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Cited by 2Microbial Natural Products and BiosynthesisMicrobial Metabolic Engineering and BioproductionMicrobial Metabolism and Applications

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Multi-layered engineering of Aspergillus terreus enhances biosynthesis of the plant-derived fungicide physcion

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Ren, Zilin, Xue, Yingying, Xu, Ning, Feng, Dandan, Geng, Ce, Wu, Yongsong, Liu, Dan, Zhao, Linshui, Zhang, Xiaoxi, Ma, Honglei, Huang, Xuenian, Qi, Feifei, Lu, Xuefeng·2025·Microbial Cell Factories
DOI 10.1186/s12934-025-02857-zPMC PMC12613595

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Abstract

Background: Emodin and its derivatives are important bioactive anthraquinones from rhubarb, with diverse pharmacological activities. Physcion, an O -methylated derivative of emodin, is a promising plant-derived fungicide and pharmaceutical lead. However, plant extraction yields are low and land-intensive, while microbial production is hampered by inefficient conversion and byproduct accumulation. Results: Here, we identify a cytochrome P450 enzyme (CYP-H6231) that, with its dedicated redox partner cytochrome P450 reductase (CPR-H10273), converts emodin to ω-hydroxyemodin in Aspergillus terreus . Deletion of CYP-H6231 increased physcion titer by 1.8-fold and significantly improved product purity. Further engineering, via 3- O -methyltransferase overexpression, SAM pathway enhancement, and enzyme fusion, yielded only modest improvement (up to 37%), likely due to compromised strain robustness from the loss of CYP-H6231 mediated detoxification. Structural modeling and mutagenesis of CYP-H6231 revealed key residues for substrate recognition and catalysis. Conclusions: This study reveals a detoxification bottleneck in anthraquinone biosynthesis and establishes two improved A. terreus platforms for scalable production of physcion and emodin, respectively, highlighting trade-offs between pathway efficiency and cellular fitness.

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Figures

Fig. 1 Engineering strategies for enhanced physcion production in second-generation A. terreus cell factory. Multiple approaches were employed including: (1) minimizing hydroxylated by-product formation through CYP-H6231 knockout, (2) improving conversion efficiency via 3-emodin-O-methyltransferase (3-EOMT) and S-adenosylmethionine synthase (SamS) overexpression, (3) enhancing precursor supply through acetyl-CoA carboxylase (ACC) overexpression, and (4) enhancing membrane transport via MFS transporter overexpression. OE: overexpression. Compound 1, emodin; compound 2, physcion; and compound 3, ω-hydroxyemodin
Fig. 1 Engineering strategies for enhanced physcion production in second-generation A. terreus cell factory. Multiple approaches were employed including: (1) minimizing hydroxylated by-product formation through CYP-H6231 knockout, (2) improving conversion efficiency via 3-emodin-O-methyltransferase (3-EOMT) and S-adenosylmethionine synthase (SamS) overexpression, (3) enhancing precursor supply through acetyl-CoA carboxylase (ACC) overexpression, and (4) enhancing membrane transport via MFS transporter overexpression. OE: overexpression. Compound 1, emodin; compound 2, physcion; and compound 3, ω-hydroxyemodinCC BY · PubMed Central
Fig. 2 By-products (ω-hydroxyemodin and fallacinol) and intermediate (emodin) yields analysis of physcion producing first-generation variant PgedA-PtaI. (A) HPLC analysis of fermented products of the A. terreus variant PgedA-PtaI on the fourth day at 440 nm. Traces ii and iii represent the authentic standards of emodin and physcion, respectively. The peaks of ω-hydroxyemodin, emodin and physcion are filled in red, orange and green, respectively. (B) The titer time courses of emodin and physcion derivatives during fed-batch fermentation of the PgedA-PtaI in a 100-liter bioreactor. Error bars represent the standard deviations of three identical replicates
Fig. 2 By-products (ω-hydroxyemodin and fallacinol) and intermediate (emodin) yields analysis of physcion producing first-generation variant PgedA-PtaI. (A) HPLC analysis of fermented products of the A. terreus variant PgedA-PtaI on the fourth day at 440 nm. Traces ii and iii represent the authentic standards of emodin and physcion, respectively. The peaks of ω-hydroxyemodin, emodin and physcion are filled in red, orange and green, respectively. (B) The titer time courses of emodin and physcion derivatives during fed-batch fermentation of the PgedA-PtaI in a 100-liter bioreactor. Error bars represent the standard deviations of three identical replicatesCC BY · PubMed Central
Fig. 3 Deciphering the type of anthraquinone hydroxylase. HPLC analysis of fermentation culture extracts of ΔgedA and ΔgedA-Δ10273 on the seventh day at 440 nm
Fig. 3 Deciphering the type of anthraquinone hydroxylase. HPLC analysis of fermentation culture extracts of ΔgedA and ΔgedA-Δ10273 on the seventh day at 440 nmCC BY · PubMed Central
Fig. 4 Transcriptome-based identification of anthraquinone hydroxylase, functional gene characterization, and metabolite titer analysis. (A) Heatmap illustrating transcript levels of the geodin BGC and 34 CYP genes. (B) Time-course accumulation of emodin, ω-hydroxyemodin, physcion, and fallacinol in the PgedA-PtaI strain during transcriptome profiling. (C) HPLC chromatograms (440 nm) showing comparative metabolite profiles of gene deletion mutants in the ΔgedA background (i–iv) and the PgedA-PtaI strain (v–vi) on day 4. Standard compounds (vii–ix): physcion (green), ω-hydroxyemodin (orange), and emodin (yellow). (D) Comparison of emodin, ω-hydroxyemodin, and total anthraquinone production (mg/g) between ΔgedA and ΔgedA-ΔH6231 strains after 4 and 7 days of cultivation. (E) Comparison of emodin, physcion, and total anthraquinone production (mg/g) between PgedA-PtaI and PgedA-PtaI-ΔH6231 strains after 4 and 7 days of cultivation
Fig. 4 Transcriptome-based identification of anthraquinone hydroxylase, functional gene characterization, and metabolite titer analysis. (A) Heatmap illustrating transcript levels of the geodin BGC and 34 CYP genes. (B) Time-course accumulation of emodin, ω-hydroxyemodin, physcion, and fallacinol in the PgedA-PtaI strain during transcriptome profiling. (C) HPLC chromatograms (440 nm) showing comparative metabolite profiles of gene deletion mutants in the ΔgedA background (i–iv) and the PgedA-PtaI strain (v–vi) on day 4. Standard compounds (vii–ix): physcion (green), ω-hydroxyemodin (orange), and emodin (yellow). (D) Comparison of emodin, ω-hydroxyemodin, and total anthraquinone production (mg/g) between ΔgedA and ΔgedA-ΔH6231 strains after 4 and 7 days of cultivation. (E) Comparison of emodin, physcion, and total anthraquinone production (mg/g) between PgedA-PtaI and PgedA-PtaI-ΔH6231 strains after 4 and 7 days of cultivationCC BY · PubMed Central
Fig. 5 Enhancement of physcion production through 3-EOMT and SamS overexpression strategy. (A) HPLC analysis (λ = 440 nm) showing the enzymatic conversion by PtaI and AwOMT in the presence of SAM and emodin. Control (-) represents the negative control without enzyme. Physcion STD shows the standard reference. (B) Schematic representation of chromosomal integration of 3-EOMT or SamS at the CYP-H6231 locus in the PgedA-PtaI-ΔpyrG strain. (C) Comparative analysis of emodin, physcion, and anthraquinone production titers (mg compound /g pellet mycelia) among different strains at day 4. Error bars indicate standard deviations from three independent replicates. (D) Comparison of emodin-to-physcion conversion rates (%) among different strains at day 4 and day 7. Error bars represent standard deviations from three independent replicates
Fig. 5 Enhancement of physcion production through 3-EOMT and SamS overexpression strategy. (A) HPLC analysis (λ = 440 nm) showing the enzymatic conversion by PtaI and AwOMT in the presence of SAM and emodin. Control (-) represents the negative control without enzyme. Physcion STD shows the standard reference. (B) Schematic representation of chromosomal integration of 3-EOMT or SamS at the CYP-H6231 locus in the PgedA-PtaI-ΔpyrG strain. (C) Comparative analysis of emodin, physcion, and anthraquinone production titers (mg compound /g pellet mycelia) among different strains at day 4. Error bars indicate standard deviations from three independent replicates. (D) Comparison of emodin-to-physcion conversion rates (%) among different strains at day 4 and day 7. Error bars represent standard deviations from three independent replicatesCC BY · PubMed Central
Fig. 6 Optimization of physcion biosynthesis through protein fusion and co-expression strategies. (A) Left: Schematic representation of the emodin to physcion conversion pathway and various protein fusion combinations between GedH and AwOMT or GedH and SamS. Right: Diagram showing chromosomal integration strategy at the CYP-H6231 locus in the PgedA-PtaI-ΔpyrG strain. (B) Quantitative analysis of metabolite production and conversion efficiency. Bar graph shows emodin, physcion, and anthraquinone titers (compound/pellet mycelia, mg/g), while the line graph indicates emodin-to-physcion transformation rates (%) for different fusion constructs at day 7. Error bars represent standard deviations from three independent replicates. (C) Cryo-scanning electron microscopy images showing morphological differences in a subset of spores among various genetic variants, including PgedA-PtaI, PgedA-PtaI-ΔH6231, P6231-PtaI, Pgpd-PtaI, P6231-AwOMT, and Pgpd-AwOMT strains
Fig. 6 Optimization of physcion biosynthesis through protein fusion and co-expression strategies. (A) Left: Schematic representation of the emodin to physcion conversion pathway and various protein fusion combinations between GedH and AwOMT or GedH and SamS. Right: Diagram showing chromosomal integration strategy at the CYP-H6231 locus in the PgedA-PtaI-ΔpyrG strain. (B) Quantitative analysis of metabolite production and conversion efficiency. Bar graph shows emodin, physcion, and anthraquinone titers (compound/pellet mycelia, mg/g), while the line graph indicates emodin-to-physcion transformation rates (%) for different fusion constructs at day 7. Error bars represent standard deviations from three independent replicates. (C) Cryo-scanning electron microscopy images showing morphological differences in a subset of spores among various genetic variants, including PgedA-PtaI, PgedA-PtaI-ΔH6231, P6231-PtaI, Pgpd-PtaI, P6231-AwOMT, and Pgpd-AwOMT strainsCC BY · PubMed Central
Fig. 7 Structural analysis, site-directed mutagenesis, and enzymatic activity of CYP-H6231. (A) Close-up view of the emodin binding pocket. Emodin (green) is positioned near the heme group (yellow), with potential key substrate-binding residues shown in cyan and labeled. Numbers indicate interaction distances (in Å). (B) Catalytic site of CYP-H6231 highlighting key residues. Emodin (green sticks) and the heme group (yellow sticks) are shown, with essential catalytic residues colored blue (E–R–R triad), yellow (oxygen activation), and orange (heme binding), as labeled. (C) Comparison of the relative transformation rates of ω-hydroxyemodin by CYP-H6231 and its emodin-binding site mutants. Error bars represent standard deviations. (D) HPLC analysis of ω-hydroxyemodin production by CYP-H6231 catalytic site single-point mutants compared to wild-type CYP-H6231
Fig. 7 Structural analysis, site-directed mutagenesis, and enzymatic activity of CYP-H6231. (A) Close-up view of the emodin binding pocket. Emodin (green) is positioned near the heme group (yellow), with potential key substrate-binding residues shown in cyan and labeled. Numbers indicate interaction distances (in Å). (B) Catalytic site of CYP-H6231 highlighting key residues. Emodin (green sticks) and the heme group (yellow sticks) are shown, with essential catalytic residues colored blue (E–R–R triad), yellow (oxygen activation), and orange (heme binding), as labeled. (C) Comparison of the relative transformation rates of ω-hydroxyemodin by CYP-H6231 and its emodin-binding site mutants. Error bars represent standard deviations. (D) HPLC analysis of ω-hydroxyemodin production by CYP-H6231 catalytic site single-point mutants compared to wild-type CYP-H6231CC BY · PubMed Central

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Authors
Ren, Z., Xue, Y., Xu, N., Feng, D., Geng, C., Wu, Y., Liu, D., Zhao, L., Zhang, X., Ma, H., Huang, X., Qi, F., & Lu, X.
Affiliations
Chinese Academy of Sciences (CN)
Corresponding author
Qi, Feifei
ORCID
Venue
Microbial Cell Factories
Publisher
Springer Science and Business Media LLC
Published
2025-11-13
Volume
24
Issue
1
Pages
234-234
ISSN
1475-2859 (ISSN-L)
DOI
10.1186/s12934-025-02857-z
PMC
PMC12613595
OpenAlex
W4416193861
Cited by
2
References
32
Citations by year
2026: 2
FWCI
1.12
Type
journal article
Language
en
Open access
Licence
Locations
  • Microbial Cell Factories (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Taishan Scholar Foundation of Shandong Province
  • National Key Research and Development Program of China (2021YFC2102600)
  • Key R&D Program of Shandong Province (2021ZDSYS02)
  • National Natural Science Foundation of China (32270062)
Topics
  • Life Sciences › Immunology and Microbiology › Applied Microbiology and Biotechnology › Microbial Natural Products and Biosynthesis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Microbial Metabolism and Applications
Keywords
physcion, emodin, anthraquinone biosynthesis, Aspergillus terreus, cytochrome P450, NADPH-cytochrome P450 reductase, detoxification, plant-derived fungicides, product purity
MeSH terms
Anthraquinones, Aspergillus, Cytochrome P-450 Enzyme System, Emodin, Fungal Proteins, Fungicides, Industrial, Metabolic Engineering

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CoChat. (2026). Citation check: Multi-layered engineering of Aspergillus terreus enhances biosynthesis of the plant-derived fungicide physcion. https://app.cochat.ai/papers/10.1186/s12934-025-02857-z
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Ren, Z., Xue, Y., Xu, N., Feng, D., Geng, C., Wu, Y., Liu, D., Zhao, L., Zhang, X., Ma, H., Huang, X., Qi, F., & Lu, X. (2025). Multi-layered engineering of Aspergillus terreus enhances biosynthesis of the plant-derived fungicide physcion. Microbial Cell Factories. https://doi.org/10.1186/s12934-025-02857-z

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