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

Abstract

Mycosporines and mycosporine-like amino acids (MAAs), including shinorine (mycosporine-glycine-serine) and porphyra-334 (mycosporine-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 described the production of MAAs and the corresponding genes involved in MAA biosynthesis from Gram-positive bacteria to date, genome mining of the Gram-positive bacterial database revealed that two microorganisms belonging to the order Actinomycetales, Actinosynnema mirum DSM 43827 and Pseudonocardia sp. strain P1, possess a gene cluster homologous to the biosynthetic gene clusters identified from cyanobacteria. When the two strains were grown in liquid culture, Pseudonocardia sp. accumulated a very small amount of MAA-like compound in a medium-dependent manner, whereas A. mirum did not produce MAAs under any culture conditions, indicating that the biosynthetic gene cluster of A. mirum was in a cryptic state in this microorganism. In order to characterize these biosynthetic gene clusters, each biosynthetic gene cluster was heterologously expressed in an engineered host, Streptomyces avermitilis SUKA22. Since the resultant transformants carrying the entire biosynthetic gene cluster controlled by an alternative promoter produced mainly shinorine, this is the first confirmation of a biosynthetic gene cluster for MAA from Gram-positive bacteria. Furthermore, S. avermitilis SUKA22 transformants carrying the biosynthetic gene cluster for MAA of A. mirum accumulated not only shinorine and porphyra-334 but also a novel MAA. Structure elucidation revealed that the novel MAA is mycosporine-glycine-alanine, which substitutes l-alanine for the l-serine of shinorine.

INTRODUCTION

Mycosporines and mycosporine-like amino acids (MAAs) are small (<400-Da), colorless, and water-soluble molecules characterized by their ability to absorb UV (1, 2). They have absorption maxima ranging from 310 to 365 nm with high molar extinction coefficients (ε = 28,100 to 50,000 M⁻¹ cm⁻¹). Mycosporines contain a cyclohexenone ring conjugated with the nitrogen substituent of an amino acid or an imino alcohol, while MAAs have a cyclohexenimine ring conjugated with two such substituents (Fig. 1). These UV-absorbing molecules are known to be produced by a wide range of organisms, including cyanobacteria, fungi, and marine micro- and macroalgae (3–5). To date, more than 30 different compounds in the mycosporine family have been identified from natural sources (1). The physiological role of mycosporines and MAAs as sunscreen compounds to protect against environmental UV radiation is well established in the producing organisms and the organism that ingests and accumulates MAAs (5, 6). In addition to their photoprotective function, mycosporines and MAAs appear to play important roles as antioxidant molecules, compatible solutes, and an intracellular nitrogen reservoir and also have roles in defense against thermal or desiccation stress and in fungal reproduction (1, 4). Moreover, mycosporines and MAAs have attracted attention in the field of cosmetics and skin care due to their effective UV-filtering capacity and ability to prevent UV-induced skin damage (7).

FIG 1.

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

Recently, the main steps in the biosynthesis of MAAs and their genetic basis in cyanobacteria were elucidated. A gene cluster with four genes ranging from ava_3855 to ava_3858 in cyanobacterium Anabaena variabilis ATCC 29413 (Fig. 2) was found to be responsible for the biosynthesis of an MAA, shinorine (Fig. 1) (8). The gene product of ava_3858, 4-deoxygadusol (DDG) synthase, catalyzes the conversion of sedoheputulose-7-phosphte, an intermediate of the pentose phosphate pathway, to demethyl 4-deoxygadusol (DDG), and DDG is converted to 4-deoxygadusol (4-DG; Fig. 1) by catalyzing Ava_3857, O-methyltransferase (O-MT). The ava_3856 gene encodes ATP-grasp family protein catalyzing the addition of glycine to 4-DG to form mycosporine-glycine (Fig. 1). A nonribosomal peptide synthetase (NRPS) homolog encoded by ava_3855 attaches serine to mycosporine-glycine to generate shinorine. However, the enzyme involved in the last biosynthetic step of shinorine in cyanobacterium Nostoc punctiforme ATCC 29133 was demonstrated to be a d-alanine (D-Ala) D-Ala ligase-like protein, NpF5597 (Fig. 2) (9). NpF5597 is responsible for the condensation of serine onto mycosporine-glycine, which is generated by DDG synthase (NpR5600), O-MT (NpR5599), and the ATP-grasp family protein (NpR5598) in N. punctiforme. Hence, the first three enzymes in the biosynthetic pathway of shinorine are conserved in these two cyanobacteria, while the last enzyme is either an NRPS homolog or a D-Ala D-Ala ligase-like protein. In the fungal genome, the genes encoding the homologues of the first three enzymes in shinorine biosynthesis are present and located in a cluster, but homologs for NRPS-like protein and D-Ala D-Ala ligase-like protein are missing (Fig. 2). This genetic context is consistent with the fact that the only fungal products reported so far are mycosporines (1).

FIG 2.

Genes involving biosynthesis of mycosporines and MAAs in Actinobacteria and other organisms. A. variabilis ATCC 29413 and N. punctiforme ATCC 29133 are 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_R5600 in N. punctiforme ATCC 29133, has been demonstrated to be both necessary and sufficient for shinorine synthesis (reference 8). The genes or gene clusters homologous to the shinorine biosynthetic genes in cyanobacteria were present in genomes of fungi, cnidarians, and dinoflagellata. The representative genes or gene 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 Heterocapsa triquetra is fused to the O-MT-encoding gene.

In the present study, we found gene clusters for biosynthesis of MAAs from two Actinomycetales microorganisms, Actinosynnema mirum DSM 43827 (10) and Pseudonocardia sp. strain P1 (11). Here we characterize the MAA biosynthetic gene clusters of the two microorganisms through heterologous expression using an engineered Streptomyces host suitable for secondary metabolite production.

MATERIALS AND METHODS

Bacterial strains and plasmid vectors.

Actinosynnema mirum DSM 43827 was obtained from the RIKEN Bioresource Center (Wako, Japan), and Pseudonocardia sp. P1 was kindly donated by Matt I. Hutchings of the University of East Anglia, United Kingdom. Streptomyces avermitilis SUKA22 (12) was used as the host for heterologous expression of the biosynthetic gene cluster. A pRED small vector (13) was used for in vivo cloning of the DNA fragment containing the gene cluster for biosynthesis of MAAs. An integrating vector, pKU492Aaac(3)IV (12), was used for subcloning the gene clusters for MAA biosynthesis. A plasmid, pKU1021 (pKU460::rpsJp; see reference 14), was used as a template DNA for the amplification of a constitutively expressed promoter of the S. avermitilis rpsJ gene encoding ribosomal protein S10. Unmethylated recombinant plasmid DNAs were prepared in Escherichia coli GM2929 hsdS::Tn10 (13) and were introduced into S. avermitilis SUKA22 using polyethylene glycol-assisted transformation (13).

Cloning of the entire gene cluster for MAA biosynthesis.

To clone the entire gene cluster for mycosporine-like amino acid (MAA) biosynthesis from A. mirum DSM 43827 and Pseudonocardia sp. P1, each chromosomal DNA of these microorganisms was digested with SfiI and MluI, respectively. No restriction sites were located inside the entire gene cluster. Fragments containing 6.3 kb (A. mirum DSM 43827) or 8.2 kb (Pseudonocardia sp. P1) were purified by agarose gel electrophoresis. Linearized cloning vector was prepared by PCR amplification with pRED as a template DNA using the designated primer pair containing both 41-nucleotide (nt) and 50-nt sequences corresponding to upstream and downstream regions of the biosynthetic gene cluster. A primer pair, forward primer 5′-TCAGGTCAGCGGAATGCCCTCCCGCACTTCCAGCCCCTGGAAGCTTTGCCAGGAAGATACTTAACAG-3′ (underlined, italicized, and bold characters indicate the downstream region of amir_4256, the HindIII site, and the region of vector pRED, respectively) and reverse primer 5′-TCAGCGCGCGGCGAGGCCGTTCTGATAGGCCCAGACCACGGAATTCCCATTCATCCGCTTATTATC-3′ (underlined, italicized, and bold characters indicate the upstream region of amir_4259, the EcoRI site, and the region of vector pRED, respectively), was used for in vivo cloning of a gene cluster from A. mirum DSM 43827. Another primer pair, forward primer 5′-CTCGACCAACGTTCCGATTGAGCCGAACAGTAGAGCGGGCATCGGGTTTCGAATTCTGCCAGGAAGATACTTAACAG-3′ (underlined, italicized, and bold characters indicate the upstream region of pseP1_01010031440, the EcoRI site, and the region of vector pRED, respectively) and reverse primer 5′-GACCGGTCGTAGAACGGTGTGATCCATCGTTCCATTCTGCGTGCACCCTGAAGCTTCCATTCATCCGCTTATTATC-3′ (underlined, italicized, and bold characters indicate the downstream region of pseP1_010100031425, the HindIII site, and the region of vector pRED, respectively), was used for the cloning of a gene cluster from Pseudonocardia sp. P1. Initial denaturation at 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 100 s) and then final incubation at 72°C for 5 min using an Expand High Fidelity PCR system (Roche Diagnostics, Tokyo, Japan) or Phusion DNA polymerase (New England BioLabs, MA). After amplification, the reaction mixture was treated with DpnI to remove template DNA. Each 1.7-kb amplicon of pRED carrying upstream and downstream sequences of the gene cluster was cotransformed into l-arabinose-induced E. coli BW25141 carrying pKD46 (13) with size-fractionated chromosomal DNA of SfiI-cut A. mirum DSM 43827 or MluI-cut Pseudonocardia sp. P1 by electroporation. The desired plasmid, pRED, carrying the entire biosynthetic gene cluster, was obtained by selection with 30 μg/ml chloramphenicol and was also confirmed by restriction digestion. The large EcoRI/HindIII fragment of each recombinant plasmid, pRED, carrying the entire biosynthetic gene cluster, was ligated with the large fragment of HindIII/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 biosynthetic gene cluster.

To introduce an alternative promoter upstream of the biosynthetic gene cluster of A. mirum DSM 43827 and Pseudonocardia sp. P1, a segment containing the promoter of rpsJ encoding ribosomal protein S10 of S. avermitilis and aac(3)I encoding an aminoglycoside N-acetyltransferase of E. coli was prepared by amplification with pKU1021 as a template DNA using the designated primer pair containing 44-nt sequences corresponding to the N-terminal region of the first gene in the cluster 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-terminal region of amir_4259, the start codon, and the region of rpsJ promoter of S. avermitilis with the ribosomal binding site, respectively) and universal reverse primer 5′-AGCAGCCCTTGCGCCCTGAGTGCTTGCGGCAGCGTGAAGCTAGCGATCTCGGCTTGAACGAATTG-3′ (underlined and bold characters indicate the upstream region of cloning site in pKU492Aaac(3)IV and the region of aac(3)I, respectively), was used for introduction of rpsJ promoter upstream of the gene cluster of A. mirum DSM 43827. Another primer pair, forward-Pse primer 5′-TCCCAACTCTCGACACGGAACTCCGTATCGGTCGCGCTGAGCATATGTACTCAGTAGTCCTTCGTCTC-3′ (underlined, italicized, and bold characters indicate N-terminal region of pseP1_010100031440, the translational start codon, and the region of rpsJ promoter of S. avermitilis with the ribosomal binding site, respectively) and the universal reverse primer described above, was used for the introduction of the rpsJ promoter upstream of the gene 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) and then final incubation at 72°C for 5 min. All amplicons were treated with DpnI to remove the template DNA. The amplicon containing rpsJ promoter and a resistance marker, aac(3)I, generated using forward-Amir and universal-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 universal-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 was controlled by the rpsJ promoter) was obtained by selection with 25 μg/ml apramycin [aac(3)IV] and 25 μg/ml fortimicin [aac(3)I] and was confirmed 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. avermitilis SUKA22.

Detection of MAAs from actinomycete strains.

A. mirum DSM 4382, Pseudonocardia sp. P1, and S. avermitilis SUKA22 transformants were grown in the vegetative medium (15) at 30°C for 2 days on a reciprocal shaker (200 strokes per min). A 0.1-ml portion of the vegetative culture was inoculated with 10 ml synthetic production medium (15) in a 125-ml flask. A. mirum DSM 4382 and Pseudonocardia sp. P1 were further examined in the following media: TSB (Trypticase soy broth), YMG (4 g yeast extract–10 g malt extract–4 g glucose–1 liter deionized water adjusted to pH 7.0 with 2 N KOH), and R4 (16). After incubation at 28°C for 8 days on a rotary shaker at 200 rpm, the whole culture was mixed with an equal volume of methanol and the mixture was subjected to a vigorous vortex procedure. The supernatant was collected by centrifugation at 3,000 rpm for 10 min, and a 2-μl portion of the supernatant was directly analyzed by high-performance liquid chromatography (HPLC)–time-of-flight mass spectrometry (TofMS) (Acquity ultraperformance LC system, Waters Xevo G2-S Tof). When necessary, the supernatant was evaporated to remove methanol and lyophilized to dissolve the residue in 1 ml water before HPLC-TofMS analysis. Analytical conditions for HPLC were as follows: column, Hypercarb (3 μm pore size; 2.1-mm inner diameter by 100 mm); mobile phase, 8% acetonitrile–0.1 M triethylammonium acetate (TEAA; pH 7.0) for 4 min, 8% to 15% acetonitrile–0.1 M TEAA (pH 7.0) for 6 min; flow rate, 0.2 ml/min; detection at 330 nm. Authentic samples of shinorine and porphyra-334 were prepared from Helioguard 365 (7). Mass spectrometry was performed in resolution mode under the following conditions: capillary voltage of 3.0 kV in positive-ion mode or 2.5 kV in negative-ion mode; cone voltage of 40 V; source temperature of 120°C; desolvation gas flow of 800 liters/h at a temperature of 450°C; and cone gas flow of 50 liters/h. For accurate mass measurements, lock mass calibration was conducted using leucine enkephalin. Mass data were acquired with collision cell energy alternating between low (6 V) and elevated (ramping from 20 to 40 V).

Purification and structural elucidation of novel MAA compound.

A 70-ml sample of the vegetative culture of S. avermitilis SUKA22 carrying pKU492Aaac(3)IV::rpsJp-mys_cluster-amir was inoculated into 7 liters of 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, the mycelium was collected by filtration, washed with tap water, and extracted with 600 ml methanol. After removal of mycelium by filtration, the methanol extract was concentrated under reduced pressure to remove methanol. The aqueous concentrate (ca. 80 ml) was diluted with water (∼400 ml) and extracted with an equal volume of dichloromethane to remove solvent-soluble impurities. The aqueous layer was lyophilized, and the residue was dissolved in a small amount of methanol to remove salts. The supernatant was concentrated to dryness, and the residue was dissolved in water. After removal of insoluble materials by suction, the clarified solution was subjected to a column containing 150 ml cation-exchange resin Amberlite IR120B [H⁺] (Organo Corporation, Japan). After the column was washed with 1,000 ml deionized water to elute shinorine, novel MAA was eluted with 300 ml of 0.5 N HCl. The eluate was neutralized by the addition of solid sodium bicarbonate, followed by lyophilization. The resulting white residue was dissolved in a small volume of methanol to remove NaCl, and the methanol-soluble material was subjected to preparative HPLC on a Hypercarb column (5 μm pore size; 10-mm inner diameter by 150 mm) developed with 8% (vol/vol) acetonitrile–0.092 M TEAA (pH 7.0) at a flow rate of 2.5 ml/min and detected at 330 nm. The fractions containing novel compound eluted at 24.5 to 30.0 min were combined and evaporated under conditions of reduced pressure to remove acetonitrile before lyophilization. The resulting residue was redissolved in water, and novel MAA was adsorbed by activated charcoal (50 ml) to remove TEAA. After washing the charcoal with 100 ml water and 100 ml of 80% (vol/vol) methanol, the novel MAA compound was eluted with 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-700 system. Nuclear magnetic resonance (NMR) (¹H, 500 MHz; ¹³C, 125 MHz) spectra were obtained using a Jeol JNM-ECP 500 FT NMR system. Chemical shifts are reported on the δ scale relative to the residual solvent signal (CD₃OD; δ_H 3.30, δ_C 49.0).

Determination of the absolute configurations of amino acids in novel MAA.

Novel MAA (0.5 mg) was hydrolyzed in 0.4 ml of 6 N HCl at 120°C for 14 h. After the reaction mixture was concentrated in vacuo, the residue was dissolved in 200 μl of 0.1 M sodium bicarbonate containing 0.2 mg N^α-(5-fluoro-2,4-dinitrophenyl)-l-alaninamide (FDAA) and was incubated at 75°C for 30 min to prepare FDAA derivatives. Authentic FDAA derivatives were also prepared from L-Ala and D-Ala by the same procedures. After the reaction was quenched by the addition of 20 μl of 1 N HCl, the reaction mixture was analyzed by HPLC-TofMS. Analytical conditions for HPLC were as follows: column, Acquity UPLC BEH C₁₈ (1.7 μm 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-ion mode as described above.

RESULTS

Bioinformatic analysis of MAA biosynthetic gene cluster in Actinomycetales.

A homology search for a gene cluster for shinorine biosynthesis of A. variabilis ATCC 29413 against public databases identified similar gene clusters from over 30 cyanobacterial genomes and over 40 fungal genomes. The corresponding homologs were also found in the genome of coral Acropora digitifera (17), sea anemone Nematostella vectensis (18), and red alga Chondrus crispus (19) and in the expressed sequence tag database of some dinoflagellates (Fig. 2) (20). The occurrence of predicted biosynthetic genes for MAAs indicates that the genes are present in organisms that are usually exposed to sunlight, such as photosynthetic cyanobacteria and marine organisms from shallow-water environments. Further genome mining in the Gram-positive bacterial database revealed that two microorganisms in the order Actinomycetales, Actinosynnema mirum DSM 43827 and Pseudonocardia sp. P1, possess the gene cluster, whose structure is similar to that of the shinorine biosynthetic genes identified in cyanobacteria. The biosynthetic gene clusters in A. mirum (amir_4256–4259) and in Pseudonocardia sp. P1 (pseP1_010100031425–010100031440) are composed of four genes encoding putative dimethyl 4-deoxygadusol (DDG) synthase, O-methyltransferase (O-MT), ATP-grasp family protein, and D-Ala D-Ala ligase homolog (Fig. 2). Three gene products of A. mirum DSM 43827 (Amir_4259–Amir_4257) and Pseudonocardia sp. P1 (PseP1_010100031440–PseP1_010100031430) were quite similar to Ava_3858–Ava_3856 of A. variabilis ATCC 29413 (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 similarity to D-Ala D-Ala ligase-like protein of N. punctiforme ATCC 29133 (Table 1). The first gene, amir_4259 (mysA), in an operon of A. mirum DSM 43827, is translationally coupled to the downstream amir_4258 (mysB) gene. Interestingly, the first three genes of Pseudonocardia sp. P1, mysA, mysB, and mysC, were translationally coupled to the downstream gene. It is interesting that two bacteria that have no photosynthesis ability and live in terrestrial environments have the potential to produce MAAs. So far, no reports have described the correlation between the production of MAAs and corresponding genes involving biosynthesis of MAAs from Gram-positive bacteria.

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 function
	ORF	aaa	Identity/similarity (%)b	ORF	aaa	Identity/similarity (%)b
mysA	Amir_4259	406	62/76	PseP1_010100031440	415	58/74	Dimethyl 4-deoxygadusol (DDG) synthase
mysB	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 protein
mysD	Amir_4256	339	50/66c	PseP1_010100031425	346	52/72c	d-Ala d-Ala ligase homolog

^aData represent numbers of amino acids (aa).

^bHomolog 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]).

^cHomology to NpF5597 (348 aa) of Nostoc punctiforme ATCC 29133.

To examine the ability to produce MAAs in A. mirum DSM 43827 and Pseudonocardia sp. P1, these two microorganisms were grown in several liquid media. HPLC analysis of methanol extracts from Pseudonocardia sp. P1 grown in TSB medium indicated the presence of a very small amount of MAA-like compound that exhibited absorbance spectra characteristic for MAAs and whose retention time was identical to that of shinorine, whereas no such compound was detected in the extract from Pseudonocardia sp. P1 grown in the other media (Fig. 3B). With respect to A. mirum DSM 43827, MAA-like compound was not accumulated under any of the medium conditions (Fig. 3A), suggesting that the putative MAA biosynthetic gene cluster might be cryptic in A. mirum DSM 43827. To elucidate the function of putative gene clusters for MAA biosynthesis in these two Actinomycetales microorganisms, we attempted to express each cluster in a heterologous host.

FIG 3.

Detection of MAAs from actinomycete strains and heterologous hosts carrying the biosynthetic gene cluster for MAA. (A) HPLC analysis of methanol extracts of the S. avermitilis SUKA22 transformants carrying the mys cluster 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 of A. mirum DSM 43827 grown in TSB medium (line d), and the authentic sample containing porphyra-334 and shinorine (line e). Methanol extracts from 0.2 μl culture were injected in each analysis. (B) HPLC analysis of methanol extracts of S. avermitilis SUKA22 carrying the mys gene cluster of Pseudonocardia sp. P1 controlled by the alternative rpsJ promoter (line f), the mys gene cluster of Pseudonocardia sp. P1 with the native promoter (line g) and vector plasmid pKU492Aaac(3)IV (line h), methanol extracts of Pseudonocardia sp. P1 grown in TSB medium (line i), production medium (line j), and YMG medium (line k), and standard samples containing porphyra-334 and shinorine (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 a HPLC-TofMS. Peaks on the chromatograms indicate shinorine (peak 1), porphyra-334 (peak 2), and novel MAA (peak 3).

Heterologous expression of gene cluster for biosynthesis of MAAs in Actinomycetales.

To clone the entire gene cluster for MAA biosynthesis from A. mirum DSM 43827, we used in vivo cloning technology mediated by λ-RED recombination. We prepared a PCR-amplified 1.7-kb pRED fragment, which contains a p15A origin and chloramphenicol resistance gene as well as 41- and 50-bp terminal homology arms upstream and downstream of the target gene cluster, respectively. After digestion of genomic DNA of A. mirum DSM 43827 with restriction enzyme to release the cluster on single DNA segment, we transformed E. coli carrying the λ-RED recombination system with the digested genomic DNA segments and amplified pRED to generate pRED::mys_cluster-amir. Digestion of the resultant plasmid with a restriction enzyme verified the direct cloning of the mys gene cluster from genomic DNA of A. mirum DSM 43827 into the pRED vector. After subcloning of the entire gene cluster into an integrating pKU492Aaac(3)IV vector, the strong and constitutively expressed promoter of a gene encoding ribosomal protein S10 was inserted in front of the first gene of the operon by λ-RED recombination, yielding pKU492Aaac(3)IV::rpsJp::mys_cluster-amir. Following the same strategy as described above, a pKU492Aaac(3)IV::rpsJp::mys_cluster-pse plasmid controlled by the rpsJ promoter was constructed. Then, each recombinant plasmid was introduced into S. avermitilis SUKA22, an engineered host suitable for heterologous expression (12). The transformants of S. avermitilis SUKA22 were grown in production medium, and methanol extracts of the whole culture were directly analyzed by HPLC-TofMS using the positive mode. The transformant carrying the mys gene cluster of A. mirum DSM 43827 did not exhibit MAA-like compounds, while the transformant carrying the mys gene cluster of A. mirum DSM 43827 placed under the control of the alternative promoter, rpsJp, gave compound 1 (λ_max = 333 nm, m/z 333.1288) and compound 2 (λ_max = 334 nm, m/z 347.1454) (Fig. 3A). Taken together with mass spectrometry fragmentation analysis using authentic samples of shinorine and porphyra-334, compounds 1 and 2 were found to be identical to shinorine and porphyra-334, respectively (see Fig. S1 in the supplemental material). Additionally, biosynthetic intermediates, 4-deoxygadusol and mycosporine-glycine, were also detected in the methanol extract (see Fig. S2 in the supplemental material) by TofMS using the negative-ion mode. The yields in the methanol extract from three independent experiments cultivating transformants carrying pKU492Aaac(3)IV::rpsJp-mys_cluster-amir were 154 ± 13.0 mg/liter shinorine and 7.2 ± 0.57 mg/ liter porphyra-334. These results demonstrated that the cryptic mys cluster of A. mirum DSM 43827 was activated by heterologous expression, which allowed us to determine that the cluster is responsible for MAA production. In addition to shinorine and porphyra-334, HPLC-TofMS analysis of the methanol extract showed the unknown compound (i.e., compound 3), which exhibited an absorbance spectrum characteristic of MAAs, to have a cyclohexenimine chromophore (λ_max = 333 nm) (Fig. 3A and 4A). The mass spectrum of compound 3 showed a major ion at m/z 317.1334, indicating a molecular formula of C₁₃H₂₁N₂O₇ (calculated mass, 317.1349), which is different that of from any MAA described previously (Fig. 4B). Further investigation of compound 3 is described in a later section.

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) of compound 3 produced by S. avermitilis SUKA22 transformants carrying pKU492Aaac(3)IV::rpsJp-mys_cluster-amir. (C) The production of compound 3 and shinorine in the transformants carrying pKU492Aaac(3)IV::rpsJp-mys_cluster-amir grown for 2, 5, and 8 days in production medium supplemented with three different concentrations of NH₄Cl. The experiments were repeated three times with similar results. AU, absorbance units.

HPLC-TofMS analysis of the methanol extract of S. avermitilis SUKA22 transformants carrying the entire mys gene cluster of Pseudonocardia sp. P1 under the control of rpsJ promoter resolved a single peak (λ_max = 333 nm, m/z 333.1288) (Fig. 3B). Comparative analysis using an authentic sample of shinorine revealed that the compound produced was identical to shinorine. The yield of shinorine in these transformants was 380 ± 33 μg/liter, which was about 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 demonstrated to 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 biosynthetic gene cluster for MAA of A. mirum DSM 43827 since the compound was predicted to be a novel MAA. Before isolation and structure elucidation of compound 3, we attempted to improve its productivity because its yield was less than 1 mg/liter. In cyanobacteria (21), dinoflagellates (22), and algae (23), the accumulation of MAA is affected by environmental factors such as UV radiation, salinity, temperature, or ammonium ions (4). As the entire biosynthetic gene cluster for MAA heterologously expressed in S. avermitilis SUKA22 is transcriptionally controlled by the alternative promoter, responses to these environmental factors were not expected in the heterologous host. However, during the course of improving productivity, we found that the addition of ammonium salts to the culture of transformants somehow induced the production of compound 3. The most marked increase of compound 3 was observed in the culture supplemented with 400 mM NH₄Cl (Fig. 4C), which led to 9.6-fold-higher production after 8 days of incubation compared to the culture without NH₄Cl. Intriguingly, the concentration of NH₄Cl had no effect on the accumulation of shinorine in the range of 0 to 400 mM. To test whether the increased production of compound 3 resulted from the increased concentration of ammonium ions, osmotic stress, or increased salinity caused by the addition of NH₄Cl, the effect of these factors on the production of compound 3 was investigated by the addition of (NH₄)₂SO₄, NH₄NO₃, NaCl, and sucrose. When the transformant was grown in production medium containing 250 mM sodium chloride or 20% sucrose, compound 3 was accumulated normally. In contrast, addition of 200 mM (NH₄)₂SO₄ or 400 mM NH₄NO₃ to the culture of the transformant improved the production of compound 3 to the same level as the addition of 400 mM ammonium chloride. These results suggested that ammonium ion stimulates the accumulation of compound 3. Since there was little difference between the counter ions of ammonium in the elevated levels of compound 3, we decided to supply 400 mM NH₄Cl to the production medium for the subsequent purification of compound 3.

To isolate compound 3, the transformant carrying pKU492Aaac(3)IV::rpsJp-mys_cluster-amir was grown in 7 liters of production medium containing 400 mM NH₄Cl for 7 days. Compound 3 was purified from the mycelia by cation-exchange chromatography on an Amberlite IR120B (H⁺ type) and by preparative reverse-phase HPLC. About four milligrams of white powder was obtained and investigated by ¹H and ¹³C NMR spectroscopy, including correlation spectroscopy (COSY), heteronuclear multiple-quantum correlation (HMQC), and heteronuclear multiple-bond correlation (HMBC), in comparison to shinorine (Fig. 5; see also Tables S1 and S2 in the supplemental material). The ¹³C NMR data obtained for compound 3 and shinorine revealed a good correlation with previously published chemical shift data for shinorine (8) and structurally similar MAAs (24, 25). The two-dimensional (2D) NMR analyses revealed the common substructure of the imino-mycosporine ring (C-1 to C-10) for compound 3. The remaining substructure was elucidated as follows. ¹H and ¹³C NMR data for compound 3 showed the presence of a methyl group (δ_C-13 19.9, δ_H-13 1.53), which is not present in shinorine. ¹H-¹H coupling between the methyl protons and a methine H-11 proton (δ_H 4.45), together with ¹H-¹³C long-range correlations from the methine proton to a carbonyl carbon C-12 (δ_C 175.0) and to an aromatic quaternary carbon C-1 (δ_C 160.8), revealed that the amino acid conjugated with the iminomycosporine ring for compound 3 is alanine. These results led to identification of compound 3 as mycosporine-glycine-alanine. To further elucidate the absolute configuration of alanine residue in compound 3, we performed acid hydrolysis of compound 3 and derivatized the hydrolysate with FDAA. HPLC-TofMS analysis of the derivatized hydrolysate and FDAA derivatives of amino acid standards allowed determination of the absolute configuration of alanine as the l-form (Fig. 6).

FIG 5.

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

FIG 6.

HPLC chromatogram (a) and MS-trace chromatograms (b and c) of FDAA amino acids from the acid hydrolysis of compound 3. Chromatograms d, e, and f correspond to authentic samples of FDAA derivatives of l-Ala, d-Ala, and Gly, respectively.

DISCUSSION

Genome sequence analysis revealed predicted gene clusters encoding the biosynthesis of MAA in A. mirum DSM 43827 and Pseudonocardia sp. P1. We directly cloned each entire gene cluster from a digested genomic DNA into a plasmid using in vivo cloning technology mediated by phage λ-RED recombinase before subcloning the gene clusters into an integrating vector. After replacement 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 were shown to overproduce shinorine as a major product (Fig. 3). These data clearly show that each gene cluster of A. mirum DSM 43827 and Pseudonocardia sp. P1 is responsible for the biosynthesis of shinorine, which is the first demonstration of genes for MAA biosynthesis in Gram-positive bacteria. In addition to shinorine, porphyra-334 and the novel MAA compound were detectable in the extracts of S. avermitilis SUKA22 transformants carrying pKU492Aaac(3)IV::rpsJp-mys_cluster-amir. The MS and NMR data demonstrated that the new compound is mycosporine-glycine-alanine. The minor products, porphyra-334 and mycosporine-glycine-alanine (where threonine and alanine, respectively, substitute for serine), might be accumulated due to the substrate ambiguity of D-Ala D-Ala ligase homolog (MysD), which catalyzes the condensation of serine to mycosporine-glycine. This production of MAAs in heterologous hosts, together with the findings that A. mirum does not produce any MAAs under laboratory conditions and that Pseudonocardia sp. P1 produces a tiny amount of shinorine when grown in a certain medium, suggested that our system for heterologous expression enabled a silent gene(s) to be awakened or activated a gene cluster which was not efficiently expressed in the original microorganism.

Furthermore, heterologous expression of mysA-mysD of A. mirum DSM 48237 in S. avermitilis SUKA22 yielded 154 mg/liter shinorine 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 cyanobacterium producing 0.03 to 0.98 mg MAA/g dry cell weight (26) and red algae from warm southern Spain, which accumulated 0.2 to 7.8 mg MAA/g dry cell weight (27), and heterologous producers such as E. coli transformants carrying shinorine biosynthetic genes of A. variabilis ATCC 29413, which were transcribed by the T7 promoter and produced 637 μg/liter 4-DG as a major product and a small amount of shinorine (145 μg/liter) (8), the S. avermitilis SUKA22 transformant carrying the mys gene cluster of A. mirum DSM 48237 exhibited much higher production of MAA. The heterologous expression of the entire mys gene clusters of A. mirum DSM 48237 and Pseudonocardia sp. P1 in S. avermitilis SUKA22 was performed under the control of alternative promoter (rpsJp). But the productivity of transformants carrying the mys gene cluster of A. mirum DSM 48237 was 400-fold higher than that of Pseudonocardia sp. P1. Since these two mys gene clusters were quite similar to each other in the sizes and transcriptional directions of the genes (Table 1), the large difference in the levels of heterologous expression of the two pathways may be involved in the kinetics of each enzyme or the stability of enzymes in the heterologous host. Similar results were observed in the production of monoterpenoid alcohol, 2-methylisoborneol, by the heterologous host (28).

Interestingly, synthesis of mycosporine-glycine-alanine was induced by ammonium ions, while shinorine production was not affected. Mycosporine-glycine-alanine (compound 3) was yielded by the condensation of L-Ala to mycosporine-glycine. In general, the intracellular concentration of L-Ala is regulated by (i) alanine dehydrogenase, 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 of l-alanine is transferred to α-ketoglutarate to produce pyruvate and glutamate. The resulting glutamates are in turn deaminated in a reversible reaction catalyzed by glutamate dehydrogenase to yield α-ketoglutarate and ammonia. Therefore, addition of an excess amount of ammonium ions to production medium may shift the equilibrium in those above-mentioned reversible reactions, which probably leads to an increased level of L-Ala in the intracellular pool of amino acids and enhances the production of mycosporine-glycine-alanine.

Actinomycetales microorganisms, including genus Streptomyces, Micromonospora, and Amycolatopsis species, produce a large number of secondary metabolites with a diverse chemical structure which encompass most classes of natural products, including polyketides, peptides, terpenes, β-lactams, aminoglycosides, and alkaloids. However, reports of MAA production by Actinomycetales microorganism are rare and the only known case so far was a marine actinobacterium, Micrococcus sp. AK-334, containing shinorine (29). In our study, gene clusters involving MAA biosynthesis from two terrestrial microorganisms in the order Actinomycetales, A. mirum DSM 43827 and Pseudonocardia sp. P1, were identified for the first time. An amino acid sequence homology search of the public database showed a putative gene cluster for MAA biosynthesis from another five Actinomycetales microorganisms, Mycobacterium chubuense NBB4, Rhodococcus sp. AW25M09, Rhodococcus sp. 29MFTsu3.1, Rhodococcus sp. 114MFTsu3.1, and Actinomycetospora chiangmaiensis DSM 45062 (see Fig. S3 and S4 in the supplemental material). Gene organization and the direction of transcription in these gene clusters for MAA biosynthesis in Actinomycetales microorganisms demonstrated that a gene encoding haloacid dehalogenase (HAD)-superfamily hydrolase is located upstream of the gene cluster and is likely to be transcriptionally coupled in six microorganisms other than A. mirum DSM 43827. The difference in levels of transcriptional units might be a plausible reason why MAA production was detected in Pseudonocardia sp. P1, whose gene cluster for MAA synthesis is transcribed by read-through from the upstream gene encoding HAD-superfamily hydrolase, but was not detected in A. mirum DSM 43827, whose gene cluster for MAA biosynthesis is not transcriptionally coupled to an upstream gene. Therefore, the other five microorganisms whose transcriptional units of MAA biosynthetic gene cluster are similar to that of Pseudonocardia sp. P1 have potential for MAA production.

Amino acid sequence alignment and phylogenetic analysis of the predicted amino acid sequence of DDG synthase, a key enzyme in MAA biosynthesis, indicated that DDG synthase from Actinomycetales microorganisms formed a clade with some DDG synthases from cyanobacteria, such as Chamaesiphon minutus PCC 6605 (WP_015160001), Oscillatoria nigro-viridis PCC 7112 (WP_015177379), and Microcystis aeruginosa (WP_012265710), indicating that the genetic origin of the DDG synthase in Actinomycetales microorganisms might be cyanobacteria (see Fig. S3 in the supplemental material). Although the response to UV irradiation in two microorganisms, A. mirum DSM 43827 and Pseudonocardia sp. P1, was unclear, Pseudonocardia sp. P1 produced shinorine in TSB medium, which contains a relatively high concentration of nitrogen source compared to the other media examined. It is possible that Pseudonocardia sp. P1 produces shinorine as a reservoir for nitrogen sources. The number of Actinomycetales microorganisms possessing the biosynthetic gene cluster for MAA corresponds to only 1% of genome-sequenced Actinomycetales microorganisms. It suggests that biosynthetic gene clusters for mycosporines and MAAs are predominantly distributed in photosynthetic Gram-negative bacteria (cyanobacteria) and rarely present in Gram-positive bacteria. The exact roles for these UV-absorbing molecules in Actinomycetales microorganisms are still unknown; however, they may have significant roles in these microorganisms, such as other secondary metabolites.