Enhanced production of phomapyrrolidone a through culture-condition optimization and morphological regulation in Didymella sp. FATR0054

Abstract

Phomapyrrolidone A (Ppd A) is a hirsutellone-type macrocyclic alkaloid derived from marine fungi, exhibiting significant activity against triple-negative breast cancer (TNBC). However, its low fermentation yield has limited further drug development. This study established a systematic optimization strategy integrating medium optimization, mycelial morphology regulation, and computational fluid dynamics (CFD) to enhance Ppd A production by the marine-derived filamentous fungus Didymella sp. FATR0054. Medium composition and fermentation parameters were optimized, increasing shake-flask production from 4.52 mg/L to 289.36 mg/L. The process was successfully scaled up in a 5-L bioreactor. Exogenous co-supplementation further increased production by 26% to 365 mg/L. Morphological regulation proved critical for productivity. Mechanical pre-grinding of the seed culture reduced mycelial pellet diameter to approximately 1 mm, creating compact and uniform pellets. This increased Ppd A production to 389.25 mg/L, representing an 86-fold improvement. CFD analysis of the interactions among the flow field, fungal pellet diameter distribution, and product formation identified 450 rpm as a candidate operating compromise for the current 5-L bioreactor, based on the simulated spatial uniformity of the Ppd A concentration field, local energy dissipation, and pellet fragmentation. This study establishes an integrated fermentation intensification strategy combining experimental optimization and flow-field modeling for large-scale production of Ppd A and process regulation in filamentous fungal fermentation.

Keywords

Phomapyrrolidone A / Marine filamentous fungus / Submerged fermentation

References

[1] Adsul MG, Dixit P, Saini JK, et al.Morphologically favorable mutant of Trichodermareesei for low viscosity cellulase production. Biotechnol Bioeng, 2022, 119: 2167-2181.

[2] Al Subeh ZY, Flores-Bocanegra L, Raja HA, et al.Embellicines C-E: macrocyclic alkaloids with a cyclopenta[b]fluorene ring system from the fungus Sarocladium sp. J Nat Prod, 2023, 86: 596-603.

[3] Ariantari NP, Ancheeva E, Frank M, et al.Didymellanosine, a new decahydrofluorene analogue, and ascolactone C from Didymella sp. IEA-3B.1, an endophyte of Terminaliacatappa. RSC Adv, 2020, 10: 7232-7240.

[4] Bellaouchi R, Hasnaoui I, Idrissi Yahyaoui M, et al.Improving dietary citric acid production by the wild-type Aspergillusniger ASP26 strain isolated from date by-product. Food Sci Nutr, 2024, 12: 4248-4258.

[5] Bergman S, Funk AN, Scherlach K, et al.Activation of a silent fungal polyketide biosynthesis pathway through regulatory cross talk with a cryptic nonribosomal peptide synthetase gene cluster. Appl Environ Microbiol, 2010, 76: 8143-8149.

[6] Betiku E, Ayinla HO, Oguntunde OA, et al.Bioconversion of breadfruit starch to citric acid by fungus Aspergillusniger: a microbial fermentation parameter optimization investigation. BioResources, 2025, 21: 1258-1273.

[7] Böl M, Schrinner K, Tesche S, et al.Challenges of influencing cellular morphology by morphology engineering techniques and mechanical induced stress on filamentous pellet systems-a critical review. Eng Life Sci, 2020, 21: 51-67.

[8] Cappello V, Plais C, Vial C, et al.Scale-up of aerated bioreactors: CFD validation and application to the enzyme production by Trichodermareesei. Chem Eng Sci, 2021, 229: 116033.

[9] Cerrone F, O’Connor KCultivation of filamentous fungi in airlift bioreactors advantages and disadvantages. Appl Microbiol Biotechnol, 2025, 10941.

[10] Chen Y, Liu Z, Huang Y, et al.Ascomylactams A–C, cytotoxic 12- or 13-membered-ring macrocyclic alkaloids isolated from the mangrove endophytic fungus Didymella sp. CYSK-4, and structure revisions of Phomapyrrolidones A and C. J Nat Prod, 2019, 821752-1758.

[11] Chen M, Zheng X, Du P, et al.Genetic context-dependent regulation of FlbE enhances morphology modulation and citric acid production in Aspergillusniger. Bioresour Technol, 2026, 439: 133350.

[12] Chu HT, Sofeo N, Jebamony JJ, et al.The Cys2-His2 zinc finger transcription factor NsdC regulates hyphal branching, conidiation and mycelial pellet morphology in Aspergillusoryzae. Fungal Genet Biol, 2025, 180104019.

[13] Costes LH, Lippi Y, Naylies C, et al.The solvent dimethyl sulfoxide affects physiology, transcriptome and secondary metabolism of Aspergillusflavus. J Fungi, 2021, 71055.

[14] Ding Y, Jiang Y, Xu S, et al.Perpyrrospirone A, an unprecedented hirsutellone peroxide from the marine-derived Penicilliumcitrinum. Chin Chem Lett, 2023, 34107562.

[15] Gong L, Yang J, Guo J, et al.Discovery of hirsutellones as nucleolin-targeting natural macrocycles for triple-negative breast cancer treatment. J Med Chem, 2026

[16] Gromozova O, Grube M, Lytvyn P, et al.Cell wall characteristics of Thielaviaterrestris dispersed and pelleted mycelia. Fungal Biol, 2025, 129101593.

[17] Isaka MNovel bioactive compounds from insect pathogenic fungi. J Synth Org Chem Jpn, 2007, 65700-708.

[18] Jiang F, Tian J, Yuan J, et al.Improved cellulase production of Trichodermareesei by regulating mycelium morphology. Fermentation, 2024, 10: 26.

[19] Kumar P, Dubey KKMycelium transformation of Streptomycestoxytricini into pellet: role of culture conditions and kinetics. Bioresour Technol, 2017, 228339-347.

[20] Lee H, Lee YM, Heo YM, et al.Optimization of fungal enzyme production by Trichodermaharzianum KUC1716 through surfactant-induced morphological changes. Mycobiology, 2017, 45: 48-51.

[21] Li X, Ear A, Nay BHirsutellones and beyond: figuring out the biological and synthetic logics toward chemical complexity in fungal PKS-NRPS compounds. Nat Prod Rep, 2013, 30: 765-782.

[22] Li T, Huang M, Lu JCancer statistics and trends in China: the potential of natural product application. Chin J Nat Med, 2024, 22: 673-675.

[23] Luo Z, Yin F, Wang X, et al.Progress in approved drugs from natural product resources. Chin J Nat Med, 2024, 22: 195-211.

[24] Meng Q, Chuai S, Chen L, et al.Effect of surfactants on the production of polysaccharides from Schizophyllumcommune through submerged fermentation. Int J Biol Macromol, 2021, 192: 210-218.

[25] Moilanen P, Laakkonen M, Aittamaa JModeling aerated fermenters with computational fluid dynamics. Ind Eng Chem Res, 2006, 45: 8656-8663.

[26] Nadal-Rey G, Mcclure DD, Kavanagh JM, et al.Computational fluid dynamics modelling of hydrodynamics, mixing and oxygen transfer in industrial bioreactors with Newtonian broths. Biochem Eng J, 2022, 177: 108265.

[27] Ohashi M, Kakule TB, Tang MC, et al.Biosynthesis of para-cyclophane-containing hirsutellone family of fungal natural products. J Am Chem Soc, 2021, 155605-5609.

[28] Ost KJ, Student M, Cord-Landwahr S, et al.Cell walls of filamentous fungi-challenges and opportunities for biotechnology. Appl Microbiol Biotechnol, 2025, 109: 125.

[29] Park E, Koike Y, Higashiyama K, et al.Effect of nitrogen source on mycelial morphology and arachidonic acid production in cultures of Mortierellaalpina. J Biosci Bioeng, 1999, 8861-67.

[30] Prasad AZinc in human health: effect of zinc on immune cells. Mol Med, 2008, 14: 353-357.

[31] Saberi A, Jalili H, Nikfarjam A, et al.Monitoring of Aspergillusterreus morphology for the lovastatin production in submerge culture by impedimetry. Biochem Eng J, 2020, 159: 107615.

[32] Sun Y, Shi X, He L, et al.Biosynthetic profile in the co-culture of Aspergillussydowii and Bacillussubtilis to produce novel benzoic derivatives. Microb Ecol, 2023, 851288-1299.

[33] Vasudhevan P, Kalaimurugan D, Ganesan S, et al.Enhanced biocatalytic laccase production using agricultural waste in solid-state fermentation by Aspergillusoryzae for p-chlorophenol degradation. Int J Biol Macromol, 2024, 281: 136460.

[34] Venkatachalam M, Mares G, Dufossé L, et al.Scale-up of pigment production by the marine-derived filamentous fungus, Talaromycesalbobiverticillius 30548, from shake flask to stirred bioreactor. Fermentation, 2023, 977.

[35] Wang Y, Lin F, Hsu T, et al.Effects of nitrogen from different sources on mycelial biomass and polysaccharide production and pellet morphology in submerged cultures of Grifolafrondosa. BioResources, 2021, 162937-2952.

[36] Wijeratne EMK, He H, Franzblau SG, et al.Phomapyrrolidones A–C, antitubercular alkaloids from the endophytic fungus Phoma sp. NRRL 46751. J Nat Prod, 2013, 76: 1860-1865.

[37] Xu M, Ming S, Cui J, et al.Harnessing mycelium-based morphology of filamentous fungi in food biomanufacturing: integrated advances, challenges, and future perspectives. Trends Food Sci Technol, 2026, 172: 105696.

[38] Yang J, Jiao RH, Yao LY, et al.Control of fungal morphology for improved production of a novel antimicrobial alkaloid by marine-derived fungus Curvularia sp IFB-Z10 under submerged fermentation. Process Biochem, 2016, 51185-194.

[39] Yang J, Xiong X, Gong L, et al.Diketopiperazines with anti-skin inflammation from marine-derived endophytic fungus Aspergillus sp. and configurational reassignment of aspertryptanthrins. Chin J Nat Med, 2025, 23: 980-989.

[40] Yao F, Liang X, Shen W, et al.Microascones, decahydrofluorene-class alkaloids from the marine-derived fungus Microascus sp. SCSIO 41821. J Nat Prod, 2024, 87: 810-819.

[41] Yatmaz E, Karahalil E, Germec M, et al.Controlling filamentous fungi morphology with microparticles to enhanced β-mannanase production. Bioprocess Biosyst Eng, 2016, 39: 1391-1399.

[42] Yoshimi A, Miyazawa K, Abe KFunction and biosynthesis of cell wall alpha-1,3-glucan in fungi. J Fungi, 2017, 363.

[43] Yuan T, Xu Q, Wang N, et al.Establishment of an efficient CRISPR-dCas9-SunTag transcriptional activation system in Aspergillusnidulans for enhancing emodin production. Chem Eng J, 2025, 522167839.

[44] Zheng G, Zhou W, Gui Y, et al.Balancing nitrogen metabolism to efficiently drive anti-tuberculosis ilamycins biosynthesis in Streptomycesatratus. Bioresour Technol, 2025, 437133099.

[45] Zhou Y, Duan Y, Chen L, et al.Engineering dispersed mycelium morphology in Aspergillusniger for enhanced mycoprotein production via CRISPR/Cas9-mediated genome editing. Bioresour Technol, 2025, 432132652.

[46] Zhou W, Zheng G, Wang J, et al.Computational fluid dynamics (CFD) modeling of Ilamycin E production in Streptomycesatratus SCSIO ZH16 submerged fermentation. Chem Eng Sci, 2027, 338124737.

[47] Zhu Y, Zheng G, Xin X, et al.Combinatorial strategies for production improvement of anti-tuberculosis antibiotics ilamycins E1/E2 from deep sea-derived Streptomyces atratus SCSIO ZH16 ΔilaR. Bioresour Bioprocess, 2022, 9111.

Funding

National key research and development program of china(2022YFC2804300)

Natural Science Foundation of Shanghai Municipality(25ZR1401076)

Shanghai Oriental Talents Program(QNJY2025001)

Rights & permissions

The Author(s)