Discovery of Gene Cluster for Mycosporine-Like Amino AcidBiosynthesis from Actinomycetales Microorganisms and Production ofa Novel Mycosporine-Like Amino Acid by Heterologous Expression

Kiyoko T. Miyamoto, Mamoru Komatsu, Haruo Ikeda

Kitasato Institute for Life Sciences, Kitasato University, Sagamihara, Kanagawa, Japan

Mycosporines and mycosporine-like amino acids (MAAs), including shinorine (mycosporine-glycine-serine) and porphyra-334 (my-cosporine-glycine-threonine), are UV-absorbing compounds produced by cyanobacteria, fungi, and marine micro- and macroalgae.These MAAs have the ability to protect these organisms from damage by environmental UV radiation. Although no reports have de-scribed the production of MAAs and the corresponding genes involved in MAA biosynthesis from Gram-positive bacteria to date, ge-nome mining of the Gram-positive bacterial database revealed that two microorganisms belonging to the order Actinomycetales, Acti-nosynnema mirum DSM 43827 and Pseudonocardia sp. strain P1, possess a gene cluster homologous to the biosynthetic geneclusters identified from cyanobacteria. When the two strains were grown in liquid culture, Pseudonocardia sp. accumulated avery small amount of MAA-like compound in a medium-dependent manner, whereas A. mirum did not produce MAAs underany culture conditions, indicating that the biosynthetic gene cluster of A. mirum was in a cryptic state in this microorganism. Inorder to characterize these biosynthetic gene clusters, each biosynthetic gene cluster was heterologously expressed in an engi-neered host, Streptomyces avermitilis SUKA22. Since the resultant transformants carrying the entire biosynthetic gene clustercontrolled by an alternative promoter produced mainly shinorine, this is the first confirmation of a biosynthetic gene cluster forMAA from Gram-positive bacteria. Furthermore, S. avermitilis SUKA22 transformants carrying the biosynthetic gene cluster forMAA of A. mirum accumulated not only shinorine and porphyra-334 but also a novel MAA. Structure elucidation revealed thatthe novel MAA is mycosporine-glycine-alanine, which substitutes L-alanine for the L-serine of shinorine.

Mycosporines and mycosporine-like amino acids (MAAs) aresmall (�400-Da), colorless, and water-soluble molecules

characterized by their ability to absorb UV (1, 2). They have ab-sorption maxima ranging from 310 to 365 nm with high molarextinction coefficients (ε � 28,100 to 50,000 M�1 cm�1). Myco-sporines contain a cyclohexenone ring conjugated with the nitro-gen substituent of an amino acid or an imino alcohol, while MAAshave a cyclohexenimine ring conjugated with two such substitu-ents (Fig. 1). These UV-absorbing molecules are known to beproduced by a wide range of organisms, including cyanobacteria,fungi, and marine micro- and macroalgae (3–5). To date, morethan 30 different compounds in the mycosporine family havebeen identified from natural sources (1). The physiological role ofmycosporines and MAAs as sunscreen compounds to protectagainst environmental UV radiation is well established in the pro-ducing organisms and the organism that ingests and accumulatesMAAs (5, 6). In addition to their photoprotective function, my-cosporines and MAAs appear to play important roles as antioxi-dant molecules, compatible solutes, and an intracellular nitrogenreservoir and also have roles in defense against thermal or desic-cation stress and in fungal reproduction (1, 4). Moreover, myco-sporines and MAAs have attracted attention in the field of cosmet-ics and skin care due to their effective UV-filtering capacity andability to prevent UV-induced skin damage (7).

Recently, the main steps in the biosynthesis of MAAs and theirgenetic basis in cyanobacteria were elucidated. A gene cluster withfour genes ranging from ava_3855 to ava_3858 in cyanobacteriumAnabaena variabilis ATCC 29413 (Fig. 2) was found to be respon-sible for the biosynthesis of an MAA, shinorine (Fig. 1) (8). Thegene product of ava_3858, 4-deoxygadusol (DDG) synthase, cat-alyzes the conversion of sedoheputulose-7-phosphte, an interme-

diate of the pentose phosphate pathway, to demethyl 4-deoxyga-dusol (DDG), and DDG is converted to 4-deoxygadusol (4-DG;Fig. 1) by catalyzing Ava_3857, O-methyltransferase (O-MT). Theava_3856 gene encodes ATP-grasp family protein catalyzing theaddition of glycine to 4-DG to form mycosporine-glycine (Fig. 1).A nonribosomal peptide synthetase (NRPS) homolog encoded byava_3855 attaches serine to mycosporine-glycine to generate shi-norine. However, the enzyme involved in the last biosynthetic stepof shinorine in cyanobacterium Nostoc punctiforme ATCC 29133was demonstrated to be a D-alanine (D-Ala) D-Ala ligase-like pro-tein, NpF5597 (Fig. 2) (9). NpF5597 is responsible for the conden-sation of serine onto mycosporine-glycine, which is generated byDDG synthase (NpR5600), O-MT (NpR5599), and the ATP-grasp family protein (NpR5598) in N. punctiforme. Hence, the firstthree enzymes in the biosynthetic pathway of shinorine are con-served in these two cyanobacteria, while the last enzyme is eitheran NRPS homolog or a D-Ala D-Ala ligase-like protein. In thefungal genome, the genes encoding the homologues of the firstthree enzymes in shinorine biosynthesis are present and located ina cluster, but homologs for NRPS-like protein and D-Ala D-Ala

Received 3 March 2014 Accepted 4 June 2014

Published ahead of print 6 June 2014

Editor: H. Nojiri

Address correspondence to Haruo Ikeda, ikeda@ls.kitasato-u.ac.jp.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.00727-14.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.00727-14

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ligase-like protein are missing (Fig. 2). This genetic context is con-sistent with the fact that the only fungal products reported so farare mycosporines (1).

In the present study, we found gene clusters for biosynthesis ofMAAs from two Actinomycetales microorganisms, Actinosynnemamirum DSM 43827 (10) and Pseudonocardia sp. strain P1 (11). Herewe characterize the MAA biosynthetic gene clusters of the two microor-ganisms through heterologous expression using an engineered Strepto-myces host suitable for secondary metabolite production.

MATERIALS AND METHODSBacterial strains and plasmid vectors. Actinosynnema mirum DSM 43827was obtained from the RIKEN Bioresource Center (Wako, Japan), andPseudonocardia sp. P1 was kindly donated by Matt I. Hutchings of theUniversity of East Anglia, United Kingdom. Streptomyces avermitilisSUKA22 (12) was used as the host for heterologous expression of thebiosynthetic gene cluster. A pRED small vector (13) was used for in vivocloning of the DNA fragment containing the gene cluster for biosynthesisof MAAs. An integrating vector, pKU492Aaac(3)IV (12), was used forsubcloning the gene clusters for MAA biosynthesis. A plasmid, pKU1021(pKU460::rpsJp; see reference 14), was used as a template DNA for theamplification of a constitutively expressed promoter of the S. avermitilisrpsJ gene encoding ribosomal protein S10. Unmethylated recombinant

FIG 1 Structures of representative mycosporines (mycosporine-glycine) andMAAs (shinorine and porphyra-334) and their biosynthetic intermediate,4-deoxygadusol.

FIG 2 Genes involving biosynthesis of mycosporines and MAAs in Actinobacteria and other organisms. A. variabilis ATCC 29413 and N. punctiforme ATCC 29133are known producers of MAAs. Each gene cluster, ranging from ava_3855 to ava_3858 in A. variabilis ATCC 29413 and ranging from npun_F5597 to npun_R5600in N. punctiforme ATCC 29133, has been demonstrated to be both necessary and sufficient for shinorine synthesis (reference 8). The genes or gene clustershomologous to the shinorine biosynthetic genes in cyanobacteria were present in genomes of fungi, cnidarians, and dinoflagellata. The representative genes orgene clusters in each organism are shown together with the putative gene cluster for MAA production in two Actinomycetales microorganisms. The O-MT-encoding gene in Aspergillus nidulans FGSC A4 is fused to the gene encoding the ATP-grasp family protein, and the DDG synthase-encoding gene in Heterocapsatriquetra is fused to the O-MT-encoding gene.

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plasmid DNAs were prepared in Escherichia coli GM2929 hsdS::Tn10 (13)and were introduced into S. avermitilis SUKA22 using polyethylene gly-col-assisted transformation (13).

Cloning of the entire gene cluster for MAA biosynthesis. To clonethe entire gene cluster for mycosporine-like amino acid (MAA) biosyn-thesis from A. mirum DSM 43827 and Pseudonocardia sp. P1, each chro-mosomal DNA of these microorganisms was digested with SfiI and MluI,respectively. No restriction sites were located inside the entire gene clus-ter. Fragments containing 6.3 kb (A. mirum DSM 43827) or 8.2 kb(Pseudonocardia sp. P1) were purified by agarose gel electrophoresis. Lin-earized cloning vector was prepared by PCR amplification with pRED as atemplate DNA using the designated primer pair containing both 41-nu-cleotide (nt) and 50-nt sequences corresponding to upstream and down-stream regions of the biosynthetic gene cluster. A primer pair, forwardprimer 5=-TCAGGTCAGCGGAATGCCCTCCCGCACTTCCAGCCCCTGGAAGCTTTGCCAGGAAGATACTTAACAG-3= (underlined, itali-cized, and bold characters indicate the downstream region of amir_4256,the HindIII site, and the region of vector pRED, respectively) and reverseprimer 5=-TCAGCGCGCGGCGAGGCCGTTCTGATAGGCCCAGACCACGGAATTCCCATTCATCCGCTTATTATC-3= (underlined, italicized,and bold characters indicate the upstream region of amir_4259, the EcoRIsite, and the region of vector pRED, respectively), was used for in vivocloning of a gene cluster from A. mirum DSM 43827. Another primer pair,forward primer 5=-CTCGACCAACGTTCCGATTGAGCCGAACAGTAGAGCGGGCATCGGGTTTCGAATTCTGCCAGGAAGATACTTAACAG-3= (underlined, italicized, and bold characters indicate the upstreamregion of pseP1_01010031440, the EcoRI site, and the region of vectorpRED, respectively) and reverse primer 5=-GACCGGTCGTAGAACGGTG T G A T C C A T C G T T C C A T T C T G C G T G C A C C C T G A A G C T TCCATTCATCCGCTTATTATC-3= (underlined, italicized, and boldcharacters indicate the downstream region of pseP1_010100031425, theHindIII site, and the region of vector pRED, respectively), was used for thecloning of a gene cluster from Pseudonocardia sp. P1. Initial denaturationat 96°C for 180 s was followed by 5 cycles of amplification (at 95°C for 30s, 50°C for 30 s, and 72°C for 100 s) and 25 cycles (at 95°C for 30 s and 68°Cfor 100 s) and then final incubation at 72°C for 5 min using an ExpandHigh Fidelity PCR system (Roche Diagnostics, Tokyo, Japan) or PhusionDNA polymerase (New England BioLabs, MA). After amplification, thereaction mixture was treated with DpnI to remove template DNA. Each1.7-kb amplicon of pRED carrying upstream and downstream sequences ofthe gene cluster was cotransformed into L-arabinose-induced E. coliBW25141 carrying pKD46 (13) with size-fractionated chromosomalDNA of SfiI-cut A. mirum DSM 43827 or MluI-cut Pseudonocardia sp. P1by electroporation. The desired plasmid, pRED, carrying the entire bio-synthetic gene cluster, was obtained by selection with 30 �g/ml chloram-phenicol and was also confirmed by restriction digestion. The largeEcoRI/HindIII fragment of each recombinant plasmid, pRED, carryingthe entire biosynthetic gene cluster, was ligated with the large fragment ofHindIII/EcoRI-pKU492Aaac(3)IV to generate pKU492Aaac(3)IV::amir_4256 – 4259 (pKU492Aaac(3)IV::mys_cluster-amir) and pKU492Aaac(3)IV::pseP1_0101003125– 0101003140 (pKU492Aaac(3)IV::mys_cluster-pse, respectively.

Introduction of the alternative promoter to express the biosyntheticgene cluster. To introduce an alternative promoter upstream of the bio-synthetic gene cluster of A. mirum DSM 43827 and Pseudonocardia sp. P1,a segment containing the promoter of rpsJ encoding ribosomal proteinS10 of S. avermitilis and aac(3)I encoding an aminoglycoside N-acetyl-transferase of E. coli was prepared by amplification with pKU1021 as atemplate DNA using the designated primer pair containing 44-nt se-quences corresponding to the N-terminal region of the first gene in thecluster and the upstream region of the cloning site in pKU492Aaac(3)IV.A primer pair, forward-Amir primer 5=-TCGTTCTCCGTGGCGGTGACCGTCGCGGTGAGGTTCGTCGTCATATGTACTCAGTAGTCCTTCGTCTC-3= (underlined, italicized, and bold characters indicate N-terminalregion of amir_4259, the start codon, and the region of rpsJ promoter of S.

avermitilis with the ribosomal binding site, respectively) and universalreverse primer 5=-AGCAGCCCTTGCGCCCTGAGTGCTTGCGGCAGCGTGAAGCTAGCGATCTCGGCTTGAACGAATTG-3= (underlined andbold characters indicate the upstream region of cloning site inpKU492Aaac(3)IV and the region of aac(3)I, respectively), was used forintroduction of rpsJ promoter upstream of the gene cluster of A. mirumDSM 43827. Another primer pair, forward-Pse primer 5=-TCCCAACTCTCGACACGGAACTCCGTATCGGTCGCGCTGAGCATATGTACTCAGTAGTCCTTCGTCTC-3= (underlined, italicized, and bold charactersindicate N-terminal region of pseP1_010100031440, the translational startcodon, and the region of rpsJ promoter of S. avermitilis with the ribosomalbinding site, respectively) and the universal reverse primer describedabove, was used for the introduction of the rpsJ promoter upstream of thegene cluster of Pseudonocardia sp. P1. Initial denaturation (96°C for 180 s)was followed by 5 cycles of amplification (at 95°C for 30 s, 50°C for 30 s,and 72°C for 100 s) and 25 cycles (at 95°C for 30 s and 68°C for 60 s) andthen final incubation at 72°C for 5 min. All amplicons were treated withDpnI to remove the template DNA. The amplicon containing rpsJ pro-moter and a resistance marker, aac(3)I, generated using forward-Amir anduniversal-reverse primers, was cotransformed into L-arabinose-induced E.coli BW25141 carrying pKD46 with EcoRI-cut pKU492Aaac(3)IV::mys_cluster-amir. Another amplicon generated using forward-Pse and univer-sal-reverse primers was also cotransformed into L-arabinose-induced E.coli BW25141 (pKD46) with EcoRI-cut pKU492Aaac(3)IV::mys_cluster-pse. The desired plasmids (expression of the biosynthetic gene cluster wascontrolled by the rpsJ promoter) was obtained by selection with 25 �g/mlapramycin [aac(3)IV] and 25 �g/ml fortimicin [aac(3)I] and was con-firmed by restriction digestion. Two recombinant plasmids,pKU492Aaac(3)IV::rpsJp-mys_cluster-amir and pKU492Aaac(3)IV::rpsJp-mys_cluster-pse, were used for heterologous expression in S. aver-mitilis SUKA22.

Detection of MAAs from actinomycete strains. A. mirum DSM 4382,Pseudonocardia sp. P1, and S. avermitilis SUKA22 transformants weregrown in the vegetative medium (15) at 30°C for 2 days on a reciprocalshaker (200 strokes per min). A 0.1-ml portion of the vegetative culturewas inoculated with 10 ml synthetic production medium (15) in a 125-mlflask. A. mirum DSM 4382 and Pseudonocardia sp. P1 were further exam-ined in the following media: TSB (Trypticase soy broth), YMG (4 g yeastextract–10 g malt extract– 4 g glucose–1 liter deionized water adjusted topH 7.0 with 2 N KOH), and R4 (16). After incubation at 28°C for 8 days ona rotary shaker at 200 rpm, the whole culture was mixed with an equalvolume of methanol and the mixture was subjected to a vigorous vortexprocedure. The supernatant was collected by centrifugation at 3,000 rpmfor 10 min, and a 2-�l portion of the supernatant was directly analyzed byhigh-performance liquid chromatography (HPLC)–time-of-flight massspectrometry (TofMS) (Acquity ultraperformance LC system, WatersXevo G2-S Tof). When necessary, the supernatant was evaporated to re-move methanol and lyophilized to dissolve the residue in 1 ml waterbefore HPLC-TofMS analysis. Analytical conditions for HPLC were asfollows: column, Hypercarb (3 �m pore size; 2.1-mm inner diameter by100 mm); mobile phase, 8% acetonitrile– 0.1 M triethylammonium ace-tate (TEAA; pH 7.0) for 4 min, 8% to 15% acetonitrile– 0.1 M TEAA (pH7.0) for 6 min; flow rate, 0.2 ml/min; detection at 330 nm. Authenticsamples of shinorine and porphyra-334 were prepared from Helioguard365 (7). Mass spectrometry was performed in resolution mode under thefollowing conditions: capillary voltage of 3.0 kV in positive-ion mode or2.5 kV in negative-ion mode; cone voltage of 40 V; source temperature of120°C; desolvation gas flow of 800 liters/h at a temperature of 450°C; andcone gas flow of 50 liters/h. For accurate mass measurements, lock masscalibration was conducted using leucine enkephalin. Mass data were ac-quired with collision cell energy alternating between low (6 V) and ele-vated (ramping from 20 to 40 V).

Purification and structural elucidation of novel MAA compound. A70-ml sample of the vegetative culture of S. avermitilis SUKA22 carryingpKU492Aaac(3)IV::rpsJp-mys_cluster-amir was inoculated into 7 liters of

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production medium containing 400 mM NH4Cl in a 10-liter fermenter(28°C, 200 rpm, 3.5 liters/min airflow). After cultivation for 7 days, themycelium was collected by filtration, washed with tap water, and extractedwith 600 ml methanol. After removal of mycelium by filtration, the meth-anol extract was concentrated under reduced pressure to remove metha-nol. The aqueous concentrate (ca. 80 ml) was diluted with water (�400ml) and extracted with an equal volume of dichloromethane to removesolvent-soluble impurities. The aqueous layer was lyophilized, and theresidue was dissolved in a small amount of methanol to remove salts. Thesupernatant was concentrated to dryness, and the residue was dissolved inwater. After removal of insoluble materials by suction, the clarified solu-tion was subjected to a column containing 150 ml cation-exchange resinAmberlite IR120B [H�] (Organo Corporation, Japan). After the columnwas washed with 1,000 ml deionized water to elute shinorine, novel MAAwas eluted with 300 ml of 0.5 N HCl. The eluate was neutralized by theaddition of solid sodium bicarbonate, followed by lyophilization. Theresulting white residue was dissolved in a small volume of methanol toremove NaCl, and the methanol-soluble material was subjected to pre-parative HPLC on a Hypercarb column (5 �m pore size; 10-mm innerdiameter by 150 mm) developed with 8% (vol/vol) acetonitrile– 0.092 MTEAA (pH 7.0) at a flow rate of 2.5 ml/min and detected at 330 nm. Thefractions containing novel compound eluted at 24.5 to 30.0 min werecombined and evaporated under conditions of reduced pressure to re-move acetonitrile before lyophilization. The resulting residue was redis-solved in water, and novel MAA was adsorbed by activated charcoal (50ml) to remove TEAA. After washing the charcoal with 100 ml water and100 ml of 80% (vol/vol) methanol, the novel MAA compound was elutedwith 1 liter of methanol. The methanol eluate was evaporated to dryness,and 4.1 mg novel MAA was obtained. The high-resolution mass spectrum(FAB) of the novel MAA compound was obtained on a Jeol JMS-700system. Nuclear magnetic resonance (NMR) (1H, 500 MHz; 13C, 125MHz) spectra were obtained using a Jeol JNM-ECP 500 FT NMR system.Chemical shifts are reported on the � scale relative to the residual solventsignal (CD3OD; �H 3.30, �C 49.0).

Determination of the absolute configurations of amino acids innovel MAA. Novel MAA (0.5 mg) was hydrolyzed in 0.4 ml of 6 N HCl at120°C for 14 h. After the reaction mixture was concentrated in vacuo, theresidue was dissolved in 200 �l of 0.1 M sodium bicarbonate containing0.2 mg N-(5-fluoro-2,4-dinitrophenyl)-L-alaninamide (FDAA) and wasincubated at 75°C for 30 min to prepare FDAA derivatives. AuthenticFDAA derivatives were also prepared from L-Ala and D-Ala by the sameprocedures. After the reaction was quenched by the addition of 20 �l of 1N HCl, the reaction mixture was analyzed by HPLC-TofMS. Analyticalconditions for HPLC were as follows: column, Acquity UPLC BEH C18

(1.7 �m pore size; 2.1-mm inner diameter by 50 mm); mobile phase, 10%to 80% acetonitrile– 0.05% formic acid for 10 min; flow rate, 0.3 ml/min;detection at 415 nm. Mass spectrometry was performed in positive-ionmode as described above.

RESULTSBioinformatic analysis of MAA biosynthetic gene cluster in Ac-tinomycetales. A homology search for a gene cluster for shino-

rine biosynthesis of A. variabilis ATCC 29413 against publicdatabases identified similar gene clusters from over 30 cyano-bacterial genomes and over 40 fungal genomes. The corre-sponding homologs were also found in the genome of coralAcropora digitifera (17), sea anemone Nematostella vectensis(18), and red alga Chondrus crispus (19) and in the expressedsequence tag database of some dinoflagellates (Fig. 2) (20). Theoccurrence of predicted biosynthetic genes for MAAs indicatesthat the genes are present in organisms that are usually exposedto sunlight, such as photosynthetic cyanobacteria and marineorganisms from shallow-water environments. Further genomemining in the Gram-positive bacterial database revealed thattwo microorganisms in the order Actinomycetales, Actinosyn-nema mirum DSM 43827 and Pseudonocardia sp. P1, possess thegene cluster, whose structure is similar to that of the shinorinebiosynthetic genes identified in cyanobacteria. The biosyntheticgene clusters in A. mirum (amir_4256 – 4259) and in Pseudonocar-dia sp. P1 (pseP1_010100031425– 010100031440) are composed offour genes encoding putative dimethyl 4-deoxygadusol (DDG)synthase, O-methyltransferase (O-MT), ATP-grasp family pro-tein, and D-Ala D-Ala ligase homolog (Fig. 2). Three gene productsof A. mirum DSM 43827 (Amir_4259–Amir_4257) and Pseudonocar-dia sp. P1 (PseP1_010100031440 –PseP1_010100031430) werequite similar to Ava_3858 –Ava_3856 of A. variabilis ATCC29413 (Table 1). The gene products of amir_4256 and pseP1_010100031425 did not show significant homology to Ava_3855(nonribosomal peptide synthetase), but these gene products,Amir_4256 and PseP1_010100031425, showed significant simi-larity to D-Ala D-Ala ligase-like protein of N. punctiforme ATCC29133 (Table 1). The first gene, amir_4259 (mysA), in an operon ofA. mirum DSM 43827, is translationally coupled to the down-stream amir_4258 (mysB) gene. Interestingly, the first three genesof Pseudonocardia sp. P1, mysA, mysB, and mysC, were transla-tionally coupled to the downstream gene. It is interesting that twobacteria that have no photosynthesis ability and live in terrestrialenvironments have the potential to produce MAAs. So far, noreports have described the correlation between the production ofMAAs and corresponding genes involving biosynthesis of MAAsfrom Gram-positive bacteria.

To examine the ability to produce MAAs in A. mirum DSM43827 and Pseudonocardia sp. P1, these two microorganisms weregrown in several liquid media. HPLC analysis of methanol extractsfrom Pseudonocardia sp. P1 grown in TSB medium indicated thepresence of a very small amount of MAA-like compound thatexhibited absorbance spectra characteristic for MAAs and whoseretention time was identical to that of shinorine, whereas no suchcompound was detected in the extract from Pseudonocardia sp. P1

TABLE 1 Deduced functions of ORFs in the biosynthetic gene cluster for shinorine of Actinosynnema mirum DSM 43827 and Pseudonocardia sp. P1

Gene

Actinosynnema mirum DSM 43827 Pseudonocardia sp. P1

Predicted functionORF aaa

Identity/similarity (%)b ORF aaa

Identity/similarity (%)b

mysA Amir_4259 406 62/76 PseP1_010100031440 415 58/74 Dimethyl 4-deoxygadusol (DDG) synthasemysB Amir_4258 284 52/64 PseP1_010100031435 261 51/67 O-Methyltransferase (O-MT)mysC Amir_4257 429 52/68 PseP1_010100031430 470 53/68 ATP-grasp family proteinmysD Amir_4256 339 50/66c PseP1_010100031425 346 52/72c

D-Ala D-Ala ligase homologa Data represent numbers of amino acids (aa).b Homolog to ORFs (Ava_3856 to Ava_3858) of Anabaena variabilis ATCC 29413 (MysA to Ava_3858 [410 aa], MysB to Ava_3857 [279 aa], and MysC to Ava_3856 [458 aa]).c Homology to NpF5597 (348 aa) of Nostoc punctiforme ATCC 29133.

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grown in the other media (Fig. 3B). With respect to A. mirumDSM 43827, MAA-like compound was not accumulated underany of the medium conditions (Fig. 3A), suggesting that the puta-tive MAA biosynthetic gene cluster might be cryptic in A. mirumDSM 43827. To elucidate the function of putative gene clusters forMAA biosynthesis in these two Actinomycetales microorganisms,we attempted to express each cluster in a heterologous host.

Heterologous expression of gene cluster for biosynthesis ofMAAs in Actinomycetales. To clone the entire gene cluster forMAA biosynthesis from A. mirum DSM 43827, we used in vivocloning technology mediated by -RED recombination. We pre-pared a PCR-amplified 1.7-kb pRED fragment, which contains ap15A origin and chloramphenicol resistance gene as well as 41-and 50-bp terminal homology arms upstream and downstream ofthe target gene cluster, respectively. After digestion of genomicDNA of A. mirum DSM 43827 with restriction enzyme to releasethe cluster on single DNA segment, we transformed E. coli carry-

ing the -RED recombination system with the digested genomicDNA segments and amplified pRED to generate pRED::mys_clus-ter-amir. Digestion of the resultant plasmid with a restriction en-zyme verified the direct cloning of the mys gene cluster fromgenomic DNA of A. mirum DSM 43827 into the pRED vector.After subcloning of the entire gene cluster into an integratingpKU492Aaac(3)IV vector, the strong and constitutively expressedpromoter of a gene encoding ribosomal protein S10 was insertedin front of the first gene of the operon by -RED recombination,yielding pKU492Aaac(3)IV::rpsJp::mys_cluster-amir. Followingthe same strategy as described above, a pKU492Aaac(3)IV::rpsJp::mys_cluster-pse plasmid controlled by the rpsJ promoter was con-structed. Then, each recombinant plasmid was introduced into S.avermitilis SUKA22, an engineered host suitable for heterologousexpression (12). The transformants of S. avermitilis SUKA22 weregrown in production medium, and methanol extracts of the wholeculture were directly analyzed by HPLC-TofMS using the positivemode. The transformant carrying the mys gene cluster of A. mirumDSM 43827 did not exhibit MAA-like compounds, while thetransformant carrying the mys gene cluster of A. mirum DSM43827 placed under the control of the alternative promoter, rpsJp,gave compound 1 (max � 333 nm, m/z 333.1288) and compound2 (max � 334 nm, m/z 347.1454) (Fig. 3A). Taken together withmass spectrometry fragmentation analysis using authentic sam-ples of shinorine and porphyra-334, compounds 1 and 2 werefound to be identical to shinorine and porphyra-334, respectively(see Fig. S1 in the supplemental material). Additionally, biosyn-thetic intermediates, 4-deoxygadusol and mycosporine-glycine,were also detected in the methanol extract (see Fig. S2 in the sup-plemental material) by TofMS using the negative-ion mode. Theyields in the methanol extract from three independent experi-ments cultivating transformants carrying pKU492Aaac(3)IV::rpsJp-mys_cluster-amir were 154 � 13.0 mg/liter shinorine and7.2 � 0.57 mg/ liter porphyra-334. These results demonstratedthat the cryptic mys cluster of A. mirum DSM 43827 was activatedby heterologous expression, which allowed us to determine thatthe cluster is responsible for MAA production. In addition to shi-norine and porphyra-334, HPLC-TofMS analysis of the methanolextract showed the unknown compound (i.e., compound 3),which exhibited an absorbance spectrum characteristic of MAAs,to have a cyclohexenimine chromophore (max � 333 nm) (Fig.3A and 4A). The mass spectrum of compound 3 showed a majorion at m/z 317.1334, indicating a molecular formula ofC13H21N2O7 (calculated mass, 317.1349), which is different thatof from any MAA described previously (Fig. 4B). Further investi-gation of compound 3 is described in a later section.

HPLC-TofMS analysis of the methanol extract of S. avermitilisSUKA22 transformants carrying the entire mys gene cluster ofPseudonocardia sp. P1 under the control of rpsJ promoter resolveda single peak (max � 333 nm, m/z 333.1288) (Fig. 3B). Compar-ative analysis using an authentic sample of shinorine revealed thatthe compound produced was identical to shinorine. The yield ofshinorine in these transformants was 380 � 33 �g/liter, which wasabout 3 times higher than that of the original Pseudonocardia sp.P1 strain. Taking the results together, the mys gene clusters of A.mirum DSM 43827 and Pseudonocardia sp. P1 were demonstratedto be responsible and sufficient for MAA synthesis.

Isolation and structure elucidation of the unknown MAAs.We were interested in the structure of compound 3 produced by S.avermitilis SUKA22 transformants carrying the entire biosyn-

FIG 3 Detection of MAAs from actinomycete strains and heterologous hostscarrying the biosynthetic gene cluster for MAA. (A) HPLC analysis of metha-nol extracts of the S. avermitilis SUKA22 transformants carrying the mys clus-ter of A. mirum DSM 43827 controlled by the alternative rpsJ promoter (line a),the mys cluster of A. mirum DSM 43827 controlled by the native promoter(line b) and vector plasmid pKU492Aaac(3)IV (line c), the methanol extract ofA. mirum DSM 43827 grown in TSB medium (line d), and the authentic sam-ple containing porphyra-334 and shinorine (line e). Methanol extracts from0.2 �l culture were injected in each analysis. (B) HPLC analysis of methanolextracts of S. avermitilis SUKA22 carrying the mys gene cluster of Pseudono-cardia sp. P1 controlled by the alternative rpsJ promoter (line f), the mys genecluster of Pseudonocardia sp. P1 with the native promoter (line g) and vectorplasmid pKU492Aaac(3)IV (line h), methanol extracts of Pseudonocardia sp.P1 grown in TSB medium (line i), production medium (line j), and YMGmedium (line k), and standard samples containing porphyra-334 and shino-rine (line l). Two microliters of 10-fold-diluted (lines a to e) and 10-fold-concentrated (lines f to l) methanol extracts was subjected to analysis using aHPLC-TofMS. Peaks on the chromatograms indicate shinorine (peak 1), por-phyra-334 (peak 2), and novel MAA (peak 3).

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thetic gene cluster for MAA of A. mirum DSM 43827 since thecompound was predicted to be a novel MAA. Before isolation andstructure elucidation of compound 3, we attempted to improve itsproductivity because its yield was less than 1 mg/liter. In cyano-bacteria (21), dinoflagellates (22), and algae (23), the accumula-tion of MAA is affected by environmental factors such as UV ra-diation, salinity, temperature, or ammonium ions (4). As theentire biosynthetic gene cluster for MAA heterologously ex-pressed in S. avermitilis SUKA22 is transcriptionally controlled bythe alternative promoter, responses to these environmental fac-tors were not expected in the heterologous host. However, duringthe course of improving productivity, we found that the additionof ammonium salts to the culture of transformants somehow in-duced the production of compound 3. The most marked increaseof compound 3 was observed in the culture supplemented with400 mM NH4Cl (Fig. 4C), which led to 9.6-fold-higher produc-tion after 8 days of incubation compared to the culture withoutNH4Cl. Intriguingly, the concentration of NH4Cl had no effect onthe accumulation of shinorine in the range of 0 to 400 mM. To testwhether the increased production of compound 3 resulted fromthe increased concentration of ammonium ions, osmotic stress, orincreased salinity caused by the addition of NH4Cl, the effect ofthese factors on the production of compound 3 was investigatedby the addition of (NH4)2SO4, NH4NO3, NaCl, and sucrose.When the transformant was grown in production medium con-taining 250 mM sodium chloride or 20% sucrose, compound 3was accumulated normally. In contrast, addition of 200 mM(NH4)2SO4 or 400 mM NH4NO3 to the culture of the transfor-mant improved the production of compound 3 to the same levelas the addition of 400 mM ammonium chloride. These resultssuggested that ammonium ion stimulates the accumulation ofcompound 3. Since there was little difference between the counter

ions of ammonium in the elevated levels of compound 3, we de-cided to supply 400 mM NH4Cl to the production medium for thesubsequent purification of compound 3.

To isolate compound 3, the transformant carrying pKU492Aaac(3)IV::rpsJp-mys_cluster-amir was grown in 7 liters of produc-tion medium containing 400 mM NH4Cl for 7 days. Compound 3was purified from the mycelia by cation-exchange chromatogra-phy on an Amberlite IR120B (H� type) and by preparative re-verse-phase HPLC. About four milligrams of white powder wasobtained and investigated by 1H and 13C NMR spectroscopy, in-cluding correlation spectroscopy (COSY), heteronuclear multi-ple-quantum correlation (HMQC), and heteronuclear multiple-bond correlation (HMBC), in comparison to shinorine (Fig. 5; seealso Tables S1 and S2 in the supplemental material). The 13C NMRdata obtained for compound 3 and shinorine revealed a goodcorrelation with previously published chemical shift data for shi-norine (8) and structurally similar MAAs (24, 25). The two-di-

FIG 4 Effect of ammonium ions on the production of novel MAA and shinorine. (A and B) Absorption spectrum (A) and MS fragmentation pattern (B) ofcompound 3 produced by S. avermitilis SUKA22 transformants carrying pKU492Aaac(3)IV::rpsJp-mys_cluster-amir. (C) The production of compound 3 andshinorine in the transformants carrying pKU492Aaac(3)IV::rpsJp-mys_cluster-amir grown for 2, 5, and 8 days in production medium supplemented with threedifferent concentrations of NH4Cl. The experiments were repeated three times with similar results. AU, absorbance units.

FIG 5 Structure of compound 3 (mycosporine-glycine-alanine). The rightpanel shows COSY (bold line) and HMBC (arrow) data for compound 3.Double arrows indicate a cross peak in HMBC.

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mensional (2D) NMR analyses revealed the common substruc-ture of the imino-mycosporine ring (C-1 to C-10) for compound3. The remaining substructure was elucidated as follows. 1H and13C NMR data for compound 3 showed the presence of a methylgroup (�C-13 19.9, �H-13 1.53), which is not present in shinorine.1H-1H coupling between the methyl protons and a methine H-11proton (�H 4.45), together with 1H-13C long-range correlationsfrom the methine proton to a carbonyl carbon C-12 (�C 175.0)and to an aromatic quaternary carbon C-1 (�C 160.8), revealedthat the amino acid conjugated with the iminomycosporine ringfor compound 3 is alanine. These results led to identification ofcompound 3 as mycosporine-glycine-alanine. To further eluci-date the absolute configuration of alanine residue in compound 3,we performed acid hydrolysis of compound 3 and derivatized thehydrolysate with FDAA. HPLC-TofMS analysis of the derivatizedhydrolysate and FDAA derivatives of amino acid standards al-lowed determination of the absolute configuration of alanine asthe L-form (Fig. 6).

DISCUSSION

Genome sequence analysis revealed predicted gene clusters en-coding the biosynthesis of MAA in A. mirum DSM 43827 andPseudonocardia sp. P1. We directly cloned each entire gene clusterfrom a digested genomic DNA into a plasmid using in vivo cloningtechnology mediated by phage -RED recombinase before sub-cloning the gene clusters into an integrating vector. After replace-ment of the native promoter with a strong constitutive promoter,the gene clusters were heterologously expressed in S. avermitilis

SUKA22. The transformants carrying each gene cluster wereshown to overproduce shinorine as a major product (Fig. 3).These data clearly show that each gene cluster of A. mirum DSM43827 and Pseudonocardia sp. P1 is responsible for the biosynthe-sis of shinorine, which is the first demonstration of genes for MAAbiosynthesis in Gram-positive bacteria. In addition to shinorine,porphyra-334 and the novel MAA compound were detectable inthe extracts of S. avermitilis SUKA22 transformants carryingpKU492Aaac(3)IV::rpsJp-mys_cluster-amir. The MS and NMRdata demonstrated that the new compound is mycosporine-gly-cine-alanine. The minor products, porphyra-334 and mycospo-rine-glycine-alanine (where threonine and alanine, respectively,substitute for serine), might be accumulated due to the substrateambiguity of D-Ala D-Ala ligase homolog (MysD), which cata-lyzes the condensation of serine to mycosporine-glycine. Thisproduction of MAAs in heterologous hosts, together with thefindings that A. mirum does not produce any MAAs under labo-ratory conditions and that Pseudonocardia sp. P1 produces a tinyamount of shinorine when grown in a certain medium, suggestedthat our system for heterologous expression enabled a silentgene(s) to be awakened or activated a gene cluster which was notefficiently expressed in the original microorganism.

Furthermore, heterologous expression of mysA-mysD of A. mi-rum DSM 48237 in S. avermitilis SUKA22 yielded 154 mg/litershinorine and 188 mg/ liter total MAAs (the sum of shinorine,porphyra-334, mycosporine-glycine, and mycosporine-glycine-alanine), which corresponds to 13.9 mg MAAs/g dry cell weight.In comparison to natural producers such as halotolerant cyano-bacterium producing 0.03 to 0.98 mg MAA/g dry cell weight (26)and red algae from warm southern Spain, which accumulated 0.2to 7.8 mg MAA/g dry cell weight (27), and heterologous producerssuch as E. coli transformants carrying shinorine biosynthetic genesof A. variabilis ATCC 29413, which were transcribed by the T7promoter and produced 637 �g/liter 4-DG as a major product anda small amount of shinorine (145 �g/liter) (8), the S. avermitilisSUKA22 transformant carrying the mys gene cluster of A. mirumDSM 48237 exhibited much higher production of MAA. The het-erologous expression of the entire mys gene clusters of A. mirumDSM 48237 and Pseudonocardia sp. P1 in S. avermitilis SUKA22was performed under the control of alternative promoter (rpsJp).But the productivity of transformants carrying the mys gene clus-ter of A. mirum DSM 48237 was 400-fold higher than that ofPseudonocardia sp. P1. Since these two mys gene clusters werequite similar to each other in the sizes and transcriptional direc-tions of the genes (Table 1), the large difference in the levels ofheterologous expression of the two pathways may be involved inthe kinetics of each enzyme or the stability of enzymes in theheterologous host. Similar results were observed in the produc-tion of monoterpenoid alcohol, 2-methylisoborneol, by the het-erologous host (28).

Interestingly, synthesis of mycosporine-glycine-alanine wasinduced by ammonium ions, while shinorine production was notaffected. Mycosporine-glycine-alanine (compound 3) was yieldedby the condensation of L-Ala to mycosporine-glycine. In general,the intracellular concentration of L-Ala is regulated by (i) alaninedehydrogenase, which catalyzes the reversible deamination of L-Ala to pyruvate and ammonia, and (ii) alanine aminotransferase,which catalyzes a reversible reaction in which the amino group ofL-alanine is transferred to -ketoglutarate to produce pyruvateand glutamate. The resulting glutamates are in turn deaminated in

FIG 6 HPLC chromatogram (a) and MS-trace chromatograms (b and c) ofFDAA amino acids from the acid hydrolysis of compound 3. Chromatogramsd, e, and f correspond to authentic samples of FDAA derivatives of L-Ala,D-Ala, and Gly, respectively.

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a reversible reaction catalyzed by glutamate dehydrogenase toyield -ketoglutarate and ammonia. Therefore, addition of an ex-cess amount of ammonium ions to production medium may shiftthe equilibrium in those above-mentioned reversible reactions,which probably leads to an increased level of L-Ala in the intracel-lular pool of amino acids and enhances the production of myco-sporine-glycine-alanine.

Actinomycetales microorganisms, including genus Streptomy-ces, Micromonospora, and Amycolatopsis species, produce a largenumber of secondary metabolites with a diverse chemical struc-ture which encompass most classes of natural products, includingpolyketides, peptides, terpenes, �-lactams, aminoglycosides, andalkaloids. However, reports of MAA production by Actinomyce-tales microorganism are rare and the only known case so far wasa marine actinobacterium, Micrococcus sp. AK-334, containingshinorine (29). In our study, gene clusters involving MAA biosyn-thesis from two terrestrial microorganisms in the order Actinomy-cetales, A. mirum DSM 43827 and Pseudonocardia sp. P1, wereidentified for the first time. An amino acid sequence homology searchof the public database showed a putative gene cluster for MAA bio-synthesis from another five Actinomycetales microorganisms,Mycobacterium chubuense NBB4, Rhodococcus sp. AW25M09,Rhodococcus sp. 29MFTsu3.1, Rhodococcus sp. 114MFTsu3.1, andActinomycetospora chiangmaiensis DSM 45062 (see Fig. S3 and S4in the supplemental material). Gene organization and the direc-tion of transcription in these gene clusters for MAA biosynthesisin Actinomycetales microorganisms demonstrated that a gene en-coding haloacid dehalogenase (HAD)-superfamily hydrolase islocated upstream of the gene cluster and is likely to be transcrip-tionally coupled in six microorganisms other than A. mirum DSM43827. The difference in levels of transcriptional units might be aplausible reason why MAA production was detected in Pseudono-cardia sp. P1, whose gene cluster for MAA synthesis is transcribedby read-through from the upstream gene encoding HAD-super-family hydrolase, but was not detected in A. mirum DSM 43827,whose gene cluster for MAA biosynthesis is not transcriptionallycoupled to an upstream gene. Therefore, the other five microor-ganisms whose transcriptional units of MAA biosynthetic genecluster are similar to that of Pseudonocardia sp. P1 have potentialfor MAA production.

Amino acid sequence alignment and phylogenetic analysis ofthe predicted amino acid sequence of DDG synthase, a key enzymein MAA biosynthesis, indicated that DDG synthase from Actino-mycetales microorganisms formed a clade with some DDG syn-thases from cyanobacteria, such as Chamaesiphon minutus PCC6605 (WP_015160001), Oscillatoria nigro-viridis PCC 7112(WP_015177379), and Microcystis aeruginosa (WP_012265710),indicating that the genetic origin of the DDG synthase in Actino-mycetales microorganisms might be cyanobacteria (see Fig. S3 inthe supplemental material). Although the response to UV irradi-ation in two microorganisms, A. mirum DSM 43827 andPseudonocardia sp. P1, was unclear, Pseudonocardia sp. P1 pro-duced shinorine in TSB medium, which contains a relatively highconcentration of nitrogen source compared to the other mediaexamined. It is possible that Pseudonocardia sp. P1 produces shi-norine as a reservoir for nitrogen sources. The number of Actino-mycetales microorganisms possessing the biosynthetic gene clus-ter for MAA corresponds to only 1% of genome-sequencedActinomycetales microorganisms. It suggests that biosyntheticgene clusters for mycosporines and MAAs are predominantly dis-

tributed in photosynthetic Gram-negative bacteria (cyanobacte-ria) and rarely present in Gram-positive bacteria. The exact rolesfor these UV-absorbing molecules in Actinomycetales microor-ganisms are still unknown; however, they may have significantroles in these microorganisms, such as other secondary metabo-lites.

ACKNOWLEDGMENTS

We thank M. I. Hutchings of the University of East Anglia, United King-dom, for kindly providing the Pseudonocardia sp. P1 strain. We also thankH. Holstein GmbH & Co. KG for providing the Helioguard 365 contain-ing shinorine and porphyra-334.

This work was supported by a research Grant-in-Aid for ScientificResearch on Innovative Areas from the Ministry of Education, Culture,Sports, Science and Technology of Japan (to H.I.) and a research Grant-in-Aid for Scientific Research from the New Energy and Industrial Tech-nology Development Organization (NEDO; to H.I.).

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