An efficient method for creation and functional analysis of libraries of hybrid type I polyketide synthases

Beom Seok Kim1,2, David H. Sherman3 and Kevin A. Reynolds1,2,4

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial type I polyketide synthases (PKSs) generate a structurally diverse group of natural products with a wide range of biological activities. Hybrid type I PKSs in which domains of one multifunctional polypeptide are replaced with components from heterologous systems have generated significant interest over the past decade. Almost invariably only one or several specific hybrids are made at a time and tested for functionality. This approach is slow, dependent upon a fortuitous choice of specific fusions points, and often leads to inactive or minimally active hybrid systems. We describe herein a method for generating and screening a library of hybrid pikAI complementation plasmids (encoding the loading domain and the first two extension domains of pikromycin PKS) able to restore pikromycin in a BB138 Streptomyces venezuelae pikAI-deletion mutant. In the first step the plasmid sequence encoding the loading domain AT0-ACP0 was replaced by a counter selectable marker, sacB. DNA family shuffling was then used to generate a diverse library of chimeric AT0-ACP0 fragments, which were used to replace sacB by {lambda}-Red-mediated in vivo recombination in an Escherichia coli host. This method resulted in the rapid and efficient generation of a large number of hybrid pikAI complementation plasmids, which were used to transform S.venezuelae BB138. A bioassay of over 4000 of these transformants successfully revealed three different PikAI hybrids which were able to lead to pikromycin production. The study suggests that most of the hybrids are not detectably functional, and underscores the need to generate and screen large and diverse libraries in which different fusion points are tried. The methodologies applied in this study address this need and can be used for directed evolution of any component of the PikPKS, and potentially other type I PKS systems.

Keywords: acyl-carrier protein/acyltransferase/erythromycin/hybrid PKS/pikromycin/typeI polyketide synthase


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Polyketides represent a large class of structurally diverse natural products that find widespread use as therapeutic and agricultural agents (Hopwood, 1997Go). The biosynthesis of the polyketides proceeds in a manner similar to that of fatty acids and involves head-to-tail condensation of acyl thioester units (typically malonyl, methylmalonyl and ethylmalonyl). Numerous types of polyketide synthases (PKSs) responsible for catalyzing these processes have been identified over the past 30 years. The type I modular PKSs identified in 1990 are large multifunctional enzymes containing discrete sets of modules or enzymes (Cortes et al., 1990Go; Donadio et al., 1991Go). Typically each module is responsible for catalyzing one round of elongation (Figure 1). An acyltransferase (AT) domain typically housed within each module is responsible for selective priming a cognate acyl carrier protein (ACP) with the acyl-derived extender unit. A ketosynthase domain (KS) within the module then catalyzes a Claisen-type condensation with this extender unit and a cysteine-bound growing acyl chain. Additional catalytic domains within the module determine the processing of the 3-keto group of the resulting product, which occurs before the next module catalyzes a subsequent extension step. In many cases a distinct loading domain responsible for priming the PKS with a specific starter unit is also present.



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 1. The pikromycin polyketide synthase and is role in generating pikromycin. Catalytic domains within each polypeptide are represented as circles. KS, ketosynthase; ACP, acyl carrier protein; KR, ketoreductase; DH, dehydratase; ER, enoyl reductase; TE, thioesterase.

 
A large number of modular type I PKSs have been cloned and sequenced since the discovery of 6-deoxyerythronolide B (6dEB) synthase (DEBS). These analyses have revealed that these systems utilize changes in substrate specificity and type of catalytic domains within a module, as well as the number of modules, to build tremendous structural diversity into their respective products. These findings also provided the inspiration and the genetic tools for creating hybrid type I PKSs, and numerous such systems have been successfully reported.

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., 1997Go). This work was first successfully reported for modules 1 and 2 by workers at Abbott Laboratories (Ruan et al., 1997Go). Independent work at Kosan Biosciences led to extension of this work to include modules 3, 5 and 6 (McDaniel et al., 1999Go), but not module 4 (Reeves et al., 2001Go). A successful switch at module 4 has been accomplished recently by a third and independent group of investigators from Biotica Technology (Petkovic et al., 2003Go). 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., 2001Go). Despite these and other successes (Stassi et al., 1998Go), many of the AT switches yielded hybrid systems that produce little or no product (Reeves et al., 2001Go). 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., 2000Go; Yoon et al., 2002Go).

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., 1998Go). Subsequently, the loading domain of DEBS1 was replaced with that from the monensin PKS (Bisang et al., 1999Go). 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., 1998Go; Bisang et al., 1999Go). 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., 1999Go; Witkowski et al., 1999Go). Attempts to use site-specific mutations to switch AT0 specificity between mono- and dicarboxylated acyl-CoA primers have not been successful (Long et al., 2002Go).



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2. Different priming mechanisms of the pikromycin polyketide synthase (Pik PKS) and 6-deoxyerythronolide B synthase (DEBS). (A) PikPKS initiates the polyketide synthesis by priming with methylmalonyl-CoA followed by decarboxylation by KSQ domain. (B) DEBS initiates 6-deoxyerythronolide B synthesis by priming directly with propionyl-CoA.

 
Hybrid type I PKSs which incorporate other changes, including insertion and deletion of other domains or even whole modules, have also been reported (McDaniel et al., 1997Go, 1999Go; Rowe et al., 2001Go; Yoon et al., 2002Go). In one case this approach using the DEBS system has been reported to provide a library of over 50 new macrolide structures (McDaniel et al., 1999Go). In the majority of the cases, however, the hybrid system made either significantly reduced levels of the new product, or no detectable product. To the best of our knowledge, all of these studies typically involve the generation and analysis of a small number of hybrid systems in which a specific fusion point or set of mutations are made. If the hybrid system does not appear functional then different fusion points, heterologous domains or a set of site-specific mutations are made, and the analyses repeated. This process is slow, and involves significant technical in vitro work with large fragments of DNA. Thus, it has taken a considerable amount of time and effort by three independent groups to complete the set of malonyl AT domain swaps in DEBS (Ruan et al., 1997Go; McDaniel et al., 1999Go; Reeves et al., 2001Go; Petkovic et al., 2003Go.

Numerous genetic techniques, including family shuffling, SCRATCHY, ITCHY and SHIPREC, have been successfully used for generating highly diversified mutant libraries (Stemmer, 1994Go; Ostermeier et al., 1999Go; Lutz et al., 2001aGo,b; Sieber et al., 2001Go; Kawarasaki et al., 2003Go). 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Reagents and standard procedures

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 manufacturer’s instructions. All other chemicals were purchased from Sigma and were of the highest available grade. Standard molecular cloning techniques were employed (Sambrook et al., 1989Go). 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., 2002Go) was used for expression of hybrid PikAI proteins and was transformed by the standard procedures (Kieser et al., 2000Go).

Generation of mutant (pBK77) and hybrid PikAI (pBK79) complementation plasmids

The plasmid pDHS722 is a pikAI complementation plasmid (Yoon et al., 2002Go), 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 EcoRI–BamHI (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 EcoRI–BamHI 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., 2002aGo). 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., 1998Go).

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, 1995Go) 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 50–300 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., 2003Go). 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 P8–P25 in Figure 5).



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 5. Depiction of shuffled DNA generated from sequence encoding AT0-ACP0 of PikAI and DEBS1. Red and purple represent the regions which encode the AT and ACP domains, respectively. Green bars and red bars represent the pikAI and eryA sequence, respectively. P1, P2 and P27 were from plasmids expressing a functional hybrid loading domain that produced pikromycin in S.venezuelae BB138. P3, P4, P5, P6 and P7 were from five separate plasmids expressing hybrid loading domains which made no detectable levels of pikromycin. P8–P25 were obtained from 17 separate E.coli transformants containing the initial family shuffled DNA.

 
In vivo recombination of shuffled DNA

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., 2003Go), which expresses the {lambda}-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 (4–6 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 5–7 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 16–20 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 2–3 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., 2002bGo) seeded with 108–109 c.f.u. of B.subtilis were overlaid onto the transformants. After 1–2 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., 2002bGo). 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, P1–P8, P27) from these nine transformants was PCR amplified (Van Dessel et al., 2003Go) for sequence analysis.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Selection of S.venezuelae and pikromycin biosynthesis as a system for directed evolution of type I PKSs

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., 2002Go). 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., 2002aGo; Yoon et al., 2002Go). In other well studied PKS systems such as DEBS, genetic manipulation of the host organism presents many technical difficulties (Katz, 1997Go) leading to most analyses being carried out in a heterologous host (Cortes et al., 1995Go; Kao et al., 1995Go; Katz, 1997Go; Kinshota et al., 2001Go; Pfeifer et al., 2001Go; Reeves et al., 2001Go; Wu et al., 2002Go). 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., 1999Go; Witkowski et al., 1999Go; Long et al., 2002Go). 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, 2001Go). 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., 2002Go). 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., 1995Go; Pacey et al., 1998Go; Lau et al., 2000Go; Kim et al., 2002bGo), 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., 2002aGo). 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, 1994Go; Ostermeier et al., 1999Go; Lutz et al., 2001aGo,b; Sieber et al., 2001Go; Kawarasaki et al., 2003Go) 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 11–16 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 60–90% identical genes were used (Crameri et al., 1998Go; Chang et al., 1999Go; Ness et al., 1999Go). 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, 1998Go; Kikuchi et al., 1999Go; Moore et al., 2001Go). 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 (P8–P25 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).



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 3. Schematic overview of the application of {lambda}-Red-mediated in vivo recombination for efficient generation of a library of hybrid pikAI complementation plasmids. (A) Family shuffling with enrichment of shuffled constructs using skewed hybrid primer sets. The 5' region of primers (shown in green) contained 39 nt of the KSA and KS1 pikAI homologous sequence. (B{lambda}-Red-mediated in vivo recombination of recipient plasmid pBK84 with selection for E.coli colonies able to grow in LB media with 5% sucrose. (C) Library of hybrid pikAI complementation plasmids from pooled E.coli colonies are transformed into S.venezuelae BB138 and screened for pikromycin production. Specific details and additional explanations are provided in the text.

 


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4. Reassembly and enrichment of fragmented DNA encoding AT0-ACP0 of PikAI and DEBS. The first and second stage cycles each comprised 21 cycles (see text for details). In the enrichment step (using 2 µl of the PCR product) different primer sets were used: lane 1, homologous primers; lane 2, pikAI forward and eryA1 reverse primers, lane 3, eryA1 forward and pikAI reverse primers (see Figure 3A for a schematic).

 
Additional challenges in the generation of hybrid type I PKS lie in the incorporation of a DNA fragment into the appropriate position of the DNA encoding a multidomain PKS. The current methods used for combinatorial biosynthesis of natural product systems generally require identification or creation of appropriate restriction sites, and the judicious use of in vitro methods of restriction and ligation. These procedures are time consuming. In addition, the in vitro cloning efficiency using a large vector such as the 28 kb pBK77 and DNA fragment is low. While this does not significantly affect the generation of a specific hybrid PKS clone (such as pBK79), it presented a significant barrier to generating a large (>3000) and thus diverse hybrid PKS library from a pool of DNA fragments. To enhance the cloning efficiency of shuffled DNA, we combined the in vivo recombination method with a positive selection using a counter selectable marker (Figure 3).

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., 1998Go; Golovliov et al., 2003Go). 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 {lambda}-Red-mediated recombination of linear DNA fragments with chromosomal or cosmid DNA in E.coli (Datsenko and Wanner, 2000Go; Yu et al., 2000Go; Gust et al., 2003Go). 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 {lambda}-Red genes on pIJ790. The {lambda}-Red functions to promote a greatly enhanced rate of homologous recombination (Datsenko and Wanner, 2000Go). 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, P3–P8) 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. P2–P7) 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 P8–P25) 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., 1999Go; Lutz et al., 2001aGo,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.


    Acknowledgements
 
We are grateful to T.Ashton Cropp for generation of the thiostrepton-resistant B.subtilis. This work was supported by an NIH grant (GM48562).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Arnold,F.H. (1998) Nat. Biotechnol., 16, 617–618.[ISI][Medline]

Bisang,C., Long,P.F., Cortes,J., Westcott,J., Crosby,J., Matharu,A.-L., Simpson,T.J. and Leadley,P.F. (1999) Nature, 401, 502–505.[CrossRef][ISI][Medline]

Chang,C.C., Chen,T.T., Cox,B.W., Dawes,G.N., Stemmer,W.P., Punnonen,J. and Patten,P.A. (1999) Nat. Biotechnol., 17, 793–797.[CrossRef][ISI][Medline]

Chen,S., Xue,Y., Sherman,D.H. and Reynolds,K.A. (2000) Chem. Biol., 7, 907–918.[CrossRef][ISI][Medline]

Cortes,J., Haydock,S.F., Roberts,G.A., Bevitt,D.J. and Leadley,P.F. (1990) Nature, 348, 176–178.[CrossRef][ISI][Medline]

Cortes,J., Wiesmann,K.E.H., Roberts,G.A., Brown,M.J., Staunton,J. and Leadley,P.F. (1995) Science, 268, 1487–1489.[ISI][Medline]

Crameri,A., Raillard,S.A., Bermudez,E. and Stemmer,W.P. (1998) Nature, 391, 288–291.[CrossRef][ISI][Medline]

Datsenko,K.A. and Wanner,B.L. (2000) Proc. Natl Acad. Sci. USA., 97, 6640–6645.[Abstract/Free Full Text]

Donadio,S., Staver,M.J., McAlpine,J.B., Swanson,S.J. and Katz,L. (1991) Science, 252, 675–679.[ISI][Medline]

Golovliov,I., Sjostedt,A., Mokrievich,A. and Pavlov,V. (2003) FEMS Microbiol. Lett., 222, 273–280.[ISI][Medline]

Gust,B., Challis,G.L., Fowler,K., Kieser,T. and Chater,K.F. (2003) Proc. Natl Acad. Sci. USA, 100, 1541–1546.[Abstract/Free Full Text]

Hopwood,D.A. (1997) Chem. Rev., 97, 2465–2497.[CrossRef][ISI][Medline]

Kao,C.M., Luo,G., Katz,L., Cane,D.E. and Khosla,C. (1995) J. Am. Chem. Soc., 117, 9105–9106.[ISI]

Katz,L. (1997) Chem. Rev., 97, 2557–2575.[CrossRef][ISI][Medline]

Kawarasaki,Y., Griswold,K.E., Stevenson,J.D., Selzer,T., Benkovic,S.J., Iverson,B.L. and Georgiou,G. (2003) Nucleic Acids Res., 31, E126.[CrossRef][Medline]

Kieser,T., Bibb,M.J., Buttner,M.J., Chater,K.F. and Hopwood,D.A. (2000) Practical Streptomyces Genetics. The John Innes Foundation, Norwich.

Kikuchi,M., Ohnishi,K. and Harayama,S. (1999) Gene, 236, 159–167.[CrossRef][ISI][Medline]

Kim,B.S., Cropp,T.A., Beck,B.J., Sherman,D.H. and Reynolds,K.A. (2002a) J. Biol. Chem., 277, 48028–48034.[Abstract/Free Full Text]

Kim,B.S., Cropp,T.A., Florova,G., Lindsay,Y., Sherman,D.H. and Reynolds,K.A. (2002b) Biochemistry, 41, 10827–10833.[CrossRef][ISI][Medline]

Kinshota,K., Willard,P.G., Khosla,C. and Cane,D.E. (2001) J. Am. Chem. Soc., 123, 2495–2502.[CrossRef][ISI][Medline]

Lau,J., Cane,D.E. and Khosla,C. (2000) Biochemistry, 39, 10514–10520.[CrossRef][ISI][Medline]

Long,P.F. et al. (2002) Mol. Microbiol., 43, 1215–1225.[CrossRef][ISI][Medline]

Lutz,S., Ostermeier,M. and Benkovic,S.J. (2001a) Nucleic Acids Res., 29, E16.[Medline]

Lutz,S., Ostermeier,M., Moore,G.L., Maranas,C.D. and Benkovic,S.J. (2001b) Proc. Natl Acad. Sci. USA, 98, 11248–11253.[Abstract/Free Full Text]

Marsden,A.F.A., Wilkinson,B., Cortes,J., Dunster,N.J., Staunton,J. and Leadley,P.F. (1998) Science, 279, 199–202.[Abstract/Free Full Text]

McDaniel,R., Kao,C.M., Fu,H., Hevezi,P., Gustafsson,C., Betlach,M., Ashley,G., Cane,D.E. and Khosla,C. (1997) J. Am. Chem. Soc., 119, 4309–4310.[CrossRef][ISI]

McDaniel,R., Thamchaipenet,A., Gustafsson,C., Fu,H., Betlach,M. and Ashley,G. (1999) Proc. Natl Acad. Sci. USA, 96, 1846–1851.[Abstract/Free Full Text]

Moore,G.L., Maranas,C.D., Lutz,S. and Benkovic,S.J. (2001) Proc. Natl Acad. Sci. USA., 98, 3226–3231.[Abstract/Free Full Text]

Ness,J.E., Welch,M., Giver,L., Bueno,M., Cherry,J.R., Borchert,T.V., Stemmer,W.P. and Minshull,J. (1999) Nat. Biotechnol., 17, 893–896.[CrossRef][ISI][Medline]

Ostermeier,M., Shim,J.H. and Benkovic,S.J. (1999) Nat. Biotechnol., 17, 1205–1209.[CrossRef][ISI][Medline]

Pacey,M.S. et al. (1998) J. Antibiot. (Tokyo), 51, 1029–1034.[ISI][Medline]

Petkovic,H., Lill,R.E., Sheridan,R.M., Wilkinson,B., McCormick,E.L., McArthur,H.A., Staunton,J., Leadlay,P.F. and Kendrew,S.G. (2003) J. Antibiot. (Tokyo). 56, 543–551.[ISI][Medline]

Pfeifer,B.A., Admiraal,S.J., Gramajo,H., Cane,D.E. and Khosla,C. (2001) Science, 291, 1790–1792.[Abstract/Free Full Text]

Pieper,R., Luo,G., Cane,D.E. and Khosla,C. (1995) J. Am. Chem. Soc., 117, 11373–11374.[ISI]

Reeves,C.D., Murli,S., Ashley,G.W., Piagentini,M., Hutchinson,C.R. and McDaniel,R. (2001) Biochemistry, 40, 15464–15470.[CrossRef][ISI][Medline]

Reyrat,J.M., Pelicic,V., Gicquel,B. and Rappuoli,R. (1998) Infect. Immun., 66, 4011–4017.[Free Full Text]

Rowe,C.J. et al. (2001) Chem. Biol., 8, 475–485.[CrossRef][ISI][Medline]

Ruan,X. et al. (1997) J. Bacteriol., 179, 6416–6425.[Abstract]

Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Schweizer,H.P. and Hoang,T.T. (1995) Gene, 158, 15–22.[CrossRef][ISI][Medline]

Sieber,V., Martinez,C.A. and Arnold,F.H. (2001) Nat. Biotechnol., 19, 456–460.[CrossRef][ISI][Medline]

Smirnova,N. and Reynolds,K.A. (2001) J. Bacteriol., 183, 2335–2342.[Abstract/Free Full Text]

Stassi,D.L. et al. (1998) Proc. Natl Acad. Sci. USA, 95, 7305–7309.[Abstract/Free Full Text]

Stemmer,W.P. (1994) Proc. Natl Acad. Sci. USA, 91, 10747–10751.[Abstract/Free Full Text]

Van Dessel,W., Van Mellaert,L., Geukens,N. and Anne,J. (2003) J. Microbiol. Methods, 53, 401–403.[CrossRef][ISI][Medline]

Witkowski,A., Joshi,A.K., Lindqvist,Y. and Smith,S. (1999) Biochemistry, 38, 11643–11650.[CrossRef][ISI][Medline]

Wu,N., Cane,D.E. and Khosla,C. (2002) Biochemistry, 41, 5056–5066.[CrossRef][ISI][Medline]

Xue,Y., Zhao,L.,H.-W.Liu and Sherman,D.H. (1998) Proc. Natl Acad. Sci. USA, 95, 12111–12116.[Abstract/Free Full Text]

Yoon,Y.J., Beck,B.J., Kim,B.S., Kang,H.Y., Reynolds,K.A. and Sherman,D.H. (2002) Chem. Biol., 9, 203–214.[CrossRef][ISI][Medline]

Yu,D., Ellis,H.M., Lee,E.C., Jenkins,N.A., Copeland,N.G. and Court,D.L. (2000) Proc. Natl Acad. Sci. USA, 97, 5978–5983.[Abstract/Free Full Text]

Received January 6, 2004; revised March 18, 2004; accepted March 24, 2004 Edited by Frances Arnold