Heterogenous biosynthesis of medicarpin using engineered Saccharomyces cerevisiae

doi:10.21203/rs.3.rs-2645193/v1

Download PDF

Research Article

Heterogenous biosynthesis of medicarpin using engineered Saccharomyces cerevisiae

https://doi.org/10.21203/rs.3.rs-2645193/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background

Medicarpin is one important bioactive compound with multiple medicinal activities, including anti-tumor, anti-osteoporosis, anti-bacterial effects. Medicarpin is assigned to pterocarpans derived from medicinal plants, such as Sophora japonica, Glycyrrhiza uralensisFisch. and Glycyrrhiza glabra L.However, these medicinal plants only contain low amounts of medicarpin. Moreover, the planting area for medicarpin-producing plants is limited, thus, current medicarpin supply cannot satisfy the great demands of medicinal markets.

Results

In this study, eight key genes involved in medicarpin biosynthesis were identified by comparative transcriptome analysis and bioinformatic analyses. In vitro and in vivoenzymatic activities assays confirmed the catalytic functions of candidate enzymes were responsible for the biosynthesis of medicarpin and medicarpin intermediates. Further engineering of these genes in Saccharomyces cerevisiae achieved the heterogenous biosynthesis of medicarpin using liquiritigenin as the substrate, and the final medicarpin titer was 0.82 ± 0.18 mg/L. By increasing the gene copy number of VRand PTS, the final titer of the medicarpin increased to 2.05 ± 0.72 mg/L.

Conclusion

This study provides a solid foundation for the economical and sustainable production of medicarpin by synthetic biology strategy.

Medicarpin

Glycyrrhiza glabra L.

Saccharomyces cerevisiae

Medicarpin biosynthesis pathway

Transcriptome sequencing

Pterocarpan

Pterocarpans belong to the group of dihydroisoflavonoids with a benzopyran-bemzofuran four ring structure skeleton. Pterocarpans are widely distributed in various medicinal plants, such as Pueraria Lobata, Hedysari Radix. Pterocarpans have many bioactivities, including anti-tumor, anti-osteoporosis, anti-bacterial, anti-inflammation, antioxidant, anti-estrogenic, inhibition of acetylcholinesterase and neuraminidase, and immunosuppressive activities[1]. Thus, pterocarpans are the potential drugs for the treatment of several different diseases.

Medicarpin, one pterocarpan compound, is widely distributed in medicinal plants, including Sophora japonica, Zollernia paraensis, Platymiscium yucatamun, Machaerium aristulatum, Platymiscium floribundum, Brazilian red propolis, Glycyrrhiza uralensis Fisch. and Glycyrrhiza glabra L.[2]. Medicarpin has anti-osteoporotic[3], bone protection activities[4, 5], and shows cytotoxic activities in human breast cancer cells and human epidermoid carcinoma cells with low IC₅₀[6]. Besides, it can lead to apoptosis of multi-drug-resistant P388 leukemia cells[7], and display antifungal[8], antibacterial properties[9, 10]. Medicapin is mainly extracted from Glycyrrhiza plants, and its composition in the G. glabra L. is 0.09%, and its composition in G. uralensis Fisch. is 0.00012%[11, 12]. The Glycyrrhiza plants mainly grow in arid and semiarid areas. Previous overharvest of Glycyrrhiza plants led to the desertification and disappearance of desertification in nature. Planting Glycyrrhiza plants occupies large amounts of field, and it takes 2–3 years obtain high-level medicarpin in Glycyrrhiza plants. Therefore, other new alternative sources to obtain sufficient amount of medicarpin is of great interest.

The chemical synthesis process of medicarpin is complex, and the reaction conditions are severe. Moreover, the yield of chemical synthesis for medicarpin is low. Plant cell culture can be used to collect biomass and extract medicarpin, but the conditions for plant cell cultivation are relatively harsh, and plant cell grow slowly, leading to the unstable yields of medicarpin. Recently, synthetic biology have been applied to produce artemisinic acid[13, 14], ginsenoside[15–17], and other complex plant natural products. Especially, some flavonoid biosynthetic pathways in plants, such as quercetin, resveratrol, kaempferol, baicalein and scutellarein, have been identified[18], and efficient production of these flavonoids in yeasts have been realized using synthetic biology and metabolic engineering strategy[19–21].

As a valuably medicinal plant, the genome and transcriptome of G.uralensis have been sequenced, and the biosynthesis pathways of the glycyrrhizin[22] and liquiritin[23] have been revealed. The liquiritin biosynthesis pathway was reconstructed in yeast, and the heterologous synthesis of the main flavonoid skeleton of G. uralensis was realized[23]. Currently, the daidzein biosynthesis was rebuilt in yeast and optimized, and the final titer of daidzein was 85.4 mg/L[24]. Further introducing of plant glycotransferases in yeast led to the production of 72.8 mg/L puerarin and 73.2 mg/L daidzin. The conversion of vestitone to medicarpin in Medicago sativa L. was catalyzed by vestitone reductase and 7,2'-dihydroxy-4'-methoxyisoflavanol dehydratase[25]. These provide the possibility to produce G. uralensis flavonoids at economically and sustainably way via synthetic biology and other cutting-edge biotechnologies. Thus, identifying the full medicarpin biosynthetic pathway in Glycyrrhiza and engineering yeasts to produce medicarpin are of great interest.

In this study, comparative analysis of Glycyrrhiza uralensis Fisch. and Glycyrrhiza glabra L. transcriptome identified some potential keystone genes involving in the biosynthesis of medicarpin from liquiritin. The functions of these genes were further verified in vitro. Then, the genes used for medicarpin biosynthesis were engineered in Saccharomyces cerevisiae, and the natural product metabolites of the engineered yeast strains were characterized. This study provides a new strategy for the sustainable production of complex bioactive natural products derived from traditional Chinese medicines using microbial cell factories.

Transcriptomic analysis of G. glabra L. and G. uralensis Fisch. root

The medicarpin was detected in the roots of the G. glabra L. and G. uralensis Fisch (Fig. S1). Though the medicarpin compositions in these two different Glycyrrhiza plants are different, their contents are low and can’t be obtained. In order to reveal the possible key enzymes for medicarpin biosynthesis in Glycyrrhiza, deep sequencing of the G. glabra L. and G. uralensis Fisch. root transcriptome was performed (Tables S5-6 and Fig. S2)[26, 27], and Glycyrrhiza genes were comprehensively annotated.

For the transcriptomic data, 35953 of the 36159 unigenes from G. glabra L. and G. uralensis Fisch. have high identities with genes in Nr database, whereas 27077 unigenes have high identities with sequences in Swiss-Prot database. All unigenes > 1000 bp has blast matches, and they are similar with homologies among several different plant species (Fig. S3). Among them, 7854 contigs were matched to Cicer arietinum, and 4899 and 4768 contigs were matched to Glycine max and Cajanus cajan, respectively. GO analysis suggested that the biological process was the most abundant (57453, 38.29%), followed by cellular component (46775, 31.17%) and molecular function (45802, 30.52%) (Fig. S4). For biological process, cellular process and metabolic process were dominant, suggesting that some important cellular processes and metabolic activities occurred in Glycyrrhiza roots. All annotated unigenes were assigned to 25 COG categories (Fig. S5-6). Amino acid transport and metabolism category is the largest group, followed by cell wall/membrane/envelop biogenesis and cytoskeleton.

To identify the active biological pathways and understand the biological gene functions, all unigenes were further analyzed by the KEGG pathway database. In summary, 8528 unigenes were assigned to six KEGG categories[28]. The metabolism category included ten sub-categories, and the four main sub-categories were carbohydrate metabolism, amino acid metabolism, lipid metabolism and energy metabolism (Fig. S7). A total of 464 unigenes were assigned to the biosynthesis of other secondary metabolites sub-category, and most of them were annotated to be the enzymes accounting for the synthesis of flavonoids, flavone and flavonol. These hinted that Glycyrrhiza roots harbored diverse natural product biosynthetic genes and might synthesize a variety of metabolites.

Prediction of medicarpin biosynthetic genes

Differentially expressed transcript analysis identified more than 2-fold changes in different gene expression and false discovery rate (FDR) < 0.05, and 1241, 1882, and 3123 genes were up-regulated, down-regulated, and differentially expressed, respectively in the roots of G. glabra L. and G. uralensis Fisch. (Fig. S8). Based on the degree of KEGG enrichment analysis, selenocompound metabolism, and other natural product biosynthetic pathway were differentially expressed in these two Glycyrrhiza roots (Fig. S9-10). The differentially expressed gene analysis based on GO, COG and KOG annotation identified some genes associated with biosynthesis, transport, and catabolism of secondary metabolites, amino acid transport and metabolism, and defense mechanisms (Fig. S11-12). Besides, 2431 unigenes were predicted to be transcription factors (Fig. S13)[29].

Medicarpin were predicted to the precursor of glybridin[30], and glybridin was mainly extracted from G. glabra L.[31–35]. Therefore, the high-level expression genes in the natural product biosynthetic pathway of G. glabra L.. may be responsible for the biosynthesis of medicarpin or its intermediates. A total of 176 genes were annotated to encode the key enzymes for medicarpin biosynthesis in the transcriptome data (Fig. 1, S2 and Table S6). Expression analysis of these candidate genes revealed that genes annotated as CHS, CHR, CHI, 2-hydroxyisoflavanone synthase (2-HIS), cytochrome P450 reductase (CPR), isoflavone 4′-O-methyltransferase (I4′OMT), 2-hydroxyisoflavanone dehydratase (HID), isoflavone 2′-hydroxylase (I2′H), isoflavone reductase (IFR), vestitone reductase (VR), and pterocarpan synthase (PTS) had relatively higher expression levels in G. glabra L. than that in G. uralensis Fisch. (Fig. 1, and Table S6).

Functional characterization of predicted medicarpin biosynthetic enzymes

The candidate genes 2-HIS, CPR, I4′OMT, HID, I2′H, IFR, VR, and PTS, were expressed in E. coli or yeast. The expressed proteins were purified for in vitro enzymatic activity assays. Incubation of liquiritigenin with 2-HIS and CPR yielded 2,7,4′-trihydroxyisoflavanone (Fig. 2A). The extraction of yeast harboring 2-HIS, CPR and I4′OMT can generate 2,7-dihydroxy-4′-methoxyisoflavanone using liquiritigenin as substrate (Fig. 2A). For the yeast strain DWY1 harboring 2-HIS, CPR and HID, the yeast extraction can catalyze liquiritigenin to daidzein[24]. When yeast DWY1 crude enzyme solution and I4′OMT protein were added into the reaction system together, can catalyze liquiritigenin to formononetin (Fig. 2B)[36, 37]. Besides, some by-product was generated in the yeast fermentation metabolites (Fig. 2B). Further analysis suggested that the by-product was generated by I4′OMT with liquiritigenin as substrate, and generated by-products were identified as liquiritigenin 4′-methyl ether and liquiritigenin 7-methyl ether by LC-MS and NMR (Fig. 2B and Fig. S14-16)[38, 39]. Thus, I4′OMT could catalyze flavonoids to novel products. The enzyme mixture of I2′H, CPR, IFR, VR and PTS could catalyze formononetin to 2′-hydroxyformononetin, vestitone, and medicarpin (Fig. 2C and Fig. S17)[40, 41]. 2-HIS, CPR, I4′OMT, HID, I2′H, IFR, VR and PTS can catalyze liquiritigenin to medicarpin in vitro, showing the biosynthetic pathway from liquiritigenin to medicarpin was recovered. Thus, introduction of the pathway in yeasts might produce medicarpin from liquiritigenin directly.

Heterologous production of medicarpin using engineered yeast

In order to verify the medicarpin synthesis in yeasts, we introduced the eight identified genes into wide-type yeasts, and three engineered yeast strains, DW08, DW09, and DW10, were constructed (Fig. 3A and Table S3). DW09 harboring 2-HIS, CPR, I4’OMT and HID genes can produce formononetin using liquiritigenin as substrate (Fig. 3B and 3C), while DW08 harboring I2’H, CPR, IFR, VR, and PTS genes can produce medicarpin using formononetin as substrate. Further shake flask analysis showed the final medicarpin title of DW08 using formononetin as substrate was 9.21 ± 1.97 mg/L (Fig. 3B and 3D). DW10 contained eight key enzyme genes of 2-HIS, CPR, I4’OMT, HID, I2’H, IFR, VR and PTS, and it can produce medicarpin directly using liquiritigenin as substrate (Fig. 3B and 3E). However, the medicarpin titer of DW10 using liquiritigenin as substrate was 0.82 ± 0.18 mg/L, which was lower than that of the DW08 using formononetin as substrate, suggesting limited formononetin was provided during the transfer from liquiritigenin to formononetin. Besides the targeted product of medicarpin, the intermediates daidzein, formononetin, and vestitone were detected in DW10 biomass. As daidzein was the completing byproduct for formononetin, I4’OMT enzyme activity should be enhanced to switch the metabolic flux to formononetin synthesis, which might increase the final product medicarpin synthesis.

The titer of vestitone produced by strain DW10 was high and the titer of medicarpin was low, suggesting the enzyme activities of VR and PTS should be increased to convert vestitone to medicarpin. Therefore, strain DW11 was further built by increasing the gene copy number of VR and PTS in DW10. After feeding liquiritigenin as substrate, the final medicarpin titer of DW11 was 2.05 ± 0.72 mg/L and it was 2.5 times of DW10 (Fig. 3B and 3F).

In the in vitro enzyme activity assay of I4′OMT, no daidzein was detected, and formononetin and LM were detected (Fig. 2B). During DW09, DW10 and DW11 fermentation with liquiritigenin as substrate, large amounts of by-product daidzein and small amounts of formononetin were detected. In the meanwhile, no LM was detected. Extra I4′OMT was available in vitro reaction system, and the I4′OMT activity might be low in vivo, suggesting I4′OMT may be the rate-limiting enzyme in medicarpin biosynthesis. Therefore, high-level expression of I4′OMT genes might increase medicarpin titer of DW11. I4′OMT was reported to be an isoflavone 4′-O-methyltransferase. In this study, I4′OMT was verified to catalyze C-4′ and C-7 hydroxyl groups of flavonoid liquiritigenin in vitro, showing that I4′OMT may have versatile bioactivity and play essential roles in the biosynthesis of several different flavonoids. Structure biology would give insights into the catalytic mechanism of I4′OMT when using liquiritigenin as its substrate, which may help to increase/strengthen I4′OMT specify and activities by protein engineering strategy.

Metabolite accumulation in the fermenter varies with the amount of cells

The starting substrates and fermentation time might affect the medicarpin production of DW11 (Fig. 4). During 120 h flask fermentation, the medicarpin titer of DW11 using formononetin as substrate was higher than that of DW11 using liquiritigenin as starting substrate. The final medicarpin titer is 4.27 ± 0.08 mg/L using formononetin as substrate (Fig. 4B), while the final medicarpin titer is 3.77 ± 0.25 mg/L using liquiritigenin as starting substrate (Fig. 4A). As the metabolic pathway from formononetin to medicarpin is shorter than that from liquiritigenin to medicarpin, more medicarpin might be produced and accumulated when using formononetin as substrate. In this study, further engineering the flavonoid biosynthetic precursors, such as p-coumaroyl-CoA and Malonyl-CoA would generate efficient yeast strains for medicarpin production.

The synthesis of liquiritigenin de novo in yeast has been realized in S. cerevisiae and Yarrowia lipolytica[42, 43]. The final liquiritigenin titer of one Y. lipolytica strain was 62.4 mg/L[42]. Though de novo production of medicarpin was not tried in this study, introducing several know plant genes and increasing liquiritigenin precursor supply in DW11 can synthesize medicarpin using glucose as sole carbon source in the future. The medicarpin was predicted to be the precursor of glabridin[30], and the medicarpin can be detected in G. glabra L. and G. uralensis Fisch.. Glabridin is only available in G. glabra L.[31–35], thus, whether medicarpin are the precursor of glabridin should be further investigated.

In this study, the comparative transcriptome analysis of G. glabra L. and G. uralensis Fisch. identified 176 genes which may function in the medicarpin synthesis. Further enzymatic assays confirmed eight key enzymes in the pathway from liquiritigenin to medicarpin. Introduction of these eight genes in S. cerevisiae led to the final medicarpin titer of 0.82 ± 0.18 mg/L; the final medicarpin titer of was further increased to 2.05 ± 0.72 mg/L in another optimized yeast strain, which lay the foundation to produce the valuable product of medicarpin using engineered yeasts. This study demonstrates that the integration of omics technology and synthetic biology strategies could recover complex plant natural product biosynthetic pathway.

Plant materials and Chemicals

The root samples of G. glabra L. and G. uralensis Fisch. were collected from Urumqi, Xinjiang province, China during the middle stage of root formation. The middle stage of root formation was two years. The root samples of G. glabra L. and G. uralensis Fisch. from the middle stage were set with three biological replicates (marked as G1, G2, G3 and W1, W2, W3, respectively). All the root samples were frozen at -80 ℃ for RNA extraction. The purity of all the chemical compounds, including liquiritigenin (CAS: 578-86-9), daidzein (CAS: 486-66-8), formononetin (CAS: 485-72-3), medicarpin (CAS: 32383-76-9), NADH (CAS: 606-68-8), and NADPH (CAS: 2646-71-1), are > 98%. All the chemical compounds were commercially available (Yuanye, Shanghai, China).

Deep Illumina sequencing and transcriptome analysis

Total RNAs were extracted from the root samples of G. uralensis Fisch. and G. glabra L. using the Column Plant RNAout2.0 (Tiandz Inc., Beijing, China). Approximately 100 mg of each sample was used to extract RNA. The RNA was evaluated using agarose gel electrophoresis, Nanodrop One (Nanodrop Technologies Inc., DE, USA), and Agilent 2100 (Agilent Technologies Inc., CA, USA) to confirm the purity, concentration, and integrity, respectively. The 260/280 nm ratios and 260/230 nm ratios of 1.8–2.2 and 1.4–1.8, respectively, from the Nanodrop were regarded as pure (Table S4). Next, the RNA library was constructed, and sequencing was performed by Genepioneer technologies corporation (Nanjing, China)[44]. Novaseq 6000 platform (Illumina Inc.) was used for high-throughput sequencing[45, 46] with pair-end 150 bp. The reference genome of G. uralensis (http://ngs-data-archive.psc.riken.jp/Gur-genome/download.pl.) was used for transcriptome analysis, and gene functions were comprehensively annotated based on the following databases of Nr (NCBI nonredundant protein sequences), Pfam (protein families), KOG/COG (clusters of orthologous groups of proteins), SwissProt (a manually annotated and reviewed protein sequence database), KEGG (Kyoto encyclopedia of genes and genomes database), GO (gene ontology), and KO (KEGG Orthology). Fragments per kilobase per million mapped reads (FPKM) value was used to estimate the expression level of the genes. DEGs between libraries were identified by DESeq2 (http://www.bioconductor.org/packages/release/bioc/html/DESeq.html). Fold change represents the ratio of expression quantity between two samples, and the Benjamini-Hochberg approach was used to adjust the P values for controlling the FDR. Unigenes with FDR < 0.05 and an absolute value log₂ (Fold change) ≥ 1 were seen as differentially expressed[47]. The full-length cDNAs of the targeted genes were amplified by PCR using High-Fidelity PCR Master Mix (Tolo Biotech, shanghai, China), and the primers were designed based on the transcriptomic data (Table S1). The transcriptomic data used in this study were submitted to the Sequence Read Archive (SRA) of NCBI database with the accession numbers as SRR22859373, SRR22859372, SRR22859371 for three biological replicates from G. glabra L. and SRR22859370, SRR22859369, SRR22859368 for three biological replicates from G. uralensis Fisch., respectively.

Based on the annotation results, seventeen candidate genes involved in the medicarpin biosynthesis pathway, including the sequences which encoding PAL (phenylalanine ammonialyase), C4H (cinnamate 4-hydroxylase), 4CL (4-coumaroyl CoA ligase), TAL (tyrosine ammonialyase), PD (pyruvate dehydrogenase), ACC (acetyl-CoA carboxylase), CHS (chalcone synthase), CHR (chalcone reductase), CHI (chalcone isomerase), 2-HIS (2-hydroxyisoflavanone synthase), CPR (cytochrome P450 reductase), I4′OMT (isoflavone 4′-O-methyltransferase), HID (2-hydroxyisoflavanone dehydratase), I2′H (isoflavone 2′-hydroxylase), IFR (isoflavone reductase), VR (vestitone reductase), PTS (pterocarpan synthase) were highly expressed in the root of G. glabra L. Since the enzyme activities of CPR and PTS from G. glabra L. were weak, CPR of soybean[24] and PTS of G. pallidiflora Maxim[48]. were selected and used for the heterogenous biosynthesis of medicarpin.

Gene expression

The genes HID, I4′OMT, IFR and PTS were amplified from the cDNAs from the roots of G. glabra L., and G. pallidiflora Maxim, respectively, and then cloned into the pET-28a vector, and the gene VR was amplified from the cDNA from the root of G. glabra L. and cloned into the pGEX-4T-1 vector using the EasyGeno Assembly Cloning kit (Tolobio, Shanghai, China). The plasmids were transferred into Escherichia coli BL21 (DE3). All primers used in vector construction are listed (Table S1). Transformants were screened on solid culture medium (containing 100 µg/mL kanamycin sulfate or ampicillin sodium), and single colonies were picked for sequencing verification.

Recombinant cells were cultured in 1 L Luria-Bertani (LB) medium (37 ℃, 165 rpm) containing 100 µg/mL kanamycin sulfate or ampicillin sodium. When OD₆₀₀ value of the reaches 0.6–0.8, isopropyl β-D-thiogalactoside (IPTG) with a final concentration of 0.5 mmol/L was added. The cultivation was further carried out at 18 ℃ for 12 hours. 1 L of bacterial suspension was centrifuged to obtain expression cells (5000 rpm, 15 min), and the precipitant was resuspended in 20 mL buffer C solution (10 mmol/L Tris-HCl, 5% glycerol, 0.2 mol/L NaCl). The cells were disrupted with a high-pressure cell crusher (ATS Engineering Limited, Suzhou, China), then centrifuged at 4 ℃ and 12000 rpm. The recombinant protein was purified using the His-Tagged Protein Purification Kit or the GST-Tagged Protein Purification Kit (Smart-Lifesciences, Changzhou, China), and the protein concentration was determined by ultraviolet spectrophotometer (Allsheng, Hangzhou, China).

Cytochrome P450 2-HIS and I2′H were first cloned from the cDNA from the roots of G. glabra L, and expressed in S. cerevisiae IMX581 using pESC-LEU vector. Cytochrome P450 reductase (CPR) was amplified from the cDNA of soybean, and expressed in IMX581 using the vector pESC-URA. The recombinant plasmid was transferred into IMX581 by electroporation, and transformants were grown on corresponding SC-Leu or -Ura media with 2% glucose at 30 ℃ for 3 days. The positive colonies were picked and shaken at 30 ℃ and 200 rpm until OD₆₀₀ value reaches about 0.8. The 2% glucose medium was replaced with an induction medium (containing 2% galactose), and cultivated at 30 ℃ and 200 rpm for 72 h.

Characterization of enzymatic activity in vitro and in vivo

The enzymatic reaction systems using liquiritigenin as substrate contained 0.1 mol/L dipotassium hydrogen phosphate, 0.5 mol/L sucrose, 0.5 mmol/L glutathione, 1 mmol/L NADPH, 1 mmol/L NADH, 5 mmol/L magnesium chloride, 100 µmol/L substrate liquiritigenin and 0.5 mmol/L S-adenosyl-L-methionine (SAM), and 500 µL crude yeast extraction supernatant solution of strain DWY1, and 30 µL I4′OMT protein derived from E. coli expression. The enzymatic reaction systems[49] using formononetin as substrate contained 0.1 mol/L potassium phosphate (pH = 8.0), 0.4 mol/L sucrose, 0.5 mmol/L glutathione, 2 mmol/L NADPH, 2 mmol/L NADH, 5 mmol/L magnesium chloride, 50 µmol/L substrate formononetin, and 250 µL crude yeast extraction supernatant solution of strain DW08.

In vitro enzymatic activity measurement mixture was incubated at 30 ℃ for 12 h with gentle shaking, and the reaction mixture was extracted with an equal volume of ethyl acetate for three times. The extracted metabolites were dried and re-dissolved in methanol. The metabolites were further purified by passing through a 0.22 µm polytetrafluoroethylene (PTFE) filter and used for UPLC-ESI-Q-TOF-MS/MS analysis.

For in vivo analysis, the yeast strains were cultivated in medium containing 20 mg liquiritigenin or formononetin at 30 ℃ and 210 rpm for 24 hours; Then 20 mg of liquiritigenin or formononetin was added to the medium, and continued to cultivate for another 72 hours. The fermentation broth was extracted with an equal volume of ethyl acetate for three times, and the final products were redissolved in methanol and used for UPLC-ESI-Q-TOF-MS/MS analysis.

UPLC‒ESI-Q-TOF-MS/MS analysis of catalytic and final products

An Acquity UPLC system coupled with a Synapt mass spectrometer (Waters Corp., Milford, MA, USA) and equipped with an electrospray ionization (ESI) device were used to analyze the catalytic products. The separation was carried out on a Waters C18 column (100 mm × 2.1 mm, 1.7 µm). The column was eluted with a gradient mobile phase which consisted of 0.1% aqueous formic acid (solvent system A) and acetonitrile (B). The mobile phases during operation were: 0–1 min, 5% B; 1–20 min, 5%-100% B; 20–30 min, 100% B; 30–31 min, 100%-5% B; 31–33 min, 5% B. The injection volume was 2 µL, and the flow rate was 0.4 mL/min.

Extraction of medicarpin and other metabolites from G. uralensis Fisch. and G. glabra L.

The dried roots of G. uralensis Fisch. or G. glabra L., were pulverized into powder, and 4 g of the powder was dissolved in 400 mL of 70% methanol. The power in the methanol was treated with ultrasonic (KQ-500DE numerical control ultrasonic cleaner, working frequency: 40 kHz, power: 500 W, Kunshan Ultrasonic Instrument Co., Ltd.) at 30 ℃ for 60 min to extract metabolites in the roots. After centrifugation, the supernatant was filtered through a 0.22 µm PTFE filter and used for qualitative analysis.

Reconstitution of the medicarpin biosynthetic pathway in yeast

The chassis strain used in this experiment was S. cerevisiae IMX581 (MATa ura3-52 can1::cas9-natNT2 TRP1 LEU2 HIS3)[50]. Heterologous genes were integrated into the targeted genomic loci via the CRISPR/Cas9 system[23]. Plasmid pMEL10 was used as gRNA vector. Phanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech Co. Ltd) was used for DNA fragment amplifications, and PrimeStar DNA polymerase (TaKaRa Bio) was used for in vitro fusion PCR. The ClonExpress MultiS One Step Cloning Kit (Vazyme Biotech Co.,Ltd) was used for in vitro fragment recombinant. FastPure Plasmid Mini Kit (Vazyme Biotech Co.,Ltd) was used for plasmid extraction. FastPure Gel DNA Extraction Mini Kit (Vazyme Biotech Co.,Ltd) was used for DNA extraction. Super Yeast Transformation Kit (Coolaber) was used for transformation. All primers, the plasmids, and the strains used in this study were listed (Table S1 -3). The genes used in this study were submitted to Genbank database with the accession numbers of OQ067102(CHS), OQ067258(CHI), OQ067259(CHR), OQ102530(2-HIS), OQ102529(HID), OQ067262(I2′H), OQ067263(IFR), OQ067264(VR), OQ067265(I4′OMT), OQ067266(C4H), OQ067267(4CL).

Fermentation and product analysis

S. cerevisiae strain DW11 was used for medicarpin biosynthesis in shake flask. DW11 was grown at 30 °C and 200 rpm for 12 h, and then transferred into 1 L of fresh medium containing 0.2 g/L liquiritigenin or formononetin. DW11 was further cultivated at 30 °C and 200 rpm for 5 days, and yeast sample was fetched every 24 h during cultivation. The collected yeast samples were disrupted with a high-pressure cell crusher, and then centrifuged at 10000 g for 20 min to collect the supernatant. The metabolites in the supernatant were extracted with the same volume of ethyl acetate (v/v = 1:1) for three times. The extraction was dried and redissolved with methanol for UPLC analysis.

Quantitative UPLC analysis

The yeast product was detected using a Waters C18 column (100 mm × 2.1 mm, 1.7 µm) on an Acquity UPLC system (Waters Corp., Milford, MA, USA) by gradient elution with a mobile phase comprising 0.1% (v/v) formic acid aqueous solution (A) and acetonitrile (B) at a flow rate of 0.4 mL/min. The mobile phases were programmed as: 0–1 min, 5% B; 1–10 min, 5%-100% B; 10–11 min, 100% B for 1 min; 11–12 min, 100%-5% B; 12–13 min, 5% B. The injection volume was 2 µL. The column temperature during operation was 25 °C. For the product vestitone with no available standard compound, the semi-preparative RP-HPLC (Shimadzu, Kyoto, Japan) was used to collect and concentrate, which was further dried to obtain solid powder by rotary evaporator (Buchi, Switzerland). The verstitone could be used as standard for quantification analysis.

Supplementary Information

The online version contains supplementary material available at

Acknowledgements

We thank the staff at the experiment center for science and technology of Nanjing University of Chinese Medicine for experimental supports. We appreciate Zhaozhu Lin, Mingming Xu, Xinyi Wei, Fulin Wang, and Jing Yang from Nanjing University of Chinese Medicine for technical assistance and discussions.

Author contributions

M.Z., YJ.W., and W.L. designed experiments. H.F., JZ. Z, R.D. and CJ.L. performed the sample collection and performed the bulk of the experiments. CJ.L. contributed to gene identification and cloning, protein expression, purification, and crystallization. CJ.L. contributed to enzymatic assay experiments. M.Z., YJ.W., and W.L. analyzed the data and wrote the manuscript. M.Z., YJ.W., and W.L. conceived the project.

Funding

This work was financially supported by the National Natural Science Foundation of China (81903526, 81991523, 82072240, and 32270192), the Open Project of Chinese Materia Medica First-Class Discipline of Nanjing University of Chinese Medicine (No. 2020YLXK008 to W.L.), the Open Project of State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (No. SIMM2205KF to W.L.), the Open Project of State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences (No. SKLMR-20220704 to W.L.), the Open Funding Project of the State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology to W.L., the Fok Ying Tung Education Foundation, and Jiangsu Specially-Appointed Professor Talent Program to W.L.

Availability of data and materials

The datasets used and analyed during the current study are available from the corresponding author upon reasonable request.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing of interests.

Wang HY, Li T, Ji R, Xu F, Liu GX, Li YL, Shang MY, Cai SQ: Metabolites of Medicarpin and Their Distributions in Rats. Molecules 2019, 24.
Yang X, Zhao Y, Hsieh MT, Xin G, Wu RT, Hsu PL, Horng LY, Sung HC, Cheng CH, Lee KH: Total Synthesis of (+)-Medicarpin. J Nat Prod 2017, 80:3284-3288.
Imran KM, Yoon D, Lee TJ, Kim YS: Medicarpin induces lipolysis via activation of Protein Kinase A in brown adipocytes. BMB Rep 2018, 51:249-254.
Imran KM, Yoon D, Kim YS: A pivotal role of AMPK signaling in medicarpin-mediated formation of brown and beige. Biofactors 2018, 44:168-179.
Mansoori MN, Raghuvanshi A, Shukla P, Awasthi P, Trivedi R, Goel A, Singh D: Medicarpin prevents arthritis in post-menopausal conditions by arresting the expansion of TH17 cells and pro-inflammatory cytokines. Int Immunopharmacol 2020, 82:106299.
Rayanil KO, Bunchornmaspan P, Tuntiwachwuttikul P: A new phenolic compound with anticancer activity from the wood of Millettia leucantha. Arch Pharm Res 2011, 34:881-886.
Gatouillat G, Magid AA, Bertin E, El btaouri H, Morjani H, Lavaud C, Madoulet C: Medicarpin and millepurpan, two flavonoids isolated from Medicago sativa, induce apoptosis and overcome multidrug resistance in leukemia P388 cells. Phytomedicine 2015, 22:1186-1194.
Mundodi SR, Watson BS, Lopez-Meyer M, Paiva NL: Functional expression and subcellular localization of the Nectria haematococca Mak1 phytoalexin detoxification enzyme in transgenic tobacco. Plant Molecular Biology 2001, 46:421-432.
Oh I, Yang WY, Chung SC, Kim TY, Oh KB, Shin J: In vitro sortase A inhibitory and antimicrobial activity of flavonoids isolated from the roots of Sophora flavescens. Arch Pharm Res 2011, 34:217-222.
Williams D, Perry D, Carraway J, Simpson S, Uwamariya P, Christian OE: Antigonococcal Activity of (+)-Medicarpin. ACS Omega 2021, 6:15274-15278.
Eerdunbayaer, Orabi MAA, Aoyama H, Kuroda T, Hatano T: Structures of New Phenolics Isolated from Licorice, and the Effectiveness of Licorice Phenolics on Vancomycin-Resistant Enterococci. Molecules 2014, 19:13027-13041.
Kitagawa I, Chen WZ, Hori K, Harada E, Yasuda N, Yoshikawa M, Ren JL: Chemical Studies of Chinese Licorice-Roots.I. Elucidation of Five New Flavonoid Constituents from the Roots of Glycyrrhiza glabra L. Collected in Xinjiang. Chemical & Pharmaceutical Bulletin 1994, 42:1056-1062.
Paddon CJ, Westfall PJ, Pitera DJ, Benjamin K, Fisher K, McPhee D, Leavell MD, Tai A, Main A, Eng D, et al: High-level semi-synthetic production of the potent antimalarial artemisinin. Nature 2013, 496:528-+.
Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL, Ndungu JM, Ho KA, Eachus RA, Ham TS, Kirby J, et al: Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 2006, 440:940-943.
Cao H, Nuruzzaman M, Xiu H, Huang J, Wu K, Chen X, Li J, Wang L, Jeong JH, Park SJ, et al: Transcriptome analysis of methyl jasmonate-elicited Panax ginseng adventitious roots to discover putative ginsenoside biosynthesis and transport genes. Int J Mol Sci 2015, 16:3035-3057.
Jayakodi M, Lee SC, Lee YS, Park HS, Kim NH, Jang W, Lee HO, Joh HJ, Yang TJ: Comprehensive analysis of Panax ginseng root transcriptomes. BMC Plant Biol 2015, 15:138.
Liu MH, Yang BR, Cheung WF, Yang KY, Zhou HF, Kwok JS, Liu GC, Li XF, Zhong S, Lee SM, Tsui SK: Transcriptome analysis of leaves, roots and flowers of Panax notoginseng identifies genes involved in ginsenoside and alkaloid biosynthesis. BMC Genomics 2015, 16:265.
Nabavi SM, Samec D, Tomczyk M, Milella L, Russo D, Habtemariam S, Suntar I, Rastrelli L, Daglia M, Xiao JB, et al: Flavonoid biosynthetic pathways in plants: Versatile targets for metabolic engineering. Biotechnology Advances 2020, 38:12.
Lyu XM, Zhao GL, Ng KR, Mark R, Chen WN: Metabolic Engineering of Saccharomyces cerevisiae for De Novo Production of Kaempferol. Journal of Agricultural and Food Chemistry 2019, 67:5596-5606.
Sun J, Sun W, Zhang G, Lv B, Li C: High efficient production of plant flavonoids by microbial cell factories: Challenges and opportunities. Metabolic engineering 2022, 70:143-154.
Volk MJ, Tran VG, Tan S-I, Mishra S, Fatma Z, Boob A, Li H, Xue P, Martin TA, Zhao H: Metabolic Engineering: Methodologies and Applications. Chemical reviews 2022.
Nomura Y, Seki H, Suzuki T, Ohyama K, Mizutani M, Kaku T, Tamura K, Ono E, Horikawa M, Sudo H, et al: Functional specialization of UDP-glycosyltransferase 73P12 in licorice to produce a sweet triterpenoid saponin, glycyrrhizin. Plant J 2019, 99:1127-1143.
Yin Y, Li Y, Jiang D, Zhang X, Gao W, Liu C: De novo biosynthesis of liquiritin in Saccharomyces cerevisiae. Acta Pharm Sin B 2020, 10:711-721.
Liu Q, Liu Y, Li G, Savolainen O, Chen Y, Nielsen J: De novo biosynthesis of bioactive isoflavonoids by engineered yeast cell factories. Nat Commun 2021, 12:6085.
Guo L, Dixon RA, Paiva NL: The 'pterocarpan synthase' of alfalfa: association and co-induction of vestitone reductase and 7,2'-dihydroxy-4'-methoxy-isoflavanol (DMI) dehydratase, the two final enzymes in medicarpin biosynthesis. FEBS letters 1994, 356:221-225.
Li YG, Mou FJ, Li KZ: De novo RNA sequencing and analysis reveal the putative genes involved in diterpenoid biosynthesis in Aconitum vilmorinianum roots. 3 Biotech 2021, 11:96.
Zhao F, Sun M, Zhang W, Jiang C, Teng J, Sheng W, Li M, Zhang A, Duan Y, Xue J: Comparative transcriptome analysis of roots, stems and leaves of Isodon amethystoides reveals candidate genes involved in Wangzaozins biosynthesis. BMC Plant Biol 2018, 18:272.
Rai M, Rai A, Kawano N, Yoshimatsu K, Takahashi H, Suzuki H, Kawahara N, Saito K, Yamazaki M: De Novo RNA Sequencing and Expression Analysis of Aconitum carmichaelii to Analyze Key Genes Involved in the Biosynthesis of Diterpene Alkaloids. Molecules 2017, 22.
Wang X, Hui F, Yang Y, Yang S: Deep sequencing and transcriptome analysis to identify genes related to biosynthesis of aristolochic acid in Asarum heterotropoides. Sci Rep 2018, 8:17850.
Simmler C, Pauli GF, Chen SN: Phytochemistry and biological properties of glabridin. Fitoterapia 2013, 90:160-184.
DONG Yalei QY, WANG Qilin, LI Li, HUANG Chuanfeng, WANG Haiyan, SUN Lei: Determination of glabridin in cosmetic by UHPLC-MS/MS method. China Surfactant Detergent & Cosmetics 2021, 51:161-165.
Hatano T, Fukuda T, Liu YZ, Noro T, Okuda T: Phenolic constituents of licorice. IV. Correlation of phenolic constituents and licorice specimens from various sources, and inhibitory effects of licorice extracts on xanthine oxidase and monoamine oxidase. Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan 1991, 111:311-321.
Ma Shu-yan MA, Bahargul Kahaer, He Qian: Study on the Preparation Technology of Isoflavon Glabridin from Glycyrrhiza glabra L. Journal of Xinjiang Medical University 2007, 30:692-694.
WANG Xu-dong SC-h, ZHOU Long, CHAI Bao-shan The Application, Extraction and Separation and Preparation Methods for Glabridin. Fine Chemical Intermediates 2020, 50:10-15.
WANG Zhongbo CM: Determination of glabridin in different species of Glycyrrhiza by HPLC. Hubei Journal of TCM 2011, 33:74-75.
Akashi T, Sawada Y, Shimada N, Sakurai N, Aoki T, Ayabe S: cDNA cloning and biochemical characterization of S-adenosyl-L-methionine: 2,7,4 '-trihydroxyisoflavanone 4 '-O-methyltransferase, a critical enzyme of the legume isoflavonoid phytoalexin pathway. Plant and Cell Physiology 2003, 44:103-112.
Liu CJ, Dixon RA: Elicitor-induced association of isoflavone O-methyltransferase with endomembranes prevents the formation and 7-O-methylation of daidzein during isoflavonoid phytoalexin biosynthesis. Plant Cell 2001, 13:2643-2658.
HANS ACHENBACH MSaMAC: flavonoid and other constituents of bauhinia manca. Phytochemistry 1988, 27:1835-1841.
Zhang W, Jiang S, Qian DW, Shang EX, Duan JA: Effect of liquiritin on human intestinal bacteria growth: metabolism and modulation. Biomedical Chromatography 2014, 28:1271-1277.
Guo L, Paiva NL: Molecular cloning and expression of alfalfa (Medicago sativa L.) vestitone reductase, the penultimate enzyme in medicarpin biosynthesis. Arch Biochem Biophys 1995, 320:353-360.
Zhao XB, Mei WL, Gong MF, Zuo WJ, Bai HJ, Dai HF: Antibacterial Activity of the Flavonoids from Dalbergia odorifera on Ralstonia solanacearum. Molecules 2011, 16:9775-9782.
Akram M, Rasool A, An T, Feng XD, Li C: Metabolic engineering of Yarrowia lipolytica for liquiritigenin production. Chemical Engineering Science 2021, 230:12.
Rodriguez A, Strucko T, Stahlhut SG, Kristensen M, Svenssen DK, Forster J, Nielsen J, Borodina I: Metabolic engineering of yeast for fermentative production of flavonoids. Bioresource Technology 2017, 245:1645-1654.
Schaarschmidt S, Fischer A, Lawas LMF, Alam R, Septiningsih EM, Bailey-Serres J, Jagadish SVK, Huettel B, Hincha DK, Zuther E: Utilizing PacBio Iso-Seq for Novel Transcript and Gene Discovery of Abiotic Stress Responses in Oryza sativa L. Int J Mol Sci 2020, 21.
Jiang Y, Wang L, Lu S, Xue Y, Wei X, Lu J, Zhang Y: Transcriptome sequencing of Salvia miltiorrhiza after infection by its endophytic fungi and identification of genes related to tanshinone biosynthesis. Pharm Biol 2019, 57:760-769.
Kim JA, Roy NS, Lee IH, Choi AY, Choi BS, Yu YS, Park NI, Park KC, Kim S, Yang HS, Choi IY: Genome-wide transcriptome profiling of the medicinal plant Zanthoxylum planispinum using a single-molecule direct RNA sequencing approach. Genomics 2019, 111:973-979.
Cui J, Shen N, Lu Z, Xu G, Wang Y, Jin B: Analysis and comprehensive comparison of PacBio and nanopore-based RNA sequencing of the Arabidopsis transcriptome. Plant Methods 2020, 16:85.
Uchida K, Akashi T, Aoki T: The Missing Link in Leguminous Pterocarpan Biosynthesis is a Dirigent Domain-Containing Protein with Isoflavanol Dehydratase Activity. Plant and Cell Physiology 2017, 58:398-408.
Chang-Jun Liu DH, Lloyd W Sumner, Richard A Dixon: Regiospecic hydroxylation of isoavones by cytochrome P450 81E enzymes from Medicago truncatula. The Plant Journal 2003, 36:471-484.
Mans R, van Rossum HM, Wijsman M, Backx A, Kuijpers NGA, van den Broek M, Daran-Lapujade P, Pronk JT, van Maris AJA, Daran JMG: CRISPR/Cas9: a molecular swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae. Yeast 2015, 32:S253-S253.

No competing interests reported.

Supplementaryfile20230301lin01.pdf

Download PDF

Version 1

posted

You are reading this latest preprint version

Heterogenous biosynthesis of medicarpin using engineered Saccharomyces cerevisiae

Status:

Version 1

Abstract

Figures

Background

Results And Discussion

Prediction of medicarpin biosynthetic genes

Functional characterization of predicted medicarpin biosynthetic enzymes

Heterologous production of medicarpin using engineered yeast

Metabolite accumulation in the fermenter varies with the amount of cells

Conclusion

Methods

Plant materials and Chemicals

Deep Illumina sequencing and transcriptome analysis

Gene expression

UPLC‒ESI-Q-TOF-MS/MS analysis of catalytic and final products

Reconstitution of the medicarpin biosynthetic pathway in yeast

Fermentation and product analysis

Quantitative UPLC analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1