Enhancing Structural Diversity of Lathyrane Derivatives through Biotransformation by the Marine-Derived Actinomycete Streptomyces puniceus BC-5GB.11

Lathyrane-type diterpenes have a wide range of biological activities. Among them, euphoboetirane A (1) exerts neurogenesis-promoting activity. In order to increase the structural diversity of this type of lathyrane and explore its potential use in neurodegenerative disorders, the biotransformation of 1 by Streptomyces puniceus BC-5GB.11 has been investigated. The strain BC-5GB.11, isolated from surface sediments collected from the intertidal zone of the inner Bay of Cadiz, was identified as Streptomyces puniceus, as determined by phylogenetic analysis using 16S rRNA gene sequence. Biotransformation of 1 by BC-5GB.11 afforded five products (3–7), all of which were reported here for the first time. The main biotransformation pathways involved regioselective oxidation at non-activated carbons (3–5) and isomerization of the ∆12,13 double bond (6). In addition, a cyclopropane-rearranged compound was found (7). The structures of all compounds were elucidated on the basis of extensive NMR and HRESIMS spectroscopic studies.


Introduction
Lathyranes are polyoxygenated macrocyclic diterpenoids with a 5/11/3-fused-ring skeleton that are widely distributed in plants from the genera Euphorbia and Jatropha [1][2][3][4][5].They stand out for their high structural diversity as well as for the variety of biological activities that they display [6].A great number of lathyrane diterpenoids have shown the capability to modulate MDR, cytotoxicity against cancer cell lines, anti-inflammatory activity, and the ability to induce cell proliferation or differentiation of neural stem cells (NSC) [6].Some structural features, such as a fused epoxy ring, the gem-dimethylcyclopropane ring, the oxidation degree, and the nature of the ester chains attached to the main skeleton, could be involved in the substrate-target biological interactions.
In previous studies, we have described that the lathyranes euphoboetirane A (1) and epoxyboetirane A (2) (Figure 1), isolated from Euphorbia boetica, increased the size of neurospheres in a dose-dependent manner in cultures stimulated with a combination of two growth factors, epidermal (EGF) and basic fibroblast (bFGF), in a synergist way.The results suggested that they act on receptors different from epidermal growth factor receptor (EGFR) or fibroblast growth factor receptor (FGFR) [7].On the other hand, it has been proven that lathyrane compounds can exert significantly different abilities to activate PKCs and promote NPC proliferation or differentiation [8,9] without knowing what the precise structural factors that determine the type of activity presented by these compounds are.Therefore, there is an interest in lathyrane-based library preparation with high structural diversity to explore its potential use in neurodegenerative disorders.
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 2 of 11 (EGFR) or fibroblast growth factor receptor (FGFR) [7].On the other hand, it has been proven that lathyrane compounds can exert significantly different abilities to activate PKCs and promote NPC proliferation or differentiation [8,9] without knowing what the precise structural factors that determine the type of activity presented by these compounds are.Therefore, there is an interest in lathyrane-based library preparation with high structural diversity to explore its potential use in neurodegenerative disorders.Microbial transformation is a really useful alternative to chemical approaches for performing highly regio-and stereoselective hydroxylations at non-activated carbons and rearrangement reactions [10][11][12][13].To the best of our knowledge, only two precedents for the biotransformation of lathyrane diterpenoids have been described.Wu [11].Recently, Euphorbia factor L1 and its deacylated derivative were biotransformed in their deoxy derivatives by Mucor polymorphosporus and Cunninghamella elegans [14].Moreover, euphorbiasteroid was glycosylated, regioselectively hydroxylated, or methyl oxidized by another C. elegans strain, bio-110930 [15].
This paper reports on the isolation and identification of a Streptomyces puniceus strain isolated from sediment samples from the intertidal zone of the inner Bay of Cadiz (Cádiz, Spain).In order to broaden the structural diversity of lathyrane diterpenoids and further study the relationship between the functionalization presented by these diterpenes and their neurogenesis-promoting activity, we also described the biotransformation of euphoboetirane A (1), a lathyrane-type diterpenoid isolated from E. boetica by Streptomyces puniceus BC-5GB.11.

Results and Discussion
Actinomycete strain BC-5GB.11was isolated from surface sediments collected in the intertidal zone of the inner Bay of Cadiz (Cádiz, Spain).This strain was sent for sequencing and identification to the identification service of the Spanish Type Culture Collection (CECT, https://www.uv.es/cect, accessed on 28 December 2023).The 16S-rRNA gene was sequenced and compared with those in NCBI databases.As a result, CECT identified strain BC-5GB.11as Streptomyces.Then, a neighbor-joining phylogenetic analysis was conducted using the Kimura two-parameter model and a bootstrap test with 5000 runs (MegAlign, DNASTAR ® La-Sergene package).A total of seventy-two sequences of the ribosomal DNA region comprising the 16S rRNA gene were downloaded from the Gen-Bank database.These sequences were selected from related species and genera in the family Streptomycetaceae and included five genera and twenty-four species.Based on all these studies, it was determined that strain BC-5GB.11 is grouped with the species S. puniceus (Figure 2).Microbial transformation is a really useful alternative to chemical approaches for performing highly regio-and stereoselective hydroxylations at non-activated carbons and rearrangement reactions [10][11][12][13].To the best of our knowledge, only two precedents for the biotransformation of lathyrane diterpenoids have been described.Wu [11].Recently, Euphorbia factor L 1 and its deacylated derivative were biotransformed in their deoxy derivatives by Mucor polymorphosporus and Cunninghamella elegans [14].Moreover, euphorbiasteroid was glycosylated, regioselectively hydroxylated, or methyl oxidized by another C. elegans strain, bio-110930 [15].
This paper reports on the isolation and identification of a Streptomyces puniceus strain isolated from sediment samples from the intertidal zone of the inner Bay of Cadiz (Cádiz, Spain).In order to broaden the structural diversity of lathyrane diterpenoids and further study the relationship between the functionalization presented by these diterpenes and their neurogenesis-promoting activity, we also described the biotransformation of euphoboetirane A (1), a lathyrane-type diterpenoid isolated from E. boetica by Streptomyces puniceus BC-5GB.11.

Results and Discussion
Actinomycete strain BC-5GB.11was isolated from surface sediments collected in the intertidal zone of the inner Bay of Cadiz (Cádiz, Spain).This strain was sent for sequencing and identification to the identification service of the Spanish Type Culture Collection (CECT, https://www.uv.es/cect, accessed on 28 December 2023).The 16S-rRNA gene was sequenced and compared with those in NCBI databases.As a result, CECT identified strain BC-5GB.11as Streptomyces.Then, a neighbor-joining phylogenetic analysis was conducted using the Kimura two-parameter model and a bootstrap test with 5000 runs (MegAlign, DNASTAR ® La-Sergene package).A total of seventy-two sequences of the ribosomal DNA region comprising the 16S rRNA gene were downloaded from the Gen-Bank database.These sequences were selected from related species and genera in the family Streptomycetaceae and included five genera and twenty-four species.Based on all these studies, it was determined that strain BC-5GB.11 is grouped with the species S. puniceus (Figure 2).Neighbor-joining tree constructed using 16S-rRNA gene sequences, comprising sequences identified in this study (highlighted in bold) and published sequences obtained from the GenBank database.The length of each branch pair reflects the distance between the respective sequence pairs.A dotted line on the tree denotes a negative branch length, while the bar indicates the number of nucleotide substitutions.Neighbor-joining tree constructed using 16S-rRNA gene sequences, comprising sequences identified in this study (highlighted in bold) and published sequences obtained from the GenBank database.The length of each branch pair reflects the distance between the respective sequence pairs.A dotted line on the tree denotes a negative branch length, while the bar indicates the number of nucleotide substitutions.
E. boetica was collected at "El Pinar del Hierro" in Chiclana de la Frontera, Cádiz (Spain).The n-hexane-soluble fraction of the methanolic extract of the aerial parts of E. boetica was fractionated and purified by silica gel column chromatography to afford the lathyrane euphoboetirane A (1), as indicated in the Section 3.
Preparative scale microbial transformation of 1 by S. puniceus BC-5GB.11furnished three new hydroxylated lathyranes 3-5, one derivative with an isomerization of the ∆ 12,13 double bond (6), and one product where a cleavage for the cyclopropane ring has occurred (7) (Figure 3).E. boetica was collected at "El Pinar del Hierro" in Chiclana de la Frontera, Cádiz (Spain).The n-hexane-soluble fraction of the methanolic extract of the aerial parts of E. boetica was fractionated and purified by silica gel column chromatography to afford the lathyrane euphoboetirane A (1), as indicated in the Materials and Methods Section.
Preparative scale microbial transformation of 1 by S. puniceus BC-5GB.11furnished three new hydroxylated lathyranes 3-5, one derivative with an isomerization of the ∆ 12,13 double bond (6), and one product where a cleavage for the cyclopropane ring has occurred (7) (Figure 3).The molecular formulas for compounds 3 and 4, C26H36O8, were identical.They were determined from [M+Na] + molecular ions in their HRESIMS spectra (m/z 499.2322 and 499.2298, respectively, calculated for C26H36O8Na 499.2308) (Figures S8 and S16) and were 16 mass units higher than that of 1, suggesting that both were monohydroxylated derivatives of euphoboetirane A (1).
Their 1 H NMR data (Table 1 and Figures S1 and S10) showed a deshielding of the signals corresponding to H-9 and H-11 (from 1.15 and 1.39 ppm for 1 [16] to 1.25 and 1.53 ppm for 3, and 1.27 and 1.59 ppm for 4, respectively).In addition, each one showed the absence of one of the singlet methyl signals and the appearance of new signals corresponding to methylenes bound to oxygen (3.46 and 3.39 ppm, d, J = 11.1 Hz, and 4.32 and 4.17 ppm, d, J = 12.2 Hz for 3 and 4, respectively).The disappearance of the signal corresponding to the methyl on the double bond (C-20) in compound 4 placed the new hydroxyl group at position C-20.In compound 3, it must be located on one of the methyls of the gem-dimethyl group.The exact location was established through the nOe effects observed between this methylene group and H-9 and H-11 (Figures 4 and S7c).
Their 1 H NMR data (Table 1 and Figures S1 and S10) showed a deshielding of the signals corresponding to H-9 and H-11 (from 1.15 and 1.39 ppm for 1 [16]   three new hydroxylated lathyranes 3-5, one derivative with an isomerization of the ∆ ble bond (6), and one product where a cleavage for the cyclopropane ring has occu (Figure 3).The molecular formulas for compounds 3 and 4, C26H36O8, were identical.Th determined from [M+Na] + molecular ions in their HRESIMS spectra (m/z 499.2 499.2298, respectively, calculated for C26H36O8Na 499.2308) (Figures S8 and S16) a 16 mass units higher than that of 1, suggesting that both were monohydroxylated tives of euphoboetirane A (1).
Their 1 H NMR data (Table 1 and Figures S1 and S10) showed a deshieldin signals corresponding to H-9 and H-11 (from 1.15 and 1.39 ppm for 1 [16] to 1.25 a ppm for 3, and 1.27 and 1.59 ppm for 4, respectively).In addition, each one sho absence of one of the singlet methyl signals and the appearance of new signal sponding to methylenes bound to oxygen (3.46 and 3.39 ppm, d, J = 11.1 Hz, and 4 4.17 ppm, d, J = 12.2 Hz for 3 and 4, respectively).The disappearance of the sign sponding to the methyl on the double bond (C-20) in compound 4 placed the n droxyl group at position C-20.In compound 3, it must be located on one of the me the gem-dimethyl group.The exact location was established through the nOe eff served between this methylene group and H-9 and H-11 (Figures 4 and S7c).
Compound 5 showed spectroscopic characteristics common to both 3 and 4 (Table 1 S25), indicated the presence of 32 additional mass units to that of compound 1, which was in agreement with the presence of two extra hydroxyl groups in the molecule.They were located on C-18 and C-20, as supported by the HMBC correlations between H 2 -18 and C-9 and between H-12 and C-20 (Figures S22 and S45).
On the other hand, compound 6 showed a molecular formula, C 26 H 36 O 7 , determined by HRMS (m/z 483.2377 [M+Na] + , calculated for C 26 H 36 O 7 Na 483,2359) (Figure S34), which was identical to that of compound 1.The HMBC correlations (Figures S31 and S45) observed indicated the same connectivity as 1, therefore it might be an isomeric compound.The principal difference observed in its 1 H NMR spectrum (Figure S27) was the large shielding of proton H-12 (from 6.48 ppm in 1 [16] to 5.28 ppm in 6, Table 2), as well as its coupling constant (d, J = 11.4Hz in 1 [16] and dd, J = 8.7, 1.6 Hz in 6).Furthermore, the 13 C NMR spectrum of 6 (Figure S28) showed a shift of the signals corresponding to C-12 and C-20 to upfield and downfield, respectively, in comparison to 1 (δ C 146.7 for C-12 and δ C 12.5 for C-20 in 1 [16]; δ C 134.8 for C-12 and δ C 22.2 for C-20 in 6).These data indicate that 6 might be the Z isomer of 1 [20].The isomerization was supported by the NOESY correlation observed between H-12 and H 3 -20 (Figures 5, S32 and S33b,c) and by the negative cotton effect (∆ε −4.06) observed in its ECD spectrum (Figure S35) at 228 nm, opposite to that shown by 1 [16].Accordingly, the structure of 6 was assigned as (12Z)-euphoboetirane A. On the other hand, compound 6 showed a molecular formula, C26H36O7, determined by HRMS (m/z 483.2377 [M+Na] + , calculated for C26H36O7Na 483,2359) (Figure S34), which was identical to that of compound 1.The HMBC correlations (Figures S31 and S45) observed indicated the same connectivity as 1, therefore it might be an isomeric compound.The principal difference observed in its 1 H NMR spectrum (Figure S27) was the large shielding of proton H-12 (from 6.48 ppm in 1 [16] to 5.28 ppm in 6, Table 2), as well as its coupling constant (d, J = 11.4Hz in 1 [16] and dd, J = 8.7, 1.6 Hz in 6).Furthermore, the 13 C NMR spectrum of 6 (Figure S28) showed a shift of the signals corresponding to C-12 and C-20 to upfield and downfield, respectively, in comparison to 1 (δC 146.7 for C-12 and δC 12.5 for C-20 in 1 [16]; δC 134.8 for C-12 and δC 22.2 for C-20 in 6).These data indicate that 6 might be the Z isomer of 1 [20].
The isomerization was supported by the NOESY correlation observed between H-12 and H3-20 (Figures 5, S32 and S33b,c) and by the negative cotton effect (∆ε −4.06) observed in its ECD spectrum (Figure S35) at 228 nm, opposite to that shown by 1 [16].Accordingly, the structure of 6 was assigned as (12Z)-euphoboetirane A.  Finally, compound 7 showed a molecular ion at m/z 481.2193 [M+Na] + (calc.for C26H34O7Na 481.2202) (Figure S43), which allowed us to deduce its molecular formula as C26H34O7.The 1 H NMR spectrum (Figure S36) of compound 7 showed similar main features for ring A to those of the previous compounds, but a significant change in rings B-C could be inferred from it.The characteristic signals for H-9 and H-11, as well as the signals Finally, compound 7 showed a molecular ion at m/z 481.2193 [M+Na] + (calc.for C 26 H 34 O 7 Na 481.2202) (Figure S43), which allowed us to deduce its molecular formula as C 26 H 34 O 7 .The 1 H NMR spectrum (Figure S36) of compound 7 showed similar main features for ring A to those of the previous compounds, but a significant change in rings B-C could be inferred from it.The characteristic signals for H-9 and H-11, as well as the signals corresponding to the gem-dimethylcyclopropane fragment, were missing (Table 2).Conversely, a spin system H 2 -7/H 2 -8/H-9/H-11/H-12 was identified in its 1 H-1 H COSY spectrum (Figure S38), which pointed to a C-10/C-11 bond cleavage of the cyclopropane ring.Indeed, proton and carbon resonances characteristic of an isopropenyl system were present.Moreover, olefinic protons located at C-10 ( 19), C-11 (12), and C-13 (20) were also observed in their NMR spectra (Figures S36 and S37).

Plant Material
The whole plants of Euphorbia boetica were collected at El Pinar del Hierro (Chiclana de la Frontera, Cádiz, Spain) in March 2020 with the permission of the national competent authorities (Dirección General de Biodiversidad, Bosques y Desertificación, Secretaría de Estado de Medio Ambiente, Ministerio para la Transición Ecológica y Reto Demográfico, reference number ESNC64, and Consejería de Agricultura, Ganadería Pesca y Desarrollo Sostenible-Delegación Territorial de Cádiz, Junta de Andalucía, reference number 201999901092011).

Microorganisms and Their Identification
The actinomycete Streptomyces puniceus BC-5GB.11was isolated from intertidal sediments collected in the inner Bay of Cadiz (Cádiz, Spain) within a Spartina spp.bed with the permission of the national competent authority (ABSCH-CNA-ES-240784-3, reference number ESNC84).Surface sediment samples were collected aseptically in the field, stored in sterile packaging, kept on ice, brought to the laboratory, and immediately processed.Sediment was diluted with sterile seawater (SSW), and aliquots were grown on potato dextrose agar (PDA) plates and marine agar plates (Condalab S.L.) and incubated at 25 • C for 5-10 d.Bacterial colonies were selected and streaked on PDA plates under axenic conditions.The isolates were maintained on PDA at 25 • C for routine experiments, and spores were stored in 60% (v/v) glycerol at −20 • C for later studies.
The BC-5GB.11 bacterial strain isolated was identified using the service of the Spanish Type Culture Collection (CECT, https://www.uv.es/cect, accessed on 28 December 2023) based on molecular techniques.Amplification and sequencing (with readings in both directions) of the 16S-rRNA gene were carried out.The sequencing of this region was compared with that in NCBI databases.The sequence was submitted to the NCBI database with the accession number PP101615.1.To study the phylogenetic relationship of our isolate, other sequences of related genera and species (72 sequences) from the family Streptomycetaceae were downloaded from the GenBank database and included in the phylogenetic trees (Figure 2).This microorganism was preserved on PDA plugs of 1 cm diameter in H 2 O at 4 • C, and it is deposited in the Bacteriological Collection of the University of Cadiz.

Extraction and Isolation of Euphoboetirane A (1)
Compound 1 was isolated from the aerial parts of E. boetica following the procedure previously described in the literature [7].The aerial parts of the fresh plant (3.0 kg) were frozen with liquid nitrogen, powdered, and extracted with MeOH (2.5 L × 3) at room temperature for 24 h.The MeOH extract was evaporated under reduced pressure to yield a crude extract, which was suspended in water (1 L) and then extracted with n-hexane (1.5 L × 3) and CH 2 Cl 2 (1.5 L × 3), sequentially.Evaporation of the solvent at reduced pressure yielded 33.3 g and 8.9 g of the n-hexaneand CH 2 Cl 2 -soluble extracts, respectively.The n-hexane extract was fractionated through silica gel column chromatography, using an increasing gradient of EtOAc in n-hexane (10-100%) to afford 21 fractions, according to TLC analysis.Fractions 4-7 were combined and further purified by column chromatography using a gradient mixture of CH 2 Cl 2 and acetone of increasing polarity (0-2%) to yield 1 (2.0 g).

Biotransformation of Euphoboetirane A (1)
Streptomyces puniceus BC-5GB.11was grown in seventeen 500-mililiter Erlenmeyer flasks containing 200 mL of ISP2 medium (4 g of yeast extract, 10 g of malt extract, and 4 g of dextrose per liter of sea water) at pH 7.3.Each flask was inoculated with 1300 µL of a fresh conidial suspension obtained by adding 25 mL of sterile ISP2 medium to seven Petri dishes (9 cm in diameter) cultured with the strain BC-5GB.11for 17 d.The flasks were shaken at 200 rpm and 25 • C under continuous white light (i.e., daylight lamp) for 3 d.A solution of euphoboetirane A (1) in DMSO (300 µL) was then added to each flask to a final concentration of 88 ppm.The culture control consisted of a fermentation flask to which 300 µL of DMSO was added.The substrate control contained sterile ISP2 medium and the same concentration of 1 dissolved in 300 µL of DMSO.All flasks were incubated under identical conditions as described above for an additional period of 11 d.The optimal biocatalysis time was established as a result of a preliminary study to determine the time at which all euphoboetirane A (1) is consumed in order to achieve maximum production of the biotransformation products.

Conclusions
Five new lathyrane-type diterpenoids were obtained by microbial transformation of euphoboetirane A (1) with Streptomyces puniceus BC-5GB.11.This strain, isolated from sediment samples from the intertidal zone of the inner Bay of Cadiz, catalyzed the regioselective hydroxylation of 1 at C-18 and C-20, as well as the isomerization of the ∆ 12,13 double bond.In addition, a rearranged cyclopropane compound was produced (7).
These derivatives have contributed to increasing the structural diversity of our lathyrane-based library, which will allow us to further investigate the relationship between the functionalization presented by these diterpenes and their neurogenesis-promoting activity.Studies are in progress to evaluate the ability of major metabolites to promote the release of transforming growth factor α (TFGα) and neuregulin 1 (NRG1) in cell cultures

Figure 2 .
Figure 2.Neighbor-joining tree constructed using 16S-rRNA gene sequences, comprising sequences identified in this study (highlighted in bold) and published sequences obtained from the GenBank database.The length of each branch pair reflects the distance between the respective sequence pairs.A dotted line on the tree denotes a negative branch length, while the bar indicates the number of nucleotide substitutions.

Figure 2 .
Figure 2.Neighbor-joining tree constructed using 16S-rRNA gene sequences, comprising sequences identified in this study (highlighted in bold) and published sequences obtained from the GenBank database.The length of each branch pair reflects the distance between the respective sequence pairs.A dotted line on the tree denotes a negative branch length, while the bar indicates the number of nucleotide substitutions.

Figure 3 .
Figure 3. Biotransformation products of euphoboetirane A (1) by Streptomyces puniceus BC-5GB.11.The molecular formulas for compounds 3 and 4, C 26 H 36 O 8 , were identical.They were determined from [M+Na] + molecular ions in their HRESIMS spectra (m/z 499.2322 and 499.2298, respectively, calculated for C 26 H 36 O 8 Na 499.2308) (Figures S8 andS16) and were 16 mass units higher than that of 1, suggesting that both were monohydroxylated derivatives of euphoboetirane A (1).Their 1 H NMR data (Table1and FiguresS1 and S10) showed a deshielding of the signals corresponding to H-9 and H-11 (from 1.15 and 1.39 ppm for 1[16] to 1.25 and 1.53 ppm for 3, and 1.27 and 1.59 ppm for 4, respectively).In addition, each one showed the absence of one of the singlet methyl signals and the appearance of new signals corresponding to methylenes bound to oxygen (3.46 and 3.39 ppm, d, J = 11.1 Hz, and 4.32 and 4.17 ppm, d, J = 12.2 Hz for 3 and 4, respectively).The disappearance of the signal corresponding to the methyl on the double bond (C-20) in compound 4 placed the new hydroxyl group at position C-20.In compound 3, it must be located on one of the methyls of the gem-dimethyl group.The exact location was established through the nOe effects observed between this methylene group and H-9 and H-11 (Figures 4 and S7c).
to 1.25 and 1.53 ppm for 3, and 1.27 and 1.59 ppm for 4, respectively).In addition, each one showed the absence of one of the singlet methyl signals and the appearance of new signals corresponding to methylenes bound to oxygen (3.46 and 3.39 ppm, d, J = 11.1 Hz, and 4.32 and 4.17 ppm, d, J = 12.2 Hz for 3 and 4, respectively).The disappearance of the signal corresponding to the methyl on the double bond (C-20) in compound 4 placed the new hydroxyl group at position C-20.In compound 3, it must be located on one of the methyls of the gem-dimethyl group.The exact location was established through the nOe effects observed between this methylene group and H-9 and H-11 (Figures 4 and S7c ).
a-c Interchangeable signals.