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Research Article

Engineered Streptomyces avermitilis Host for Heterologous
Expression of Biosynthetic Gene Cluster for Secondary Metabolites
Mamoru Komatsu,† Kyoko Komatsu,† Hanae Koiwai,† Yuuki Yamada,† Ikuko Kozone,§ Miho Izumikawa,§
Junko Hashimoto,§ Motoki Takagi,§ Satoshi Omura,‡ Kazuo Shin-ya,⊥ David E. Cane,¶
and Haruo Ikeda*,†

Kitasato Institute for Life Sciences, Kitasato University, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0373, Japan
Japan Biological Informatics Consortium, 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan

Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan

National Institute of Advanced Industrial Science and Technology, 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan

Department of Chemistry, Box H, Brown University, Providence, Rhode Island 02912-9108, United States
S Supporting Information

ABSTRACT: An industrial microorganism, Streptomyces
avermitilis, which is a producer of anthelmintic macrocyclic
lactones, avermectins, has been constructed as a versatile
model host for heterologous expression of genes encoding
secondary metabolite biosynthesis. Twenty of the entire
biosynthetic gene clusters for secondary metabolites were
successively cloned and introduced into a versatile model host
S. avermitilis SUKA17 or 22. Almost all S. avermitilis
transformants carrying the entire gene cluster produced
metabolites as a result of the expression of biosynthetic gene
clusters introduced. A few transformants were unable to
produce metabolites, but their production was restored by the
expression of biosynthetic genes using an alternative promoter
or the expression of a regulatory gene in the gene cluster that controls the expression of biosynthetic genes in the cluster using an
alternative promoter. Production of metabolites in some transformants of the versatile host was higher than that of the original
producers, and cryptic biosynthetic gene clusters in the original producer were also expressed in a versatile host.
KEYWORDS: secondary metabolism, heterologous expression, biosynthesis, Streptomyces, genome

A prominent property of members of the actinomycete
microorganisms belonging to the order Actinomycetales is
the ability to produce numerous secondary metabolites,
tases (NRPSs), bacteriocins, terpenoids, shikimate-derived
metabolites, aminoglycosides, and other natural products.
The identification and detailed characterization of secondary
including antibiotics and other biologically active compounds metabolic gene clusters have proved to be an invaluable tool for
of proven value in human and veterinary medicine or elucidation of the biosynthetic mechanism of secondary
agriculture or useful as biochemical tools. These structurally metabolites. Furthermore, the results show a rich potential
diverse metabolites possess not only antibacterial, antifungal, source of cryptic metabolites, which appear to be encoded by
antiviral, and antitumor activity but also antihypertensive and silent biosynthetic pathways. Genetically engineered biosyn-
immunosuppressant properties. Streptomyces are a rich source thetic gene clusters have in certain cases allowed the production
of pharmaceutical compounds in which common cellular of novel analogues by combinatorial biosynthesis or the
intermediates, including sugars, fatty acids, amino acids, and production of novel intermediates by specific in-frame
terpenes, are combined to give more complex structures by deletion(s), respectively. A critical requirement for these
defined biochemical pathways. In this century, genome analysis applications is the availability of the relevant biosynthetic
of Streptomyces microorganisms S. avermitilis MA 4680,4,5 S. gene clusters controlling the production of a secondary
bingchenggensis BCW-1,6 S. cattleya NRRL 8057,7 S. clavuligerus metabolite of interest as well as appropriate genetic systems
ATCC 27064,8 S. coelicolor A3(2),9 S. griseus IFO 13350,10 S. for the in vivo manipulation of the corresponding genes in
scabiei 87.22,11 and S. venezuelae ATCC 1071212 has revealed
that these microorganisms each have a large linear chromosome
Special Issue: Natural Products: Tools and More
that harbors over 20 secondary metabolic gene clusters
encoding the biosynthesis of polyketides by polyketide Received: September 30, 2012
synthases (PKSs), peptides by non-ribosomal peptide synthe- Published: January 10, 2013

© 2013 American Chemical Society 384 | ACS Synth. Biol. 2013, 2, 384−396

1 12.2 0.13 0.39 12. It microorganism has already proved to be a highly efficient has been proved that these chromosomes are linear.39 0.39 12.56 vphd 0.39 0. tsr.5e 1. vinaceus.56 0. which biochemical energy for the formation of biosynthesis of are generally from tens to hundreds of kilobases.39 0. In consideration of the above concept.5e 1. The sequence secondary metabolites.1 12. bald (bld) mutants were counted. avermitilis.5 3. hygroscopicus. avermitilis MA-4680 49 1/2675 (0.56 0.56 0.39 12. aac(3)IV.78 200 >100 0. streptomycin 3″-adenylyltransferase/spectinomycin 9-adenylyltransferase gene of E.13 This pioneer production of secondary metabolites.1 12.39 0.13 0.1 12. since all secondary metabolites are ultimately Not only loss of the development of morphological differ- derived from primary metabolic building blocks and require an entiation but also the marked reduction or loss of secondary adequate source of energy and reducing equivalents derived metabolite production has often been observed in many from primary metabolism such as ATP and NAD(P)H.1 1.56 ermEd <0. we have constructed a Streptomyces strains.563 nt in the wild-type chromosome. and yet their origins remain production of streptomycin. and feasibility of using genetically engineered S. dS.39 12.56 SUKA22c <0.5 3.1 25 6.5e 1.6) 908/2367 (38. cephamycin C.39 12.25 >100 0. coli. aad(3″). hph.15) S. 2013.1 12.56 aac(3)IVd 0. vph. coelicolor A3(2) 21.13 0. Furthermore. eMIC values were obtained from S. azureus.39 0.56 0.56 0.16−18 The genetic instability of producing model host for heterologous expression of biosynthetic gene microorganisms is a serious problem in the industrial clusters using engineered S. The timing and flux between primary and secondary cellular genetic instability of Streptomyces strains has been reported.1 12.595.15) S.56 aad(3″)d <0.5 6.56 hphd 0.39 0. heterologous hosts.1 25 3. The right side of the deletion region of SUKA22 (corresponding to 8.455− 1. Large-Deletion Mutants and Their Transformants Carrying Resistance Gene minimum inhibitory concentration (MIC. and pladienlide in unclear. respectively. ments (high temperature or hypertonic incubation). hygromycin B 7″-phosphotransferase gene of S. The complete genome the important anthelmintic agent avermectin14 and the sequences of some Streptomyces strains have been revealed. Biol.1 1. avermitilis Wild Type. avermitilis SUKA5 carrying ermE.39 0.2 0.78 25 12.2 0.1 3.1 1.1 1.925.56 a Spores (ca.5e 1.910 26/2138 (1.2 0.39 25 >200 3.43) 197/2781 (7.56 tsrd <0.39 12.5 3.56 0.25 0. however.2) 893/2013 (44. Genetic Instability of Genome-Sequenced Streptomycetaceae Microorganisms Containing Linear Chromosomes under High Temperature Growth appearance of bld mutantsa microorganism TIR (bp) 28 °C (%) 37 °C (%) S.5 6. 2. After each plate had been incubated at 28 or 37 °C for 6 days.1 12.1 1.414 nt in wild-type chromosome) was replaced by a mutant-type loxP sequence to prevent recombination between another wild-type loxP sequence on the left side of the deletion region corresponding to S.39 0.2 >100 12. aminoglycoside 3′-phosphotransferase gene of Tn5. 385 dx.5 SUKA5 <0.13 0. the morphology and secondary metabolite heterologous genetically engineered hosts.13 0. successful production of the desired products requires the optimum relationship of the ■ RESULTS AND DISCUSSION Properties of S. viomycin phosphotransferase gene of S.4) a About 200 spores of each microorganism were inoculated onto M4 (inorganic salts-starch agar) plate. Table 2.56 aph(3′)d 25 >200 0. In this paper. Mutants affecting morphological 20 biosynthetic gene clusters for exogenous secondary differentiation are sometimes generated in stressful environ- metabolites.148 54/2064 (2. avermitilis as a Versatile Host.56 SUKA17 <0. griseus IFO 13350 132.39 >200e 1. 104) were spotted onto YMS agar medium containing the designated concentration of antibiotic and grown at 30 °C for 3 days.15 metabolism. μg/mL)a neomycin ribostamycin apramycin streptomycin spectinomycin hygromycin B viomycinb thiostrepton lincomycinb lasalocide wild-type <0.13 0.ACS Synthetic Biology Research Article Table 1. we have shown the of TIRs appears to be divergent among different species.5 6.5 3.13 0.5 shorter heterologous host by the effective expression and production of than other Streptomyces TIRs. Antibiotic Susceptibility of S. avermitilis SUKA22 is isogenic to SUKA17. avermitilis is only 49-bp long.2 0. aminoglycoside 3-N-acetyltransferase type IV gene of E.56 0.56 0. rRNA adenosine-2′-O-methyltransferase gene of S.1 1.doi. and two producing microorganism of secondary metabolites. cS.653 9/2056 (0.2 0. avermitilis SUKA22 carrying aph(3′).5e 1.5 >200 0.2 0.1 1. and ermE.39 >200 1.25 0. Streptomyces efficient supply of primary metabolite precursors and chromosomes contain terminal inverted repeats (TIRs).5e 1.1 12. avermitilis SUKA5 production of S.39 0. coli. rRNA aminoadenine-6-N-methyltransferase gene of Saccharomyces erythraea).025−8.1 1. b Viomycin and lincomycin replaced tuberactinomycin N and erythromycin.39 >200 1. it seems telomeres capped by covalently bound terminal proteins are that the microorganism has been already optimized for the located at both ends of the linear chromosome. These phenomena have heterologous expression system has been confirmed by the been well documented for years. avermitilis is used for industrial production of with other Streptomyces strains.1021/sb3001003 | ACS Synth.2 0.2 0. avermitilis is somewhat stable in comparison and 17.25 0.5 1. Since S.4) Kitasatospora setae KM-6054 127. avermitilis as a the length of TIRs in S. 384−396 .03) 4/2567 (0.886.

9 generated bald mutants at the frequency of 0. S.4%. trans- formants or exoconjugants generated by transformation or conjugation are selected by antibiotic resistance conferred by a vector marker. aac(3)IV.7-fold that of the wild-type strain (Figure 2). Large-deletion derivatives could containing ribostamycin was extremely low. Another compared with the wild-type strain. viomycin or tuberactinomycin N resistance). coelicolor A3(2). the targets of which are different from that of examined for the appearance of bald mutants. also showed a similar response between the wild- to form aerial hyphae and/or spores. Furthermore.15% in grew well on the sporulation medium and their morphological high temperature incubation (37 °C). Ribostamycin was suitable for the formed aerial hyphae and started sporulation after 3 days of selection of transformants carrying aph(3′) because the growth. Both Streptomycetaceae development on the medium was slightly faster than that of the microorganisms. raea. avermitilis were more stable than those of S. setae KM-6054. the compound from inside of the cells because helvolic acid and three Streptomyces and one related genus Kitasatospora. 2013. and K.doi. avermitilis derivatives. S. from the wild-type strain in liquid minimum medium (Figure thiostrepton resistance). these SUKA series derivatives were resistant to lincomycin/ In large-deletion derivatives of S. transformants carrying these genes responded to resistance to neomycin/ribostamycin. avermitilis chromosome included no resistant to ribostamycin. and it seemed that genetic stability might depend on the length of TIRs on the chromosome. large-deletion derivatives had the ability to grow faster isms or genetic recombination with S. avermitilis wild-type and its large-deletion mutants on (A) YMS medium at 30 °C for 3 days and on (B) MM coli. griseus IFO 13350 and K. all biosynthetic erythromycin. incubation (Table 1). in high temperature type and SUKA mutants. In studies of the regulation of the biosynthetic gene cluster or combinatorial biosynthesis to generate hybrid metabolites. vph (S. setae KM-6054 was markedly increased to 44. but the growth vinaceus. azureus. In these resistance grow on minimum medium without any supplements because genes. the wild-type strain 6054.4-Mb region from the differentiation in Streptomyces and has relatively long TIRs of 9.653 Interestingly. the appearance of bald mutants from S. The growth rates of large-deletion derivatives SUKA4. wild-type strain. which has been used Since all of the large-deletion derivatives of S. The growth rate on liquid four resistance genes derived from actinomycetes. which was replaced with the biosynthetic continued to grow until 44 h and their biomass contents gene cluster of neopentalenolactone. than the wild-type strain. respectively) generated more bald mutants than S. avermitilis. coelicolor A3(2). 384−396 . were fusidic acid.910 and 127. large-deletion derivatives of S. but all of the large-deletion derivatives fully sporulated background growth of nontransformant progenies in the plate and formed abundant spores. respectively. Since the wild-type strain. avermitilis (SUKA10− strain increased until 24 h.19 containing more than 100-kb TIRs (132. erythromycin or lincomycin resistance). and filipin.ACS Synthetic Biology Research Article Four genome-sequenced Streptomycetaceae microorganisms. each transformant carrying aph(3′) or essential genes (Figure 1B). S. avermitilis were more sensitive to a polyether compound. which are unable lasalocide. All of the large-deletion derivatives (SUKA1−17) under normal culture conditions. which elevated to 7.1021/sb3001003 | ACS Synth. by high temper- ature incubation. avermitilis was quite low. respectively. their growth rates were aac(3)IV were selected by neomycin and apramycin.148 bp. their growth characteristics were 21.43% examined. setae KM. were removed to avoid not only lasalocide. On the other hand. Interestingly. To evaluate the heterologous expression of genes concerning secondary metabolite biosynthesis. Since the development of aac(3)IV was able to be selected by neomycin or apramycin. characteristics of large-deletion derivatives obviously differed hygroscopicus. 5.20 hph (S.55 In general.54. morphological differentiation in large-deletion derivatives was respectively. Although the resistance complex analysis and separation of products derived from mechanism of lasalocide in the wild-type strain has not been heterologous expression of the biosynthetic gene cluster and examined.21 tsr (S. apramycin and streptomycin/specti- nomycin. 2). and agar medium supplemented with 1% glucose at 30 °C for 6 days. although transformants carrying aac(3)IV were also the regions deleted from S. the biomass of the wild-type some large-deletion derivatives of S. griseus IFO 13350.03-Mb linear chromosome. The three resistance genes derived from Escherichia Figure 1. gene clusters for endogenous major metabolites avermectin. a large DNA fragment containing the entire biosynthetic gene cluster is ligated with an appropriate vector and introduced into the host. avermitilis were as a model microorganism for studying morphological constructed to remove more than a 1. the frequency of bald mutants of S. the mechanism might be involved in the exclusion of endogenous major products but also precursor competition 386 dx. were well expressed.23 were also used and 17 until 24 h incubation were similar and were faster than as selective markers for transformation and conjugation. as a selection marker after were about 1. Thus. oligomycin.22 and ermE (Saccharopolyspora eryth. aph(3′). but large-deletion derivatives 17)13 contain ermE. and transformants carrying both aph(3′) and faster than that of the wild-type strain. and increase their biomass. complex medium was not clearly different. griseus IFO 1335010 and K. interspecific conjugation with other Streptomyces microorgan. hygromycin B resistance). S. The growth character- istics of S. and aad(3″). Biol. Growth of S.4% and 38. coelicolor A3(2). 2. avermitilis was sensitive to various antibiotics (Table 2). As shown in Figure 1A. more than two vectors carrying genes derived from different microorganisms are convenient for the construction.

To clone. A2b. B2a. B1a. competition between the precursors would occur in the cell. and SUKA17. Methanol extract from mycelium was directly applied to ODS-HPLC (HyperSil 3 μm. Wild-type S. These technologies are involved in short sequence- avermitilis produced a large amount of eight components of driven homologous recombination and site-specific recombina- avermectins (B2b. incubated at 28 °C with shaking (180 rpm). 4. bet.6 mL/min and detection at 246 nm. A2a. failed to produce technology could be applied to clone up to a ∼15-kb DNA Figure 3. This suggests that the deletion of biosynthetic gene 100-fold by TSB and incubated with shaking for 24 h at 30 °C. Spores were cultured on TSB at 30 °C for 48 h. Figure 2. respectively. Solid and dotted lines indicate the production profiles of the wild-type strain and aveR mutant. 2013. Vegetative culture was diluted to synthesis. Heterologous Expression of Biosynthetic Gene Clusters of Secondary Metabolites. and modify biosynthetic gene clusters in heterologous hosts. A 0. in liquid culture. which are building blocks of these compounds. therefore. coli recE/recT gene products25 aveR gene. avermitilis wild-type and its large-deletion These acyl-CoAs will be preferably used for oligomycin mutants. avermitilis wild-type strain and its aveR deletion mutant. A1b. was also detected in the mutant (Figure 3). The latter possibility was confirmed in the production of recent technology will be useful for manipulation of the gene endogenous products oligomycin and avermectin. the gene product of which activates the expression requires at least 40 homologous nucleotides to recombine.1% yeast extract and by heterologous expression of the biosynthetic gene cluster. and exo a very small amount of oligomcyin A.4 clusters of endogenous major secondary metabolites is mL portion of the culture was inoculated into 20 mL of MM liquid preferred for the synthesis of exogenous secondary metabolites medium supplemented with 1% glucose and 0. 2. B1b. Each strain was cultured in avermectin production medium for 168 h at 28 °C. HPLC profiles of mycelial extracts of S. oligomcyin productivity was markedly increased more than 50-fold that of the wild-type strain. Growth of S. malonyl-CoA and methylmalonyl-CoA are not consumed by avermectin synthesis because the gene product of aveR acts as an activator of the expression of genes involved in avermectin biosynthesis. SUKA5. between exogenous and endogenous biosynthetic pathways. Recombination by λ phage gam.doi. cluster. This of genes involving avermectin biosynthesis. 387 dx.ACS Synthetic Biology Research Article avermectins.6 ϕ × 100 mm) and developed with acetonitrile/ methanol/water (65:10:25) at a flow rate of 0. whereas deletion of the gene products (λ-Red)24 or E. express. and a minor component. almost all malonyl-CoA and methylmalonyl-CoA are consumed by avermectin synthesis and the supply of acyl-CoA for oligomycin synthesis is very low. 384−396 . Both avermectin and oligomycin are macrocyclic lactones synthesized from malonyl- CoA and methylmalonyl-CoA by each Biol. Since these two biosynthetic pathways share the same precursors. and A1a) and tion (Figure 4). In the aveR deletion mutant. SUKA4. However. oligomycin C (14-deoxyoligomycin A).1021/sb3001003 | ACS Synth.

a DNA segment containing a resistant marker clusters.doi. cephamycin C. 2. When more than a 15-kb DNA segment was 500-bp homologous regions corresponding to (MVA).595. or methylerythritol (MEP) pathways. already successful in the expression of biosynthetic gene Red homologous recombination.1021/sb3001003 | ACS Synth. ATCC 2129428 was cloned by a cosmid vector. and pladienolide.025−8. and a “Gibson’s assembling”. would be a powerful tool for the streptidine-containing aminoglycoside. has been produced by a S.563 nt in the wild-type chromosome.−1. shikimate and mevalonate flanking 125. The resultant sequence on the left side of the deletion region corresponding transformants produced ribostamycin in the culture broth.13 deletion derivatives. In vitro assembling of DNA fragments. λ-Red and Cre site-specific recombination26 was 1. avermitilis large. Genome analysis of S. avermitilis revealed that the gene and alternative promoter sequence was amplified by PCR microorganism harbored more than 30 biosynthetic gene using a pair of primers consisting of a 24-nt sequence of the 5′ clusters and many biosynthetic gene clusters for polyketide. a vector polyketide.ACS Synthetic Biology Research Article Figure 4. precursors that are building blocks of the metabolite. glycoside. ribostamycin (2) (Figure 5A). This recombination system clusters for streptomycin.13 The right side of the deletion region of aminoglycoside. ribosidif icus SUKA17 (corresponding to 8. and a 24-nt reversed sequence linear chromosome. peptide (amino acid). ure 5A). and shikimate-derived terminal region of the first gene in the gene operon. We have been upstream and downstream of the gene cluster was used for λ. 388 dx.414 nt in wild. avermitilis SUKA22 was The biosynthetic gene cluster for deoxystreptamine-containing isogenic to SUKA17. Aminoglyco- (Figure 4C). avermitilis large-deletion Heterologous expression of genes involved in secondary derivative carrying the gene cluster for streptomycin biosyn- metabolite biosynthesis was examined in S. and a ∼35-kb type chromosome) was replaced by a wild-type loxP sequence. thesis (Supplementary Figure S1-A) of S. and (D) construction of synthetic operon by Gibson’s method. griseus IFO 13350. The and their productivity was about 8 mg/L. Biol. segment from chromosomal DNA preparation and a vector secondary metabolites could be classified into at least five carrying two ∼41-bp homologous sequences corresponding to classes on the basis of the pathway for the generation of upstream and downstream of the gene cluster (Figure 4A). (C) introduction of alternative promoter by λ- Red recombination. and terpene compounds located in the upstream of the gene cluster. Procedures concerning manipulation of the gene cluster for secondary metabolite biosynthesis. S. segment containing the entire set of the gene cluster but SUKA22 was used as a mutant-type loxP sequence to (Supplementary Figure1-B) was subcloned into the integrating prevent recombination between another wild-type loxP vector pKU465cos13 to generate pRSM1.925. sugar. Expression of the Biosynthetic Gene Clusters for combined to construct an in-frame deletion in the gene operon Compounds Derived from the Sugar Pathway. streptomycin (1) (Fig- construction of an artificial gene operon. (B) construction of in-frame deletion in the gene cluster using λ-Red recombination.27 called side antibiotics are synthesized from the sugar pathway. In this for use as heterologous hosts for more biosynthetic gene insertion process. compounds were found. to 79. of S. end and ∼41-nt homologous sequence corresponding to non-ribosomal peptide. polyketide-peptide hybrid. No biosynthetic gene clusters for amino- of the 3′ end and a ∼41-nt homologous sequence of the N.13 could be also applied to insert and replace an alternative Large-deletion derivatives should be confirmed to be applicable promoter upstream of the gene operon (Figure 4B). (A) In vivo cloning by λ-Red recombination. SUKA17 or 22. In general.886. 384−396 . 2013.

AB360380) cyclitol. and abietatriene (25). Structures of secondary metabolites derived from the (A) sugar pathway: streptomycin (1). taxa-4. erythromycin A (8). than biosynthetic gene clustsers for other aminoglycoside cyclitol and two or more molecules of neutral or amino sugars antibiotic (about 20-kb). nemadectin α (10).ACS Synthetic Biology Research Article Figure holomycin (14). the size of the gene cluster was smaller produced about 5 mg/L kasugamycin in the culture broth. pentalenolactone (21). pladienolide B (7). kasugamycin (3). myo-inositol. Biol. levopimaradiene (24).4-diene (22). Transformants carrying the entire such as streptomycin and ribostamycin. aminoglycoside antibiotics consist of one molecule of amino. resistomycin (6). and clavulanic acid (16). (C) amino acid pathway: cephamycin C (13). and pholipomycin (4). aureothin (11). lactacystin (15). and leptomycin (12). bafilimycin B1 (9). and (E) MVA or MEP pathway: 2- methylisoborneol (20). amorpha-1.doi. (B) polyketide pathway: oxytetracycline (5). The 389 dx. ribostamycin (2). 2013. kasugaensis MB 273 (accession no.1021/sb3001003 | ACS Synth. (D) shikimate pathway: rebeccamycin (17). novobiocin (18). and chloramphenicol (19). Since kasugamycin (3) gene cluster for kasugamycin biosynthesis (Supplementary (Figure 5A) consists of one amino sugar and one neutral Figure1-C) of S. 2. 384−396 .11-diene (23).

respectively. 6-deoxyerythronolide B.34 was isolated. and the transformants carrying respectively. pKU465aurP2-A6.31 Macrocyclic lactones are generated by type-I PKSs cluster for leptomycin37 was estimated to be larger than 40 kb. and export of oxytetracycline was could transform a ∼100-kb BAC clone without detectable subcloned into the integrating vector.8 The 26-kb region had 17 protein-coding genes (Supplementary Figure1-Q) of Kitasatospora setae KM-605419 (Supplementary Figure1-D) that were highly homologous to was cloned into BAC vector. 17 genes in the 30-kb region of S. and nosokomycin. and the desired plasmid resultant BAC clone. avermitilis biosynthesis. The transformants produced 16 mg/L bafilomycin clavuligerus ATCC 27064 has not been reported to date. pKU503nemP11-L5. Expression of the Biosynthetic Gene Clusters for tetracycline (5) (Figure 5B) in the culture broth. and the desired deletions involved in the highly homologous region of each recombinant plasmid was introduced into S. nemadectin bacterial peptidoglycan synthesis at the transglycosylation stage. III PKSs. 2. and a BAC clone. transformants produced 1. A similar of a broad spectrum of Gram-positive pathogenic bacteria. carrying the entire Compounds Derived from the Polyketide Pathway. pKU503lepP5-L8. we have already shown the heterologous expression in S. the resultant transformants produced a large Cephamycin C (13) (Figure 5C). avermitilis SUKA22.32 A 25-kb region containing 22 genes involved in the the mycelium. did not produce pholipomycin in any compounds aureothin (11) and leptomycin (12) were cloned culture conditions. A cosmid clone (pOTC1) BAC vector. during several rounds of ATCC 27064. biosynthetic process for peptide bonds of peptide compounds resistomycin. was isolated. prasinomycin. gene cluster for macrocyclic lactone. 40 kb. and linear polyketides aureothin (11) and leptomycin Cloning of the gene cluster for moenomcyin biosynthesis in S.6 mg/L nemadectin α (10) and a and a very small amount of pholipomycin was detected from trace amount of related components in the mycelium. A ∼28-kb cloned segment was cyanogenus NRRL 15774 was cloned into BAC vector. to clone the entire 75-kb quebemycin. and the resultant BAC clone. the size and organization of genes in about a 26. Two the culture broth. clavuligerus the culture broth. peptide bonds are generated by non-ribosomal least 18 genes involved in resistomycin biosynthesis (Supple. and inhibit clusters for erythromycin (8). containing the entire (pPHM1) was introduced into S. simple modification of the amino residue of amino acid. clavuligerus ATCC avermectin. large-deletion mutants of S. We used an integrating BAC (bacterial artificial A number of phosphoglycolipid compounds. avermitilis The production of phosphoglycolipid compounds from S. EM52 was cloned by clinical field as an antibacterial agent. Anthelmintic desired region containing these 17 genes was cloned by λ-Red macrolide nemadectin α35 (10) is structurally related to recombination using a cosmid library of S. and and the productivity was about 360 mg/L. derived from three amino amount of resistomycin (6) (Figure 5B) in the culture broth. derived from amino acids is accomplished by at least three NA97. Since the biosynthetic gene bacteria. avermitilis SUKA22. pKU503eryP4-A14 produced about 4 mg/L erythromycin A in From bioinformatic analysis of genome data of S. MM 2336 produced 116 mg/L polyketide synthases (PKSs). quite similar to those of a 30-kb region of S. moenomycin. α (10). pKU503bafP3-P20 (pBFM1). and the organization of each gene in the gene 27064 and pRED vector13 carrying 500-bp homologous regions cluster was also quite similar to that in the avermectin gene cluster. rimosus NRRL resultant transformants produced 5 mg/L leptomycin (12) in 2234. pKU503. productivity was reduced. 384−396 . Thus. (Supplementary Figure1-P) of Sacchropolyspora erythraea kb and 5-kb regions contained 20 and 3 protein-coding genes. is a typical peptide compound generated by NRPS. acids.29 Interestingly. the entire gene cluster for discoid polyketide. and they accumulated kb region of mega-plasmid pSCL4 in this microorganism were the aglycone moiety of erythromycin. peptide synthetase (NRPS). pholipo. entire biosynthetic gene clusters for linear polyketide clavuligerus ATCC 27064. Oxytetracycline is widely used in the mentary Figure1-T) of Streptomyces sp. was introduced into S. ghanaenesis ATCC 14772. The B1 (9) and a trace amount of bafilomycin A1. the size of the gene encoding modular PKS is When kasT was expressed in the multicopy plasmid that gave quite large in comparison with genes encoding type-II or type- the desired gene-dosage effect or by the alternative promoter. resultant transformants produced ∼20 mg/L pholipomycin30 AB363939). Polyke. A BAC clone. ribosomal peptide synthesis. Expression of the Biosynthetic Gene Clusters for carrying a cosmid clone. Transformants produced about 20 mg/L oxy. Biol. self-resistance. in which 30. was introduced into S. avermitilis module in the biosynthetic gene cluster. type I.ACS Synthetic Biology Research Article expression of the kasugamycin biosynthetic genes was As modular PKS contains multicatalytic domains on a controlled by the gene product of regulatory gene kasT. A cosmid clone. Unfortunately. cyaneogriseus subsp.1021/sb3001003 | ACS Synth. The ligated with the integrating vector. biosynthesis33 was cloned from Streptomyces sp. and type III in aureothin (11) in the culture broth. 390 dx. propagation. The biosynthesis (Supplementary Figure1-E) of S. Interestingly. and mentary Figure1-F). chromosome) vector. containing contained a putative entire gene cluster for the oxytetracycline the entire 80-kb gene cluster for leptomycin. Interestingly. SUKA17. pladienolide B (7) (Figure mycin. 35-kb gene cluster for aureothin biosynthesis (Supplementary tide compounds are synthesized by at least three types of Figure1-S) of Streptomyces sp. technique was applied to clone the entire biosynthetic gene including multidrug resistant Staphylococcus aureus. pKU503eryP4-A14. ghanaensis ATCC The entire 72-kb gene cluster for bafilomycin B1 biosynthesis 14672. (12) (Figure 5B). 3. are required for the biosynthesis of moenomycin. and the entire biosynthetic gene clusters are at least productivity was improved (7 to 12 mg/L). avermitilis SUKA22. macarbomycin. non- and sclav_p1290. bafilomycin B1 (9). SUKA22. The 80-kb gene cluster for nemadectin biosynthesis (accession no. respectively. containing ghanaenesis ATCC 14672 was first reported for this group of the entire 60-kb gene cluster for erythromycin biosynthesis compounds. into cosmid and BAC vectors. which was mainly accumulated in mycelium. The second example. (modular PKS). Transformants 2. In the Compounds Derived from the Amino Acid Pathway. S.doi. diumycin. containing at methods. are potent inhibitors for the growth 5B). NRRL 2338. the original microorganism. two distinct clusters. polypeptide. type II. The gene cluster for nemadectin biosynthesis corresponding to upstream and downstream of sclav_p1274 (Supplementary Figure1-R) of S. and the (4) (Figure 5A). and aromatic compounds are produced by the entire biosynthetic gene cluster for leptomycin (Supple- type-II or type-III PKSs. biosynthesis13 (Supplementary Figure1-O). 2013. was introduced into S.

synthetase. avermitilis have region was amplified by PCR and the amplicon was introduced been deleted naturally during evolution.5-kb segment containing ccaR and its promoter volved in 2-methylisoborneol biosynthesis of S. Expression of the Biosynthetic Gene Clusters for cluster for holomycin biosynthesis (Supplementary Figure1-H) Compounds Derived from the Shikimate Pathway. Many pKU492novP-G18-1n. Biol. geosmin. Indolocarbazoles are potent inhibitors of produced 8 mg/L holomycin (14) (Figure 4C) and 2 mg/L protein kinases and the compounds were synthetized from two deacetylated derivative.42 a ∼1. carrying Lactacystine39 (15) (Figure 5C). clavuligerus ATCC 27064. the regions corresponding to upstream and downstream of biosynthetic process of which does not require activation of the sven_0913 and sven_0931. clavuligerus the culture broth. clavuligerus ATCC 27064 by λ-Red Compounds Derived from the MVA or MEP Pathway.44 and the size. produced 1 mg/ was introduced into the promoter region of the first gene in the L novobiocin (18) (Figure 5D) in the culture broth. organization and transcriptional biosynthetic gene cluster for lactacystin. clavuligerus ATCC 27064. caeruleus NCBI 11891 was the same gene cluster formed a polycistron. and the resultant Figure1-J) is located adjacent to the gene cluster for transformants produced abundant chloramphenicol (250 mg/ cephamycin C biosynthesis in S. which is an important intermediate in the shikimate downstream of the gene cluster. only by many actinomycete microorganisms but also by strated that the expression of the biosynthetic gene cluster for Cyanobacteria and Myxobacteria.13 Two other peptide compounds. which contained a minimum and tryptophan are generated from intermediates derived from set of the biosynthetic gene cluster for holomycin. lactacystinaeus OM. holothine. anulatus 3533-SV4 GM95. recombinant plasmid (pHLM1). The entire 18-kb gene 4. pCML1. 384−396 .43 in which 10 protein-coding genes are located. clavulanic acid was pAH91 by λ-Red recombination using 500-bp homologous generated from L-arginine and glyceraldehyde-3-phosphate. of S. The biosynthetic gene cluster for novobiocin of that activate the expression of genes involved in biosynthesis in S. A 25-kb region (sclav_4180−sclav_4197) was cloned from the 5. 12-kb (pLTC1). Since all genes in the direction of each gene of S. coelicolor A3(2) mutants acid (16) (Figure 5C). whereas clavulanic acid was not produced by ATCC 27064. holomycin transformants carrying the entire 25-kb biosynthetic gene (14). avermitilis SUKA22. are derived from 41-bp homologous sequences corresponding to upstream and chorismate.41 L) in the culture broth.38 in which 13 protein-coding genes including compounds derived from the shikimate genes are located. but not all transformants SV4 GM95 was cloned by a cosmid vector and a cosmid clone. belongs to the β-lactam compounds. anulatus 3533- SUKA22 using an integrating vector. rpsJ13) transformants carrying pKU492novP-G18-1n. was gene cluster for clavulanic acid biosynthesis (Supplementary introduced into S. (Supplementary Figure1-K) of Lechevalieria aerocolonigenes PKS hybrid. lactacystinaeus OM-6519 by λ-Red recombination using novobiocin (18) and chloramphenicol (19). camphor-like odor is the (sclav_4204) of the gene cluster for cephamycin C biosyn. produced lactacystin in both mycelium and culture broth. The genes in- thesis. and the trans- 6519 suggested that the biosynthetic gene cluster for lactacystin formants produced 7 mg/L rebeccamycin (17) (Figure 5D) in was ∼12-kb. was thought to be synthesized by the NRPS. carrying 19 protein-coding genes. Aromatic amino acids phenylalanine. avermitilis biosynthesis (Supplementary Figure1-L) of S. pREB1. was cloned from the cosmid library of S. was introduced into S. avermitilis genome. molecules of L-tryptophan. avermitilis SUKA22 promoter of the gene encoding ribosomal protein an alternative promoter (a as that of S. The entire 29-kb gene cluster for novobiocin cluster. in the culture broth. Although these antibacterial penam a cosmid clone of S. was the glycolytic and pentose phosphate pathways by the introduced into S. The entire 24-kb and cephem compounds are synthesized from three amino gene cluster for chloramphenicol biosynthesis was cloned from acids (α-aminoadipate. pathway were not found in the S. and the transformants shikimate pathway. venezuelae ATCC 1071245 A strong inhibitor against bacterial β-lactamases. NRPS-PKS hybrid. venezuelae ATCC 10712. the clavuligerus ATCC 27064 by λ-Red recombination using 500-bp heterologous expression of the biosynthetic gene cluster for homologous regions corresponding to upstream and down. Since of S. The entire gene cluster for lactacystin biosynthesis the mycelium. and lactacystin (15). A cosmid clone. previously. (Supplementary Figure1-M) of S. The resultant transformants methylisoborneol synthase and GPP 2-methyltransferase carrying the entire 25-kb biosynthetic gene cluster for clavulanic (Supplementary Figure1-N) in Saccharopolyspora erythraea 391 dx.doi. tyrosine. synthetized by NRPS. is produced not respectively. Expression of the Biosynthetic Gene Clusters for cosmid library of S. respectively. cysteine.1021/sb3001003 | ACS Synth. clavulanic was isolated and expressed in S. Genes encoding 2- into pCLV1 to generate pCLV1*. avermitilis SUKA22. 2. The desired microorganism. respectively. containing a self-resistance gene (gryBr). Another common metabolite clavulanic acid was controlled by the gene product of ccaR with a characteristic musty. synthesized by cluster alone (pCLV1). was introduced into S. monoterpenoid alcohol 2-methylisoborneol. degraded sesquiterpenoid alcohol. and 23 biosynthetic genes one or more genes encoding transcriptional regulatory proteins (novA−novW). which is cephamycins) compounds. which is respon- ing to upstream and downstream of sclav_4180 and sclav_4197. The entire operon to generate pLTC1*. Transformants carrying pLTC1* gene cluster for chloramphenicol (19) (Figure 5D) biosynthesis produced 30 mg/L lactacystin (15) in the culture broth. a specific inhibitor of the entire 16-kb gene cluster for rebeccamycin biosynthesis proteasomes. were examined. sequence data of lactacystin-producing S. compounds derived from shikimate was quite interesting in this stream of sclav_5267 and sclav_5278. to generate pCLV1. sible for the characteristic odor of moist soil. spheroides) NCBI 11891 was cloned the gene cluster. and valine). The desired recombi- amino acid for the formation of peptide bonds by NRPS. Since it has been demon. (Supplementary Figure1-I) was cloned from the cosmid library The aromatic ring moieties of antibacterial compounds.46 We subcloned the minimum set of the gene such as penam (penicillins) and cephem (cephalosporines and cluster for chloramphenicol biosynthesis from pAH91. A recombination using 500-bp homologous regions correspond.ACS Synthetic Biology Research Article avermitilis SUKA17 of the gene cluster for cephamycin C acid and ccaR gene produced 16 mg/L clavulanic acid (16) in biosynthesis (Supplementary Figure1-G) of S. The minimum set of the gene pathway. biosynthetic gene clusters for secondary metabolites contain which was insensitive to novobiocin. S. caeruleus (former name. but no regulatory genes were found in the previously.40 The nant plasmid. 2013. avermitilis SUKA17. Bioinformatic analysis of the draft ATCC 39243.

and non-heme iron-dependent. pentalenolactone biosynthetic gene clusters by introducing pKU462::ptlH-ptlG-ptlF-ptlB-ptlA-ptlI into S. did not produce pentalenolactone but Figure 6. amorpha-4. which catalyzes the cyclization of farnesyl diphosphate to the parent sesquiterpenoid hydrocarbon pentalenene. and transformants produced abundant 2- methylisoborneol47 (20) (Figure 5E). by PtlF.ACS Synthetic Biology Research Article NRRL 2338 and S.1021/sb3001003 | ACS Synth.56 Each synthetic operon ketoglutarate. production was encoding taxa-4. PenA.doi. avermitilis. introducing a copy of the native S. the gene product of which catalyzed the native of S. 2013. avermitilis were identified from established producers of the antibiotic. and antiviral sesquiterpenoid antibiotic produced by many Streptomyces microorganisms. geranylgeranyl diphosphate synthase upstream of the synthetic lenolactone D. 2. and transformants produced abundant introduced into engineered S. ambofaciens ATCC 15154 were expressed in S. two products with mass [M+] m/z 272 (C20H32) tone54 (21). (ii) The resultant recombinants were further corresponding to C20H32 was detected in the n-hexane extract introduced into penM. and penM was expressed as an operon with fdxD (sav3129) previously characterized gene products in the S. Similarly.52 Thereafter. respectively. the gene product of which was of transformants carrying synthetic tds and the product was cytochrome P450. avermitilis under control of efficiently expressed as a synthetic operon with the gene the rpsJ promoter that was expressed constitutively. avermitilis mutants. avermitilis ΔptlE ΔptlD. avermitilis ptl and f prD (sav5675) encoding ferredoxin and ferredoxin reductase. arenae TÜ 469 (pnt stepwise introduction of pKU464aac(3)IV::pntE-pntD and gene cluster). 384−396 . All gene cassettes were controlled by ermE coding genes. S. which Q947C4). two-step desatu. We biosynthetic gene clusters showed efficient expression. The encoding farnesyl diphosphate synthase or geranylgeranyl requisite farnesyl diphosphate precursor was provided by diphosphate synthase. Q41594) avermitilis SUKA17 carrying ptl cluster (Figure 6). Biol. an isomer of the expected product pentalenolactone (26) producer (S. These two neopentalenoketolactone. avermitilis ptlB gene Productivity of Secondary Metabolites in a Heterol- (sav2997) encoding farnesyl diphosphate synthase downstream ogous Host. The following stepwise process was introduced into S.4-kb ptl gene cluster encoding 13 predicted protein coding sequences that we initially thought might represent the set of biosynthetic genes for pentalenolactone itself. avermitilis. avermitilis. (lps) of taxol producer Taxus brevifolia (accession no. avermitilis crtE gene (sav1022) encoding region-specific Baeyer−Villiger oxidation exclusively to penta. was for expression in S. and pntD-encoded dioxygenase catalyzed the α. avermitilis SUKA16 ΔptlE ΔptlD) was constructed F. Artemisia annua. Ultimately. The final recombinant strain. or ϕBT1. S. of plant origin. Two synthetic genes the transformants did not produce metabolites. Construction of engineered pentalenolactone biosynthesis in S.49 Pentalenene synthase. avermitilis SUKA17 by transformation. 11-oxopentalenic acid (26). pKU460aac(3)IV. have now extended this observation to the expression of plant Although a few gene clusters were inefficiently expressed and diterpene synthase genes in S. including close orthologues of each of the promoter. but their productivity was pntD-pntE55 and penM produced pentalenolactone but not lower than that of transformants carrying tds. was first isolated from S. in which codon usage was optimized produces a key intermediate. S.and diterpenoid the gene encoding sesquiterpene synthase. intermediate. (i) The and ginkgolide A producer Ginkgo biloba (accession no. S.11-diene (tds) and levopimaradine synthases successfully restored by controlling the expression of the 392 dx. More than 20 biosynthetic gene clusters were of the ads gene. pen and pnt gene clusters.51 The ptlA gene is itself located within a 13. were prepared and joined with transformed by pntE. SUKA16. and pKU464hph each gene cluster. Production of the We have recently examined the heterologous expression of parent intermediates of plant sesqui. which is the natural product of S. diterpene synthase gene. TG1. Pentalenolactone (21) (Figure 5E) is an antibacterial. was introduced into S. data. Each of the two gene clusters contained a pKU460hph::penM-fdxD-f prD into S. Construction steps were as follows: (i) an intermediate of produced a novel sesquiterpenoid compound. avermitilis and [M+] m/z 270 (C20H30) were observed from the extract of SUKA17 carrying the ptl cluster (ΔptlE ΔptlD) processed transformants carrying synthetic lpd. pentalenene by PtlA. which catalyzed the unusual oxidative identical to taxa-1.48. respectively. (ii) Pentalenolactone (21) producer was constructed by exfoliatus UC5319 (pen gene cluster) and S. respectively. double deletion mutant.53 PenF or PntF was identical in ptl. in which the expression of ptl cluster was controlled by an alternative promoter.11-diene compounds by S. The biosynthetic process from the formation of contained an attachment site and integrase gene of bacteriophages. respectively.4-diene (22) (Figure 5E) in the mycelium. 11-oxo-1-deoxypentalenic acid tolactone. and each terpene synthase gene was synthetic gene was expressed in S. pKU462. and many of these amorpha-1. pentalenolactone-family metabolites. antifungal. exfoliatus U5319 and subsequently cloned and expressed in E.50 The sav2998 gene of S. avermitilis SUKA17 transformants was synthase (ads). ΔptlD recombinant. or PntA to the formation of a key ϕC31. avermitilis SUKA16 ΔptlE homologous set of 10 unidirectionally transcribed protein. avermitilis. products were thought to be levopimaradine (24) and avermitilis. avermitilis SUKA17. avermitilis. exfoliatus U5319.13 A the addition of precursors. abietatriene (25) (Figure 5E). in which codon observed by growth in a simple synthetic medium without usage was optimized for expression in S. 11-oxo-1-deoxypentalenic acid (26). A ration/epoxidation of pentalenolactone D to pentalenolactones single major product with mass [M+ + 1] m/z 273 E and F (27). neopentalenoke.4-diene (23) (Figure 5E) in several spectrum rearrangement of pentalenolactone F (27) to pentalenolac. avermitilis encodes a 336-aa protein (PtlA) with 76% identity to the well- characterized pentalenene synthase of S. respectively.

Similarly.5-fold higher than that of the original (promoter of rpsJ). ribosidif icus ATCC 21294. metabolites derived from sugar and shikimate pathways. 384−396 . but no compounds. S. pPHM1. and the productivity secondary metabolites derived from polyketide. The production of these secondary metabolites in S. both the wild-type and SUKA22 was 7. pLTC1*. Production profiles of (A) ribostamycin (2). (D) lactacystin (15). Individual productivity was calculated by duplicated experiments.5−4% productivity of that of S. The productivity of these metabolites in S.doi. 2. 45- side and phosphoglycolipid antibiotics. As described above. These two biosynthetic gene clusters might be inefficiently NRPS. Bafilomycin B1 produc- Transformants of S. although at least eight biosynthetic gene clusters polyketide compounds synthesized by type-I PKSs (avermectin. Not all the original producer S. but lactacystin production was extremely improved by improved. avermitilis produced aminoglyco. As mentioned above. venezuelae ATCC 10712. avermitilis. 2013.ACS Synthetic Biology Research Article Figure 7. about 12-fold higher than avermitilis wild-type and its large deletion mutant SUKA17 that of the original producer S. lactacystinaeus OM-6159. setae KM-6054. setae KM-6054 (Figure 7C). pHLM1.5. 100-kb) in Streptomyces strains was large-deletion derivatives of S. ribosidif icus ATCC 27065 was less than 0. (C) bafilomycin B1 (9).13 Both transformants of S. avermitilis SUKA17 and its deacetylated derivative holothin in both S. griseus IFO 13350 or S. S. but no biosynthetic gene clusters for secondary expressed in S. avermitilis 393 dx. K. avermitilis wild-type produced principal endogenous Furthermore. clavuligerus transformants carrying the entire gene cluster for lactacystin ATCC 27065.38 ATCC 21294 produced small amount of ribostamycin (Figure Holomycin production in wild-type S. and filipin). avermitilis wild-type strain by pBFM1 were not obtained. and vertical lines indicate standard deviation. respectively. Since S.1021/sb3001003 | ACS Synth. avermitilis wild-type and SUKA17 or SUKA22 carrying each biosynthetic gene cluster (pRSM1. pOTC1.2 mg/ 7-fold higher than that of wild-type expression of the gene cluster using an alternative promoter transformants and 2. oligomycin. ribosidificus ATCC 27065 was extremely low level and variable. S. ribostamycin (2). Transformation or conjugation effi- avermitilis is interesting. produced about half of the amount of metabolite as the avermitilis harbors many biosynthetic gene clusters for original producer S. clavuligerus containing aminoglycoside. lactacystinaeus OM-6159 (Figure carrying biosynthetic gene cluster for deoxystreptamine. and (G) chloramphenicol (19) in original producers S. polyketide compounds. (E) holomycin (14). transformants of wild-type appeared. clavuligerus ATCC 7A. rimosus NRRL 2234. biosynthesis of S. being 4. extremely low in comparison to that by cosmid clone (ca. rimosus NRRL 2234. lactacystinaeus OM-6159 produced lactacys- avermitilis large-deletion mutant SUKA17 transformants was tin. and peptide compounds. A few transformants of SUKA22 were obtained. amino acid of S. The production of holomycin 21294). transformants of ciency by BAC clone (ca. 7D). and S. transformants of S. (F) pholipomycin (4). we were quite interested in no peptide compounds were detected under any culture heterologous expression of biosynthetic gene cluster for conditions. avermitilis wild-type transformants was inefficient (Figure pathways. shikimate-derived kb). Lactacystin production in transformants of producers as described previously. (B) oxytetracycline (5). avermitilis wild-type strain carrying the tion in the SUKA22 transformants carrying the entire gene biosynthetic gene cluster for streptomycin (1) or cephamycin C cluster for bafilomycin biosynthesis was 4-fold higher than that (13) produced about half of the amount of each metabolite as of the original producer K. avermitilis genome. the backbones of which were synthesized by PKS and 7B). harboring NRPS genes are located in the S. of S. pBFM1. S. or pCML1). clavuligerus ATCC 27065. 2. regulatory gene or directly expressing the biosynthetic gene carrying the biosynthetic gene cluster for oxytetracycline operon using an alternative promoter. Holomycin production in the wild-type S. Transformants of S.

The productivity of these MgSO4·7H2O. 0.0 g (NH4)2SO4. being 2. low in the semisynthetic medium. S. clavuligerus ATCC 27065. The lactone. Two minimum media (MM).0 g agar per liter metabolites in S. setae KM. instability and the genetically stable characteristics of S. the original producer S. 394 dx. lactacystin. The characteristics of structure was confirmed by spectral data (1H and 13C NMR.13 In almost all cases. IL.0 g heterologous production of secondary metabolites derived from glucose (pH 12-fold higher than that of gene clusters. cyaneogriseus subsp. SUKA17 and SUKA22. 10. S.0). production of bafilomycin B1.0).13 Secondary metabolites Using large-deletion derivatives of S. 2. The large-deletion derivatives of S. S. These results indicate and three codon-optimized genes encoding plant terpenoid that S. holomycin. noncyanogenus Streptomyces microorganisms and could be an important driving NRRL 15774 and S. of S. linear plasmid SAP2) was used as the wild-type strain.003 g FeSO4·7H2O. the biosynthetic cluster would be cryptic in S. has a stable phenotype in comparison with construction of the genomic library using a cosmid or BAC other Streptomycetaceae microorganisms. source of chromosomal DNA preparation. rimosus NRRL 2234 were from the force in evolution.2 g and S. 200 mg FeCl3. clavuligerus The feasibility of using large-deletion derivatives of S. 384−396 . pholipomycin. and 15. US Department of Streptomyces microorganisms genome-sequenced to date. Furthermore. ribosidif icus since each precursor for biosynthesis is different from that of ATCC 21294 (JCM 4923). 0. avermitilis. avermitilis make it especially suitable as a source of medium was used for the production.1 M phosphate biochemical energy of the host are apparently efficiently used buffer (pH7. 10 mg CuCl2·2H2O. therefore. Cultivation for Secondary Metabolite Production. avermitilis are fundamentally productivity of holomycin in SUKA22 transformants was similar to those of the wild-type strains. in the stationary phase was enhanced. This phenomenon disagrees with the Agricultural Research Service Culture Collection (National industrial production of secondary metabolites. avermitilis. clavuligerus ATCC 27064 the principal endogenous polyketide metabolites. 0. The productivity of lactacystin. large-deletion derivatives did not authentic sample. respectively (Figure 7F). and its Among them. USA). 0. avermitilis. and chloramphenicol was extremely heterologous expression of biosynthetic gene clusters for efficient.0 mL of trace elements solution (40 mg transformants. agar medium containing 1. transformants of both the avermitilis as a heterologous host has been shown by the wild-type and SUKA22 carrying the entire biosynthetic gene effective expression of 18 intact biosynthetic gene clusters. were used to assess the growth characteristics in these metabolites. Other microorganisms were from avermitilis. clavuligerus ATCC 27065 (Figure 7E). On the other hand. metabolites. the natural precursors and (NH4)2MoO24·4H2O per liter). avermitilis. large-deletion derivatives.0) per liter was used when the productivity was exogenous biosynthetic gene clusters. Biol. The intrinsic genetically stable characteristics of were described previously. Because S. venezuelae ATCC 10712. it has been planned to construct the heterologous derivatives of S.ACS Synthetic Biology Research Article wild-type and SUKA22 was relatively stable (Figure 7E). S. 10 mg Na 2 B 4 O 7 ·10H 2 O. Bioresource Center (Wako. producer of an industrial anthelmintic macrocyclic our culture collection. 2013. Peoria. Streptomyces precursors of the shikimate pathway are also efficiently supplied avermitilis K13947 (isogenic to MA-4680 but lacking a large to chloramphenicol synthesis in S. In lactacystin. medium57 at 30 °C for 3−5 days. the produc. Each spore preparation for the mycin. Actinomycete microorganisms were used as the and chloramphenicol production of S. semisynthetic S. avermitilis wild-type trans.doi. S. It seems that the biosynthetic gene cluster for chloramphenicol of S. avermitilis are due to the shortest TIRs in the linear Conditions for vegetative culture and fermentation culture chromosome. venezuelae ATCC 10712 (Figure 7G). and chloramphenicol in S. Japan) and Saccharopolyspora Conclusion. isolated from soil samples.0 g malt extract and 10. (JCM 4710).0 g yeast extract. pholipomycin. avermitilis SUKA22 lacks the ZnCl2·7H2O. which is observed in many producer S. because large-deletion derivatives the control of the expression of the gene cluster for of S.0 g (NH4)2SO4. of large-deletion derivatives was more developed pholipomycin production has never been detected from the than that of the wild-type. 6054. and the produce endogenous main metabolites. Production of chloramphenicol. avermitilis will possess the upper regulatory system for intermediates. and 10 mg metabolites of S. pholipo. and 20 mL of 0. Since Streptomyces. lactacystinaeus OM-6159. The metabolite was purified. 10 mg biosynthetic gene clusters for the principal endogenous MnCl 2 ·4H 2 O. Among the Center for Agricultural Utilization Research. and another medium endogenous secondary metabolites and as a host for the containing 4. Agriculture. avermitilis SUKA22 transformants was (pH 7. in transformants of both exploited to generate precursors of exogenous biosynthetic the wild-type and SUKA22 was 6.5 g K2HPO4. avermitilis no longer produced major endogenous pholipomycin biosynthesis.1021/sb3001003 | ACS Synth. venezuelae ATCC 10712 is efficiently expressed. secondary metabolites. two cluster for pholipomycin produced 9 and 20 mg/L biosynthetic gene clusters controlled by an alternative promoter pholipomycin. from which caused by introduction of the exogenous entire biosynthetic biosynthetic gene clusters for endogenous major products were gene cluster for secondary metabolites into large-deletion removed.5-fold higher than that of wild-type g MgSO4·7H2O.6 improved. and liquid medium containing 2. Genetic instability has been observed in many erythraea NRRL 2338. were used for 2. Not only improved to at least 40-fold higher than that of the original morphological differentiation. avermitilis transformants. and ■ METHODS Bacterial Strains and Growth Conditions. and S. ATCC 27065. primary metabolism seemed to be efficiently derived from the shikimate pathway. holomycin. avermectin. avermitilis were detected by HPLC−MS expression of biosynthetic gene clusters for secondary analysis and/or biological activity with comparison with each metabolites. clavuligerus ATCC 27065 in any culture conditions. production and storage was obtained by growth on YMS formants was higher than that of original producer K. Comparative analysis vector and in vivo cloning of the entire biosynthetic gene cluster of these genome data suggests that the size of TIRs found at the by λ-RED recombination are described in “Supporting end of the linear chromosome ends correlated with genetic Information methods”. venezuelae ATCC 10712 (JCM 4526) tivity of these metabolites in the wild-type strain was not too were obtained from the culture collection of the RIKEN but also the growth rate and biomass culture of S. 1. S.

(2003) (6) Wang. A. T... Oiwa. isolation. ■ ACKNOWLEDGMENTS We thank I... H. A. M. Energy and Industrial Technology Development Organization R. S.. X. Squares.. Chandra. M... Hattori. J.. Ikenoya. S. H. D.. P. J. Biotechnol. H. Deciphering tuberactinomycin biosynthesis: isolation. S.A.. H. 2. Horinouchi. Natl. a research grant from the (1979) Avermectins. H. figure and references. Sports. B. M. microorganism Streptomyces avermitilis. Liu. ■ Parkhill. Goble.S. 522−529... Osonoe. 21.. and M.. J. Currie. hygromycin B phosphotransferase gene from Streptomyces hygro- Segurens. Bibb for kindly BMC Genomics 12. Hanamoto. (1986) Nucleotide sequence of the (7) Barbe. W. C.. J. Bacteriol. Complete genome sequence of Streptomyces cattleya NRRL 8057. Y. Science 118. (2011) scopicus. J.. H. T. Kishi. aerial mycelium-negative isolates of streptomycetes. REFERENCES M.. providing gene clusters for kasugamycin biosynthesis. Huguet-Tapia.. the productivity of the 224.. Ikeda. Deducing the ability of producing secondary metabolites. J. C. (20) Thomas.. Toyama Prefecture University. Challis. Agents Chemother. Takahashi. S.. T. M. Sasagawa. Fukui. Kikuchi. S. R. W. J. Japan (to H.. J. Sakaki.. A.. Chambers. N. Hara. Proc. Chater. H. H. (2008) ls. Zhang. C... H.). K. Ohnishi. Oliver. T. A.. Vallenet. Kuzuyama... (23) Uchiyama. C.. 891−899. Tsai.. S. H. Huang. (2005) Bioactive microbial metabolites . Nierman. metabolite(s) was measured from 4−6 transformants. Trends Genet. Wallick. Wietzorrek. properties.. Notes (11) Bignell.. (2001) Genome Watanabe. new family of potent anthelmintic agents: Institute for Fermentation. (17) Fishman. and Cohen. Badet. A. Mol. A. (21) Zalacain. L. Bacteriol.. 18. Burghardt. (12) Pullan. (1987) Genetic instability and view. Zhang. T. Culture. G. Osaka. Shiba. Plant-Microbe Interact.... Heijne.. Proc.. E. J. Genet. Horikawa. 212− purchased or obtained. T. E. R. K. S. A. 361−367.. Alam.. 4526−4527. S. 4796− (4) Omura. (19) Ichikawa. *Tel: +81-42-778-9345... A... genes of bacteriophage ϕBT1...). Rabbinowitsch. R.. 26−36. 14. 526−531. a (22) Bibb. S. AUTHOR INFORMATION 141−147. J. The authors declare no competing financial interest. sequencing. E. DNA amplification in Streptomyces lividans 66. (16) Redshaw. J. Larke. M. B.. Cane. Pauker. Putter. 23... Nature 417. (2003) Complete evolutionary snapshot of the family Streptomycetaceae. Ikeda. Taylor. Ishikawa. This material is available free of charge Rutter.1021/sb3001003 | ACS Synth..kitasato-u.. (2002) Once the circle has been broken: dynamics and evolution of (NEDO.. Seipke. A. G. to K. * S Supporting Information Howarth. Bouzon.. and Loria. Zhu. S. Fax: +81-42-778-9930. Sharp. and Fujita. Bacteriol. Mangenot.. Seeger. L. Hornsby.. Yan. and Merrick.. A. and Hattori.K. Huang... Hanamoto..... (2010) Genome sequence of Kitasatospora setae NBRC 14216T: an Shiba. 17.I... Y... M. Mattaliano. O. U. W. Y. 175 (2011). M. Jiang.. (2) Stackebrandt.. J. 2. M. G. 459−466. 4803. (2002) Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). S. S.. J. Oguchi.. Uchiyama. J. T. Huang. M. Kovalchuk.. Berg. and Hippe. M. B. Genome sequence of the streptomycin-producing microorganism Streptomyces griseus IFO rebecca. Z.. T. Murphy. E. F... L. S... Miller. (1985) producer of antibiotics and fluorometabolites. J. K. R. E. 1−26... G.I. R. Stapley. S.. Yamashita. Quail.. B. Saunders. (5) Ikeda.S. 259−266. J. H. Arisawa. C. Rainey. Smith of the expression of secondary metabolism.. Hartman. M. 12215−12220. Corresponding Author (10) Ohnishi.A personal (18) Dyson. H. Brown.. C. U. Y. U. Tian. 137... Biol. Chen.acs.. A.. J. M. McCann. B. and Y. Wu. Malpartida. Yin. In each case.. Chandra. J. mycin biosynthesis. R. milbemycin-producing bacterium Streptomyces bingchenggensis. R. via the Internet at http://pubs. A.. 15. S. W. M. Bibb. L. E. G... H. H. A.. Genome Biol. and chloramphenicol biosynthesis H. Y. W. E. Evol. J. M. and Hopwood. Lee. M. and L. V. B. Marlière. Baker. Antimicrob. L. Bacteriol. Bacteriol. Bateman. J.. We also thank M. Sproer.. 190.. Y. M... Harper. Nihira. and Omura. Takahashi... Tanikawa. H. respectively. An. Cronin... Natl. D... D. S. (3) Berdy. N. P. S... and Jimenez. Hidalgo.I. R. S.. Sakaki. A. Thomson. 47... (8) Medema. D. Breitling. K.. S. F. (2010) Genome sequence of the Agents Chemother.. J. J. A.doi. and utilization. J. (1979) Simultaneous loss of multiple differentiated functions in (1) Waksman.. Gao. 193. I... Chan.. N. M. H... H. and Ozanick. Parry. University of Aberdeen. 98. Hattori.A. C. M. A. and Weisblum.. Rutherford. and Horinouchi. Olson. and Weissenbach.. This work was supported by a (14) Burg. P. Areas from the Ministry of Education. R. H. L. D. and Omura... F.. H. Warren. Rajandream. James.. Guerrero. and H.. E... C. Barrell. J. C. S...). Birnbaum.. D. Nat.. (1953) Streptomycin: Background. and Technology of Japan (to H. A. T. H. Squares. C. Omura. Nucleic Acids Res. Katano. Nakazawa. 5055− Nucleotide sequences encoding and promoting expression of three 5056... M. M.. M.. M. 155. Kikuchi. Otoguro. 199. Kieser.. and Takano.. H. Y. W. Chen. H. and Pogell. H. M. Onaka of 161−175. Nomoto. 1565−1581.. Acad. J. Kitani.. Wang.. A. R. J. X. D. S.. 1134−1139. Ankai. Takahashi. E. (1983) Amplified DNA O.. Bacteriol. antibiotic resistance genes indigenous to Streptomyces. S. Ishikawa. P. Kamata. ASSOCIATED CONTENT Collins. Monaghan. E. Science Tunac. Y.ACS Synthetic Biology Research Article MS analyses and so on) when authentic samples could not be reservoir of secondary metabolic pathways. M... ■ L.. Hayakawa.... D. Müller.. Harris.. G. S. supplementary O’Neil.. J. F. A. M. U. research Grant-in-Aid for Scientific Research on Innovative A. (2010) Genome-minimized Streptomyces host for the heterologous (pAH91). M. Shinose. C.. N.. DNA Res.. Bibb. L.. H. 384−396 .. research Grant-in-Aid for Scientific Research from the New (15) Chen. genome sequence and comparative analysis of the industrial 393−406. Acad. Details of the constructs used in this study. S. Trefzer. 4050−4060... Shinose.. (2010) Mol. A. B. Suzuki. A. Gen. sequence of an industrial microorganism Streptomyces avermitilis: A. T. in Streptomyces f radiae. Ronning... M.. J. Gonzalez.. and Xiang. (2010) The sequence Streptomyces erythraeus that confers resistance to the macrolide- of a 1. J. Pentella. K... K. Y. E-mail: ikeda@ Ikeda. Y. Cerdeno-Tarraga. Nakamura. A. R.. A.. 2823−2830... H.. B. Woodward. Ishikawa. and Kirby.. A.. M. Fraser. 58. F. Y.. Sci. Syst.. J..8-Mb bacterial linear plasmid reveals a rich evolutionary lincosamide-streptogramin B antibiotics: amino acid sequence and its 395 dx. and a producing organism and fermentation. Ward.. Bacteriol. J. Y. Antibiot. J. X. D. M. (1997) Int. 47. ■ Streptomyces chromosomes. 2013. S. S. (9) Bentley.S. L.. J. Sci. G. Kong. 192. annotation of the viomycin biosynthetic gene cluster. X. R. C. and Ikeda.. Antimicrob. S. J. Hashimoto. H. M. Poulain. M. 169. for provision of attP and integrase 2646−2651. C. A... R. A. 107. A. Kojima of Akita Prefecture University. D. J. and Schrempf. (2011) Genome-wide analysis of the role of GlnR in Streptomyces venezuelae provides new insights into global nitrogen regulation in actinomycetes. Kieser. J.. Miura.. (1985) N-Methyl transferase of Bovenberg. (13) Komatsu.

metabolite. biosynthesis are encoded by two sets of paralogous genes in Streptomyces clavuligerus. E. 257−267.. M.. comparison with other members of this family of antibiotics. Mol. Acad. cocci. Yoshida. M. Biotechnol. Igarashi. K... Nagai. P. E. 2.. M.. C. venezuelae ISP5230 includes novel shikimate pathway homologues Cells 20. S. Piraee. Nakashima. T. H. and identification of a new biosynthetic (36) Ueda. H.. S. characterization and structures of LL. II new antibiotic. Biochemistry 48. Muyrers. Antimicrob. and Cane. Bacteriol. Biochemistry 45. H. J. 1314−1317. Agents Chemother. Arch.. S. J. A. (43) Onaka. Tetrahedron Lett. Anzai... (2005) The ribostamycin biosynthetic gene cluster in gene cluster for chloramphenicol biosynthesis in Streptomyces Streptomyces ribosidif icus: comparison with butirosin biosynthesis. Fujimoto. Isolation and chemical properties.S. D.. Microbiol. A. Micro- (29) Ostash. You. (37) Hu. 67. Wong.. M. L. S. 44. Discovery of an unprecedented P450- derivative.. M. 2737−2740. (47) Komatsu. S. Moriguchi. Hertz. A. (2007) New aureothin mining in Streptomyces. 343− cluster of Streptomyces spheroides NCIB 11891. Takahashi. 225−233. (2003) (26) Suzuki. S. Omura. 720−726. W. Tetzlaff. 133. Scott.. N.. E. Williams. Cane. (2005) Cre/ Characterization of the biosynthetic gene cluster rebeccamycin from loxP-mediated deletion system for large genome rearrangements in Lechevalieria aerocolonigenes ATCC 39243. NRRL 23338. (2011) Genome Natsume. and Hutchison. Takeuchi. Y. 515. D. J. F.. Appl. G.. and Jensen. H. Lee. S.. P.. K. K. Ed. and Ikeda. 103−110.. A. J. T. G. S... Li. H. Proc. M. Biotechnol.A. (41) Paradkar. (28) Subba. Streptomyces coelicolor for heterologous expression of secondary 14. T. E. Z. R. and Shin-ya.. (2012) Diversity and (39) Omura. Antimicrob. B. Annu. 30.. Biol. F. a new member of (2008) Identification and functional analysis of genes controlling phosphoglycolipid antibiotics. N. Biochem. G. Agents DNA cloning by homologous recombination in Escherichia coli. U. J. (49) Takeuchi. J. B.. and Ikeda. 2298−2299. Biotechnol.. S. J. T.. K. C. and Tang. Siegel.. (24) Datsenko. E. Antibiot. Testa.. (1977) Pholipomycin. Bacteriol. 126. Aidoo. (52) Jiang. Curr. O. and Sasaki. Appl. and Cane. (1991) Lactacystin. intermediate. K. 7. L. Dickens. J. N..S. Methods 6. Microbiol. Harayama. Molecular cloning and assignment of macrocyclic lactones. Kim.. K. Soc. 169. (2010) Identification of the gene cluster ase/oxygenases in the final steps of the biosynthesis of pentaleno- for the dithiolopyrrolone antibiotic holomycin in Streptomyces lactone and neopentalenolactone. Chem. H. clavuligerus. H. and Cane. K. 207−215. S. Acad. You. M.. 6431−6440. L. New York.. (30) Arai. and Furumai. C. D. B. 60. (2009) Genome mining in Streptomyces genome sequence of the erythromycin-producing bacterium Saccha.. H. Am. Streptomyces avermitilis. Venter. (48) Koe. H. J. Morton. 44. a (31) Shen. new branch of the pentalenolactone family tree. Biotechnol. biochemical function to PtlF. Isolation. III. Nakayama. 58. Z. Soc. Nat.. (2000) Streptomyces clavuligerus. E. D.. Kotaki.. Sci. Elucidation of the role of Baeyer- Antibiot. 2013.. and a monomodular non-ribosomal peptide synthetase gene. M. and III polyketide synthase paradigms. H. M. Omura. J... (46) Gomez-Escribano. Takagi...A. and Celmer. 19731−19735. 4. (2007) A streamlined biology 147. Iwatsuki.... Y.. Antibiot.. (1983) Pentalenene malonamyl-specific initiation module from the oxytetracycline biosynthesis and the enzymatic cyclization of farnesyl pyrophosphate. S. Cane. E.. F. Tsuda. R. D. B. and Walsh. L. M. B. activator of cephamycin C and clavulanic acid production in (25) Zhang. C.. 1055−1059. 107. T. (34) Oliynyk. A. K. Chemother. James. Ikeda.). Reid. J. 625−633. G... Young. and Torikata. 384−396 . G. MM23. and Leadlay. X.. Microb. Biochem. D. Nat... R. Chemother. (2006) A gene cluster for biosynthesis of the sesqui- polyketide cyclization beyond linear and angucyclic patterns.. 122−124. A. B. Doi. A. Komaki. H.. (56) Yamada. S. (2000) Enzymes catalyzing the early steps of clavulanic acid 5615−5621. 127−138.. S. (2000) One-step (42) Bignell. Proc. D... Aidoo. Tsai. Chapter 7. Magarvey. P. K. M... metabolite gene clusters.. Y. (2003) Polyketide biosynthesis beyond the type I. M. A. encoding the positive PCR products. Seo. H. U. (2006) of pentalenolactone (PA-132).1021/sb3001003 | ACS Synth. 2573−2580. 1739−1754. D. K. Antimicrob. Natl. K. Endo. C. Gene 38.. (54) Zhu. H. J. I. Natl. (2005) The leptomycin gene (55) Seo.. O. K. M... A. (1987) Genetic studies of (40) Jensen. A. 49. Acad. 6179−6186. B. C. Muhlenweg. C.. 18. R. and Bibb. Z. Genome mining in Streptomyces. Antibiot. (2009) Enzymatic assembly of Heide.. 90−96. Samborskyy. R. and Vining. S. Chem. A. K. (1957) PA 132. Soc. S. using the (50) Cane. Environ. D.. 1214−1222.. 113−116. terpenoid antibiotic pentalenolactone in Streptomyces avermitilis. 233−240.. exploring and Ikeda. Tanaka. A.. Tahlan.A.. Mironenko. Part A. Inui. (2007) LL-F28249 antibiotic complex: A new family of antiparasitic Pentalenolactone biosynthesis. Engineered biosynthesis of a novel amidated polyketide. Taniguchi. Kharel. H. J.. (2011) cluster and its heterologous expression in Streptomyces lividans. K. M. E. (1988) (53) Cane. S.. biosynthesis of pentalenolactone. Nat. Biosci. Sobin. Otoguro. B. and Stewart. K. Agents 345. J. 285− 672−675. Liou. K. Ogawa. N... Y.. R. resistomycin biosynthesis in Streptomyces resistomycif icus. Antibiot.. Academic Press. (44) Steffensky. C. Antibiot. and Borders. J. (33) Jakobi... Ikeda. Natl. U. Y. Zhu. pp 123−166. (2005) Expression of ccaR. and Wanner. J.. and Gramajo. a novel microbial thesis by Microorganisms and Plants.. and (45) He. 459. Saghatelian. Sci. 519−529.. Villiger monooxygenases and non-heme iron-dependent dehydrogen- (38) Li.. J. γ. Nagasawa. D. D. Wang.. Z. S. Ames. (2000) Identification of the novobiocin biosynthetic gene DNA molecules up to several hundred kilobases. D. from Streptomyces sp. Colvin. (2001) The Sohng. R. a short-chain dehydrogenase from 28249 α. H. Oikawa. Chem. Chem. A. Omura. Physico-chemical properties and biosynthesis of 2-methylisoborneol.. In Natural Product Biosyn- R. β. D. H. M... (57) Ikeda.. Ikeda. Biol. catalyzed oxidative rearrangement that is the final step in the 321−324. S. polyketide synthase... 6266−6274. M. J. 295. Takamatsu. (2010) Engineering metabolic pathway for the biosynthesis of moenomycin A. Am. Jensen. induces neurogenesis of neuroblastoma cells. C. Lester... 44. S. Chem. C. M. Biophys. W.... (1996) Molecular analysis of a β-lactam resistance gene encoded within the 396 dx. 2817−2829. and Walker. T. Sci... B. 97.. II. Biochemistry 50.ACS Synthetic Biology Research Article homology to cognate R-factor enzymes from pathogenic bacilli and cephamycin gene cluster of Streptomyces clavuligerus. Y. Y. λ. Opin. and Ikeda. Am. Haydock. Matsuzaki. P. Chuang.doi. Komatsu. N. J.S.. 105. Tsuge. K. K. E. and Yukawa. J. Y. Takamatsu.. alloaureothin. J. (27) Gibson. A. T. (35) Carter. Teramoto. M. (2004) A gene cluster encoding (51) Tetzlaff. 2128−2131. J. D. Analysis of Bacterial Terpene Synthases. 178... T. H. 447−453. 105. Proc. and Tillman. 1529−1541.. J.. S. D. avermitilis: A biochemical Baeyer-Villiger reaction and discovery of a ropolyspora erythraea. B... D. Nietsche. Biol. avermectin biosynthesis in Streptomyces avermitilis. J. H. 7422−7427. A. T. Methods in Enzymology Vol. (Hopwood. and Paradkar. and Smith. D. M.. 67. and Yonehara... S. 41. and Hertweck. (2007) Complete M. elder. (1969) The structure (32) Zhang. S... 6640−6645. E. H... 72.. J.. Corynebacterium glutamicum. and inactivation of chromosomal genes in Escherichia coli K-12 using Leskiw. A.. 25.. B. and Omura. is dependent on bldG. Biotechnol. Hashimoto.