Metabolic engineering of a high-light tolerant cyanobacterium Synechocystis sp. PCC 6803 for efficient biosynthesis of β-farnesene from CO₂

Abstract

As a naturally occurring substance derived from plants, β-farnesene represents a significant volatile sesquiterpene with potential applications in the fields of pest control and pharmaceutical intermediates. Nevertheless, the trace and transient production of farnesene from plants imposes limitations on its utilisation. Cyanobacteria, the sole oxygenic photosynthetic bacteria, are optimal chassis cells for farnesene synthesis from CO₂. In order to achieve efficient and sustained release of β-farnesene, a synthetic pathway based on the endogenous methylerythritol phosphate (MEP) pathway was designed and created in a fast-growing Synechocystis sp. PCC 6803, which is high-light-tolerant and was recently found (named as HL6803). The β-farnesene synthase gene (AaFS) and isopentenyl diphosphate isomerase gene (AaIDI) from Artemisia annua were introduced into the cyanobacterial strain HL6803 for β-farnesene production. A combination of basic engineering strategies resulted in a β-farnesene productivity of up to 2.0±0.4 mg/L/day. This is the highest productivity reported using similar engineering strategies. This work contributes to the engineering cyanobacteria for farnesene production from CO₂, as well as providing a novel fast-growing Synechocystis strain for the production of useful chemicals from CO₂.

Keywords

Β-Farnesene / Biosynthesis from CO₂ / Metabolic engineering / Fast-growing Synechocystis 6803 / Cyanobacterium

References

[1] Tang R, Wen Q, Li M, Zhang W, Wang Z, Yang J. Recent advances in the biosynthesis of Farnesene using metabolic engineering. J Agr Food Chem. 2021, 69(51): 15468-83

[2] Shi T, Li Y, Zhu L, Tong Y, Yang J, Fang Y, Wang M, Zhang J, Jiang Y, Yang S. Engineering the oleaginous yeast Yarrowia lipolytica for β-farnesene overproduction. Biotechnol J. 2021, 16(7): e2100097

[3] Liu J, Zhao X, Zhan Y, Wang K, Francis F, Liu Y. New slow release mixture of (E)-β-farnesene with Methyl salicylate to enhance aphid biocontrol efficacy in wheat ecosystem. 2021; 77(7):3341–48.

[4] Ye Z, Shi B, Huang Y, Ma T, Xiang Z, Hu B, Kuang Z, Huang M, Lin X, Tian Z, et al. . Revolution of vitamin E production by starting from microbial fermented Farnesene to isophytol. Innovation. 2022, 3(3): 100228

[5] Arslan ME, Hasan T, Mardinoğlu A. In vitro neuroprotective effects of Farnesene sesquiterpene on alzheimer’s disease model of differentiated neuroblastoma cell line. Int J Neurosci. 2021, 131(8): 745-54

[6] Banda-Villanueva A, González-Zapata JL, Martínez-Cartagena ME, Magaña I, Córdova T, López R, Valencia L, Medina SG, Rodríguez AM, Soriano F, et al. . Synthesis and vulcanization of polymyrcene and Polyfarnesene bio-based rubbers: influence of the chemical structure over the vulcanization process and mechanical properties. Polymers. 2022, 14(7): 1406

[7] Akutagawa S, Taketomi T, Kumobayashi H, Takayama K, Someya T, Otsuka S. Metal-assisted terpenoid synthesis. V. The catalytic trimerization of isoprene to trans-β-farnesene and its synthetic applications for terpenoids. B CHEM SOC JPN. 2006, 51(4): 1158-62

[8] Meadows AL, Hawkins KM, Tsegaye Y, Antipov E, Kim Y, Raetz L, Dahl RH, Tai A, Mahatdejkul-Meadows T, Xu L, et al. . Rewriting yeast central carbon metabolism for industrial isoprenoid production. Nature. 2016, 537(7622): 694-7

[9] Picaud S, Brodelius M, Brodelius PE. Expression, purification and characterization of Recombinant (E)-β-farnesene synthase from Artemisia annua. Phytochem. 2005, 66(9): 961-7

[10] Chandran SS, Kealey JT, Reeves CD. Microbial production of isoprenoids. Process Biochem. 2011, 46(9): 1703-10

[11] Wang X, Ort DR, Yuan JS. Photosynthetic terpene hydrocarbon production for fuels and chemicals. Plant Biotechnol J. 2015, 13(2): 137-46

[12] Zhou J, Yang F, Zhang F, Meng H, Zhang Y, Li Y. Impairing photorespiration increases photosynthetic conversion of CO₂ to isoprene in engineered cyanobacteria. Bioresour Bioprocess. 2021, 8(1): 42

[13] Englund E, Shabestary K, Hudson EP, Lindberg P. Systematic overexpression study to find target enzymes enhancing production of terpenes in Synechocystis PCC 6803, using isoprene as a model compound. Metab Eng. 2018, 49: 164-77

[14] Blanc-Garin V, Chenebault C, Diaz-Santos E, Vincent M, Sassi JF, Cassier-Chauvat C, Chauvat F. Exploring the potential of the model Cyanobacterium Synechocystis PCC 6803 for the photosynthetic production of various high-value terpenes. Biotechnol Biofuels Bioprod. 2022, 15(1): 110

[15] Sun J, Xu X, Wu Y, Sun H, Luan G, Lu X. Conversion of carbon dioxide into Valencene and other sesquiterpenes with metabolic engineered Synechocystis sp. PCC 6803 cell factories. GCB Bioenergy. 2023, 15(9): 1154-65

[16] Lee HJ, Lee J, Lee SM, Um Y, Kim Y, Sim SJ, Choi JI, Woo HM. Direct conversion of CO₂ to α-farnesene using metabolically engineered Synechococcus elongatus PCC 7942. J Agric Food Chem. 2017, 65(48): 10424-8

[17] Pattharaprachayakul N, Lee HJ, Incharoensakdi A, Woo HM. Evolutionary engineering of cyanobacteria to enhance the production of α-farnesene from CO₂. J Agric Food Chem. 2019, 67(49): 13658-64

[18] Lee HJ, Choi JI, Woo HM. Biocontainment of engineered Synechococcus elongatus PCC 7942 for photosynthetic production of α-farnesene from CO₂. J Agric Food Chem. 2021, 69(2): 698-703

[19] Rautela A, Yadav I, Gangwar A, Chatterjee R, Kumar S. Photosynthetic production of α-farnesene by engineered Synechococcus elongatus UTEX 2973 from carbon dioxide. Bioresour Technol. 2024, 396: 130432

[20] Vincent M, Blanc-Garin V, Chenebault C, Cirimele M, Farci S, Garcia-Alles LF, Cassier-Chauvat C, Chauvat F. Impact of carbon fixation, distribution and storage on the production of Farnesene and limonene in Synechocystis PCC 6803 and Synechococcus PCC 7002. Int J Mol Sci. 2024, 25(7): 3827

[21] Lin PC, Saha R, Zhang F, Pakrasi HB. Metabolic engineering of the Pentose phosphate pathway for enhanced limonene production in the Cyanobacterium Synechocysti s sp. PCC 6803. Sci Rep. 2017, 7(1): 17503

[22] Wang X, Liu W, Xin C, Zheng Y, Cheng Y, Sun S, Li R, Zhu X-G, Dai SY, Rentzepis PM, et al. . Enhanced limonene production in cyanobacteria reveals photosynthesis limitations. Proc Natl Acad Sci USA. 2016, 113(50): 14225-30

[23] Englund E, Pattanaik B, Ubhayasekera SJ, Stensjö K, Bergquist J, Lindberg P. Production of squalene in Synechocystis sp. PCC 6803. PLoS ONE. 2014, 9(3): e90270

[24] Yu J, Liberton M, Cliften PF, Head RD, Jacobs JM, Smith RD, Koppenaal DW, Brand JJ, Pakrasi HB. Synechococcus elongatus UTEX 2973, a fast-growing cyanobacterial chassis for biosynthesis using light and CO₂. Sci Rep. 2015; 5.

[25] Lou W, Tan X, Song K, Zhang S, Luan G, Li C, Lu X. A specific single nucleotide polymorphism in the ATP synthase gene significantly improves environmental stress tolerance of Synechococcus elongatus PCC 7942. Appl Environ Microbiol. 2018, 84(18): e01222-18

[26] Sengupta S, Jaiswal D, Sengupta A, Shah S, Gadagkar S, Wangikar PP. Metabolic engineering of a fast-growing Cyanobacterium Synechococcus elongatus PCC 11801 for photoautotrophic production of succinic acid. Biotechnol Biofuels. 2020, 13: 89

[27] Meng H, Zhang W, Zhu H, Yang F, Zhang Y, Zhou J, Li Y. Over-expression of an electron transport protein OmcS provides sufficient NADH for d-lactate production in Cyanobacterium. Biotechnol Biofuels. 2021, 14(1): 109

[28] Zhou J, Zhang H, Zhang Y, Li Y, Ma Y. Designing and creating a modularized synthetic pathway in Cyanobacterium Synechocystis enables production of acetone from carbon dioxide. Metab Eng. 2012, 14(4): 394-400

[29] Zhou J, Zhou JX, Yang HM, Yan CS, Huang F. Characterization of a sodium-regulated glutaminase from Cyanobacterium Synechocystis sp. PCC 6803. Sci China Ser C. 2008, 51: 1066-75

[30] Zhou J, Zhang H, Meng H, Zhang Y, Li Y. Production of optically pure D-lactate from CO₂ by blocking the PHB and acetate pathways and expressing D-lactate dehydrogenase in Cyanobacterium Synechocystis sp. PCC 6803. Process Biochem. 2014, 49(0): 2071-77

[31] Srivastava V, Amanna R, Rowden SJL, Sengupta S, Madhu S, Howe CJ, Wangikar PP. Adaptive laboratory evolution of the fast-growing Cyanobacterium Synechococcus elongatus PCC 11801 for improved solvent tolerance. J Biosci Bioeng. 2021, 131(5): 491-500

[32] Zhou J, Zhang F, Meng H, Zhang Y, Li Y. Introducing extra NADPH consumption ability significantly increases the photosynthetic efficiency and biomass production of cyanobacteria. Metab Eng. 2016, 38: 217-27

[33] Ma D, Li G, Alejos-Gonzalez F, Zhu Y, Xue Z, Wang A, Zhang H, Li X, Ye H, Wang H, et al. . Overexpression of a type-I isopentenyl pyrophosphate isomerase of Artemisia annua in the cytosol leads to high Arteannuin B production and Artemisinin increase. Plant J. 2017, 91(3): 466-79

[34] Zhou J, Zhang H, Meng H, Zhu Y, Bao G, Zhang Y. Discovery of a super-strong promoter enables efficient production of heterologous proteins in cyanobacteria. Sci Rep. 2014, 4: 4500

[35] Victoria AJ, Astbury MJ, McCormick AJ. Engineering highly productive cyanobacteria towards carbon negative emissions technologies. Curr Opin Biotechnol. 2024, 87: 103141

[36] Halfmann C, Gu L, Gibbons W, Zhou R. Genetically engineering cyanobacteria to convert CO₂, water, and light into the long-chain hydrocarbon Farnesene. Appl Microbiol Biotechnol. 2014, 98(23): 9869-77

RIGHTS & PERMISSIONS

Jiangnan University