High-Titer Heterologous Production in E. coli of Lyngbyatoxin, aProtein Kinase C Activator from an Uncultured MarineCyanobacteriumSarah E. Ongley,† Xiaoying Bian,‡ Youming Zhang,§ Rocky Chau,† William H. Gerwick,∥ Rolf Muller,*,‡

and Brett A. Neilan*,†

†School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney 2052, Australia‡Department of Microbial Natural Products, Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre forInfection Research and Department of Pharmaceutical Biotechnology, Saarland University, Saarbrucken 66041, Germany§Shandong University-Helmholtz Joint Institute of Biotechnology, State Key Laboratory of Microbial Technology, ShandongUniversity, Shanda Nanlu 27, 250100 Jinan, P. R. China∥Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, and Skaggs School of Pharmacy andPharmaceutical Science, University of California-San Diego, La Jolla, California 92093, United States

*S Supporting Information

ABSTRACT: Many chemically complex cyanobacterial poly-ketides and nonribosomal peptides are of great pharmaceuticalinterest, but the levels required for exploitation are difficult toachieve from native sources. Here we develop a framework forthe expression of these multifunctional cyanobacterialassembly lines in Escherichia coli using the lyngbyatoxinbiosynthetic pathway, derived from a marine microbialassemblage dominated by the cyanobacterium Mooreaproducens. Heterologous expression of this pathway affordedhigh titers of both lyngbyatoxin A (25.6 mg L−1) and itsprecursor indolactam-V (150 mg L−1). Production, isolation,and identification of all expected chemical intermediates oflyngbyatoxin biosynthesis in E. coli also confirmed the previously proposed biosynthetic route, setting a solid chemical foundationfor future pathway engineering. The successful production of the nonribosomal peptide lyngbyatoxin A in E. coli also opens thepossibility for future heterologous expression, characterization, and exploitation of other cyanobacterial natural product pathways.

Cyanobacteria are prolific producers of natural productsincluding nonribosomal peptides,1 polyketides,2 terpe-

noids, alkaloids, and ribosomally synthesized peptides.3

Cyanobacterial natural products exhibit a diverse range ofbioactivities, ensuring they are highly valued as leadpharmaceutical and industrial compounds.4 Large quantitiesof compound are required for the medicinal use, character-ization, or generation of new structural analogues viasemisynthetic chemistry. However, the cyanobacteria thatproduce these compounds exhibit slow growth rates and areintractable to genetic manipulation. Consequently, there is asupply shortage of these exploitable compounds from the nativeproducing microorganisms, which can be circumvented by theuse of heterologous overproduction.Cyanobacterial complex biosynthetic pathway character-

ization noticeably lags behind that of other phyla, such asStreptomyces and Proteobacteria, due to an inability toheterologously express entire pathways in a readily culturableand genetically amenable host that would additionally offeropportunities for production optimization.5−14 Biosyntheticallymuch less complex ribosomal peptide natural products from

cyanobacteria, typically featuring extensive post-translationalmodifications, have previously been expressed in Escherichiacoli.15−18 Furthermore, the expression of small stand-alonetailoring enzymes from cyanobacterial nonribosomal peptidesynthetase (NRPS) and polyketide synthase (PKS) pathwayshas also been achieved allowing characterization of theirbiochemistry in vitro.19−22 However, the heterologousexpression of a complete cyanobacterial PKS or NRPSbiosynthetic pathway to yield the final biosynthetic producthas proven very difficult to attain. The hybrid PKS/NRPSbarbamide derivative, 4-O-demethylbarbamide, is to ourknowledge the only successful heterologous production of acyanobacterial compound of this type,23 although yield in aStreptomyces host was very low (<1 μg L−1).While not impossible, the heterologous expression of

proteins in a distantly related organism can be troublesome

Received: March 30, 2013Accepted: June 10, 2013

Letters

pubs.acs.org/acschemicalbiology

© XXXX American Chemical Society A dx.doi.org/10.1021/cb400189j | ACS Chem. Biol. XXXX, XXX, XXX−XXX

and requires a significant amount of optimization.24,25 Here, weselected the nonribosomal peptide-terpene hybrid lyngbyatoxinA (LTX, 1) as a model to identify suitable conditions for theheterologous expression of a cyanobacterial nonribosomalpeptide in E. coli. LTX is an indole alkaloid and a tumorpromoter that elicits its bioactivity via potent activation ofprotein kinase C26 and is structurally related to the actino-bacterial compounds teleocidin and methylpendolmycin(Figure 1). The indole ring is highly nucleophilic, and all

positions are susceptible to electrophilic attack, making this aninteresting precursor for semisynthetic chemistry.27 Indolac-tam-V (ILV, 2), the structural core of these molecules, inducescellular differentiation of stem cells to pancreatic cells,28 withstructural analogues investigated for the treatment of neuro-degeneration and cancer.29,30

LTX was originally isolated from a cyanobacterial bloomdominated by Lyngbya majuscula (Supplementary Video),31,32

which has since been taxonomically reclassified as Mooreaproducens.33 Despite numerous attempts, we have been unableto establish a culture of an LTX-producing strain of Mooreaproducens. Consequently, environmental sources remain theonly available routes for accessing both the compound and thegenetic basis for biosynthesis. Elucidation of the lyngbyatoxinbiosynthetic pathway22 revealed four open reading frames(ltxA−D) spanning 11.3 kb, thus comprising the smallestcyanobacterial NRPS reported to date (Figure 2). The LtxAprotein is a bimodular NRPS that condenses L-methyl-valineand L-tryptophan to form the linear dipeptide N-methyl-L-valyl-L-tryptophan, which is released from the synthetase via aNADPH-dependent reductive cleavage to form the correspond-ing alcohol (NMVT, 3) rather than the usual thioesterasemechanism of NRPSs, which typically couple chain release andcyclization.34 A P450-dependent monooxygenase (LtxB) thencatalyzes the formation of ILV through the oxidation andcyclization of the dipeptide.21 The final step of LTXbiosynthesis is performed by LtxC, a reverse prenyltransferasethat transfers a geranyl pyrophosphate (GPP) to the C7position of ILV. The isoforms lyngbyatoxin B (4) and C (5) arepresumed to be formed though the action of an oxidoreductase(LtxD). Earlier endeavors to express the entire lyngbyatoxingene cluster in Streptomyces coelicolor were not successful due topremature transcriptional termination of the ltxA gene.35

However, the tailoring enzymes LtxB and LtxC have beenexpressed individually in both E. coli21,22 and S. coelicolor.35

We rationally adapted methodology, developed through thesuccessful heterologous expression of NRPS and PKS geneclusters from diverse phyla,13,14 to perform expression of theLTX biosynthetic gene cluster in E. coli. Previous accounts ofthe heterologous expression of cyanobacterial NRPS35 orhybrid PKS/NRPS23 biosynthesis pathways in Streptomyceshave demonstrated limited success, which is probably at least inpart a result of the disparity in GC content between

Figure 1. Structures of nine-membered indolactam alkaloids.

Figure 2. Lyngbyatoxin biosynthetic genes and proposed pathway for lyngbyatoxin A (1), B (4), and C (5). LtxB, P450-dependent monooxygenase;LtxC, prenyltransferase; LtxD, oxidoreductase; GPP, geranyl pyrophosphate; PPi, pyrophosphate.

ACS Chemical Biology Letters

dx.doi.org/10.1021/cb400189j | ACS Chem. Biol. XXXX, XXX, XXX−XXXB

biosynthetic genes and the host genome. Genomic GC contentdetermines bacterial codon usage,36 and the median GCcontent of 38 cyanobacterial genomes is ∼45%. As thelyngbyatoxin biosynthetic genes exhibit a GC content of 46%,E. coli was a logical host with a genomic GC content of 50%.While the functionality of cyanobacterial promoters from

biosynthetic pathways has been studied in E. coli using reportergenes,37 initial attempts to produce LTX from the nativelyngbyatoxin biosynthesis pathway yielded no detectable levelsof the compound or any intermediates, irrespective of growthtemperature or media constituents. This suggested that thenative lyngbyatoxin transcriptional regulatory regions are notsufficiently functional to facilitate heterologous expression ofthe pathway in E. coli. However, the transcription of thepathway under the control of native promoters in E. coli wasnot investigated further.The E. coli GB05-MtaA host carries a heterologous

myxobacterial PPTase with broad-range substrate activity38

and is capable of expressing a range of NRPS clusters at 30 °Cfrom the tetO tetracycline-inducible promoter (PtetO).

13,14 Weutilized a similar approach for the heterologous expression ofthe lyngbyatoxin biosynthetic pathway; however, under thesame conditions at 30 °C, production of LTX or any pathwayintermediates was not observed (Supplementary Figure 1). Inlight of our experience and other NRPS expression studiesdescribed in the literature,9,39 we reduced the expressiontemperature to 18 °C, which yielded substantial levels of LTXin the crude methanol extract (Supplementary Figure 2). Inaddition to LTX, the biosynthetic intermediates NMVT andILV were also detectable in the methanol extracts of E. colifermentations (Figure 3, Supplementary Figure 1 and 3a−c).The total production levels of both ILV and LTX werequantified using HPLC by comparison to a standard curvederived from known quantities of analytical standards(Supplementary Figures 4 and 5). NMVT was not quantifieddue to lack of a standard. The average total yields, in the crudeextract of cells and media, from three independent cultures of

E. coli GB05-MtaA harboring tetracycline-induced pCC-Ptet-ltxwere 150 ± 4.36 mg L−1 of ILV and 25.6 ± 0.77 mg L−1 ofLTX. HPLC−ESI-HRMS analysis of the extract also showedvery low abundance peaks corresponding to the expectedmasses of the lyngbyatoxin B or C isoforms (SupplementaryFigure 6) in all induced cultures but were absent in theuninduced culture. To confirm the absolute identity of thecompounds quantified, ILV and LTX were isolated andanalyzed by NMR. The 1H NMR data for LTX and ILVwere in agreement with published spectra (SupplementaryTables 1 and 2).40,41 In comparison to the amount expectedfrom directly scaled cultures, the yields obtained fromunoptimized large-scale cultures totaled 30% of ILV andapproximately 10% of LTX. This reduced yield correlated witha reduction in the culture growth rate and likely losses duringpurification. LTX yields from the native producer represented0.02% of the extracted environmental material.31 As the nativeproducer has yet to be established in culture, production in E.coli is a necessary measure to enable exploitation of thesecompounds.The soluble protein fraction was visualized by SDS-PAGE

(Supplementary Figure 7) to shed light as to whether the highproduction levels were due to an overexpression of thebiosynthesis enzymes. The 276 kDa LtxA NRPS was faintlyvisible as a minor constituent of the total soluble protein on day4 of the cultivation. The tailoring enzymes LtxB (52 kDa) andLtxC (43 kDa) were difficult to differentiate from endogenoushost proteins in the soluble fraction, presumably due to theirlow relative abundance. Therefore, we speculate that the highcompound levels may be influenced by the relatively lowburden that production of the small product places on the cellfollowing the initial synthesis of the biosynthetic machinery andthe very high culture density achieved during cultivation.Despite the absence of ILV from M. producens extracts, it has

been isolated from teleocidin-producing strains of Streptomy-ces.42 Due to the structural relatedness of the lyngbyatoxins andteleocidin, ILV was predicted to be a biosynthetic intermediateand was confirmed via in vitro prenylation by LtxC.22 The levelsof ILV present in the heterologous host suggest an inefficientprenylation reaction with only 10% of ILV being converted toLTX. Due to the hybrid NRPS-terpenoid nature of LTX, yieldswould most likely be improved by augmentation of theisopentyl diphosphate (IPP) and dimethylallyl diphosphate(DMAPP) availability. This can be achieved via an enhance-ment of flux though the deoxyxylulose 5-phosphate (DXP)pathway43 or heterologous expression of a mevalonatepathway44 to increase the levels of available geranylpyrophosphate for prenylation. However, our present focuswas to create a foundation for cyanobacterial secondarymetabolite heterologous biosynthesis and was beyond thescope of this study. In future, LTX yield optimization using ametabolically engineered E. coli strain may be possible;however, we also observe that ILV is a useful candidate forsemisynthetic chemistry and was produced in abundance bythis system.Extracellular export of intermediates prior to conversion is an

alternate reasoning for their accumulation, and hence the levelsof NMVT, ILV, and LTX in the cell-free spent culture mediawere examined and compared to the total production levels.Extractions of spent cell-free culture media showed thatapproximately 50% of the total yield of NMVT is extracellular,while approximately 20% of both ILV and LTX wereextracellular (Figure 4). The large proportion of extracellular

Figure 3. Production of LTX in E. coli. HPLC−ESI-HRMSchromatograms of the tetracycline-induced and uninduced cultures.EIC mass ranges m/z 302−305 and m/z 435−440. Compounds andexperimentally observed masses ([M + H+]) are indicated with theirrespective peaks. Retention times (min) are NMVT 3.3, ILV 7.2, andLTX 13.5. Theoretical exact masses (amu) are NMVT 303.1947, ILV301.1790, and LTX 437.3042.

ACS Chemical Biology Letters

dx.doi.org/10.1021/cb400189j | ACS Chem. Biol. XXXX, XXX, XXX−XXXC

NMVT relative to ILV/LTX indicates that this dipeptide maybe actively exported from the cell and may negatively influencethe conversion of NMVT to ILV. However, this does not seemto be the case with ILV and LTX, with the poor conversion ratemost likely due to geranyl pyrophosphate limitation.The replacement of the native promoter and regulatory

regions of the lyngbyatoxin biosynthetic pathway with aninducible promoter and new ribosome-binding site resulted inthe successful heterologous expression of LTX in thegenetically unrelated host E. coli. Following successfulproduction of LTX from the modified pathway, LTXproduction from the native pathway was reassessed in parallelwith identical conditions to confirm the necessity for thesemodifications (Supplementary Figure 4). The inability to detectLTX production under the control of the native promotersupports the notion that these elements do not function in E.coli; however, the possibility remains that suitable inductionconditions were simply not achieved. Additional features, whichwe speculate may have been vital for our success, include theuse of a host with a GC content similar to that of thebiosynthetic genes. The moderate production levels rather thanoverexpression of biosynthetic enzymes, in particular the NRPSLtxA,24 was additionally influenced by the intentional use of avery low copy number and, by our experience, a stable plasmidbackbone, as well as the combination of using a low growthtemperature, to allow time for the correct folding of the largeNRPS, with a rich buffered growth media providing anabundance of essential amino acids for biosynthesis. Whilenone of these factors were shown to be unequivocally essential,the combination of these factors in addition to the presence ofthe heterologous phosphopantetheinyl transferse MtaA,38

ensuring efficient posttranslational activation of theNRPS,13,14,45 proved sufficient for the heterologous expressionof the cyanobacterial NRP LTX.Due to fast growth rates, extensive characterization, the

availability of a suite of genetic tools, and a relatively robusttolerance to exogenous proteins, E. coli is considered thepreferred option for heterologous expression. The relativesimplicity of the lyngbyatoxin biosynthetic pathway comparedto most NRPSs makes it an ideal choice for the production ofnew compounds targeting protein kinase C. For the generationof new compound analogues it is essential that all downstreamenzymatic reactions tolerate any upstream changes to thesubstrate.46,47 The tailoring enzymes, LtxB and LtxC, exhibitrelaxed specificity for NMVT analogues where the valyl group

is substituted by other aliphatic groups.21 Therefore, our E. coliheterologous system provides an extremely feasible route forthe generation of new ILV-based protein kinase C modulators.Cyanobacteria are prolific producers of natural products

exhibiting a wide range of biological activities. We havedemonstrated the biological synthesis, via a multienzymethiotemplate mechanism, and overproduction of such mole-cules in a heterologous expression system. This technologicaldevelopment will allow the exploitation of molecules withcommercial significance from sources whereby previously theonly feasible route was synthetic chemistry. The next step forcyanobacterial natural products will utilize the amenability ofheterologous hosts to genetically engineer pathways for thegeneration of structural analogues with modified pharmaco-logical properties.

■ METHODSBacterial Strains and Culture Conditions. E. coli strain GB05-

red13 was used for propagation and recombineering, cultured inLuria−Bertani (LB) medium 37 °C supplemented with theappropriate antibiotic (chloramphenicol 15 μg mL−1, gentamicin 2μg mL−1). E. coli strain GB05-MtaA, with the promiscuous PPTaseMtaA from the myxobacterium Stigmatella aurantiaca chromosomallyintegrated via transposition,13 was used for expression and cultured in50 mL of Terrific Broth (TB) supplemented with antibiotics in 250mL Erlenmeyer flasks stoppered with a cotton bung.

Expression Vector Construction. The fosmid fos-DE3-8622

carries ∼35 kb of genomic DNA from Moorea producens, including the11.3 kb lyngbyatoxin biosynthesis pathway, within a pCC1FOS(Epicenter) backbone. The tetracycline-inducible promoter cassette,consisting of gentamicin resistance (aacC1), tetracycline repressor(tetR), and PtetO regions (gentaR-tetR-PtetO) from plasmid pR6K-Tps-Genta14 was inserted immediately upstream of the ltxA ATG startcodon of fos-DE3-86 (Supplementary Figure 8) using the primers ltx-genta5 (5′-TATTCCTTATACCATCAAGATCCTTTGATTTTG-AGGGCTAGAAGGCACGAACCCAGTTGAC-3′) and ltx-tetR3(5′-TATCGCCCGCCTACTTCCACTCCAAGGTTGATTCAT-AATCATGTCGATCCTCTTCTCTATCACTGATAGG-3′). Se-quences homologous to fos-DE3-86, to facilitate recombineering, areunderlined. This new construct was named pCC-Ptet-ltx. In detail,PCR amplification was performed using Phusion DNA polymerase(New England Biolabs). The product was purified and concentratedby ethanol precipitation. E. coli GB-red harboring plasmid fos-DE3-86was inoculated (1:40 dilution) from an overnight culture and grown ina thermomixer (Eppendorf) at 37 °C shaking 950 rpm. At OD600 0.2,0.2% (w/v) L-arabinose was added to induce expression ofrecombinases. Cells were cultured to an OD600 of 0.4, harvested,washed three times with ice-cold dH2O, and resuspended in 40 μL ofdH2O. The cells were mixed with 0.2−0.3 μg of PCR product andtransferred to a sterile 1 mm electroporation cuvette. Electroporationwas performed at 1300 V using an Electroporator 2510 (Eppendorf),followed by the immediate addition of 1 mL of LB media. The cellswere transferred to 37 °C shaking 950 rpm for 75 min, concentrated,and plated on LB agar plates containing 15 μg mL−1 chloramphenicoland 1 μg mL−1 gentamicin. The plates were incubated overnight at 37°C. Correct recombinants were determined by restriction digestion.

Expression and Analysis of Lyngbyatoxin A Production.Overnight cultures of four different clones of GB05-MtaA harboringthe pCC-Ptet-ltx construct were inoculated into 50 mL of TB mediaand grown at 30 °C, shaking at 200 rpm to an OD600 of 0.17−0.18.Cultures were transferred to 18 °C, shaking at 200 rpm, for 30 minprior to induction of three of the cultures at an OD600 of ∼0.2 by theaddition of 0.5 μg mL−1 tetracycline (final concentration). Cultureswere transferred to 18 °C, shaking at 200 rpm, for 5 days, and 2% (v/v) Amberlite XAD-16 polymeric adsorbent resin (Sigma) was added toadsorb extracellular compounds. Cultures were returned to 18 °C foran additional 48 h prior to harvesting of the cells and resin bycentrifugation. Cells and resin were extracted with methanol and

Figure 4. Production levels of N-methyl-L-valyl-L-tryptophanol(NMVT), indolactam-V (ILV), and lyngbyatoxin A (LTX) in E. coli.Striped bars, total culture production levels; unfilled bars, extracellularlevels. ILV and LTX were quantified by HPLC using analyticalstandards, and NMVT levels are depicted as the area under therespective HPLC trace peak. Values represent the average levels fromthree independent cultures (N = 3) depicted with SEM.

ACS Chemical Biology Letters

dx.doi.org/10.1021/cb400189j | ACS Chem. Biol. XXXX, XXX, XXX−XXXD

evaporated to dryness. Extracts were redissolved in 1 mL of methanoland analyzed by HPLC−ESI-HRMS/MS. Separation was performedon a Dionex UltiMate 3000 RSLC system using a Waters BEHC18(100 × 2.1 mm, 1.7 μm dp) column by injection of 2 μL ofconcentrated sample. Separation was achieved using a linear gradientwith (A1) water + 0.1% formic acid to (B1) acetonitrile + 0.1% formicacid at a flow rate of 550 μL min−1 and 45 °C. The gradient wasinitiated by a 0.39 min isocratic step with 5% B1 followed by anincrease to 95% B1 within 18 min. HPLC was coupled to a ThermoFisher Orbitrap via an Advion Triversa Nanomate nano-ESI system.Mass spectra were acquired in centroid mode with a range of 200−2000 m/z at a resolution of R = 30000. Fragmentation by collision-induced dissociation was performed on ions corresponding to 302.20,304.20, 438.31 m/z. Extracts from induced cultures were compared toan uninduced negative control. The yields of ILV and LTX werequantified in duplicate by comparing to a standard curve(Supplementary Figure 5) derived from known amounts of eachcompound on a HP Agilent series 1100 HPLC using an AgilentZorbax SB-C18 (4.6 × 250 mm) column. Separation was achievedusing a linear gradient with (A2) water + 0.1% trifluoroacetic acid to(B2) acetonitrile at a flow rate of 1.1 mL min−1 and 27.5 °C. Thegradient was initiated with 5% B2 and increased to 95% B2 within 30min, followed by 95% B2 for 10 min. Results were analyzed, andproduction levels with SEM were determined using Prism 5.0 for Mac(GraphPad Software).

■ ASSOCIATED CONTENT*S Supporting InformationThis material is available free of charge via the Internet athttp://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: b.neilan@unsw.edu.au; rom@helmholtz-hzi.de.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe would like to kindly thank A. Jones and D. Edwards forproviding valuable assistance towards the preparation of thismanuscript, L. Gerwick for provision of strains and analyticalstandards, and R. C. Coates for M. producens photographicmaterial. T. Hoffmann’s contributions to this work regardingHPLC−ESI-HRMS Orbitrap measurements in Saarbrucken arehighly appreciated. This work was funded by the AustralianResearch Council (grants FF0883440 and LP110201096) andDiagnostic Technology P/L (B.A.N.), as well as NIHCA108874 (W.H.G.). Work in R.M.’s laboratory was supportedby the Deutsche Forschungsgemeinschaft and the Bundesmi-nisterium fur Bildung und Forschung. S.E.O. was supported byan Australian Postgraduate Award and an Adrian Lee TravelScholarship. X.B. is grateful to the China Scholarship Council(CSC) for a Ph.D. scholarship.

■ REFERENCES(1) Sieber, S., and Marahiel, M. (2005) Molecular mechanismsunderlying nonribosomal peptide synthesis: approaches to newantibiotics. Chem. Rev. 105, 715−738.(2) Hertweck, C. (2009) The biosynthetic logic of polyketidediversity. Angew. Chem., Int. Ed. 48, 4688−4716.(3) Arnison, P. G., Bibb, M. J., Bierbaum, G., Bowers, A. A., Bugni, T.S., Bulaj, G., Camarero, J. A., Campopiano, D. J., Challis, G. L., Clardy,J., Cotter, P. D., Craik, D. J., Dawson, M., Dittmann, E., Donadio, S.,Dorrestein, P. C., Entian, K.-D., Fischbach, M. A., Garavelli, J. S.,Goransson, U., Gruber, C. W., Haft, D. H., Hemscheidt, T. K.,Hertweck, C., Hill, C., Horswill, A. R., Jaspars, M., Kelly, W. L.,

Klinman, J. P., Kuipers, O. P., Link, A. J., Liu, W., Marahiel, M. A.,Mitchell, D. A., Moll, G. N., Moore, B. S., Muller, R., Nair, S. K., Nes, I.F., Norris, G. E., Olivera, B. M., Onaka, H., Patchett, M. L., Piel, J.,Reaney, M. J. T., Rebuffat, S., Ross, R. P., Sahl, H.-G., Schmidt, E. W.,Selsted, M. E., Severinov, K., Shen, B., Sivonen, K., Smith, L., Stein, T.,Sussmuth, R. D., Tagg, J. R., Tang, G.-L., Truman, A. W., Vederas, J.C., Walsh, C. T., Walton, J. D., Wenzel, S. C., Willey, J. M., and van derDonk, W. A. (2013) Ribosomally synthesized and post-translationallymodified peptide natural products: overview and recommendations fora universal nomenclature. Nat. Prod. Rep. 30, 108−160.(4) Burja, A. M., Banaigs, B., Abou-Mansour, E., Burgess, J. G., andWright, P. C. (2001) Marine cyanobacteria−a prolific source of naturalproducts. Tetrahedron 57, 9347−9377.(5) Gomez-Escribano, J. P., and Bibb, M. J. (2012) Streptomycescoelicolor as an expression host for heterologous gene clusters. MethodsEnzymol. 517, 279−300.(6) Wang, P., Kim, W., Pickens, L. B., Gao, X., and Tang, Y. (2012)Heterologous expression and manipulation of three tetracyclinebiosynthetic pathways. Angew. Chem., Int. Ed. 51, 11136−11140.(7) Zhang, H., Boghigian, B. A., Armando, J., and Pfeifer, B. A.(2010) Methods and options for the heterologous production ofcomplex natural products. Nat. Prod. Rep. 28, 125−151.(8) Wenzel, S., and Muller, R. (2005) Recent developments towardsthe heterologous expression of complex bacterial natural productbiosynthetic pathways. Curr. Opin. Biotechnol. 16, 594−606.(9) Wenzel, S. C., Gross, F., Zhang, Y., Fu, J., Stewart, A. F., andMuller, R. (2005) Heterologous expression of a myxobacterial naturalproducts assembly line in pseudomonads via Red/ET recombineering.Chem. Biol. 12, 349−356.(10) Chai, Y., Shan, S., Weissman, K. J., Hu, S., Zhang, Y., and Muller,R. (2012) Heterologous expression and genetic engineering of thetubulysin biosynthetic gene cluster using Red/ET recombineering andinactivation mutagenesis. Chem. Biol. 19, 361−371.(11) Fu, J., Wenzel, S. C., Perlova, O., Wang, J., Gross, F., Tang, Z.,Yin, Y., Stewart, A. F., Muller, R., and Zhang, Y. (2008) Efficienttransfer of two large secondary metabolite pathway gene clusters intoheterologous hosts by transposition. Nucleic Acids Res. 36, e113.(12) Binz, T. M., Wenzel, S. C., Schnell, H.-J., Bechthold, A., andMuller, R. (2008) Heterologous expression and genetic engineering ofthe phenalinolactone biosynthetic gene cluster by using Red/ETrecombineering. ChemBioChem 9, 447−454.(13) Fu, J., Bian, X., Hu, S., Wang, H., Huang, F., Seibert, P. M.,Plaza, A., Xia, L., Muller, R., Stewart, A. F., and Zhang, Y. (2012) Full-length RecE enhances linear-linear homologous recombination andfacilitates direct cloning for bioprospecting. Nat. Biotechnol. 30, 440−446.(14) Bian, X., Huang, F., Stewart, A. F., Xia, L., Zhang, Y., and Muller,R. (2012) Direct cloning, genetic engineering and heterologousexpression of the syringolin biosynthetic gene cluster in E. coli throughRed/ET recombineering. ChemBioChem 13, 1946−1952.(15) Schmidt, E., Nelson, J., Rasko, D. A., Sudek, S., Eisen, J. A.,Haygood, M. G., and Ravel, J. (2005) Patellamide A and Cbiosynthesis by a microcin-like pathway in Prochloron didemni, thecyanobacterial symbiont of Lissoclinum patella. Proc. Natl. Acad. Sci.U.S.A. 102, 7315−7320.(16) Long, P., Dunlap, W., Battershill, C. N., and Jaspars, M. (2005)Shotgun cloning and heterologous expression of the patellamide genecluster as a strategy to achieving sustained metabolite production.ChemBioChem 6, 1760−1765.(17) Ziemert, N., Ishida, K., Quillardet, P., Bouchier, C., Hertweck,C., de Marsac, N. T., and Dittmann, E. (2008) Microcyclamidebiosynthesis in two strains of Microcystis aeruginosa: from structure togenes and vice versa. Appl. Environ. Microbiol. 74, 1791−1797.(18) Ziemert, N., Ishida, K., Weiz, A., Hertweck, C., and Dittmann, E.(2010) Exploiting the natural diversity of microviridin gene clusters fordiscovery of novel tricyclic depsipeptides. Appl. Environ. Microbiol. 76,3568−3574.(19) Muenchhoff, J., Siddiqui, K. S., and Neilan, B. A. (2012)Identification of two residues essential for the stringent substrate

ACS Chemical Biology Letters

dx.doi.org/10.1021/cb400189j | ACS Chem. Biol. XXXX, XXX, XXX−XXXE

specificity and active site stability of the prokaryotic L-arginine:glycineamidinotransferase CyrA. FEBS J. 279, 805−815.(20) Muenchhoff, J., Siddiqui, K. S., Poljak, A., Raftery, M. J., Barrow,K. D., and Neilan, B. A. (2010) A novel prokaryotic L-arginine:glycineamidinotransferase is involved in cylindrospermopsin biosynthesis.FEBS J. 277, 3844−3860.(21) Huynh, M., Elston, M., Hernandez, N. M., Ball, D. B., Kajiyama,S.-i., Irie, K., Gerwick, W. H., and Edwards, D. J. (2010) Enzymaticproduction of (−)-indolactam V by LtxB, a cytochrome P450monooxygenase. J. Nat. Prod. 73, 71−74.(22) Edwards, D., and Gerwick, W. (2004) Lyngbyatoxin biosyn-thesis: sequence of biosynthetic gene cluster and identification of anovel aromatic prenyltransferase. J. Am. Chem. Soc. 126, 11432−11433.(23) Kim, E. J., Lee, J. H., Choi, H., Pereira, A. R., Ban, Y.-H., Yoo, Y.J., Kim, E., Park, J. W., Sherman, D. H., Gerwick, W. H., and Yoon, Y.J. (2012) Heterologous production of 4-O-demethylbarbamide, amarine cyanobacterial natural product. Org. Lett. 14, 5824−5827.(24) Mutka, S. C., Carney, J. R., Liu, Y., and Kennedy, J. (2006)Heterologous production of epothilone C and D in Escherichia coli.Biochemistry 45, 1321−1330.(25) Menzella, H. G., Reisinger, S. J., Welch, M., Kealey, J. T.,Kennedy, J., Reid, R., Tran, C. Q., and Santi, D. V. (2005) Redesign,synthesis and functional expression of the 6-deoxyerythronolide Bpolyketide synthase gene cluster. J. Ind. Microbiol. Biotechnol. 33, 22−28.(26) Basu, A., Kozikowski, A. P., and Lazo, J. S. (1992) Structuralrequirements of lyngbyatoxin A for activation and downregulation ofprotein kinase C. Biochemistry 31, 3824−3830.(27) Williams, R. M., Stocking, E. M., and Sanz-Cervera, J. F. (2000)Biosynthesis of prenylated alkaloids derived from tryptophan. Top.Curr. Chem. 209, 97−173.(28) Chen, S., Borowiak, M., Fox, J. L., Maehr, R., Osafune, K.,Davidow, L., Lam, K., Peng, L. F., Schreiber, S. L., Rubin, L. L., andMelton, D. (2009) A small molecule that directs differentiation ofhuman ESCs into the pancreatic lineage. Nat. Chem. Biol. 5, 258−265.(29) Nakagawa, Y. (2012) Artificial analogs of naturally occurringtumor promoters as biochemical tools and therapeutic leads. Biosci.Biotechnol. Biochem. 76, 1262−1274.(30) Nelson, T. J., and Alkon, D. L. (2009) Neuroprotective versustumorigenic protein kinase C activators. Trends Biochem. Sci. 34, 136−145.(31) Cardellina, J. H., Marner, F. J., and Moore, R. E. (1979)Seaweed dermatitis: structure of lyngbyatoxin A. Science 204, 193−195.(32) Aimi, N., Odaka, H., Sakai, S., Fujiki, H., Suganuma, M., Moore,R. E., and Patterson, G. M. (1990) Lyngbyatoxins B and C, two newirritants from Lyngbya majuscula. J. Nat. Prod. 53, 1593−1596.(33) Engene, N., Rottacker, E. C., Kastovsky, J., Byrum, T., Choi, H.,Ellisman, M. H., Komarek, J., and Gerwick, W. H. (2012) Mooreaproducens gen. nov., sp. nov. and Moorea bouillonii comb. nov., tropicalmarine cyanobacteria rich in bioactive secondary metabolites. Int. J.Syst. Evol. Microbiol. 62, 1171−1178.(34) Read, J. A., and Walsh, C. T. (2007) The lyngbyatoxinbiosynthetic assembly line: chain release by four-electron reduction ofa dipeptidyl thioester to the corresponding alcohol. J. Am. Chem. Soc.129, 15762−15763.(35) Jones, A. C., Ottilie, S., Eustaquio, A. S., Edwards, D. J., Gerwick,L., Moore, B. S., and Gerwick, W. H. (2012) Evaluation of Streptomycescoelicolor A3(2) as a heterologous expression host for thecyanobacterial protein kinase C activator lyngbyatoxin A. FEBS J.279, 1243−1251.(36) Lightfield, J., Fram, N. R., and Ely, B. (2011) Across bacterialphyla, distantly-related genomes with similar genomic GC contenthave similar patterns of amino acid usage. PLoS One 6, e17677.(37) Jones, A. C., Gerwick, L., Gonzalez, D., Dorrestein, P. C., andGerwick, W. H. (2009) Transcriptional analysis of the jamaicamidegene cluster from the marine cyanobacterium Lyngbya majuscula andidentification of possible regulatory proteins. BMC Microbiol. 9, 247.(38) Gaitatzis, N., Hans, A., Muller, R., and Beyer, S. (2001) ThemtaA gene of the myxothiazol biosynthetic gene cluster from

Stigmatella aurantiaca DW4/3−1 encodes a phosphopantetheinyltransferase that activates polyketide synthases and polypeptidesynthetases. J. Biochem. 129, 119−124.(39) Watanabe, K., Hotta, K., Praseuth, A. P., Koketsu, K., Boddy, C.N., Wang, C. C. C., Oguri, H., and Oikawa, H. (2006) Totalbiosynthesis of antitumor nonribosomal peptides in Escherichia coli.Nat. Chem. Biol. 2, 423−428.(40) Endo, Y., Shudo, K., Itai, A., Hasegawa, M., and Sakai, S.-i.(1986) Synthesis and stereochemistry of indolactam-V, an activeragnent of teleocidins. Structural requirements for tumor-promotingactivity. Tetrahedron 42, 5905−5924.(41) Izumikawa, M., Khan, S. T., Komaki, H., Takagi, M., and Shin-ya, K. (2010) JBIR-31, a new teleocidin analog, produced by salt-requiring Streptomyces sp. NBRC 105896 isolated from a marinesponge. J. Antibiot. 63, 33−36.(42) Irie, K., Hirota, M., Hagiwara, N., Koshimizu, K., Hayashi, H.,Murao, S., Tokuda, H., and Ito, Y. (1984) The Epstein-Barr virus earlyantigen inducing indole alkaloids, (-)-indolactam V and its relatedcompounds produced by Actinomyces. Agric. Biol. Chem. 48, 1269−1274.(43) Reiling, K. K., Yoshikuni, Y., Martin, V. J. J., Newman, J.,Bohlmann, J., and Keasling, J. D. (2004) Mono and diterpeneproduction in Escherichia coli. Biotechnol. Bioeng. 87, 200−212.(44) Martin, V. J. J., Pitera, D. J., Withers, S. T., Newman, J. D., andKeasling, J. D. (2003) Engineering a mevalonate pathway inEscherichia coli for production of terpenoids. Nat. Biotechnol. 21,796−802.(45) Lai, J., Koglin, A., and Walsh, C. (2006) Carrier proteinstructure and recognition in polyketide and nonribosomal peptidebiosynthesis. Biochemistry 45, 14869−14879.(46) Han, J. W., Kim, E. Y., Lee, J. M., Kim, Y. S., Bang, E., and Kim,B. S. (2012) Site-directed modification of the adenylation domain ofthe fusaricidin nonribosomal peptide synthetase for enhancedproduction of fusaricidin analogs. Biotechnol. Lett. 34, 1327−1334.(47) Villiers, B., and Hollfelder, F. (2011) Directed evolution of agatekeeper domain in nonribosomal peptide synthesis. Chem. Biol. 18,1290−1299.

ACS Chemical Biology Letters

dx.doi.org/10.1021/cb400189j | ACS Chem. Biol. XXXX, XXX, XXX−XXXF