loading didomain. There are 52 separate regions containing 5 bp or more of contiguous identical sequence in these two DNA fragments. Thus, despite their low homology, this method created a diverse shuffled DNA library which explored an odd number of discrete crossovers along the entire length and was subsequently analyzed for ability to make a functional hybrid PikAI (Figure 3).
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The sacB gene and its native promoter were used as a positive selection marker and were cloned into the BamHI sites of pBK77 to give pBK84. The sacB gene from Bacillus spp. is a levan-sucrase, the presence of which is toxic to Gram-negative bacteria grown in the presence of sucrose (Reyrat et al., 1998; Golovliov et al., 2003
). We were able to demonstrate that under the appropriate conditions, E.coli harboring pBK84 has impaired growth on sucrose LB media, as compared to pBK77. Thus, the sacB product encoded by pBK84 was able to hinder the E.coli growth and was appropriate for use as a counter selectable marker. In the subsequent step, in vivo replacement of sacB in pBK84 by DNA fragments encoding the hybrid Pik-DEBS AT0-ACP0 could be readily detected by restoration of the ability to grow effectively on LB-sucrose (step B in Figure 3). The two skewed primer pairs (Figure 4A) used to enrich the shuffled DNA pool contained 39 nt of a 5'-targeting sequence tail homologous to the pikAI sequence encoding KSA (forward primers) and KS1 (reverse primers). This 39 bp region of DNA has been shown to be sufficient for
-Red-mediated recombination of linear DNA fragments with chromosomal or cosmid DNA in E.coli (Datsenko and Wanner, 2000
; Yu et al., 2000
; Gust et al., 2003
). The enriched DNA fragments were introduced by electroporation into E.coli BW25113/pIJ790 competent cells prepared from the LB culture, supplemented with 10 mM arabinose to induce the
-Red genes on pIJ790. The
-Red functions to promote a greatly enhanced rate of homologous recombination (Datsenko and Wanner, 2000
). A subsequent selection step on LB-sucrose produced a library of pBK84 derivatives. Restriction analysis of the pBK84 derivatives revealed no detectable sacB inserts. The gene replacement was highly efficient and was calculated to produce 27 500 clones containing the desired derivative from only 5 µg of the shuffled DNA fragment. Attempts to clone shuffled DNA fragments into the BamHI sites of pBK77 proved to be less efficient by several orders of magnitude. Equally important, the presence of sacB, once cloned into a region of DNA, can be used to carry out in vivo recombination at any flanking location by simply using the appropriate 39 nt of homologous sequence at either end of the linear DNA fragment. Thus, the in vivo recombination method is not only highly efficient, but also does not require specific restriction sites for insertion of either heterologous or shuffled DNA fragments. A plasmid such as pBK84 can also be used to replace the entire loading domain (including the region encoding KSA) and pick any fusion point after the 5' region of the KS1 domain sequence.
DNA family shuffling provides activity hybrid PikAI molecules containing regions of the DEBS1 AT0-ACP0
The hybrid pikAI complementation plasmids derived from pBK84 were pooled and used to transform S.venezuelae BB138. A bioassay using a thiostrepton-resistant B.subtilis revealed that three (2.1 cm inhibition zones) out of 4400 S.venezuelae colonies (P1, P2 and P27) produced pikromycin. These three transformants produced comparable levels of pikromycin to that observed with BB138/pBK77 (the KSA complementation plasmid) in SCM liquid culture. The remaining transformants, like BB138/pBK79, produced no detectable level of pikromycin, suggesting that the majority of the hybrid PikAI proteins are not functional.
DNA sequencing of P1 and P2 (Figure 5) revealed that these shuffled DNA fragments were generated from the skewed PCR primer pair containing the forward eryAI primer. These two functional hybrids had a single crossover to the PikAI sequence at the 5'-end and resulted in hybrids containing either seven or 14 amino acids encoded by the eryAI sequence. As this DNA region is reasonably conserved between the two sequences this insertion of a small region of eryAI led to only three or four amino acid mutations of KSA-PikAI. The third functional plasmid, P27, was derived from the opposite skewed primer set and contained almost exclusively the entire pikAI AT0-ACP0 region, with just four amino acid substitutions in the beginning of the KS1 domain. The three complementation plasmids, P1, P2 and P27, all produced significantly higher levels of pikromycin than the other hybrid PikAI complementation plasmids, including pBK79. Thus, the use of DNA family shuffling, in vivo recombination with a counter selectable marker, and screening for pikromycin production led to the identification of hybrid systems with activity. DNA sequencing of plasmids (Figure 5, P3P8) from five randomly chosen BB138 transformants that did not produce pikromycin revealed a mixture of hybrids containing either one or three crossovers along the entire DNA region. This analysis indicated that the genetic diversity obtained in the DNA family shuffling experiment was maintained in the subsequent genetic steps through to the final complementation experiment.
Despite this success, the functional PikAI hybrid molecules contain almost entirely the homologous AT0-ACP0 region and likely initiate pikromycin biosynthesis in the same manner as the KSA-PikAI mutant. Hybrids that contained more of the DEBS AT0 (e.g. P2P7) did not produce any detectable pikromycin. While this initial study did not provide a hybrid that efficiently primed by direct loading of propionyl-CoA, it allowed us to rapidly determine that the other fusion points explored in the remaining 4400 clones (which likely included those shown for P8P25) produce a non-functional hybrid PKS component.
There are numerous possible reasons why an efficient functional AT0-ACP0 exchange was not observed in this preliminary study. The use of eryAI primers in both skewed primer sets (essential for avoiding reconstituting the parental sequences) fixes one of the DEBS-PikAI fusion points and allows only fragments containing an odd number of crossovers to be obtained. Other limitations on the diversity of the library were that the crossovers were limited to areas of contiguous identical sequence. The presence of PikAI KSA may also have a negative structural mechanistic impact. As described above, pBK84 is an excellent host for replacing KSA, as well as inserting a more diverse library of chimeric DNA fragments obtained by sequence homology independent methods (Ostermeier et al., 1999; Lutz et al., 2001a
,b).
Concluding comments
Despite significant interest and efforts in the generation and analysis of hybrid PKS over the past decade, directed evolution using molecular breeding techniques, such as the DNA family, have not been reported, due in part to a number of technical issues. Current methods still rely on trial and error approaches with generation of single and, often, inactive hybrid systems based on selected fusion points. To the best of our knowledge this proof of principle study represents the first reported application of DNA shuffling on a type I PKS. The method implements an in vivo recombination method in E.coli using a sacB as a counter selectable marker, and allows rapid, efficient and precise integration of shuffled DNA into a PKS complementation plasmid. Transformation of the resulting library into the appropriate S.venezuelae host allows functional hybrid proteins to be rapidly identified. In the example used, the data have provided clear evidence (not unexpectedly) that most of the hybrid systems generated are not functional. The approach developed in this work is suitably flexible to allow molecular breeding techniques to be applied to make additional and functional changes to PikAI and other components of the PikPKS. In principle, the techniques can be applied to other modular type I PKS systems.
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Acknowledgements |
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Received January 6, 2004; revised March 18, 2004; accepted March 24, 2004 Edited by Frances Arnold