自工業革命以來，文明的進步和科技發展導致大量的二氧化碳釋放至大氣中，引起全球範圍的溫室效應和對生態環境威脅。微藻具有高效吸收二氧化碳和光合作用的能力，已成為新興的綠色生物製造平台以應對全球暖化。
小球藻Chlorella sorokiniana在生物量和適應環境脅迫等方面的表現優異，是近年的研究焦點；然而，高鹽的培養環境抑制了小球藻的整體表現。為減少培養微藻所需的淡水資源與經濟發展之間的衝突，本研究在高鹽系統中添加來自谷胺酸鈉生產的γ-胺基丁酸 (GABA) 改善小球藻在逆境中生長。研究結果顯示，在相當於60%海水鹽度的培養條件下添加10 mM GABA，獲得了4.56 g/L生物量、2.07 g/L蛋白質以及51.48 mg/L的總色素。
在解決負碳排與淡水資源的前提下，本研究進一步探討GABA補料的時間及濃度。發現在初期添加GABA使蛋白產量增加至1.4倍，而在培養中期進行補料可分別提高至1.58倍生物量以及2.13倍色素含量。然而，添加過多GABA對小球藻的生長和化學品製造造成了抑制效果。另一方面，在10 g/L到20 g/L鹽環境中優化GABA的補料策略，結果顯示在長時間批次培養中搭配5 mM的GABA補料成功將生物量、蛋白質以及色素分別提高至6.74 g/L、3.24 g/L以及49.87 mg/L。
本研究第三部分更一步嘗試降低小球藻的培養成本，透過CRISPRa基因干擾技術刺激小球藻內源的谷胺酸合成酶(GS)表達，並搭配味精(MSG)的補料，成功提高35%的生物量和120%總色素。最後，將微藻萃取物加入益生菌的培養系統中，明顯提高了益生菌的生物量。本研究通過探討GABA對小球藻Chlorella sorokiniana的培養條件優化、補料策略和CRISPRa基因調控，明顯提高了微藻在鹽的逆境生長，獲取最高的產量，進一步達成負碳排與再生資源的利用。

Microalgae have emerged prominent attention for their potential to achieve carbon negativity and applications in diverse fields. However, enhancing biomass and productivity cost-effectively remains a challenge. This study explored the potential of Chlorella sorokiniana (CS) to exhibit high biomass, protein, and pigment yields under halo-tolerance with γ-aminobutyric acid (GABA) supplement. We applied CS in the presence of GABA, obtained through in-vitro biotransformation of monosodium glutamate (MSG), under salt stress condition. At 20 g/L NaCl system, chlorophyll content was initially suppressed, leading to a significant decline in biomass, protein, and pigment synthesis.
Following the addition of 10 mM GABA successfully elevated biomass, protein, and pigment contents to 4.56 g/L, 2.07 g/L and 51.48 mg/L, respectively. Regarding to the feeding strategy, initial addition of GABA resulted in 1.4-fold increase in protein content, whereas periodic supplementation resulted in 1.58-fold and 2.13-fold biomass and pigment content enhancement, respectively. Under halophilic conditions, repeated feeding with 5 mM GABA achieved impressive results, with biomass, protein, and pigment reaching 6.74 g/L, 3.24 g/L, and 49.87 mg/L. Furthermore, systematic MSG feeding strategy and CRISPRa technology mediated on glutamate synthase (GS) enhancement facilitated CS performance. Additionally, protein from CS was successfully used as prebiotic to cultivate Escherichia coli Nissle 1917 and Lactobacillus rhamnosus ZY. We provide a carbon negative and bioresources application for producing biomass and valuable products using Chlorella sorokiniana relying on GABA addition, feeding strategy, and CRISPRa technology at halophilic condition.

[1] Amorim, M. L., Soares, J., Coimbra, J. S. D. R., Leite, M. D. O., Albino, L. F. T., & Martins, M. A. Microalgae proteins: Production, separation, isolation, quantification, and application in food and feed. Crit Re. Food Sc. Nutr, 61(12), 1976-2002, 2021.
[2] Arora, N., Nanda, M., & Kumar, V. Sustainable algal biorefineries: capitalizing on many benefits of GABA. Trends Biotechnol, 41(5), 600-603, 2023.
[3] Arriola, M. B., Velmurugan, N., Zhang, Y., Plunkett, M. H., Hondzo, H., & Barney, B. M. Genome sequences of Chlorella sorokiniana UTEX 1602 and Micractinium conductrix SAG 241.80: implications to maltose excretion by a green alga. Plant J, 93(3), 566-586, 2018.
[4] Asadi, P., Rad, H. A., & Qaderi, F. Comparison of Chlorella vulgaris and Chlorella sorokiniana pa. 91 in post treatment of dairy wastewater treatment plant effluents. Environ Sci Pollut Res, 26, 29473-29489, 2019.
[5] Baek, K., Kim, D. H., Jeong, J., Sim, S. J., Melis, A., Kim, J. S., Jin, E., & Bae, S. DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins. Sci Rep, 6(1), 1-7, 2016.
[6] Barone, R. S. C., Sonoda, D. Y., Lorenz, E. K., & Cyrino, J. E. P. Digestibility and pricing of Chlorella sorokiniana meal for use in tilapia feeds. Sci Agric, 75, 184-1, 2018.
[7] Begum, H., Yusoff, F. M., Banerjee, S., Khatoon, H., & Shariff, M. Availability and utilization of pigments from microalgae. Crit Rev Food Sci Nutr, 56(13), 2209-2222, 2016.
[8] Beheshtipour, H., Mortazavian, A. M., Haratian, P., & Darani, K. K. Effects of Chlorella vulgaris and Arthrospira platensis addition on viability of probiotic bacteria in yogurt and its biochemical properties. Eur Food Res Technol, 235(4), 719-728, 2012.
[9] Belhaj, K., Chaparro-Garcia, A., Kamoun, S., Patron, N. J., & Nekrasov, V. Editing plant genomes with CRISPR/Cas9. Curr Opin Biotechnols 32, 76-84, 2015.
[10] Bhowmik, D., Dubey, J., & Mehra, S. Probiotic efficiency of Spirulina platensis-stimulating growth of lactic acid bacteria. World J. Dairy Food Sci, 4(2), 160-163, 2009.
[11] Buono, S., Langellotti, A. L., Martello, A., Rinna, F., & Fogliano, V. Functional ingredients from microalgae. Food Funct, 5(8), 1669-1685, 2014.
[12] Chapman, R. L. Algae: the world’s most important “plants”- an introduction. Mitig Adapt Strateg Glob Chang, 18(1), 5-12, 2013.
[13] Chen, C. Y., Hsieh, C., Lee, D. J., Chang, C. H., & Chang, J. S. Production, extraction and stabilization of lutein from microalga Chlorella sorokiniana MB-1. Bioresour Technol, 200, 500-505, 2016.
[14] Chen, C. Y., Kuo, E. W., Nagarajan, D., Ho, S. H., Dong, C. D., Lee, D. J., & Chang, J. S. Cultivating Chlorella sorokiniana AK-1 with swine wastewater for simultaneous wastewater treatment and algal biomass production. Bioresour Technol, 302, 122814, 2020.
[15] Chen, C. Y., Lu, J. C., Chang, Y. H., Chen, J. H., Nagarajan, D., Lee, D. J., & Chang, J. S. Optimizing heterotrophic production of Chlorella sorokiniana SU-9 proteins potentially used as a sustainable protein substitute in aquafeed. Bioresour Technol, 370, 128538, 2023.
[16] Chen, F., Leng, Y., Lu, Q., & Zhou, W. The application of microalgae biomass and bio-products as aquafeed for aquaculture. Algal Res, 60, 102541. 2021.
[17] Chen, H., & Wang, Q. Regulatory mechanisms of lipid biosynthesis in microalgae. Biol Rev, 96(5), 2373-2391, 2021.
[18] Chen, H., Zheng, Y., Zhan, J., He, C., & Wang, Q. Comparative metabolic profiling of the lipid-producing green microalga Chlorella reveals that nitrogen and carbon metabolic pathways contribute to lipid metabolism. Biotechnol Biofuels, 10, 1-20, 2017.
[19] Chen, J. H., Chen, C. Y., & Chang, J. S. Lutein production with wild-type and mutant strains of Chlorella sorokiniana MB-1 under mixotrophic growth. J Taiwan Inst Chem Eng, 79, 66-73, 2017.
[20] Chen, J. H., Kato, Y., Matsuda, M., Chen, C. Y., Nagarajan, D., Hasunuma, T., Kondo, A. & Chang, J. S. Lutein production with Chlorella sorokiniana MB-1-M12 using novel two-stage cultivation strategies–metabolic analysis and process improvement. Bioresour Technol, 334, 125200, 2021.
[21] Chen, Q., Chen, Y., Hu, Q., & Han, D. Metabolomic analysis reveals astaxanthin biosynthesis in heterotrophic microalga Chromochloris zofingiensis. Bioresour Technol, 374, 128811, 2023.
[22] Cho, S., Shin, J., & Cho, B. K. Applications of CRISPR/Cas system to bacterial metabolic engineering. Int J Mol Sci, 19(4), 1089. 2018.
[23] Coda, R., Melama, L., Rizzello, C. G., Curiel, J. A., Sibakov, J., Holopainen, U., Pulkkinen, M. & Sozer, N. Effect of air classification and fermentation by Lactobacillus plantarum VTT E-133328 on faba bean (Vicia faba L.) flour nutritional properties. Int J Food Microbiol, 193, 34-42, 2015.
[24] Cordero, B. F., Obraztsova, I., Couso, I., Leon, R., Vargas, M. A., & Rodriguez, H. Enhancement of lutein production in Chlorella sorokiniana (Chorophyta) by improvement of culture conditions and random mutagenesis. Mar Drugs, 9(9), 1607-1624. 2011.
[25] Daneshvar, E., Wicker, R. J., Show, P. L., & Bhatnagar, A. Biologically-mediated carbon capture and utilization by microalgae towards sustainable CO2 biofixation and biomass valorization-A review. Chem Eng J, 427, 130884, 2022.
[26] Dao, L. H., & Beardall, J. Effects of lead on growth, photosynthetic characteristics and production of reactive oxygen species of two freshwater green algae. Chemosphere, 147, 420-429, 2016.
[27] de Winter, L., Klok, A. J., Franco, M. C., Barbosa, M. J., & Wijffels, R. H. The synchronized cell cycle of Neochloris oleoabundans and its influence on biomass composition under constant light conditions. Algal Res, 2(4), 313-320, 2013.
[28] Demmig-Adams, B., López-Pozo, M., Stewart, J. J., & Adams III, W. W. Zeaxanthin and lutein: Photoprotectors, anti-inflammatories, and brain food. Molecules, 25(16), 3607, 2020.
[29] Diprat, A. B., Thys, R. C. S., Rodrigues, E., & Rech, R. Chlorella sorokiniana: A new alternative source of carotenoids and proteins for gluten-free bread. LWT, 134, 109974, 2020.
[30] Doudna, J. A., & Charpentier, E. The new frontier of genome engineering with CRISPR-Cas9. Science, 346(6213), 1258096, 2014.
[31] Dudgeon, D., Arthington, A. H., Gessner, M. O., Kawabata, Z. I., Knowler, D. J., Lévêque, C., Naiman, R. J., Prieur-Richard, A. H., Soto, D., Stiassny, M. L. J. & Sullivan, C. A. Freshwater biodiversity: importance, threats, status and conservation challenges. Biol Rev, 81(2), 163-182, 2006.
[32] Dumont, S., Bykova, N. V., Pelletier, G., Dorion, S., & Rivoal, J. Cytosolic triosephosphate isomerase from Arabidopsis thaliana is reversibly modified by glutathione on cysteines 127 and 218. Front Plant Sci, 7, 1942, 2016.
[33] Elrayies, G. M. Microalgae: prospects for greener future buildings. Renew Sust Energ Rev, 81, 1175-1191, 2018.
[34] Escapa, C., Coimbra, R. N., Paniagua, S., García, A. I., & Otero, M. Nutrients and pharmaceuticals removal from wastewater by culture and harvesting of Chlorella sorokiniana. Bioresour Technol, 185, 276-284, 2015.
[35] Fait, A., Fromm, H., Walter, D., Galili, G., & Fernie, A. R. Highway or byway: the metabolic role of the GABA shunt in plants. Trends Plant Sci, 13(1), 14-19, 2008.
[36] Forde, B. G., & Roberts, M. R. Glutamate receptor-like channels in plants: a role as amino acid sensors in plant defence? F1000 Med Rep, 6, 2014.
[37] Gao, F., Zhang, X. L., Zhu, C. J., Huang, K. H., & Liu, Q. High-efficiency biofuel production by mixing seawater and domestic sewage to culture freshwater microalgae. Chem Eng J, 443, 136361, 2022.
[38] Gasiunas, G., Barrangou, R., Horvath, P., & Siksnys, V. Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci, 109(39), E2579-E2586, 2012.
[39] Geada, P., Moreira, C., Silva, M., Nunes, R., Madureira, L., Rocha, C. M., Pereira, R. N., Vicente, A. A., & Teixeira, J. A. Algal proteins: Production strategies and nutritional and functional properties. Bioresour Technol, 332, 125125, 2021.
[40] González-Vega, R. I., Cárdenas-López, J. L., López-Elías, J. A., Ruiz-Cruz, S., Reyes-Díaz, A., Perez-Perez, L. M., Cinco-Moroyoqui, F. J., Robles-Zepeda, R. E., Borboa-Flores, J., & Del-Toro-Sánchez, C. L. Optimization of growing conditions for pigments production from microalga Navicula incerta using response surface methodology and its antioxidant capacity. Saudi J Biol Sci, 28(2), 1401-1416, 2021.
[41] Grossmann, L., Hinrichs, J., Goff, H. D., & Weiss, J. Heat-induced gel formation of a protein-rich extract from the microalga Chlorella sorokiniana. Innov Food Sci Emerg Technol, 56, 102176, 2019.
[42] Hajdukiewicz, P., Svab, Z., & Maliga, P. The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol, 25, 989-994, 1994.
[43] Hamed, S. M., Zinta, G., Klöck, G., Asard, H., Selim, S., & AbdElgawad, H. Zinc-induced differential oxidative stress and antioxidant responses in Chlorella sorokiniana and Scenedesmus acuminatus. Ecotoxicol Environ Saf, 140, 256-263, 2017.
[44] Han, P., Lu, Q., Fan, L., & Zhou, W. A review on the use of microalgae for sustainable aquaculture. Appl Sci, 9(11), 2377, 2019.
[45] Hepsomali, P., Groeger, J. A., Nishihira, J., & Scholey, A. Effects of oral gamma-aminobutyric acid (GABA) administration on stress and sleep in humans: a systematic review. Front Neurosci, 14, 923, 2020.
[46] Hinton, T., Jelinek, H. F., Viengkhou, V., Johnston, G. A., & Matthews, S. Effect of GABA-fortified oolong tea on reducing stress in a university student cohort. Front nutr, 6, 27, 2019.
[47] Ho, S. H., Huang, S. W., Chen, C. Y., Hasunuma, T., Kondo, A., & Chang, J. S. Characterization and optimization of carbohydrate production from an indigenous microalga Chlorella vulgaris FSP-E. Bioresour Technol, 135, 157-165, 2013.
[48] Hu, Q., Sommerfeld, M., Jarvis, E., Ghirardi, M., Posewitz, M., Seibert, M., & Darzins, A. Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J, 54(4), 621-639, 2008.
[49] IEA. Global Energy Review 2021, 2021, https://www.iea.org/reports/global-energy-review-2021.
[50] Inokuchi, R., Kuma, K. I., Miyata, T., & Okada, M. Nitrogen‐assimilating enzymes in land plants and algae: phylogenic and physiological perspectives. Physiol Plant, 116(1), 1-11, 2002.
[51] Jantaro, S., & Kanwal, S. Low-molecular-weight nitrogenous compounds (GABA and polyamines) in blue–green algae. Algal Green Chemistry, Elsevier, 149-169, 2017.
[52] Jiang, W., Brueggeman, A. J., Horken, K. M., Plucinak, T. M., & Weeks, D. P. Successful transient expression of Cas9 and single guide RNA genes in Chlamydomonas reinhardtii. Eukaryot Cell, 13(11), 1465-1469, 2014.
[53] Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science, 337(6096), 816-821, 2012.
[54] Kamani, M. H., Eş, I., Lorenzo, J. M., Remize, F., Roselló-Soto, E., Barba, F. J., Clark, J., & Khaneghah, A. M. Advances in plant materials, food by-products, and algae conversion into biofuels: use of environmentally friendly technologies. Green Chem, 21(12), 3213-3231, 2019.
[55] Kandasamy, S., Vlasova, A. N., Fischer, D., Kumar, A., Chattha, K. S., Rauf, A., Shao, L., Langel, S. N., Rajashekara, G., & Saif, L. J. Differential effects of Escherichia coli Nissle and Lactobacillus rhamnosus strain GG on human rotavirus binding, infection, and B cell immunity. J Immunol, 196(4), 1780-1789, 2016.
[56] Kao, C. Y., Chen, T. Y., Chang, Y. B., Chiu, T. W., Lin, H. Y., Chen, C. D., Chang, J.-S., & Lin, C. S. Utilization of carbon dioxide in industrial flue gases for the cultivation of microalga Chlorella sp. Bioresour Technol, 166, 485-493, 2014.
[57] Karuppanapandian, T., Moon, J. C., Kim, C., Manoharan, K., & Kim, W. Reactive oxygen species in plants: their generation, signal transduction, and scavenging mechanisms. Aust J Crop Sci, 5(6), 709-725, 2011.
[58] Khanra, S., Mondal, M., Halder, G., Tiwari, O. N., Gayen, K., & Bhowmick, T. K. Downstream processing of microalgae for pigments, protein and carbohydrate in industrial application: A review. Food Bioprod Process, 110, 60-84, 2018.
[59] Khemiri, S., Khelifi, N., Nunes, M. C., Ferreira, A., Gouveia, L., Smaali, I., & Raymundo, A. Microalgae biomass as an additional ingredient of gluten-free bread: Dough rheology, texture quality and nutritional properties. Algal Res, 50, 101998, 2020.
[60] Khoshnamvand, M., Ashtiani, S., Chen, Y., & Liu, J. Impacts of organic matter on the toxicity of biosynthesized silver nanoparticles to green microalgae Chlorella vulgaris. Environ Res, 185, 109433, 2020.
[61] Kim, G. Y., Roh, K., & Han, J. I. The use of bicarbonate for microalgae cultivation and its carbon footprint analysis. Green Chem, 21(18), 5053-5062, 2019.
[62] Koyande, A. K., Chew, K. W., Rambabu, K., Tao, Y., Chu, D. T., & Show, P. L. Microalgae: A potential alternative to health supplementation for humans. Food Sci Hum Wellness, 8(1), 16-24, 2019.
[63] Krimech, A., Helamieh, M., Wulf, M., Krohn, I., Riebesell, U., Cherifi, O., Mandi, L., & Kerner, M. Differences in adaptation to light and temperature extremes of Chlorella sorokiniana strains isolated from a wastewater lagoon. Bioresour Technol, 350, 126931, 2022.
[64] Kumar, K., Dasgupta, C. N., & Das, D. Cell growth kinetics of Chlorella sorokiniana and nutritional values of its biomass. Bioresour Technol, 167, 358-366, 2014.
[65] Kumbhar, A. N., He, M., Rajper, A. R., Memon, K. A., Rizwan, M., Nagi, M., Woldemicael, A. G., Li, D., Wang, C., & Wang, C. The use of urea and kelp waste extract is a promising strategy for maximizing the biomass productivity and lipid content in Chlorella sorokiniana. Plants, 9(4), 463, 2020.
[66] Labboun, S., Tercé-Laforgue, T., Roscher, A., Bedu, M., Restivo, F. M., Velanis, C. N., Skopelitis, D. S., Moshou, P. N., Roubelakis-Angelakis, K. A., Suzuki, A., & Hirel, B. Resolving the role of plant glutamate dehydrogenase. I. in vivo real time nuclear magnetic resonance spectroscopy experiments. Plant Cell Physiol, 50(10), 1761-1773, 2009.
[67] Lan, Y. J., Tan, S. I., Cheng, S. Y., Ting, W. W., Xue, C., Lin, T. H., Cai, M.-Z., Chen, P.-T., & Ng, I. S. Development of Escherichia coli Nissle 1917 derivative by CRISPR/Cas9 and application for gamma-aminobutyric acid (GABA) production in antibiotic-free system. Biochem Eng J, 168, 107952, 2021.
[68] Lavecchia, R., & Zuorro, A. Shelf stability of lutein from marigold (Tagetes erecta L.) flowers in vegetable oils. Chem Eng Trans, 14(199), e204, 2008.
[69] Li, Q., Zhao, Y., Ding, W., Han, B., Geng, S., Ning, D., Ma, T., & Yu, X. Gamma-aminobutyric acid facilitates the simultaneous production of biomass, astaxanthin and lipids in Haematococcus pluvialis under salinity and high-light stress conditions. Bioresour Technol, 320, 124418, 2021.
[70] Li, S. Y., Cheng, Q. X., Wang, J. M., Li, X. Y., Zhang, Z. L., Gao, S., Cao, R. B., Zhao, G.-P., & Wang, J. CRISPR-Cas12a-assisted nucleic acid detection. Cell Discov, 4(1), 20, 2018.
[71] Li, X., Zhang, X., Zhao, Y., & Yu, X. Cross-talk between gama-aminobutyric acid and calcium ion regulates lipid biosynthesis in Monoraphidium sp. QLY-1 in response to combined treatment of fulvic acid and salinity stress. Bioresour Technol, 315, 123833, 2020.
[72] Lim, Y. A., Chong, M. N., Foo, S. C., & Ilankoon, I. M. S. K. Analysis of direct and indirect quantification methods of CO2 fixation via microalgae cultivation in photobioreactors: A critical review. Renewable Sustainable Energy Rev, 137, 110579, 2021.
[73] Lin, J. Y., Lin, W. R., & Ng, I. S. CRISPRa/i with A daptive S ingle G uide A ssisted R egulation D NA (ASGARD) mediated control of Chlorella sorokiniana to enhance lipid and protein production. Biotechnol J, 17(10), 2100514, 2022.
[74] Lin, J. Y., Xue, C., Tan, S. I., & Ng, I. S. Pyridoxal kinase PdxY mediated carbon dioxide assimilation to enhance the biomass in Chlamydomonas reinhardtii CC-400. Bioresour Technol, 322, 124530, 2021.
[75] Lin, W. R., & Ng, I. S. Development of CRISPR/Cas9 system in Chlorella vulgaris FSP-E to enhance lipid accumulation. Enzyme Microb, 133, 109458, 2020.
[76] Liu, M., Yu, Z., Jiang, L., Hou, Q., Xie, Z., Ma, M., Yu, S., & Pei, H. Monosodium glutamate wastewater assisted seawater to increase lipid productivity in single-celled algae. Renew Energy, 179, 1793-1802, 2021.
[77] Liu, Y., Cao, Y., Zhang, Q., Li, X., & Wang, S. A cytosolic triosephosphate isomerase is a key component in XA3/XA26-mediated resistance. Plant Physiol, 178(2), 923-935, 2018.
[78] Lizzul, A. M., Lekuona-Amundarain, A., Purton, S., & Campos, L. C. Characterization of Chlorella sorokiniana, UTEX 1230. Biol, 7(2), 25, 2018.
[79] Ma, R., Zhang, Z., Tang, Z., Ho, S. H., Shi, X., Liu, L., Xie, Y., & Chen, J. Enhancement of co-production of lutein and protein in Chlorella sorokiniana FZU60 using different bioprocess operation strategies. Bioresour Bioprocess, 8(1), 1-12, 2021.
[80] Maeder, M. L., Linder, S. J., Cascio, V. M., Fu, Y., Ho, Q. H., & Joung, J. K. CRISPR RNA–guided activation of endogenous human genes. Nat Methods, 10(10), 977-979, 2013.
[81] Mallick, N., & Mohn, F. H. Reactive oxygen species: response of algal cells. J Plant Physiol, 157(2), 183-193, 2000.
[82] Masojídek, J., Torzillo, G., & Koblížek, M. Photosynthesis in microalgae. Handbook of microalgal culture: applied phycology and biotechnology, 21-36, 2013.
[83] Mathiot, C., Ponge, P., Gallard, B., Sassi, J. F., Delrue, F., & Le Moigne, N. Microalgae starch-based bioplastics: Screening of ten strains and plasticization of unfractionated microalgae by extrusion. Carbohydr Polym, 208, 142-151, 2019.
[84] Mayerhofer, R., Koncz‐Kalman, Z., Nawrath, C., Bakkeren, G., Crameri, A., Angelis, K., Redei, G. P., Schell, J., Hohn, B., & Koncz, C. T‐DNA integration: a mode of illegitimate recombination in plants. EMBO J, 10(3), 697-704, 1991.
[85] Mohamed, A. G., Abo-El-Khair, B. E., & Shalaby, S. M. Quality of novel healthy processed cheese analogue enhanced with marine microalgae Chlorella vulgaris biomass. World Appl Sci J, 23(7), 914-925, 2013.
[86] Nagappan, S., Devendran, S., Tsai, P. C., Dahms, H. U., & Ponnusamy, V. K. Potential of two-stage cultivation in microalgae biofuel production. Fuel, 252, 339-349, 2019.
[87] Napolitano, G., Fasciolo, G., Salbitani, G., & Venditti, P. Chlorella sorokiniana dietary supplementation increases antioxidant capacities and reduces ros release in mitochondria of hyperthyroid rat liver. Antioxidants, 9(9), 883, 2020.
[88] Ngo, D. H., & Vo, T. S. An updated review on pharmaceutical properties of gamma-aminobutyric acid. Molecules, 24(15), 2678, 2019.
[89] Nymark, M., Sharma, A. K., Sparstad, T., Bones, A. M., & Winge, P. A CRISPR/Cas9 system adapted for gene editing in marine algae. Sci Rep, 6(1), 24951, 2016.
[90] Oketch-Rabah, H. A., Madden, E. F., Roe, A. L., & Betz, J. M. United States Pharmacopeia (USP) safety review of gamma-aminobutyric acid (GABA). Nutrients, 13(8), 2742, 2021.
[91] Okumoto, S., Funck, D., Trovato, M., & Forlani, G. Amino acids of the glutamate family: Functions beyond primary metabolism. Front Plant Sci, 7, 318, 2016.
[92] Pérez-Pérez, M. E., Lemaire, S. D., & Crespo, J. L. Reactive oxygen species and autophagy in plants and algae. Plant Physiol, 160(1), 156-164, 2012.
[93] Pan, Y., Shen, Y., Zhang, H., Ran, X., Xie, T., Zhang, Y., & Yao, C. Fine-tuned regulation of photosynthetic performance via γ-aminobutyric acid (GABA) supply coupled with high initial cell density culture for economic starch production in microalgae. Bioresour Bioprocess, 9(1), 52, 2022.
[94] Park, S. J., Kim, E. Y., Noh, W., Oh, Y. H., Kim, H. Y., Song, B. K., Cho, K. M., Hong, S. H., Lee, S. H., & Jegal, J. Synthesis of nylon 4 from gamma-aminobutyrate (GABA) produced by recombinant Escherichia coli. Bioprocess Biosyst Eng, 36, 885-892, 2013.
[95] Peters, G. P., Andrew, R. M., Canadell, J. G., Friedlingstein, P., Jackson, R. B., Korsbakken, J. I., Le Quéré, C., & Peregon, A. Carbon dioxide emissions continue to grow amidst slowly emerging climate policies. Nat Clim Change, 10(1), 3-6, 2020.
[96] Potocki, L., Oklejewicz, B., Kuna, E., Szpyrka, E., Duda, M., & Zuczek, J. Application of green algal Planktochlorella nurekis biomasses to modulate growth of selected microbial species. Molecules, 26(13), 4038, 2021.
[97] Rajkumar, R., Ezhumalai, G., & Gnanadesigan, M. A green approach for the synthesis of silver nanoparticles by Chlorella vulgaris and its application in photocatalytic dye degradation activity. Environ Technol Innov, 21, 101282, 2021.
[98] Ramesh, S. A., Tyerman, S. D., Xu, B., Bose, J., Kaur, S., Conn, V., Domingos, P., Ullah, S., Wege, S., Shabala, S., Feijó, J. A., Ryan, P. R., & Gilliham, M. GABA signalling modulates plant growth by directly regulating the activity of plant-specific anion transporters. Nat Commun, 6(1), 7879, 2015.
[99] Ran, W., Xiang, Q., Pan, Y., Xie, T., Zhang, Y., & Yao, C. Enhancing photosynthetic starch production by γ-aminobutyric acid addition in a marine green microalga Tetraselmis subcordiformis under nitrogen stress. Ind Eng Chem Res, 59(39), 17103-17112, 2020.
[100] Rathod, J. P., Vira, C., Lali, A. M., & Prakash, G. Metabolic engineering of Chlamydomonas reinhardtii for enhanced β-carotene and lutein production. Appl Biochem Biotechnol, 190, 1457-1469, 2020.
[101] Ren, Y., Sun, H., Deng, J., Huang, J., & Chen, F. Carotenoid production from microalgae: biosynthesis, salinity responses and novel biotechnologies. Mar Drugs, 19(12), 713, 2021.
[102] Reyes-Becerril, M., Angulo, C., Estrada, N., Murillo, Y., & Ascencio-Valle, F. Dietary administration of microalgae alone or supplemented with Lactobacillus sakei affects immune response and intestinal morphology of Pacific red snapper (Lutjanus peru). Fish Shellfish Immunol, 40(1), 208-216, 2014.
[103] Ricroch, A. E., & Hénard-Damave, M. C. Next biotech plants: new traits, crops, developers and technologies for addressing global challenges. Crit Rev Biotechnol, 36(4), 675-690, 2016.
[104] Sarasa, S. B., Mahendran, R., Muthusamy, G., Thankappan, B., Selta, D. R. F., & Angayarkanni, J. A brief review on the non-protein amino acid, gamma-amino butyric acid (GABA): its production and role in microbes. Curr Microbiol, 77, 534-544, 2020.
[105] Ścieszka, S., Gorzkiewicz, M., & Klewicka, E. Innovative fermented soya drink with the microalgae Chlorella vulgaris and the probiotic strain Levilactobacillus brevis ŁOCK 0944. LWT, 151, 112131, 2021.
[106] Seddon, J. M., Ajani, U. A., Sperduto, R. D., Hiller, R., Blair, N., Burton, T. C., Farber, M. D., Gragoudas, E. S., Haller, J., Miller, D. T., & Willett, W. Dietary carotenoids, vitamins A, C, and E, and advanced age-related macular degeneration. Jama, 272(18), 1413-1420, 1994.
[107] Seifikalhor, M., Aliniaeifard, S., Hassani, B., Niknam, V., & Lastochkina, O. Diverse role of γ-aminobutyric acid in dynamic plant cell responses. Plant Cell Rep, 38, 847-867, 2019.
[108] Shalem, O., Sanjana, N. E., Hartenian, E., Shi, X., Scott, D. A., Mikkelsen, T. S., Heckl, D., Ebert, B. L., Root, D. E., Doench, J. G., & Zhang, F. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science, 343(6166), 84-87, 2014.
[109] Sharma, Y. C., Singh, B., & Korstad, J. A critical review on recent methods used for economically viable and eco-friendly development of microalgae as a potential feedstock for synthesis of biodiesel. Green Chem, 13(11), 2993-3006, 2011.
[110] Shelp, B. J., Aghdam, M. S., & Flaherty, E. J. γ-Aminobutyrate (GABA) regulated plant defense: Mechanisms and opportunities. Plants, 10(9), 1939, 2021.
[111] Shetty, P., Gitau, M. M., & Maróti, G. Salinity stress responses and adaptation mechanisms in eukaryotic green microalgae. Cells, 8(12), 1657, 2019.
[112] Shimada, M., Hasegawa, T., Nishimura, C., Kan, H., Kanno, T., Nakamura, T., & Matsubayashi, T. Anti-hypertensive effect of γ-aminobutyric acid (GABA)-rich Chlorella on high-normal blood pressure and borderline hypertension in placebo-controlled double blind study. Clin Exp Hypertens, 31(4), 342-354, 2009.
[113] Shin, Y. S., Jeong, J., Nguyen, T. H. T., Kim, J. Y. H., Jin, E., & Sim, S. J. Targeted knockout of phospholipase A2 to increase lipid productivity in Chlamydomonas reinhardtii for biodiesel production. Bioresour Technol, 271, 368-374, 2019.
[114 Solovchenko, A. E. Physiological role of neutral lipid accumulation in eukaryotic microalgae under stresses. Russ J Plant Physiol, 59, 167-176, 2012.
[115] Song, I., Kim, J., Baek, K., Choi, Y., Shin, B., & Jin, E. The generation of metabolic changes for the production of high-purity zeaxanthin mediated by CRISPR-Cas9 in Chlamydomonas reinhardtii. Microb Cell Factories, 19, 1-9, 2020.
[116] Stringham, J. M., Stringham, N. T., & O’Brien, K. J. Macular carotenoid supplementation improves visual performance, sleep quality, and adverse physical symptoms in those with high screen time exposure. Foods, 6(7), 47, 2017.
[117] Subhadra, B., & Edwards, M. An integrated renewable energy park approach for algal biofuel production in United States. Energy Policy, 38(9), 4897-4902, 2010.
[118] Takayama, M., & Ezura, H. How and why does tomato accumulate a large amount of GABA in the fruit? Front Plant Sci, 6, 612, 2015.
[119] Tan, S. I., & Ng, I. S. Stepwise optimization of genetic RuBisCO-equipped Escherichia coli for low carbon-footprint protein and chemical production. Green Chem, 23(13), 4800-4813, 2021.
[120] Teng, C. S., Xue, C., Lin, J. Y., & Ng, I. S. Towards high-level protein, beta-carotene, and lutein production from Chlorella sorokiniana using aminobutyric acid and pseudo seawater. Biochem Eng J, 184, 108473, 2022.
[121] The Arabidopsis Genome Initiative. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature, 408(6814), 796-815, 2000.
[122] Tsai, T. H., Lin, J. Y., & Ng, I. S. Cooperation of phytoene synthase, pyridoxal kinase and carbonic anhydrase for enhancing carotenoids biosynthesis in genetic Chlamydomonas reinhardtii. J Taiwan Inst Chem Eng, 137, 104184, 2022.
[123] Vadrale, A. P., Dong, C. D., Haldar, D., Wu, C. H., Chen, C. W., Singhania, R. R., & Patel, A. K. Bioprocess development to enhance biomass and lutein production from Chlorella sorokiniana Kh12. Bioresour Technol, 128583, 2023.
[124] Valletta, S., Dolatshad, H., Bartenstein, M., Yip, B. H., Bello, E., Gordon, S., Yu, Y., Shaw, J., Roy, S., Scifo, L., Schuh, A., Pellagatti, A., Fulga, T. A., Verma, A., & Boultwood, J. ASXL1 mutation correction by CRISPR/Cas9 restores gene function in leukemia cells and increases survival in mouse xenografts. Oncotarget, 6(42), 44061, 2015.
[125] Vincill, E. D., Bieck, A. M., & Spalding, E. P. Ca2+ conduction by an amino acid-gated ion channel related to glutamate receptors. Plant Physiol, 159(1), 40-46, 2012.
[126] Wang, H., Guo, Y., Wang, C., Jiang, X., Liu, H., Yuan, A., Yan, J., Hu, Y., & Wu, J. Light-controlled oxygen production and collection for sustainable photodynamic therapy in tumor hypoxia. Biomater, 269, 120621, 2021.
[127] Wei, D., Zhang, W., Wang, C., Meng, Q., Li, G., Chen, T. H., & Yang, X. Genetic engineering of the biosynthesis of glycinebetaine leads to alleviate salt-induced potassium efflux and enhances salt tolerance in tomato plants. Plant Sci, 257, 74-83, 2017.
[128] Wu, M., Zhang, H., Sun, W., Li, Y., Hu, Q., Zhou, H., & Han, D. Metabolic plasticity of the starchless mutant of Chlorella sorokiniana and mechanisms underlying its enhanced lipid production revealed by comparative metabolomics analysis. Algal Res, 42, 101587, 2019.
[129] Xue, C., & Ng, I. S. Sustainable production of 4-aminobutyric acid (GABA) and cultivation of Chlorella sorokiniana and Chlorella vulgaris as circular economy. Bioresour Technol, 343, 126089, 2022.
[130] Yadavalli, R., Ratnapuram, H., Peasari, J. R., Reddy, C. N., Ashokkumar, V., & Kuppam, C. Simultaneous production of astaxanthin and lipids from Chlorella sorokiniana in the presence of reactive oxygen species: a biorefinery approach. Biomass Convers Biorefin, 1-9, 2021.
[131] Yamatsu, A. The Beneficial Effects of Coffee on Stress and Fatigue can be Enhanced by the Addition of GABA―A Randomized, Double‒blind, Placebo Controlled, Crossover‒designed Study―. 薬理と治療, 43(4), 515-519, 2015.
[132] Yamatsu, A., Yamashita, Y., Pandharipande, T., Maru, I., & Kim, M. Effect of oral γ-aminobutyric acid (GABA) administration on sleep and its absorption in humans. Food Sci Biotechnol, 25(2), 547-551, 2016.
[133] Yi, Y. C., & Ng, I. S. Redirection of metabolic flux in Shewanella oneidensis MR-1 by CRISPRi and modular design for 5-aminolevulinic acid production. Bioresour Bioprocess, 8(1), 1-11, 2021.
[134] Zhang, L., Liao, C., Yang, Y., Wang, Y. Z., Ding, K., Huo, D., & Hou, C. Response of lipid biosynthesis in Chlorella pyrenoidosa to intracellular reactive oxygen species level under stress conditions. Bioresour Technol, 287, 121414, 2019.
[135] Zhao, Y., Li, Q., Gu, D., Yu, L., & Yu, X. The synergistic effects of gamma-aminobutyric acid and salinity during the enhancement of microalgal lipid production in photobioreactors. Energy Convers Manag, 267, 115928, 2022.
[136] Zhao, Y., Song, X., Zhong, D. B., Yu, L., & Yu, X. γ-Aminobutyric acid (GABA) regulates lipid production and cadmium uptake by Monoraphidium sp. QLY-1 under cadmium stress. Bioresour Technol, 297, 122500, 2020.
[137] Zheng, Y., Li, T., Yu, X., Bates, P. D., Dong, T., & Chen, S. High-density fed-batch culture of a thermotolerant microalga Chlorella sorokiniana for biofuel production. Appl Energy, 108, 281-287, 2013.
[138] Zhu, L., Peng, Q., Song, F., Jiang, Y., Sun, C., Zhang, J., & Huang, D. Structure and regulation of the gab gene cluster, involved in the γ-aminobutyric acid shunt, are controlled by a σ54 factor in Bacillus thuringiensis. J Bacteriol Res, 192(1), 346-355, 2010.