Phototemtide A, a Cyclic Lipopeptide Heterologously Expressed from Photorhabdus temperata Meg1, Shows Selective Antiprotozoal Activity

Abstract A new cyclic lipopeptide, phototemtide A (1), was isolated from Escherichia coli expressing the biosynthetic gene cluster pttABC from Photorhabdus temperata Meg1. The structure of 1 was elucidated by HR‐ESI‐MS and NMR experiments. The absolute configurations of amino acids and 3‐hydroxyoctanoic acid in 1 were determined by using the advanced Marfey's method and comparison after total synthesis of 1, respectively. Additionally, three new minor derivatives, phototemtides B–D (2–4), were identified by detailed HPLC–MS analysis. Phototemtide A (1) showed weak antiprotozoal activity against Plasmodium falciparum, with an IC50 value of 9.8 μm. The biosynthesis of phototemtides A–D (1–4) was also proposed.

An ew cyclic lipopeptide, phototemtide A( 1), was isolated from Escherichiac oli expressing the biosynthetic gene cluster pttABC from Photorhabdus temperata Meg1. The structure of 1 was elucidated by HR-ESI-MS and NMR experiments.T he absolute configurations of amino acids and 3-hydroxyoctanoic acid in 1 were determined by using the advanced Marfey'sm ethod and comparison after total synthesis of 1,r espectively.A dditionally,t hree new minor derivatives, phototemtides B-D (2)(3)(4), were identified by detailed HPLC-MS analysis. Phototemtide A (1)s howed weak antiprotozoal activity against Plasmodium falciparum,w ith an IC 50 value of 9.8 mm.T he biosynthesis of phototemtides A-D (1-4)w as also proposed.
Cyclic lipopeptides (CLPs) are ac lass of structurally diversen atural products mainly produced by aw ide variety of microorganisms, including cyanobacteria, [1] bacteria, [2] actinobacteria, [3] and fungi. [4] They are generally composed of af atty acid tail linked to the Nterminus of as hort oligopeptide and the Cterminus of the oligopeptide forms al actoneo rlactam with ah ydroxy,p henol, or amino functional group of the side chains of peptideo rp art of the lipid moiety. [5] These compounds can be considered as amphiphiles due to the existence of hydrophobic lipid tails and hydrophilic amino acids or peptides, which endow them with ideal biosurfactant properties. [6] Although the biological activity of CLPs is often reduced to theseb iosur-factantp roperties, [5] they also displayed potent and selective antibacterial, [7] antifungal, [8] antiprotozoal, [9] and cytotoxic activities. [10] Daptomycin, isolated from Streptomycesr oseoporus,i s the first clinically used CLP antibiotic with an ew structural type and uniquem echanismo fa ction. [11] It was approved by FDA in 2003 for the nontopicalt reatment of complicated skin and skin structure infections causedb yG ram-positive pathogens. [11] As one of the few newly approved antibiotics, the recents uccess of daptomycin highlightst he evolvingr ole of CLPs as important pharmaceutical lead compounds. [5] The entomopathogenic bacteria of the genera Photorhabdus and Xenorhabdus that live in symbiosis with nematodes are a rich source of bioactive naturalp roducts for killing the insect, nematode development, and protecting the insectc adaver against food competitors. [12][13][14][15] Several CLPs, such as xenematides, [16] xefoampeptides, [17] chaiyaphumines, [9] taxlllaids, [18] and xentrivalpeptides, [19] have been identified previously.D uring our further investigation on new natural products from Photorhabdus and Xenorhabdus strains, we found an ew family of CLPs, namedp hototemtides (Scheme 1), after their biosynthetic gene cluster (BGC) pttABC from Photorhabdus temperata Meg1 was heterologously expressed in Escherichia coli. Here, we report the discovery, structurale lucidation, biosynthesis, total synthesis, and bioactivity of the major compound phototemtide A( 1). In addition, three minor derivatives, phototemtides B-D (2-4), were identified by detailed HPLC-MS analysis.
Genome miningo fP. temperata Meg1 (accession No. NZ_ JGVH01000010) showedt hat the strain encodes aB GC with two nonribosomal peptides ynthetases (NRPSs), termedP ttB (MEG1_RS04970) and PttC (MEG1_RS04975). Detailed analysis identified that PttBC consist of five modules with overall 17 domains,including astarter condensation (C start )domain in the initiation module of PttB (Figure 1), which could load af atty acid to the Nterminus of oligopeptide as shown in many lipopeptides, such as anikasin, [5] daptomycin, [20] and taxlllaids. [18] PttBC was thuse xpected to produce al ipodepsipeptide containing five amino acids.H owever,n os uch peptide could be identified in extracts of P. temperata Meg1 under standard laboratory conditions. Heterologous expression of an intact BGC in surrogate hosth as been proven ag ood strategy to bypass the endogenous regulatory controla nd activate silent pathways for the productiono fm any interesting natural products, [21] therefore this approach was applied to activate pttBC in well-characterized E. coli. Notably,o ne MbtH-encodingg ene, termed pttA (MEG1_RS04960,1 95 bp) is located 0.8 kb upstream from pttB. The MbtH proteins have been proposed to play an important role in stimulating adenylation reactions that are required for biosynthesis of some nonribosomal peptides. [22,23] Our effort expressing pttBC together with pttA in E. coli resulted in successfulp roduction of phototemtides A-D ( Figure S1 in the Supporting Information). Indeed, E. coli was found incapable of producing phototemtides withoutM btH-encoding gene pttA ( Figure S1).
To identify the structures of 1-4,t he major compound phototemtide A( 1)w as isolated as aw hite solid (4 mg) from the XAD-16 extracts of 4Lcultures of E. coli expressing pttABC. The molecular formula of 1 was determined to be C 34  ,a nd two oxygenated sp 3 carbon signals (d C = 71.9, 66.1 ppm) were observed. In addition, one phenylg roup was identified on the basis of the typical chemical shifts of d H = 7.30-7.13 ppm with total integration of five protons in the 1 HNMR spectrum and typical chemical shifts of d C = 137.9-126.2 ppm in the 13 CNMR spectrum.B ecause six carbonyl carbons ando ne phenyl ring accounted for ten of the eleven degrees of unsaturation, compound 1 shouldb eam onocyclic peptide. Combining 1 H, 1 HCOSY and 1 H, 13 CHSQC NMR data, six partial structuresi n 1 were constructed as glycine, valine, phenylalanine, threonine, isoleucine, and 3-hydroxyoctanoic acid (3-HOA).T he connectiv-  (Table S1); the examples of an amino acid different from that predicted in the BGC analysisa re not uncommon. [24,25] Thereby,c ompound 1 was unequivocally elucidated to be aC LP as depicted in Scheme 1, showing fatty acidi nvolved in al actone ring formation, as it is rare in Photorhabdus and Xenorhabdus strains and so far has only been observed forthe xefoampeptides. [17] Analysis of MS 2 fragmentation pattern of 1 further confirmed its structure ( Figure S2).E ster bond of 1 was first cleaved to give [M + H] + precursor ion at m/z 660 according to McLafferty rearrangement. [26] Subsequently,t he peak at m/z 642 was obtained after the loss of H 2 Of rom m/z 660. Further peaks at m/z 529, 428, 281, 182, and 125 indicatedt he consecutive losses of Ile, Thr,P he, Val, and Gly,w hich is identical with amino acid compositiona nd sequence of 1.W ith these fragmentation regularities in hand, the minor derivatives 2-4 were identified by detailed analysiso ft heir MS 2 fragmentation pat-terns ( Figure S2). They display similar constitution to 1 but are different in length of fatty acid chains (Scheme 1). As E. coli is not capable of producing iso-or anteiso-branched fatty acids due to am issing branched-chain keto acid dehydrogenase complex, [27] we only expect linear fatty acids. Considering that odd-numbered fatty acid containingn atural products are uncommon in E. coli,alabeling experiment in lysogeny broth (LB) mediumwith deuterated propionic acid fed as the biosynthetic precursors of odd-chain fatty acids [28] was performed (Figure S3), revealingt he incorporation of odd-numbered fatty acid building block (C9) in 4.T hat is to say, E. coli DH10BM taA used in this study is capable of synthesizing odd-numbered fatty acids when cultivated in LB medium, albeit in trace amounts.
To determine the absolutec onfigurations of amino acids, compound 1 was hydrolyzed with 6 m HCl as previously described [29] and was analyzed accordingt ot he advanced Marfey's method, [30] resulting in the absolute configurations for Val (l), Phe (d), Thr (l), and Ile (l;F igure S4). As nonproteinogenic amino acids l-allo-Thr and l-allo-Ile are relativelyu ncommon in natural peptides produced by organisms, [31,32] compound 1 was more likely to feature l-Thr and l-Ile, which was confirmed by subsequents ynthesis of 1.T he d-Phe also matches the presence of epimerization (E) domain within module 3. The absolutec onfigurations of amino acids in 2-4 were assumed to be the same as those of 1 because of their common biosynthesis origin.
However,t he information regarding the stereochemistry of 3-OH fatty acid was still missing. To address this problem, we conducted the total synthesis of two epimeric 1a and 1b containing( R)-and (S)-3-HOA moiety,r espectively (Scheme 2). Due to lack of commercial availability,t he synthetic work started with preparation of the chiral 3-HOA using samarium(II) iodide (SmI 2 )m ediated Reformatsky-type reaction of (R)-or (S)-4-  The key on-resine sterification of 3-HOA was then achieved with Fmoc-Ile-OH using am odified Yamaguchi esterification conditions with benzoyl chloride (BzCl), triethylamine (Et 3 N), 4-(dimethylamino)pyridine( DMAP) in dichloromethane (DCM) after several attempts. [34] Subsequently,t he Fmoc-protecting group was removed via treatment with piperidine in DMF,a nd the peptide was cleaved from the resin with hexafluoroisopropanol (HFIP) in DCM in order to preserve the side-chain protecting group of Thr. [35] Without purification, we next performed the key macrolactamization step in solution with HATU, HOAt, and DIPEA in DMF,a ssistedb ym icrowavei rradiation. [35] Finally,t he remaining side-chainp rotecting group of peptidew as removed by using trifluoroacetic acid (TFA), followed by purification using semipreparative HPLC to give 1a and 1b in 6.1 and 1.8 %o verall yield based on initially actual loading of the resin, respectively.W ith lipid tail isomers 1a and 1b available, we assigned the absolute configuration of 3-HOA in natural 1 to be R by comparison of the retentiont ime with synthetic standards since natural 1 showedt he identical retention time with synthetic 1a containing (R)-3-HOAm oiety,w hile 1b containing (S)-3-HOAmoiety eluted prior to 1/1a ( Figure S5). The absolute configurations of 3-OH fatty acids in 2-4 were assumed to be the same with that of 1.Besides,the total synthesis also identified l-Thr and l-Ile, not l-allo-Thr and l-allo-Ile incorporated in 1.
In summary,anew antimalarial CLP phototemtide A( 1), with three minor derivatives, phototemtides B-D (2-4), was identified from entomopathogenic P. temperata Meg1 after the silent NRPS-encoding gene cluster pttABC was activated by heterologous expression in E. coli. The structurale lucidationw as accomplished by combining spectroscopica nalysisa nd chemical methods including atotal synthesis of phototemtide A. Recent-Scheme2.To tal synthesiso fepimeric 1a and 1b.A)Synthesiso fc hiral 3-HOA, B) synthesis of epimeric 1a and 1b. ly,n ew peptided rugs have emerged due to the low toxicity, easy synthesis, rapid elimination, and less side effects, [36] but there is currently no approved peptidealternative for the treatment of malaria.T he number of marketed active ingredients is limited andm ost of them face challenges, such as newly observed resistance [37,38] and unpleasant side effects. [39,40] Althought he bioactivity of 1 against P. falciparum is only weak, it might be as tartingp oint towardaselective P. falciparum compound, as it shows no activity against any other tested organisms. With an efficient approacho ft otal synthesis in hand, furtheri nvestigationc ould focus on structure-activity relationships and subsequenti nvivo experiments of this new family of CLPs. This work is also the first example of the importance of MbtH for peptidep roduction in Photorhabdus andw ea re currentlys tudying the role of PttA in the originalp roducer and related strains.