De novo production of bioactive sesterterpenoid ophiobolins in Saccharomyces cerevisiae cell factories.
Saccharomyces cerevisiae
Metabolic engineering
Ophiobolin
Sesterterpenoid
Whole-cell transformation
Journal
Microbial cell factories
ISSN: 1475-2859
Titre abrégé: Microb Cell Fact
Pays: England
ID NLM: 101139812
Informations de publication
Date de publication:
06 May 2024
06 May 2024
Historique:
received:
15
01
2024
accepted:
25
04
2024
medline:
7
5
2024
pubmed:
7
5
2024
entrez:
6
5
2024
Statut:
epublish
Résumé
Sesterterpenoids are rare species among the terpenoids family. Ophiobolins are sesterterpenes with a 5-8-5 tricyclic skeleton. The oxidized ophiobolins exhibit significant cytotoxic activity and potential medicinal value. There is an urgent need for large amounts of ophiobolins supplication for drug development. The synthetic biology approach has been successfully employed in lots of terpene compound production and inspired us to develop a cell factory for ophiobolin biosynthesis. We developed a systematic metabolic engineering strategy to construct an ophiobolin biosynthesis chassis based on Saccharomyces cerevisiae. The whole-cell biotransformation methods were further combined with metabolic engineering to enhance the expression of key ophiobolin biosynthetic genes and improve the supply of precursors and cofactors. A high yield of 5.1 g/L of ophiobolin F was reached using ethanol and fatty acids as substrates. To accumulate oxidized ophiobolins, we optimized the sources and expression conditions for P450-CPR and alleviated the toxicity of bioactive compounds to cells through PDR engineering. We unexpectedly obtained a novel ophiobolin intermediate with potent cytotoxicity, 5-hydroxy-21-formyl-ophiobolin F, and the known bioactive compound ophiobolin U. Finally, we achieved the ophiobolin U titer of 128.9 mg/L. We established efficient cell factories based on S. cerevisiae, enabling de novo biosynthesis of the ophiobolin skeleton ophiobolin F and oxidized ophiobolins derivatives. This work has filled the gap in the heterologous biosynthesis of sesterterpenoids in S. cerevisiae and provided valuable solutions for new drug development based on sesterterpenoids.
Sections du résumé
BACKGROUND
BACKGROUND
Sesterterpenoids are rare species among the terpenoids family. Ophiobolins are sesterterpenes with a 5-8-5 tricyclic skeleton. The oxidized ophiobolins exhibit significant cytotoxic activity and potential medicinal value. There is an urgent need for large amounts of ophiobolins supplication for drug development. The synthetic biology approach has been successfully employed in lots of terpene compound production and inspired us to develop a cell factory for ophiobolin biosynthesis.
RESULTS
RESULTS
We developed a systematic metabolic engineering strategy to construct an ophiobolin biosynthesis chassis based on Saccharomyces cerevisiae. The whole-cell biotransformation methods were further combined with metabolic engineering to enhance the expression of key ophiobolin biosynthetic genes and improve the supply of precursors and cofactors. A high yield of 5.1 g/L of ophiobolin F was reached using ethanol and fatty acids as substrates. To accumulate oxidized ophiobolins, we optimized the sources and expression conditions for P450-CPR and alleviated the toxicity of bioactive compounds to cells through PDR engineering. We unexpectedly obtained a novel ophiobolin intermediate with potent cytotoxicity, 5-hydroxy-21-formyl-ophiobolin F, and the known bioactive compound ophiobolin U. Finally, we achieved the ophiobolin U titer of 128.9 mg/L.
CONCLUSIONS
CONCLUSIONS
We established efficient cell factories based on S. cerevisiae, enabling de novo biosynthesis of the ophiobolin skeleton ophiobolin F and oxidized ophiobolins derivatives. This work has filled the gap in the heterologous biosynthesis of sesterterpenoids in S. cerevisiae and provided valuable solutions for new drug development based on sesterterpenoids.
Identifiants
pubmed: 38711040
doi: 10.1186/s12934-024-02406-0
pii: 10.1186/s12934-024-02406-0
doi:
Substances chimiques
Sesterterpenes
0
ophiobolins
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
129Subventions
Organisme : National Key R&D Program of China
ID : 2018YFC0311000
Organisme : Zhongnan Hospital of Wuhan University
ID : ZNJC202234
Informations de copyright
© 2024. The Author(s).
Références
Tian W, Deng Z, Hong K. The biological activities of sesterterpenoid-type ophiobolins. Mar Drugs. 2017;15:1–21.
doi: 10.3390/md15070229
Wen H, Zhong Y, Yin Y, Qin K, Yang L, Li D, et al. A marine-derived small molecule induces immunogenic cell death against triple-negative breast cancer through ER stress-CHOP pathway. Int J Biol Sci. 2022;18:2898–913.
pubmed: 35541893
pmcid: 9066120
doi: 10.7150/ijbs.70975
Rowley M, Tsukamoto M, Kishi Y. Total synthesis of (+)-ophiobolin C. J Am Chem Soc. 1989;111:2737–39.
doi: 10.1021/ja00189a069
Thach DQ, Brill ZG, Grover HK, Esguerra KV, Thompson JK, Maimone TJ. Total synthesis of (+)-6-epi‐ophiobolin A. Angew Chem Int Ed. 2020;132:1548–52.
doi: 10.1002/ange.201913150
Brill ZG, Grover HK, Maimone TJ. Enantioselective synthesis of an ophiobolin sesterterpene via a programmed radical cascade. Science. 2016;352:1078–82.
pubmed: 27230373
pmcid: 5319821
doi: 10.1126/science.aaf6742
Tsuna K, Noguchi N, Nakada M. Convergent total synthesis of (+)-ophiobolin A. Angew Chem Int Ed. 2011;123:9624–7.
doi: 10.1002/ange.201104447
Yan J, Pang J, Liang J, Yu W, Liao X, Aobulikasimu A, et al. The biosynthesis and transport of ophiobolins in aspergillus ustus 094102. Int J Mol Sci. 2022;23(3):1903.
pubmed: 35163826
pmcid: 8836403
doi: 10.3390/ijms23031903
Liu Z, Huang M, Chen H, Lu X, Tian Y, Hu P, et al. Metabolic engineering of Yarrowia Lipolytica for high-level production of squalene. Bioresour Technol. 2024;394:130233.
pubmed: 38141883
doi: 10.1016/j.biortech.2023.130233
Ma Y, Liu N, Greisen P, Li J, Qiao K, Huang S, et al. Removal of lycopene substrate inhibition enables high carotenoid productivity in Yarrowia Lipolytica. Nat Commun. 2022;13:572.
pubmed: 35102143
pmcid: 8803881
doi: 10.1038/s41467-022-28277-w
Ye Z, Shi B, Huang Y, Ma T, Xiang Z, Hu B, et al. Revolution of vitamin E production by starting from microbial fermented farnesene to isophytol. Innovation. 2022;3(3):100228.
pubmed: 35373168
pmcid: 8968663
Paddon CJ, Westfall PJ, Pitera DJ, Benjamin K, Fisher K, McPhee D, et al. High-level semi-synthetic production of the potent antimalarial artemisinin. Nature. 2013;496:528–32.
pubmed: 23575629
doi: 10.1038/nature12051
Zhang J, Hansen LG, Gudich O, Viehrig K, Lassen LMM, Schrübbers L, et al. A microbial supply chain for production of the anti-cancer drug vinblastine. Nature. 2022;609:341–7.
pubmed: 36045295
pmcid: 9452304
doi: 10.1038/s41586-022-05157-3
Gao J, Zuo Y, Xiao F, Wang Y, Li D, Xu J, et al. Biosynthesis of catharanthine in engineered Pichia pastoris. Nat Synthesis. 2023;2:231–42.
doi: 10.1038/s44160-022-00205-2
Kong X, Wu Y, Yu W, Liu Y, Li J, Du G, et al. Efficient synthesis of limonene in Saccharomyces cerevisiae using combinatorial metabolic engineering strategies. J Agric Food Chem. 2023;71:7752–64.
pubmed: 37189018
doi: 10.1021/acs.jafc.3c02076
Chai H, Yin R, Liu Y, Meng H, Zhou X, Zhou G, et al. Sesterterpene ophiobolin biosynthesis involving multiple gene clusters in aspergillus ustus. Sci Rep. 2016;6:27181.
pubmed: 27273151
pmcid: 4895135
doi: 10.1038/srep27181
Chiba R, Minami A, Gomi K, Oikawa H. Identification of ophiobolin F synthase by a genome mining approach: a sesterterpene synthase from Aspergillus Clavatus. Org Lett. 2013;15:594–7.
pubmed: 23324037
doi: 10.1021/ol303408a
Narita K, Chiba R, Minami A, Kodama M, Fujii I, Gomi K, et al. Multiple oxidative modifications in the ophiobolin biosynthesis: P450 oxidations found in genome mining. Org Lett. 2016;18:1980–3.
pubmed: 27116000
doi: 10.1021/acs.orglett.6b00552
Yuan W, Lv S, Chen L, Zhao Y, Deng Z, Hong K. Production of sesterterpene ophiobolin by a bifunctional terpene synthase in Escherichia coli. Appl Microbiol Biotechnol. 2019;103:8785–97.
pubmed: 31515597
doi: 10.1007/s00253-019-10103-x
Mai W, Hong K. Heterologous expression of a fungal cytochrome P450 in Escherichia coli. Microbiol China. 2019;46(5):1092–9.
Arendt P, Miettinen K, Pollier J, De Rycke R, Callewaert N, Goossens A. An endoplasmic reticulum-engineered yeast platform for overproduction of triterpenoids. Metab Eng. 2017;40:165–75.
pubmed: 28216107
doi: 10.1016/j.ymben.2017.02.007
Yee DA, DeNicola AB, Billingsley JM, Creso JG, Subrahmanyam V, Tang Y. Engineered mitochondrial production of monoterpenes in Saccharomyces cerevisiae. Metab Eng. 2019;55:76–84.
pubmed: 31226348
pmcid: 6717016
doi: 10.1016/j.ymben.2019.06.004
Chatzivasileiou AO, Ward V, Edgar SM, Stephanopoulos G. Two-step pathway for isoprenoid synthesis. Proc Natl Acad Sci U S A. 2019;116:506–11.
pubmed: 30584096
doi: 10.1073/pnas.1812935116
Siemon T, Wang Z, Bian G, Seitz T, Ye Z, Lu Y, et al. Semisynthesis of plant-derived englerin a enabled by microbe engineering of Guaia-6,10(14)-diene as building block. J Am Chem Soc. 2020;142:2760–5.
pubmed: 31999448
doi: 10.1021/jacs.9b12940
Cao X, Yu W, Chen Y, Yang S, Zhao ZK, Nielsen J, et al. Engineering yeast for high-level production of diterpenoid sclareol. Metab Eng. 2023;75:19–28.
pubmed: 36371032
doi: 10.1016/j.ymben.2022.11.002
Chen Y, Daviet L, Schalk M, Siewers V, Nielsen J. Establishing a platform cell factory through engineering of yeast acetyl-CoA metabolism. Metab Eng. 2013;15:48–54.
pubmed: 23164578
doi: 10.1016/j.ymben.2012.11.002
Yocum H, Bassett S, Silva NA, Da. Enhanced production of acetyl-CoA based products via peroxisomal surface display in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 2022;119:1–13.
doi: 10.1073/pnas.2214941119
Kwak S, Yun EJ, Lane S, Oh EJ, Kim KH, Jin Y. Redirection of the glycolytic flux enhances isoprenoid production in Saccharomyces cerevisiae. Biotechnol J. 2019;15:1900173.
doi: 10.1002/biot.201900173
Verho R, Londesborough J, Penttilä M, Richard P. Engineering Redox cofactor regeneration for improved pentose fermentation in Saccharomyces cerevisiae. Appl Environ Microbiol. 2003;69:5892–7.
pubmed: 14532041
pmcid: 201209
doi: 10.1128/AEM.69.10.5892-5897.2003
Lee SY, Kim HU. Systems strategies for developing industrial microbial strains. Nat Biotechnol. 2015;33(10):1061–72.
pubmed: 26448090
doi: 10.1038/nbt.3365
Lin B, Tao Y. Whole-cell biocatalysts by design. Microb Cell Fact. 2017;16:106.
pubmed: 28610636
pmcid: 5470193
doi: 10.1186/s12934-017-0724-7
Niu F-X, He X, Wu YQ, Liu JZ. Enhancing production of pinene in Escherichia coli by using a combination of tolerance, evolution, and modular co-culture engineering. Front Microbiol. 2018;9:1–14.
doi: 10.3389/fmicb.2018.01623
Naito Y, Hino K, Bono H, Ui-Tei K. CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites. Bioinformatics. 2015;31:1120–3.
pubmed: 25414360
doi: 10.1093/bioinformatics/btu743
Zhang Y, Wang J, Wang Z, Zhang Y, Shi S, Nielsen J, et al. A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae. Nat Commun. 2019;10:1053.
pubmed: 30837474
pmcid: 6400946
doi: 10.1038/s41467-019-09005-3
Gietz RD, Schiestl RH. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc. 2007;2:31–4.
pubmed: 17401334
doi: 10.1038/nprot.2007.13
Ricci-Tam C, Ben-Zion I, Wang J, Palme J, Li A, Savir Y, et al. Decoupling transcription factor expression and activity enables dimmer switch gene regulation. Science. 2021;372:292–5.
pubmed: 33859035
pmcid: 8173539
doi: 10.1126/science.aba7582
Ma B, Liu M, Li ZH, Tao X, Wei DZ, Wang FQ. Significantly enhanced production of patchoulol in metabolically engineered Saccharomyces cerevisiae. J Agric Food Chem. 2019;67:8590–8.
pubmed: 31287301
doi: 10.1021/acs.jafc.9b03456
Faulkner A, Chen X, Rush J, Horazdovsky B, Waechter CJ, Carman GM, et al. The lpp1 and dpp1 gene products account for most of the isoprenoid phosphate phosphatase activities in Saccharomyces cerevisiae. J Biol Chem. 1998;274(21):14831–7.
doi: 10.1074/jbc.274.21.14831
Zhang C, Liu J, Zhao F, Lu C, Zhao GR, Lu W. Production of sesquiterpenoid zerumbone from metabolic engineered Saccharomyces cerevisiae. Metab Eng. 2018;49:28–35.
pubmed: 30031850
doi: 10.1016/j.ymben.2018.07.010
Trikka FA, Nikolaidis A, Athanasakoglou A, Andreadelli A, Ignea C, Kotta K, et al. Iterative carotenogenic screens identify combinations of yeast gene deletions that enhance sclareol production. Microb Cell Fact. 2015;14:60.
pubmed: 25903744
pmcid: 4413541
doi: 10.1186/s12934-015-0246-0
Chen Y, Wang Y, Liu M, Qu J, Yao M, Li B, et al. Primary and secondary metabolic effects of a key gene deletion (∆YPL062W) in metabolically engineered terpenoid-producing Saccharomyces cerevisiae. Appl Environ Microbiol. 2019;85(7):e01990–18.
pubmed: 30683746
pmcid: 6585493
doi: 10.1128/AEM.01990-18
Chen R, Gao J, Yu W, Chen X, Zhai X, Chen Y, et al. Engineering cofactor supply and recycling to drive phenolic acid biosynthesis in yeast. Nat Chem Biol. 2022;18:520–9.
pubmed: 35484257
doi: 10.1038/s41589-022-01014-6
Yang J, Liang J, Shao L, Liu L, Gao K, Zhang JL, et al. Green production of silybin and isosilybin by merging metabolic engineering approaches and enzymatic catalysis. Metab Eng. 2020;59:44–52.
pubmed: 32004707
doi: 10.1016/j.ymben.2020.01.007
Hou J, Lages NF, Oldiges M, Vemuri GN. Metabolic impact of redox cofactor perturbations in Saccharomyces cerevisiae. Metab Eng. 2009;11:253–61.
pubmed: 19446033
doi: 10.1016/j.ymben.2009.05.001
Hiltunen JK, Mursula AM, Rottensteiner H, Wierenga RK, Kastaniotis AJ, Gurvitz A. The biochemistry of peroxisomal β-oxidation in the yeast Saccharomyces cerevisiae. FEMS Microbiol Rev. 2003;27:35–64.
pubmed: 12697341
doi: 10.1016/S0168-6445(03)00017-2
Sun Y, Sun L, Shang F, Yan G. Enhanced production of β-carotene in recombinant Saccharomyces cerevisiae by inverse metabolic engineering with supplementation of unsaturated fatty acids. Process Biochem. 2016;51:568–77.
doi: 10.1016/j.procbio.2016.02.004
Kunau WH, Dommes V, Schulzt H. β-oxidation of fatty acids in mitochondria, peroxisomes, and bacteria: a century of continued progress. Prog Lipid Res. 1995;34(4):267–342.
pubmed: 8685242
doi: 10.1016/0163-7827(95)00011-9
Van Roermund CWT, Elgersma Y, Singh N, Wanders RJA, Tabak HF. The membrane of peroxisomes in Saccharomyces cerevisiae is impermeable to NAD(H) and acetyl-CoA under in vivo conditions. EMBO J. 1995;14:3480–6.
pubmed: 7628449
pmcid: 394415
doi: 10.1002/j.1460-2075.1995.tb07354.x
Halbach A, Landgraf C, Lorenzen S, Rosenkranz K, Volkmer-Engert R, Erdmann R, et al. Targeting of the tail-anchored peroxisomal membrane proteins PEX26 and PEX15 occurs through C-terminal PEX19-binding sites. J Cell Sci. 2006;119:2508–17.
pubmed: 16763195
doi: 10.1242/jcs.02979
de Carvalho CCCR. Whole cell biocatalysts: essential workers from Nature to the industry. Microb Biotechnol. 2017;10(2):250–63.
pubmed: 27145540
doi: 10.1111/1751-7915.12363
Sugawara F, Strobel G, Strange RN, Siedow JN, Van Duyne GD, Clardy J. Phytotoxins from the pathogenic fungi Drechslera maydis and Drechslera sorghicola. Proc Natl Acad Sci U S A. 1987;84:3081–5.
pubmed: 16593832
pmcid: 304811
doi: 10.1073/pnas.84.10.3081
Li E, Clark AM, Rotella DP, Hufford CD. Microbial metabolites of ophiobolin a and antimicrobial evaluation of ophiobolins. J Nat Prod. 1995; 58, (1).
Bladt TT, Durr C, Knudsen PB, Kildgaard S, Frisvad JC, Gotfredsen CH, et al. Bio-activity and dereplication-based discovery of ophiobolins and other fungal secondary metabolites targeting leukemia cells. Molecules. 2013;18:14629–50.
pubmed: 24287995
pmcid: 6290568
doi: 10.3390/molecules181214629
Urlacher VB, Girhard M. Cytochrome P450 monooxygenases in biotechnology and synthetic biology. Trends Biotechnol. 2019;37(8):882–97.
pubmed: 30739814
doi: 10.1016/j.tibtech.2019.01.001
Bernhardt R, Urlacher VB. Cytochromes P450 as promising catalysts for biotechnological application: chances and limitations. Appl Microbiol Biotechnol. 2014;98:6185–203.
pubmed: 24848420
doi: 10.1007/s00253-014-5767-7
Gold ND, Fossati E, Hansen CC, DIfalco M, Douchin V, Martin VJJ. A combinatorial approach to study cytochrome P450 enzymes for de novo production of steviol glucosides in baker’s yeast. ACS Synth Biol. 2018;7:2918–29.
pubmed: 30474973
doi: 10.1021/acssynbio.8b00470
Milne N, Thomsen P, Mølgaard Knudsen N, Rubaszka P, Kristensen M, Borodina I. Metabolic engineering of Saccharomyces cerevisiae for the de novo production of psilocybin and related tryptamine derivatives. Metab Eng. 2020;60:25–36.
pubmed: 32224264
pmcid: 7232020
doi: 10.1016/j.ymben.2019.12.007
Wolfger H, Mamnun YM, Kuchler K. Fungal ABC proteins: pleiotropic drug resistance, stress response and cellular detoxification. Res Microbiol. 2001;152:375–89.
pubmed: 11421285
doi: 10.1016/S0923-2508(01)01209-8
Jungwirth H, Kuchler K. Yeast ABC transporters-A tale of sex, stress, drugs and aging. FEBS Lett. 2006;580(4):1131–8.
pubmed: 16406363
doi: 10.1016/j.febslet.2005.12.050