Extending the Structural Diversity of Labdane Diterpenoids from Marine-Derived Fungus Talaromyces sp. HDN151403 Using Heterologous Expression

Heterologous biosynthesis has become an effective means to activate fungal silent biosynthetic gene clusters (BGCs) and efficiently utilize fungal genetic resources. Herein, thirteen labdane diterpene derivatives, including five undescribed ones named talarobicins A–E (3–7), were discovered via heterologous expression of a silent BGC (labd) in Aspergillus nidulans. Their structures with absolute configurations were elucidated using extensive MS and NMR spectroscopic methods, as well as electronic circular dichroism (ECD) calculations. These labdanes belong to four skeleton types, and talarobicin B (4) is the first 3,18-dinor-2,3:4,18-diseco-labdane diterpene with the cleavage of the C2–C3 bond in ring A and the decarboxylation at C-3 and C-18. Talarobicin B (4) represents the key intermediate in the biosynthesis of penioxalicin and compound 13. The combinatorial heterologous expression and feeding experiments revealed that the cytochrome P450 enzymes LabdC, LabdE, and LabdF were responsible for catalyzing various chemical reactions, such as oxidation, decarboxylation, and methylation. All of the compounds are noncytotoxic, and compounds 2 and 8 displayed inhibitory effects against methicillin-resistant coagulase-negative staphylococci (MRCNS) and Bacillus cereus.

In this study, an attractive gene cluster (named labd) containing PvCPS homologous protein and various tailoring enzymes was found in the Antarctic sponge-derived fungus Talaromyces sp.HDN151403 based on bioinformatic analysis.Due to no labdane-related diterpene detected in the wild-type, reconstructing the entire biosynthetic pathway in genetically treatable fungal hosts may address this issue.Genetically tractable filamentous fungi such as Aspergillus nidulans have compatible transcription, translation, post-translational modification, and secretion machineries for the expression of foreign fungal genes [21], and therefore, the heterologous expression of the labd cluster in Aspergillus nidulans A1145 was attempted, which led to the production of 13 labdane diterpene derivatives, including five undescribed ones (namely talarobicins A-E).Structurally, these diterpenoids could be classified into four types, 3,18-dinor-2,3:4,18-diseco-, 18-nor-4,18-seco-and 3-nor-2,3-seco-labdane, and classical bicyclic labdane diterpenoids.Talarobicin B (4) is the first 3,18-dinor-2,3:4,18diseco-labdane diterpene and served as the key intermediate in generation of labdane diterpenes with ring A cleaved and rearranged.Based on biosynthetic pathway reconstruction and feeding experiments in A. nidulans, the biosynthetic pathway of these compounds was proposed, which involved P450-catalyzed multiple oxidation.

Results and Discussion
2.1.Prediction of the Labdane-Related Diterpenes BGC in Talaromyces sp.HDN151403 By using the PvCPS gene (GenBank: LC316181.1)as a query to LocalBlast on our sequenced fungal genomes, a series of conservative homologous gene clusters were found in several fungi (Figure 1).The orthologous gene cluster was also conserved in Talaromyces and Penicillium fungi when using the remote search tool cblaster [22] to search the online database (Figures S2 and S3).These clusters contain two conserved genes encoding a phosphatase and a bifunctional terpene synthase, adjacent to various post-modification genes (Figure S3).Among them, a gene cluster (labd) from Talaromyces sp.HDN151403 is attractive for the abundant oxidoreductase.The labd gene cluster contains a bifunctional diterpene synthase LabdA, a phosphatase LabdB, a short-chain dehydrogenase/reductase (SDR) LabdG, an aldehyde dehydrogenase LabdD, two P450 monooxygenases LabdE and LabdF, and a dual domain P450-methyltransferase gene labdC (Figure 1, Table S3).Detailed bioinformatics analysis indicate that the diterpene synthase LabdA possesses both type II terpene cyclase and prenyltransferase domains with 89% identity to PvCPS and 59% identity to PfCPS.The sequence similarity search results indicate that the P450s LabdC, LabdE, and LabdF have high similarity with many sequences in the public database, but the functions are unknown (Figures S6-S8).Further phylogenetic analysis indicated that LabdE and LabdF may have the ability to catalyze multiple oxidation reactions (Figure S4), while the domain analysis of LabdC suggested that it may have both P450 and methyltransferase catalytic activities (Figure S5).[23].Among them, the genome sequence of P. verruculosum TPU1311 (GenBank: GCA_001305275.1) was accessed from the NCBI database, and the genome sequence of P. fellutanum ATCC 48694 was accessed from the JGI Genome Portal [24].

Heterologous Reconstitution of the Labdane Diterpenes Biosynthetic Pathway
To investigate the biosynthetic pathway of isolated compounds, we sought to reconstitute its biosynthesis in A. nidulans.Firstly, compounds 1 and 2 were produced from the transformant AN-labdAB (Figure 5A, trace iii).Structurally, 2 is the product of the reduction of the C13/C14 double bond of 1, which is inferred to be catalyzed by the endogenous oxidoreductase of A. nidulans.Subsequently, 5 and 8 were produced from the two threegene-expressing transformants AN-labdABE and AN-labdABF, respectively (Figure 5A, traces iv and v).Compounds 3 and 6-12 were produced by the four-gene-expressing transformants labdABEF (Figure 5B, trace iii).In addition, trace amounts of 4 and 13 were produced by strains AN-labdABDEF and AN-labdABCDEF, respectively (Figure 5B, traces iv and v).Only 5 was produced from the transformant strain AN-labdABCDEG (Figure 5B, trace vi), while 8 was extensively produced in the strain AN-labdABCDFG (Figure 5A, trace xi), indicating that the two genes labdE and labdF catalyze the most critical reaction in the biosynthesis pathway of other compounds.
Next, we aimed to reconstitute the complete biosynthetic pathway using A. nidulans feeding experiments.As shown in Figure 6A,B, LabdE can catalyze the conversion from 6 to 7 and 8 to 9-12, respectively, indicating that LabdE is a multifunctional cytochrome P450 enzyme that catalyzes multiple oxidization.The strain AN-labdG fed 11 and produced 9, indicating that LabdG can catalyze the reduction of the ketone group at C-2 in 11 (Figure 6A, traces iii and iv).To identify whether 4 and 13 are products of the labd gene cluster, strain AN-labdCDEFG was constructed and fed 8 and 9 separately.As a result, only 8 was successfully converted to 4 and 9-13 (Figure 6B, traces i and ii).Aside from that, 4 and 13 were also successfully produced from strain AN-labdCDEFG fed 10 (Figure 6A, traces v and vi), confirming that 10 was the direct precursor for the synthesis of 4 and 13.

The Proposed Biosynthetic Pathways
On the basis of the heterologous reconstitution of 1-13, a plausible biogenetic pathway is proposed (Scheme 1).Firstly, the PT domain of LabdA catalyzes the condensation of DMAPP with three successively added IPPs to form GGPP, which is then cyclized to form copalyl diphosphate by the type II TC domain of LabdA [15].After that, an endogenous phosphatase in A. nidulans or the phosphatase LabdB could convert copalyl diphosphate into (+)-CPP.The (+)-CPP is converted into compound 1 through intermediate A under the catalysis of endogenous oxidase in A. nidulans.Moreover, 1 can be converted into 2 under the catalysis of endogenous reductase in A. nidulans.Compound 5 is derived from the multi-step oxidation of intermediate A catalyzed by the P450 LabdE.The P450 LabdF can catalyze the hydroxylation of Me-18 in A, followed by oxidation to aldehydes and finally, dehydrogenates to 8.During this cascade reaction, LabdE can catalyze the continued oxidation reaction of hydroxylation intermediate B to generate 3, 6, and 7.

The Proposed Biosynthetic Pathways
On the basis of the heterologous reconstitution of 1-13, a plausible biogenetic pathway is proposed (Scheme 1).Firstly, the PT domain of LabdA catalyzes the condensation of DMAPP with three successively added IPPs to form GGPP, which is then cyclized to form copalyl diphosphate by the type II TC domain of LabdA [15].After that, an endogenous phosphatase in A. nidulans or the phosphatase LabdB could convert copalyl diphosphate into (+)-CPP.The (+)-CPP is converted into compound 1 through intermediate A under the catalysis of endogenous oxidase in A. nidulans.Moreover, 1 can be converted into 2 under the catalysis of endogenous reductase in A. nidulans.Compound 5 is derived from the multi-step oxidation of intermediate A catalyzed by the P450 LabdE.The P450 LabdF can catalyze the hydroxylation of Me-18 in A, followed by oxidation to aldehydes On the one hand, compound 8 can be converted to 9-12 under the catalysis of LabdE.Compound 11 can also be reduced to 9 under the catalysis of the SDR LabdG.In the process of converting 8 to 12, it is speculated that LabdE catalyzes multi-step reactions, including oxidation and decarboxylation.On the other hand, 10 undergoes a ring-opening reaction under the catalysis of LabdE to form aldehyde intermediate F, which is catalyzed by aldehyde dehydrogenase LabdD to form intermediate G. Since G is unstable, it will oxidize and decarboxylate spontaneously or under the catalysis of LabdE to produce 4. The Intermediate H is catalyzed by LabdE to form I, which can further spontaneously or enzymatically form the compound penioxalicin.Bioinformatic analysis showed that the two P450s LabdE and LabdF share 39% and 20% identity to LabE, which is responsible for oxidizing the C-20 methyl group of raimonol in Streptomyces sp.KIB 015 to an aldehyde or carboxyl group, respectively [33].Therefore, it is speculated that penioxalicin undergoes multi-step oxidation and methylation reactions under the catalysis of LabdE and LabdC, respectively, to generate 13.On the one hand, compound 8 can be converted to 9-12 under the catalysis of LabdE.Compound 11 can also be reduced to 9 under the catalysis of the SDR LabdG.In the process of converting 8 to 12, it is speculated that LabdE catalyzes multi-step reactions, including oxidation and decarboxylation.On the other hand, 10 undergoes a ring-opening reaction under the catalysis of LabdE to form aldehyde intermediate F, which is catalyzed by aldehyde dehydrogenase LabdD to form intermediate G. Since G is unstable, it will oxidize and decarboxylate spontaneously or under the catalysis of LabdE to produce 4. The Intermediate H is catalyzed by LabdE to form I, which can further spontaneously or enzymatically form the compound penioxalicin.Bioinformatic analysis showed that the two P450s LabdE and LabdF share 39% and 20% identity to LabE, which is responsible for oxidizing the C-20 methyl group of raimonol in Streptomyces sp.KIB 015 to an aldehyde or carboxyl group, respectively [33].Therefore, it is speculated that penioxalicin undergoes multi-step oxidation and methylation reactions under the catalysis of LabdE and LabdC, respectively, to generate 13.

General Experimental Procedures
NMR spectra were recorded on a JEOLJN M-ECP 600 spectrometer (JEOL, Tokyo, Japan), an Agilent DD2-500 spectrometer, or a Bruker Avance NEO 400 MHz spectrometer (Bruker, Karlsruhe, Germany) using tetramethylsilane (TMS) as an internal standard.Specific rotations were obtained on a JASCO P-1020 digital polarimeter.UV-vis spectra were recorded on UFLC system (Shimadzu, Tokyo, Japan).HRESIMS spectra were obtained on a Thermo Scientific LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Scheme 1. Proposed biosynthetic pathways of compounds 1-13.

Materials and Culture Conditions
The fungal strains used in this study are listed in Table S1.The fungus, Talaromyces sp.HDN151403, was isolated from an unidentified sponge sample collected from Prydz Bay, Antarctica (−624 m, 75.5 • E, 69.7 • S) and identified based on internal transcribed spacer DNA sequencing (GenBank: MW888514) [34].Talaromyces sp.HDN151403 was cultured on potato dextrose agar (PDA, BD) at 28 • C for 5-7 days, and then the mycelia were transferred into potato dextrose broth (PDB, BD) for the isolation of genomic DNA, the isolation of total RNA, or the OSMAC analysis of secondary metabolites.Escherichia coli XL1-Blue was used for DNA manipulation.Heterologous expression was carried out with Aspergillus nidulans A1145 as the host.Saccharomyces cerevisiae BJ5464-NpgA was used for plasmid construction.

Gene Cloning, Plasmid Construction, and Genetic Manipulation
The plasmids utilized in this work are listed in Table S1.The oligonucleotide sequences for PCR primers are given in Table S2.The Q5 ® High-fidelity DNA Polymerase and Restriction endonucleases used for all DNA manipulation were from New England Biolabs (NEB).The Frozen-EZ Yeast Transformation II Kit (Zymo Research) and the Zymoprep Yeast Plasmid Miniprep I Kit (Zymo Research) were used for transformation and plasmid recombination in yeast.
Mycelia were collected from the liquid medium PDB via centrifugation.After grinding the mycelia in liquid nitrogen with a mortar, genomic DNA was extracted using cetyltrimethylammonium bromide (CTAB) buffer and phenol-chloroform extraction methods.All seven genes and their native terminators (300-500 bp downstream from the stop codon) were amplified via PCR using the gDNA of the original host as the template.Then, the recombinant plasmids were constructed according to the description from Yee and Tang [35].The glaA, gpdA, and amyB promoters were amplified from vectors pANU, pANR, and pANP by using primer pairs glaA-F/glaA-R, gpdA-F/gpdA-R, and amyB-F/amyB-R, respectively.The labd genes were amplified via PCR using primer pairs as listed in Table S1 and then combined with the PacI-linearized vector pANU, pANP, or pANR in vivo using yeast homologous recombination to yield several plasmids.The obtained yeast colonies were characterized using PCR.Yeast plasmids were isolated by using a Zymoprep Yeast Plasmid Miniprep I Kit and transformed into E. coli XL1 Blue.All the plasmids were confirmed via restriction enzyme digestion and DNA sequencing.

RNA Extraction and Reverse Transcriptase PCR (RT-PCR)
The RNA manipulations were carried out using the Direct-zol™ RNA Microprep Kit (Zymo Research) and the HiScript ® II 1st Strand cDNA Synthesis Kit (Vazyme Biotech Co., Ltd., Nanjing, China).The spores of Talaromyces sp.HDN151403 were inoculated into 100 mL of several media and cultivated at 28 • C for 14 days under dark conditions with two replicates each.Then, the mycelia were harvested and the total RNA was extracted using the Direct-zol™ RNA Microprep Kit according to the instructions.To evaluate the transcription of the labd gene cluster under different fermentation conditions, singlestranded cDNAs were synthesized using the HiScript ® II 1st Strand cDNA Synthesis Kit.The synthetic cDNA was used as a template to amplify gene fragments via PCR using primer pairs labdA-F/labdA-R, labdB-F/labdB-R, labdC-F/labdC-R, labdD-F/labdD-R, labdE-F/labdE-R, labdF-F/labdF-R, and labdG-F/labdG-R.The target gene fragment synthesized via PCR using gDNA as a template was used as a control.Then, we checked whether the target gene was expressed via DNA sequencing.The PCR primer pairs are listed in Table S2.

Transformation of A. nidulans
A. nidulans A1145 was used as the recipient host.Fungal protoplast preparation and transformation were carried out as described by Yee and Tang [35].PEG-mediated protoplast transformation was employed to construct the 12 A. nidulans transformant strains AN-labdA, AN-labdE, AN-labdG, AN-labdAB, AN-labdABE, AN-labdABF, AN-labdABEF, AN-labdABDEF, AN-labdABEFG, AN-labdABCDEF, AN-labdABCDEG, and AN-labdABCDEFG.Transformation with three empty pANU, pANR, and pANP vectors was used as the control (AN-empty vectors).Transformants were verified using PCR.

Extraction, Isolation, and Purification
The large-scale solid fermentation culture (CD-ST medium, 5 L) of the transformant strain AN-labdAB was extracted four times with EtOAc.The crude extract (9 g) was subjected to a C18 column using a stepped gradient elution of MeOH/H 2 O, yielding five subfractions (Fr.1-Fr.5, 30% to 70%).Fr.3 was purified on a preparative C18 HPLC column with a gradient of MeOH/H 2 O (72:28) to yield 2 (10.5 mg, retention time (t R ) = 22 min).Fr.4 was purified on a preparative C18 HPLC column with a gradient of MeOH/H 2 O (75:25) to yield 1 (12.0 mg, t R = 16.5 min).

A. nidulans Feeding Experiments
The A. nidulans transformant strain was grown on CD-ST liquid culture (50 mL) at 28 • C 180 rpm.After growth for 2 days, substrate (1-2 mg) dissolved in DMSO (50 µL) was added to the culture.After further incubation for 3 days, the mycelium was collected via filtration and extracted with acetone, while the media was extracted with ethyl acetate.The crude extracts were reduced to dryness in vacuo and re-dissolved in methanol for LC-MS analysis.

ECD Calculations
Conformation searches based on molecular mechanics with MMFF force fields were performed for stereoisomers to obtain stable conformers.All the stable conformers were further optimized using the density functional theory (DFT) method at the B3LYP/6-31G(d)-GD3BJ level via Gaussian 16 program package [36].The ECD were calculated using time-dependent density functional theory (TDDFT) at a B3LYP/6-311+G(d,p) level in methanol with IEFPCM model.The calculated ECD curves were all generated using the SpecDis 1.71 program package, and the calculated ECD data of all conformers were Boltzmann averaged using Gibbs free energy [37].

Antimicrobial Activity Assay
The isolated compounds were assayed against the pathogenic bacteria methicillinresistant coagulase-negative staphylococci (MRCNS), methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter baumannii, Bacillus cereus, Pseudomonas aeruginosa, and S. aureus, as well as the pathogenic fungus Canidia albicans.The detailed methodologies for biological testing were performed as previously described [38,39].Ciprofloxacin or Nystatin was used as the positive control.

Cytotoxicity Assay
Five human cancer cell lines, K562 (using the MTT method), ASPC-1, MDA-MB-231, H69AR, and L-02 (using the SRB method) cells, were used in the cytotoxic assay.Adriamycin (Sigma-Aldrich Inc., Washington, USA) was used as the positive control.The detailed methodologies for biological testing were performed as previously described [40,41].

Conclusions
Fungi-derived secondary metabolites have always been an important source of natural medicines [42].However, the success rate for discovering new compounds from fungi is hindered by the gene being silent or poorly expressed under laboratory conditions [43].In this study, a total of 13 labdane diterpenes belonging to four skeleton types, including five undescribed analogs, were obtained by heterologous expression in A. nidulans.Compounds 3-13 were all produced under the catalysis of tailoring enzymes in the BGC labd.Moreover, in the in vivo conversion process of isolated compounds, the three P450s LabdC, LabdE, and LabdF were disclosed for the first time to be responsible for catalyzing multi-step chemical reactions, such as hydroxylation, methylation, decarboxylation, etc.It is remarkable that 4 possessed a unique 3,18-dinor-2,3:4,18-diseco-labdane core structure which could serve as a key intermediate in the biosynthetic route of a group of ring A-cleaved and rearranged labdane diterpene derivatives, such as penioxalicin and 13.

Figure 1 .
Figure 1.(A) The labd BGC cluster from Talaromyces sp.HDN151403.(B) Comparison of conserved gene clusters across different sequenced fungi were visualized via clinker[23].Among them, the genome sequence of P. verruculosum TPU1311 (GenBank: GCA_001305275.1) was accessed from the NCBI database, and the genome sequence of P. fellutanum ATCC 48694 was accessed from the JGI Genome Portal[24].

7 iFigure 2 .
Figure 2. Expression of the labd cluster in Aspergillus nidulans (AN).HPLC traces of the EtOAc extracts derived from the A. nidulans wild-type and mutant strains (i−vi).The chromatograms were recorded at 220 nm in comparison with the separated and purified compounds (vii, separated compounds marked with numbers 1-13 and different colors).

Figure 2 .
Figure 2. Expression of the labd cluster in Aspergillus nidulans (AN).HPLC traces of the EtOAc extracts derived from the A. nidulans wild-type and mutant strains (i−vi).The chromatograms were recorded at 220 nm in comparison with the separated and purified compounds (vii, separated compounds marked with numbers 1-13 and different colors).

Figure 5 .
Figure 5. Combinatorial heterologous expression of the labd cluster in A. nidulans (AN).(A) LC-MS profiles of the EtOAc extracts derived from the A. nidulans transformants (i-xii) in comparison with the separated and purified compounds (xiii).(B) LC-MS traces of extracts from mutant strains (ivi).The chromatograms were recorded at 220 nm on the Shimadzu UFLC system connected in series with the Shimadzu LCMS-2020 mass spectrometer.Separated compounds marked with numbers 1-13 and different colors.

Figure 5 .Figure 6 .
Figure 5. Combinatorial heterologous expression of the labd cluster in A. nidulans (AN).(A) LC-MS profiles of the EtOAc extracts derived from the A. nidulans transformants (i-xii) in comparison with the separated and purified compounds (xiii).(B) LC-MS traces of extracts from mutant strains (i-vi).The chromatograms were recorded at 220 nm on the Shimadzu UFLC system connected in series with the Shimadzu LCMS-2020 mass spectrometer.Separated compounds marked with numbers 1-13 and different colors.

Figure 6 .
Figure 6.Dissecting the biosynthetic pathway of isolated compounds.(A,B) LC-MS traces of extracts from A. nidulans mutants after feeding with compounds 6, 8, 9, 10, and 11.The chromatograms in all cases are extracted from mass spectra of the base peak for each compound.Separated compounds marked with numbers 4 and 6-13 and different colors.