Combinatorial metabolic engineering of Escherichia coli for high-level production of 3-dehydroshikimic acid

Abstract

3-dehydroshikimic acid (3-DHS), which is recognized as a key precursor of the shikimate pathway, has been regarded as an important platform compound for the biosynthesis of a wide range of aromatic products. However, microbial production is often constrained by imbalanced pathway expression and the accumulation of by-products, resulting in reduced cellular activity and loss of the target product. In this study, combinatorial metabolic engineering was applied for the production of 3-DHS. The downstream pathway was blocked through the deletion of aroE, after which engineering targets were screened and cumulatively integrated to enhance the basal metabolic flux. Subsequently, the biosynthetic pathway was optimized using a modular design strategy. On this basis, a multidimensional fine-tuning regulation strategy was implemented, by which the accumulation of the by-product gallic acid was reduced by 74.3%. Furthermore, carbon source utilization efficiency was enhanced through the combination of reinforced non-PTS glucose uptake and an optimized feeding strategy. As a result, a 3-DHS titer of 147.5 g/L was achieved in a 5 L bioreactor, with a yield of 0.6 g/g glucose and a productivity of 3.69 g/L/h, all of which represent the highest values reported to date. Overall, this study is expected to provide an important reference for the scalable biomanufacturing of 3-DHS and its high-value derivatives.

Keywords

3-dehydroshikimic acid / Escherichia coli / Metabolic engineering / Modular pathway optimization / Byproduct control

References

[1] Albino M, Gargalo CL, Nadal-Rey G, Albæk MO, Krühne U, Gernaey KV. Hybrid modeling for on-line fermentation optimization and scale-up: a review. Processes, 2024, 12(8): 1635

[2] An N, Xie C, Zhou SB, Wang J, Sun XX, Yan YJ, et al. . Establishing a growth-coupled mechanism for high-yield production of β-arbutin from glycerol in Escherichia coli. Bioresour Technol, 2023, 369: 128491

[3] Balderas-Hernández VE, Treviño-Quintanilla LG, Hernández-Chávez G, Martinez A, Bolívar F, Gosset G. Catechol biosynthesis from glucose in Escherichia coli anthranilate-overproducer strains by heterologous expression of anthranilate 1,2-dioxygenase from Pseudomonas aeruginosa PAO1. Microb Cell Fact, 2014, 13: 136

[4] Borges A, Ferreira C, Saavedra MJ, Simoes M. Antibacterial activity and mode of action of ferulic and gallic acids against pathogenic bacteria. Microb Drug Resist, 2013, 19(4): 256-265

[5] Candeias NR, Assoah B, Simeonov SP. Production and synthetic modifications of shikimic acid. Chem Rev, 2018, 118(20): 10458-10550

[6] Chandran SS, Yi J, Draths KM, von Daeniken R, Weber W, Frost JW. Phosphoenolpyruvate availability and the biosynthesis of shikimic acid. Biotechnol Prog, 2003, 19(3): 808-814

[7] Chen Y, Nielsen J. Advances in metabolic pathway and strain engineering paving the way for sustainable production of chemical building blocks. Curr Opin Biotechnol, 2013, 24(6): 965-972

[8] Chen Z, Liu H, Zong QJ, Liang TX, Sun J, Xu T, et al. . Engineering strategies enabled protocatechuic acid production from lignin by Pseudomonas putida KT2440. ACS Sustain Chem Eng, 2024, 12(49): 17726-17738

[9] Choi SS, Seo SY, Park SO, Lee HN, Song JS, Kim JY, et al. . Cell factory design and culture process optimization for dehydroshikimate biosynthesis in Escherichia coli. Front Bioeng Biotechnol, 2019, 7: 241

[10] Cravens A, Payne J, Smolke CD. Synthetic biology strategies for microbial biosynthesis of plant natural products. Nat Commun, 2019, 10: 2142

[11] Deng LH, Ping JR, Liu ZY, Linghu K, Zhang H, Shan XY, et al. . Efficient synthesis of L-DOPA in Escherichia coli via cofactor and enzyme engineering. Synth Syst Biotechnol, 2026, 11: 226-236

[12] Gao C, Hou JS, XuD P, Guo L, Chen XL, Hu GP, et al. . Programmable biomolecular switches for rewiring flux in Escherichia coli. Nat Commun, 2019, 10: 3751

[13] Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, Smith HO. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods, 2009, 6(5): 343-345

[14] Gosset G. Improvement of Escherichia coli production strains by modification of the phosphoenolpyruvate: sugar phosphotransferase system. Microb Cell Fact, 2005, 4: 14

[15] Holtz WJ, Keasling JD. Engineering static and dynamic control of synthetic pathways. Cell, 2010, 140(1): 19-23

[16] Hou JS, Gao C, Guo L, Nielsen J, Ding Q, Tang WX, et al. . Rewiring carbon flux in Escherichia coli using a bifunctional molecular switch. Metab Eng, 2020, 61: 47-57

[17] Hu GP, Gao C, Li XM, Song W, Wu J. Microbial engineering for monocyclic aromatic compounds production. FEMS Microbiol Rev, 2025, 49: fuaf003

[18] Hung YHR, Sauvageau D, Bressler DC. An adaptive, continuous substrate feeding strategy based on evolved gas to improve fed-batch ethanol fermentation. Appl Microbiol Biotechnol, 2025, 109(1): 64

[19] Jiang M, Zhang HR. Engineering the shikimate pathway for biosynthesis of molecules with pharmaceutical activities in E. coli. Curr Opin Biotechnol, 2016, 42: 1-6

[20] Jiang Y, Pei J, Zheng Y, Miao YJ, Duan BZ, Huang LF. Gallic acid: a potential anti-cancer agent. Chin J Integr Med, 2022, 28(7): 661-671

[21] Ko YS, Kim JW, Lee JA, Han T, Kim GB, Park JE, et al. . Tools and strategies of systems metabolic engineering for the development of microbial cell factories for chemical production. Chem Soc Rev, 2020, 49(14): 4615-4636

[22] Konzock O, Nielsen J. TRYing to evaluate production costs in microbial biotechnology. Trends Biotechnol, 2024, 42(11): 1339-1347

[23] Li K, Mikola MR, Draths KM, Worden RM, Frost JW. Fed-batch fermentor synthesis of 3-dehydroshikimic acid using recombinant Escherichia coli. Biotechnol Bioeng, 1999, 64(1): 61-73

[24] Li YF, Lin ZQ, Huang C, Zhang Y, Wang ZW, Tang YJ, et al. . Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. Metab Eng, 2015, 31: 13-21

[25] Li ZD, Gao C, Ye C, Guo L, Liu J, Chen XL, et al. . Systems engineering of Escherichia coli for high-level shikimate production. Metab Eng, 2023, 75: 1-11

[26] Liu LN, Ma XL, Bilal M, Wei LL, Tang SJ, Luo HZ, et al. . Toxicity and inhibition mechanism of gallic acid on physiology and fermentation performance of Escherichia coli. Bioresour Bioprocess, 2022, 9(1): 76

[27] Liu DM, Wang L, Ma LL, Wang XY, Li S, Zhou JW. Metabolic network rewiring and temperature-dependent regulation for enhanced 3-dehydroshikimate production in Escherichia coli. Bioresour Technol, 2024, 412: 131403

[28] Lu HY, Villada JC, Lee PKH. Modular metabolic engineering for biobased chemical production. Trends Biotechnol, 2019, 37(2): 152-166

[29] Martau GA, Calinoiu LF, Vodnar DC. Bio-vanillin: towards a sustainable industrial production. Trends Food Sci Technol, 2021, 109: 579-592

[30] Muir RM, Ibáñez AM, Uratsu SL, Ingham ES, Leslie CA, McGranahan GH, et al. . Mechanism of gallic acid biosynthesis in bacteria (Escherichia coli) and walnut (Juglans regia). Plant Mol Biol, 2011, 75(6): 555-565

[31] Nishikura-Imamura S, Matsutani M, Insomphun C, Vangnai AS, Toyama H, Yakushi T, et al. . Overexpression of a type II 3-dehydroquinate dehydratase enhances the biotransformation of quinate to 3-dehydroshikimate in Gluconobacter oxydans. Appl Microbiol Biotechnol, 2014, 98(7): 2955-2963

[32] Pan XW, Jiang SR, Tang M, Wang N, Sun QS, Yang TW, et al. . Biosynthesis of high-value chorismate derivatives in Escherichia coli: Recent advances and perspectives. Biotechnol Adv, 2026, 87: 108762

[33] Richman JE, Chang YC, Kambourakis S, Draths KM, Almy E, Snell KD, et al. . Reaction of 3-dehydroshikimic acid with molecular oxygen and hydrogen peroxide: products, mechanism, and associated antioxidant activity. J Am Chem Soc, 1996, 118(46): 11587-11591

[34] Shende VV, Bauman KD, Moore BS. The shikimate pathway: gateway to metabolic diversity. Nat Prod Rep, 2024, 41(4): 604-648

[35] Sheng Q, Yi LX, Zhong B, Wu XY, Liu LM, Zhang B. Shikimic acid biosynthesis in microorganisms: current status and future direction. Biotechnol Adv, 2023, 62: 108073

[36] Song J, He YN, Luo CH, Feng B, Ran F, Xu H, et al. . New progress in the pharmacology of protocatechuic acid: a compound ingested in daily foods and herbs frequently and heavily. Pharmacol Res, 2020, 161: 105109

[37] Tang M, Zhang WZ, Tian YJ, Qiao JJ, Li XB, Li WG, et al. . A review of the progress in the microbial biosynthesis of prenylated aromatic compounds. Molecules, 2025, 30(19): 3931

[38] Tian DG, Qin Z, Liu WL, Caiyin Q, Li WG, Zhao GR, et al. . Engineering a biosensor based high-throughput screening platform for high-yield caffeic acid production in Escherichia coli. Metab Eng, 2026, 93: 128-144

[39] Xu P, Gu Q, Wang WY, Wong L, Bower AGW, Collins CH, et al. . Modular optimization of multi-gene pathways for fatty acids production in E. coli. Nat Commun, 2013, 4: 1409

[40] Yi J, Draths KM, Li K, Frost JW. Altered glucose transport and shikimate pathway product yields in E-coli. Biotechnol Prog, 2003, 19(5): 1450-1459

[41] Zou YS, Zhang JL, Wang J, Gong XY, Jiang T, Yan YJ. A self-regulated network for dynamically balancing multiple precursors in complex biosynthetic pathways. Metab Eng, 2024, 82: 69-78

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