Alternative method for site-directed mutagenesis of complex polyketide synthase in Streptomyces albus JA3453

Original Paper

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Acta Biochim Biophys

Sin 2008, 40: 319-326

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‘-ACGTCGAGGCGGCCGTGG­TCGCCGGCGTCAC­CC­T­G­CTGCattccggggatccgtcgacc-3‘

(KS10-1-FP1) and 5‘-GT­CCCC­GCCGGTCGAAGAGGC­GGTGCCC­TCCCGCGT­C­GGtgtaggctggagctgcttc-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.

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‘-ACGTCGAGGCGGCCGTGG­TCGCCGGCGTCAC­CC­T­G­CTGCattccggggatccgtcgacc-3‘

(KS10-1-FP1) and 5‘-GT­CCCC­GCCGGTCGAAGAGGC­GGTGCCC­TCCCGCGT­C­GGtgtaggctggagctgcttc-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‘-AGTGC­GAG­GTC­G­C­CGTCGCGGGCG­GCGTCAACCTCTCGC­attccg­ggg­atccgtcgacc-3‘

(KS10-2-FP1) and 5‘-TGCCC­AC­G­G­C­T­CC­G­TAGGTG­CGGTACTTGCCC­GGGTGCAtgt­aggct­gga­gc­tg­cttc-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‘-TCCACT­GCG­T­TT­C­TGCGTGCTGTTC-3‘

(KS10-1-FP2) and 5‘-GCCG­TGG­A­AGTCGCAGACGAA 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‘-GGTTACGCCCCCGACG­AG­CTGAAGG-3‘

(KS10-2-FP2) and 5‘-GACGGCCTTGACC­A­G­T­CGCAGCAG-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‘-GGTCGTGGACACCGCC­­­­­­G­G­ATCCTCCGCGCTCGTGGCCCT-3‘

(KS10-1-FP3) and 5‘-AGGGCCACGAGCGCGGAGG­ATCCGG­CGGT­GT­C­C­ACGACC-3‘

(KS10-1-RP3); and for KS10-2, 5‘-GAC­G­G­T­G­GACACCCTG­GGATCCTCCTCGCTCACCGCGC-3‘

(KS10-2-FP3) and 5‘-GCGCGGTGAGCGAGGAGGA­­­­T­C­C­C­AGGGTGTCCACCGTC-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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