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DOI: 10.1055/s-0043-1776291
Squalene Biosynthesis in Engineered Escherichia Coli
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Abstract
Squalene is a natural triterpenoid that is important in medicine, and daily-use chemical and nutraceutical industries. The demand for squalene remains high and constant. Shark liver oil is the source of the compound; however, the source is unsustainable due to the protection of the gradually extinct animal. This study aimed to construct an engineered Escherichia coli strain to produce squalene in a green and sustainable manner. In this study, the activity of several squalene synthases from different species was screened. Gas chromatography-mass spectrometry and high-performance liquid chromatography were used for chemical structure identification. The mevalonate pathway (MVA) was constructed into BL21(DE3) to enhance the supply of farnesyl diphosphate to obtain squalene at a concentration of 69.3 mg/L. IspH was overexpressed in BL21(DE3) to decrease squalene production due to the accumulation of dimethylallyl diphosphate. By balancing the overexpression of ispH and ispG, the production of squalene increased to 298.3 mg/L. The genes of the MVA pathway were further separated upstream and downstream, and constructed into two plasmids with different origins to balance the metabolic flux. By optimizing the expression strength of genes in MVA and 2-C-methyl-d-erythritol-4-phosphate pathway, an engineered strain was finally obtained with squalene production of 974.3 mg/L. By replacing chassis cell BL21(DE3) with XL1-Blue, squalene production was further increased to 1,274 mg/L.
Publication History
Received: 13 April 2023
Accepted: 30 September 2023
Article published online:
01 December 2023
© 2023. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. (https://creativecommons.org/licenses/by/4.0/)
Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany
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References
- 1 Ro DK, Paradise EM, Ouellet M. et al. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 2006; 440 (7086): 940-943
- 2 Ajikumar PK, Xiao WH, Tyo KE. et al. Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli. Science 2010; 330 (6000): 70-74
- 3 Westfall PJ, Pitera DJ, Lenihan JR. et al. Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin. Proc Natl Acad Sci U S A 2012; 109 (03) E111-E118
- 4 Popa O, Băbeanu NE, Popa I, Niță S, Dinu-Pârvu CE. Methods for obtaining and determination of squalene from natural sources. BioMed Res Int 2015; 2015: 367202
- 5 Danner H, Boeckler GA, Irmisch S. et al. Four terpene synthases produce major compounds of the gypsy moth feeding-induced volatile blend of Populus trichocarpa. Phytochemistry 2011; 72 (09) 897-908
- 6 Li Y, Wang G. Strategies of isoprenoids production in engineered bacteria. J Appl Microbiol 2016; 121 (04) 932-940
- 7 Ghimire GP, Thuan NH, Koirala N, Sohng JK. Advances in biochemistry and microbial production of squalene and its derivatives. J Microbiol Biotechnol 2016; 26 (03) 441-451
- 8 Xu W, Ma X, Wang Y. Production of squalene by microbes: an update. World J Microbiol Biotechnol 2016; 32 (12) 195
- 9 Katabami A, Li L, Iwasaki M, Furubayashi M, Saito K, Umeno D. Production of squalene by squalene synthases and their truncated mutants in Escherichia coli. J Biosci Bioeng 2015; 119 (02) 165-171
- 10 Wang J, Xiong Z, Li S, Wang Y. Exploiting exogenous MEP pathway genes to improve the downstream isoprenoid pathway effects and enhance isoprenoid production in Escherichia coli. Process Biochem 2015; 50 (01) 24-32
- 11 Yang C, Gao X, Jiang Y, Sun B, Gao F, Yang S. Synergy between methylerythritol phosphate pathway and mevalonate pathway for isoprene production in Escherichia coli. Metab Eng 2016; 37: 79-91
- 12 Furubayashi M, Li L, Katabami A, Saito K, Umeno D. Construction of carotenoid biosynthetic pathways using squalene synthase. FEBS Lett 2014; 588 (03) 436-442
- 13 Sun C, Ding Y, Cheng B, Zong Y. Using engineered Escherichia coli to synthesize squalene with optimized manipulation of squalene synthase and mevalonate pathway. bioRxiv. [preprint]. 2020.10.28.360032. Available at: https://doi.org/10.1101/2020.10.28.360032 . Accessed October 12, 2023
- 14 Xu W, Chai C, Shao L, Yao J, Wang Y. Metabolic engineering of Rhodopseudomonas palustris for squalene production. J Ind Microbiol Biotechnol 2016; 43 (05) 719-725
- 15 Meng H, Wang Y, Hua Q, Zhang S, Wang X. In silico analysis and experimental improvement of taxadiene heterologous biosynthesis in Escherichia coli. Biotechnol Bioprocess Eng 2011; 16 (02) 205-215
- 16 Li Q, Fan F, Gao X. et al. Balanced activation of IspG and IspH to eliminate MEP intermediate accumulation and improve isoprenoids production in Escherichia coli. Metab Eng 2017; 44: 13-21
- 17 Kim SW, Keasling JD. Metabolic engineering of the nonmevalonate isopentenyl diphosphate synthesis pathway in Escherichia coli enhances lycopene production. Biotechnol Bioeng 2001; 72 (04) 408-415
- 18 Boghigian BA, Salas D, Ajikumar PK, Stephanopoulos G, Pfeifer BA. Analysis of heterologous taxadiene production in K- and B-derived Escherichia coli. Appl Microbiol Biotechnol 2012; 93 (04) 1651-1661
- 19 Green MR, Sambrook J. Molecular Cloning: A Laboratory Manual. 4th ed.. New York, NY: Cold Spring Harbor Laboratory Press; 2012: 1-1881
- 20 Furubayashi M, Ikezumi M, Kajiwara J. et al. A high-throughput colorimetric screening assay for terpene synthase activity based on substrate consumption. PLoS One 2014; 9 (03) e93317
- 21 Liu H, Han S, Xie L. et al. Overexpression of key enzymes of the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway for improving squalene production in Escherichia coli. Afr J Biotechnol 2017; 16 (50) 2307-2316
- 22 George KW, Thompson MG, Kim J. et al. Integrated analysis of isopentenyl pyrophosphate (IPP) toxicity in isoprenoid-producing Escherichia coli. Metab Eng 2018; 47: 60-72
- 23 Martin VJ, Pitera DJ, Withers ST, Newman JD, Keasling JD. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat Biotechnol 2003; 21 (07) 796-802
- 24 Sivy TL, Fall R, Rosenstiel TN. Evidence of isoprenoid precursor toxicity in Bacillus subtilis. Biosci Biotechnol Biochem 2011; 75 (12) 2376-2383
- 25 Niu FX, Lu Q, Bu YF, Liu JZ. Metabolic engineering for the microbial production of isoprenoids: Carotenoids and isoprenoid-based biofuels. Synth Syst Biotechnol 2017; 2 (03) 167-175
- 26 Li M, Nian R, Xian M, Zhang H. Metabolic engineering for the production of isoprene and isopentenol by Escherichia coli. Appl Microbiol Biotechnol 2018; 102 (18) 7725-7738
- 27 Liu H, Wang Y, Tang Q, Kong W, Chung WJ, Lu T. MEP pathway-mediated isopentenol production in metabolically engineered Escherichia coli. Microb Cell Fact 2014; 13: 135
- 28 Zhou K, Zou R, Stephanopoulos G, Too HP. Metabolite profiling identified methylerythritol cyclodiphosphate efflux as a limiting step in microbial isoprenoid production. PLoS One 2012; 7 (11) e47513
- 29 Yang L, Wang C, Zhou J, Kim SW. Combinatorial engineering of hybrid mevalonate pathways in Escherichia coli for protoilludene production. Microb Cell Fact 2016; 15: 14
- 30 Gohil N, Bhattacharjee G, Khambhati K, Braddick D, Singh V. Engineering strategies in microorganisms for the enhanced production of squalene: advances, challenges and opportunities. [published correction appears in Front Bioeng Biotechnol 2019;7:114] Front Bioeng Biotechnol 2019; 7: 50
- 31 Peralta-Yahya PP, Ouellet M, Chan R, Mukhopadhyay A, Keasling JD, Lee TS. Identification and microbial production of a terpene-based advanced biofuel. Nat Commun 2011; 2: 483
- 32 Pongsupasa V, Anuwan P, Maenpuen S, Wongnate T. Rational-design engineering to improve enzyme thermostability. Methods Mol Biol 2022; 2397: 159-178
- 33 Guo JY, Hu KL, Bi CH, Li QY, Zhang XL. Construction of an alternative glycerol-utilization pathway for improved β-carotene production in Escherichia coli. J Ind Microbiol Biotechnol 2018; 45 (08) 697-705
- 34 Wang SY, Wang HR, Huang ZH. et al. Efficient synthesis of pinene by using genetically engineered Escherichia coli. [In Chinese]. Letters Biotechnol 2019; 30 (04) 455-463