Enhancing squalene production in Aurantiochytrium Ch25 via integrated optimization strategy and genome-scale metabolic modeling

Abstract

Squalene, a high-value terpenoid, can be sustainably produced by Aurantiochytrium Ch25. This study integrated experimental optimization using Taguchi design with genome-scale metabolic modeling (GEM) to investigate its biosynthesis. The Taguchi L9 orthogonal array identified a promising culture condition (glucose 15 g/L, yeast extract 3 g/L, seawater 20% v/v, agitation 140 r/min), resulting in a squalene titer of 238 mg/L after 98 h. A validated GEM was used to simulate metabolic fluxes and explore potential engineering targets. Flux balance analysis (FBA) indicated that ammonia limitation favors biomass formation, while flux variability analysis (FVA) estimated squalene flux at 0.0014 mmol gDCW⁻¹ h⁻¹. Dynamic FBA simulations aligned well with experimental glucose uptake and biomass profiles. Single-reaction deletion analysis identified 13 essential and 35 semi-essential reactions, with inorganic diphosphatase (PPase) and F-type ATP synthase highlighted as critical for squalene biosynthesis. OptKnock predicted two mutant strains: Mutant A (deletion of 3-Oxoacyl-ACP reductase and Acyl-CoA oxidase) increased simulated squalene flux to 0.00994 mmol gDCW⁻¹ h⁻¹, and Mutant B (additional deletions) further raised it to 0.01274 mmol gDCW⁻¹ h⁻¹. OptGene suggested phosphoenolpyruvate synthase as a key deletion target to redirect flux toward the mevalonate pathway. This integrative approach paves the way for future metabolic engineering efforts and bioprocess optimization in this industrially relevant thraustochytrid.

Keywords

Aurantiochytrium / Squalene / Integrated optimization strategy / Genome-scale metabolic model

References

[1] Ardalani O, Motamedian E, Hamedi J. Reconstruction and validation of genome-scale metabolic model of L. lactis subsp. lactis NCDO 2118 and in silico analysis for succinate and gamma-aminobutyric acid overproduction. Biochem Eng J, 2021, 170 Article ID: 107967

[2] Berger PD, Maurer RE, Celli GB. Introduction to Taguchi methods. In: Berger PD, Maurer RE, Celli GB, editors. Experimental design. Cham: Springer; 2018. pp. 449–480. https://doi.org/10.1007/978-3-319-64583-4_13

[3] Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol, 1959, 37: 911-917

[4] Burgard AP, Pharkya P, Maranas CD. OptKnock: A bilevel programming framework for identifying gene knockout strategies for microbial strain optimization. Biotechnol Bioeng, 2003, 84: 647-657

[5] Cheng L, Ji T, Zhang M, et al.. Recent advances in squalene: biological activities, sources, extraction, and delivery systems. Trends Food Sci Technol, 2024, 146 Article ID: 104392

[6] Cho SH, Toth K, Kim D, et al.. Activation of the plant mevalonate pathway by extracellular ATP. Nat Commun, 2022, 13 Article ID: 450

[7] Cuevas DA, Edirisinghe J, Henry CS, et al.. From DNA to FBA: How to build your own genome-scale metabolic model. Front Microbiol, 2016, 7: 907

[8] Du F, Wang YZ, Xu YS, et al.. Biotechnological production of lipid and terpenoid from thraustochytrids. Biotechnol Adv, 2021, 48 Article ID: 107725

[9] Esmizade M, Shakeri S, Maleki M. Effect of carbon and nitrogen sources on carotenoids production by native strain of Aurantiochytrium Ch25. J Microb Biol, 2016, 5(18): 67-82

[10] Fracchia-Durán AG, Ramos-Zambrano E, Marquez-Rocha FJ, et al.. Bioprocess conditions and regulation factors to optimize squalene production in thraustochytrids. World J Microbiol Biotechnol, 2023, 39 Article ID: 251

[11] Furlan JM, Centenaro GS, Bittencourt FM, et al.. Thraustochytrium sp. and Aurantiochytrium sp.: Sustainable alternatives for squalene production. Mar Drugs, 2025, 23 Article ID: 132

[12] Galea AM, Brown AJ. Special relationship between sterols and oxygen: were sterols an adaptation to aerobic life?. Free Radic Biol Med, 2009, 47: 880-889

[13] García-Contreras R, de la Mora J, Mora-Montes HM, et al.. The inorganic pyrophosphatases of microorganisms: a structural and functional review. PeerJ, 2024, 12 Article ID: e17496

[14] Gohil N, Bhattacharjee G, Khambhati K, et al.. Engineering strategies in microorganisms for the enhanced production of squalene: advances, challenges and opportunities. Front Bioeng Biotechnol, 2019, 7 Article ID: 50

[15] Gomez JA, Hoffner K, Barton PI. DFBAlab: a fast and reliable MATLAB code for dynamic flux balance analysis. BMC Bioinformatics, 2014, 15 Article ID: 409

[16] Heggeset TMB, Ertesvåg H, Liu B, et al.. Lipid and DHA production in Aurantiochytrium sp.: Responses to nitrogen starvation and oxygen limitation revealed by analyses of production kinetics and global transcriptomes. Sci Rep, 2019, 9 Article ID: 19470

[17] Heirendt L, Arreckx S, Pfau T, et al.. Creation and analysis of biochemical constraint-based models using the COBRA Toolbox v3.0. Nat Protoc, 2019, 14: 639-702

[18] Hisam MW, Dar AA, Elrasheed MO, et al.. The versatility of the Taguchi method: optimizing experiments across diverse disciplines. J Stat Theory Appl, 2024, 23: 365-389

[19] Iqbal N, Masood A, Mubeen H, et al.. ATP synthase subunits: Structure and role in plants under stress conditions. Pak J Bot, 2023, 55: 1661-1668

[20] Ishibashi Y, Tanimura R, Ataka Y, et al.. Transgene-free protein-based genome editing in thraustochytrids enables customizable modulation of long-chain polyunsaturated fatty acid profiles. Chem Eng J, 2025, 526 Article ID: 171156

[21] Kim M, Park BG, Kim E-J, et al.. In silico identification of metabolic engineering strategies for improved lipid production in Yarrowia lipolytica by genome-scale metabolic modeling. Biotechnol Biofuels, 2019, 12: 187

[22] Kuttiraja M, Douha A, Valero JR, et al.. Elucidating the effect of glycerol concentration and C/N ratio on lipid production using Yarrowia lipolytica SKY7. Appl Biochem Biotechnol, 2016, 180: 1586-1600

[23] Liu TT, Xiao H, Xiao JH, et al.. Impact of oxygen supply on production of terpenoids by microorganisms: State of the art. Chin J Chem Eng, 2021, 30: 46-53

[24] Liu L, Zhu X, Ye H, et al.. Low dissolved oxygen supply functions as a global regulator of the growth and metabolism of Aurantiochytrium sp. PKU# Mn16 in the early stages of docosahexaenoic acid fermentation. Microb Cell Fact, 2023, 22 Article ID: 52

[25] Lopes HJS, Bonturi N, Kerkhoven EJ, et al.. C/N ratio and carbon source-dependent lipid production profiling in Rhodotorula toruloides. Appl Microbiol Biotechnol, 2020, 104: 2639-2649

[26] Man Z, Guo J, Zhang Y, et al.. Regulation of intracellular ATP supply and its application in industrial biotechnology. Crit Rev Biotechnol, 2020, 40: 1151-1162

[27] Matsuura H, Watanabe MM, Kaya K. Squalene quantification using octadecylbenzene as the internal standard. Procedia Environ Sci, 2012, 15: 43-46

[28] Menzorov AG, Iukhtanov DA, Naumenko LG, et al.. Thraustochytrids: evolution, ultrastructure, biotechnology, and modeling. Int J Mol Sci, 2024, 25 Article ID: 13172

[29] Micera M, Botto A, Geddo F, et al.. Squalene: More than a step toward sterols. Antioxidants (Basel), 2020, 9 Article ID: 688

[30] Min KH, Lee HH, Anbu P, et al.. The effects of culture condition on the growth property and docosahexaenoic acid production from Thraustochytrium aureum ATCC 34304. Korean J Chem Eng, 2012, 29: 1211-1215

[31] Miziorko HM. Enzymes of the mevalonate pathway of isoprenoid biosynthesis. Arch Biochem Biophys, 2011, 505: 131-143

[32] Nakazawa A, Matsuura H, Kose R, et al.. Optimization of culture conditions of the thraustochytrid Aurantiochytrium sp. strain 18W-13a for squalene production. Bioresour Technol, 2012, 109: 287-291

[33] Nakazawa A, Kokubun Y, Matsuura H, et al.. TLC screening of thraustochytrid strains for squalene production. J Appl Phycol, 2014, 26: 29-41

[34] Nazir Y, Shuib S, Kalil MS, et al.. Optimization of culture conditions for enhanced growth, lipid and docosahexaenoic acid (DHA) production of Aurantiochytrium SW1 by response surface methodology. Sci Rep, 2018, 8 Article ID: 8909

[35] Neupane P, Bhuju S, Thapa N, et al.. ATP synthase: Structure, function and inhibition. Biomol Concepts, 2019, 10: 1-10

[36] Orth JD, Thiele I, Palsson BO. What is flux balance analysis?. Nat Biotechnol, 2010, 28: 245-248

[37] Øyås O, Stelling J. Genome-scale metabolic networks in time and space. Curr Opin Syst Biol, 2018, 8: 51-58

[38] Paramasivan K, Mutturi S. Recent advances in the microbial production of squalene. World J Microbiol Biotechnol, 2022, 38 Article ID: 91

[39] Parichehreh R, Gheshlaghi R, Mahdavi MA, et al.. Optimization of lipid production in Chlorella vulgaris for biodiesel production using flux balance analysis. Biochem Eng J, 2019, 141: 131-145

[40] Park JM, Kim TY, Lee SY. Constraints-based genome-scale metabolic simulation for systems metabolic engineering. Biotechnol Adv, 2009, 27: 979-988

[41] Patel A, Rova U, Christakopoulos P, et al.. Mining of squalene as a value-added byproduct from DHA-producing marine thraustochytrid cultivated on food waste hydrolysate. Sci Total Environ, 2020, 736 Article ID: 139691

[42] Patil KR, Rocha I, Förster J, et al.. Evolutionary programming as a platform for in silico metabolic engineering. BMC Bioinformatics, 2005, 6: 308

[43] Pomraning KR, Kim YM, Nicora CD, et al.. Multi-omics analysis reveals regulators of the response to nitrogen limitation in Yarrowia lipolytica. BMC Genomics, 2016, 17 Article ID: 138

[44] Rau E-M, Ertesvåg H. Method development progress in genetic engineering of thraustochytrids. Mar Drugs, 2021, 19(9): Article ID: 515

[45] Schütte L, Hanisch PG, Scheler N, et al.. Squalene production under oxygen limitation by Schizochytrium sp. S31 in different cultivation systems. Appl Microbiol Biotechnol, 2024, 108 Article ID: 201

[46] Shakeri S, Amoozyan N, Fekrat F, et al.. Antigastric cancer bioactive Aurantiochytrium oil rich in docosahexaenoic acid: From media optimization to cancer cells cytotoxicity assessment. J Food Sci, 2017, 82: 2706-2718

[47] Shene C, Paredes P, Flores L, et al.. Dynamic flux balance analysis of biomass and lipid production by Antarctic thraustochytrid Oblongichytrium sp. RT2316-13. Biotechnol Bioeng, 2020, 117: 3006-3017

[48] Shene C, Leyton A, Flores L, et al.. Genome-scale metabolic modeling of Thraustochytrium sp. RT2316-16: Effects of nutrients on metabolism. Biotechnol Bioeng, 2024, 121: 1986-2001

[49] Simensen V, Voigt A, Almaas E. High-quality genome-scale metabolic model of Aurantiochytrium sp. T66. Biotechnol Bioeng, 2021, 118: 2105-2117

[50] Suen YL, Tang H, Huang J, et al.. Enhanced production of fatty acids and astaxanthin in Aurantiochytrium sp. by the expression of Vitreoscilla hemoglobin. J Agric Food Chem, 2014, 62: 12392-12398

[51] Tang WY, Wang DP, Tian Y, et al.. Metabolic engineering of Yarrowia lipolytica for improving squalene production. Bioresour Technol, 2021, 323 Article ID: 124652

[52] van Hoek P, van Dijken JP, Pronk JT. Regulation of fermentative capacity and levels of glycolytic enzymes in chemostat cultures of Saccharomyces cerevisiae. Enzyme Microb Technol, 2000, 26: 724-736

[53] Vyas S, Bettiga M, Rova U, et al.. Structural and molecular characterization of squalene synthase belonging to the marine thraustochytrid species Aurantiochytrium limacinum using bioinformatics approach. Mar Drugs, 2022, 20 Article ID: 180

[54] Wang C, Liu J, Liu H, et al.. A genome-scale dynamic flux balance analysis model of Streptomyces tsukubaensis NRRL18488 to predict the targets for increasing FK506 production. Biochem Eng J, 2017, 123: 45-56

[55] Xu C, Liu L, Zhang Z, et al.. Genome-scale metabolic model in guiding metabolic engineering of microbial improvement. Appl Microbiol Biotechnol, 2013, 97: 519-539

[56] Xu W, Yao J, Liu L, et al.. Improving squalene production by enhancing the NADPH/NADP+ ratio, modifying the isoprenoid-feeding module and blocking the menaquinone pathway in Escherichia coli. Biotechnol Biofuels, 2019, 12 Article ID: 68

[57] Yang T, Juntila DJ, Fujihara N, et al.. Enhancement of squalene production by constitutive expression of the 3-hydroxy-3-methylglutaryl-CoA reductase in Aurantiochytrium sp. 18W-13a. Mar Biotechnol, 2022, 24: 733-743

[58] Yarkent Ç, Oncel SS. Recent progress in microalgal squalene production and its cosmetic application. Biotechnol Bioprocess Eng, 2022, 27: 295-305

[59] Ye C, Qiao W, Yu X, et al.. Reconstruction and analysis of the genome-scale metabolic model of Schizochytrium limacinum SR21 for docosahexaenoic acid production. BMC Genomics, 2015, 16: 799

[60] Yen JY, Tanniche I, Fisher AK, et al.. Designing metabolic engineering strategies with genome-scale metabolic flux modeling. Adv Genomics Genet, 2015, 5: 93-105

[61] Yin L, Zhou Y, Ding N, et al.. Recent advances in metabolic engineering for the biosynthesis of phosphoenolpyruvate–oxaloacetate–pyruvate-derived amino acids. Molecules, 2024, 29 Article ID: 2893

[62] Zhang A, Xie Y, He Y, et al.. Bio-based squalene production by Aurantiochytrium sp. through optimization of culture conditions, and elucidation of the putative biosynthetic pathway genes. Bioresour Technol, 2019, 287 Article ID: 121415

[63] Zhang A, Mernitz K, Wu C, et al.. ATP drives efficient terpene biosynthesis in marine thraustochytrids. Mbio, 2021, 12 Article ID: e00881-21

[64] Zhang A, He Y, Sen B, et al.. Optimal NaCl medium enhances squalene accumulation in Thraustochytrium sp. ATCC 26185 and influences the expression levels of key metabolic genes. Front Microbiol, 2022, 13 Article ID: 900252

[65] Zhang L, Tsui TH, Tong YW, Liu R, Aggarangsi P. Microbial lipid technology based on oleaginous thraustochytrids. In: Zhang L, Tsui TH, Tong YW, Liu R, editors. Microbial lipids and biodiesel technologies. Singapore: Springer; 2025. pp. 119–137. https://doi.org/10.1007/978-981-96-0982-6_6

[66] Zhu ZT, Du MM, Gao B, et al.. Metabolic compartmentalization in yeast mitochondria: burden and solution for squalene overproduction. Metab Eng, 2021, 68: 232-245

Funding

Center for International Scientific Studies & Collaborations(A.1402.373)

RIGHTS & PERMISSIONS

Jiangnan University