具備光合作用能力以及高值化產品合成能力，微藻被視為極佳的綠色製造原料。在本研究當中採用基因表達與調控兩種策略促進類胡蘿蔔素之生產。本研究首先過表達類胡蘿蔔素代謝途徑中重要的八氫番茄紅素合成酶(psy)、輔助生長的吡哆醛激酶(pdxY)及碳酸酐酶(CA)，整合於萊茵衣藻促進類胡蘿蔔素與生物量；因碳酸酐酶具有促進碳酸根與二氧化碳轉化之能力，研究進一步採用雙層培養裝置具有較高二氧化碳，使碳酸酐酶發揮作用。結果含Sulfurihydrogenibium yellowstonense碳酸酐酶的藻株在雙層裝置培養下有9.2毫克/升的葉黃素以及19.83毫克/升的beta胡蘿蔔素產出。另一方面，來自Mesorhizobium loti碳酸酐酶的藻株儘管在生物量具有突破，卻較不穩定。過去因萊茵衣藻之生物量較低，在三基因共表達的作用下，不只增加類胡蘿蔔素產量，亦提昇了生物量。
代謝的調控提供了重新導流到所需產物或改變特定機制的機會。在本研究的第二部分，採用CRISPR干擾法下調自噬蛋白和茄紅素epsilon環化酶(LCYe) 的基因表達，均可提高類胡蘿蔔素的產量。使用錐形瓶培養時，LCYe 和 ATG8 抑制藻株(iATG8)的 beta胡蘿蔔素濃度分別提高到 9.6 毫克/升 和 12.9 毫克/升。接著改變培養基、裝置、藻種等方法均可增加生物量，然而在雙層裝置中培養 iATG8 藻株卻導致beta胡蘿蔔素減少，這表明 iATG8 難以在 CC-400 進行放大生產。另外在小球藻Chlorella sorokiniana以CRISPRi下調ATG8對類胡蘿蔔素的生產改善有限，但在雙層裝置中培養LCYe抑制之藻株其生成β-胡蘿蔔素之性能優於ATG8抑制之藻株，在GABA或5-ALA添加條件下分別有15.83毫克/升及20.38毫克/升的產出，而ATG8抑制之藻株則為11.56毫克/升及10.92毫克/升。
本研究中採用了基因工程及CRISPRi技術，以培養條件和裝置來解決生物量及類胡蘿蔔生產的瓶頸，結果表明由適當的設計，基改過後的藻株之細胞密度以及類胡蘿蔔素產量皆得到了提升。

Microalgae are a group of photosynthesis microorganisms with great potential as green hosts for the synthesis of valuable chemicals. Herein improvement on production of organic pigments carotenoid was achieved from both gene overexpression and regulation of metabolism. For overexpression of genes, the crucial gene in carotenoid synthesis pathway psy was cooperated with pdxY and CA gene for enhancing carotenoid content and biomass accumulation, resulting in increment in both biomass and carotenoid titer. Under CO2 supply condition in two-layer photoreactor, engineered strains also exhibited higher cell density compared to that of wild type, thus being a strain superior in carotenoids production and CO2 assimilation with CA gene catalyzing interconversion of bicarbonate and carbon dioxide. Strain SPPY, in which SyCA was integrated, gave 9.2 mg/L of lutein and 19.83 mg/L of beta-carotene titer in two-layer photoreactor cultivation. On the other hand, integration of MlCA established a strain with high cell density under CO2 supply but carotenoid production in this strain is unstable. This result implied that MlCA in CC-400 directs CO2 into the cells but also bring in stress. Genetic engineering on microalgae contributes to improve biochemical synthesis. As the biomass of C. reinhardtii is relatively low, application of this model organism is limited. Thus, we also seek to enhance cell growth with genes related to biomass accumulation and carbon uptake ability.
Regulation of metabolism by CRISPR interference offers the opportunity of redirecting to the desired products or changes on certain mechanisms. In the second part of this study, expression of autophagy protein and LCYe were individually downregulated by CRISPRi aiming to enhance carotenoid production. In flask cultivation, beta-carotene titer was improved to 9.6 mg/L and 12.9 mg/L, respectively for LCYe and ATG8 knockdown strain. The potential of increasing cell density in culture was investigate in several approaches including medium, device, and host. However, cultivation in two-layer photoreactor for iATG8 strain caused a decrease on beta-carotene, indicating that it might be difficult for iATG8 to scale-up in CC-400 and extending ATG8-downregulation to Chlorella sorokiniana has minor effect on carotenoid production. On the other hand, downregulation of LCYe was applied in two-layer photoreactor and the performance was better than that for iATG8. In condition where GABA or 5-ALA was included in medium, iLCYe strain has 15.83 or 20.38 mg/L of beta-carotene titer whereas iATG8 has 11.56 or 10.92 mg/L of beta-carotene titer. Finally, knockdown of LCYe was adopted in engineered strains to improve its performance, establishing strains with higher cell density and carotenoid titer in microalgae.
Despite the achievement in the previous study for enhancing carotenoid content in C. reinhardtii, production was limited due to low biomass concentration. In this study, genetic engineering, culture condition, and instrument were considered to deal with this issue, and the results obtained indicated that with appropriate design, cell density and carotenoid production could be improved in the genetic engineered strains.

[1] Ahmad, I., A. K. Sharma, H. Daniell and S. Kumar "Altered lipid composition and enhanced lipid production in green microalga by introduction of brassica diacylglycerol acyltransferase 2." Plant Biotechnol J 13(4): 540-550. (2015).145
[2] Arias, R. S., F. E. Dayan, A. Michel, J. L. Howell and B. E. Scheffler "Characterization of a higher plant herbicide-resistant phytoene desaturase and its use as a selectable marker." Plant Biotechnology Journal 4(2): 263-273. (2006).130
[3] Atikij, T., Y. Syaputri, H. Iwahashi, T. Praneenararat, S. Sirisattha, H. Kageyama and R. Waditee-Sirisattha "Enhanced Lipid Production and Molecular Dynamics under Salinity Stress in Green Microalga Chlamydomonas reinhardtii (137C)." Mar Drugs 17(8). (2019).105
[4] Baier, T., N. Jacobebbinghaus, A. Einhaus, K. J. Lauersen and O. Kruse "Introns mediate post-transcriptional enhancement of nuclear gene expression in the green microalga Chlamydomonas reinhardtii." PLoS Genet 16(7): e1008944. (2020).134
[5] Barbosa, J. M., N. K. Singh, J. H. Cherry and R. D. Locy "Nitrate uptake and utilization is modulated by exogenous γ-aminobutyric acid in Arabidopsis thaliana seedlings." Plant Physiology and Biochemistry 48(6): 443-450. (2010).156
[6] Barrangou, R., C. Fremaux, H. Deveau, M. Richards, P. Boyaval, S. Moineau, D. A. Romero and P. Horvath "CRISPR provides acquired resistance against viruses in prokaryotes." Science 315(5819): 1709-1712. (2007).93
[7] Barsanti, L. and P. Gualtieri "Is exploitation of microalgae economically and energetically sustainable?" Algal Research 31: 107-115. (2018).107
[8] Becker, E. W. "Micro-algae as a source of protein." Biotechnol Adv 25(2): 207-210. (2007).120
[9] Bogaert, K. A., E. Perez, J. Rumin, A. Giltay, M. Carone, N. Coosemans, M. Radoux, G. Eppe, R. D. Levine, F. Remacle and C. Remacle "Metabolic, Physiological, and Transcriptomics Analysis of Batch Cultures of the Green Microalga Chlamydomonas Grown on Different Acetate Concentrations." Cells 8(11). (2019).3
[10] Brouns, S. J., M. M. Jore, M. Lundgren, E. R. Westra, R. J. Slijkhuis, A. P. Snijders, M. J. Dickman, K. S. Makarova, E. V. Koonin and J. van der Oost "Small CRISPR RNAs guide antiviral defense in prokaryotes." Science 321(5891): 960-964. (2008).94
[11] Cecchin, M., S. Benfatto, F. Griggio, A. Mori, S. Cazzaniga, N. Vitulo, M. Delledonne and M. Ballottari "Molecular basis of autotrophic vs mixotrophic growth in Chlorella sorokiniana." Scientific Reports 8(1): 6465. (2018).166
[12] Chen, C. Y., A. L. Kao, Z. C. Tsai, Y. M. Shen, P. H. Kao, I. S. Ng and J. S. Chang "Expression of Synthetic Phytoene Synthase Gene to Enhance beta-Carotene Production in Scenedesmus sp. CPC2." Biotechnol J 12(11). (2017).18
[13] Cordero, B. F., I. Couso, R. León, H. Rodríguez and M. A. Vargas "Enhancement of carotenoids biosynthesis in Chlamydomonas reinhardtii by nuclear transformation using a phytoene synthase gene isolated from Chlorella zofingiensis." Applied microbiology and biotechnology 91(2): 341-351. (2011).140
[14] Cordero, B. F., I. Obraztsova, I. Couso, R. Leon, M. A. Vargas and H. Rodriguez "Enhancement of lutein production in Chlorella sorokiniana (Chorophyta) by improvement of culture conditions and random mutagenesis." Mar Drugs 9(9): 1607-1624. (2011).84
[15] Couso, I., M. Vila, H. Rodriguez, M. A. Vargas and R. León "Overexpression of an exogenous phytoene synthase gene in the unicellular alga Chlamydomonas reinhardtii leads to an increase in the content of carotenoids." Biotechnology Progress 27(1): 54-60. (2011).141
[16] Deltcheva, E., K. Chylinski, C. M. Sharma, K. Gonzales, Y. Chao, Z. A. Pirzada, M. R. Eckert, J. Vogel and E. Charpentier "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Nature 471(7340): 602-607. (2011).98
[17] Deng, X., J. Cai, Y. Li and X. Fei "Expression and knockdown of the PEPC1 gene affect carbon flux in the biosynthesis of triacylglycerols by the green alga Chlamydomonas reinhardtii." Biotechnol Lett 36(11): 2199-2208. (2014).104
[18] Diaz-Santos, E., M. de la Vega, M. Vila, J. Vigara and R. Leon "Efficiency of different heterologous promoters in the unicellular microalga Chlamydomonas reinhardtii." Biotechnol Prog 29(2): 319-328. (2013).122
[19] Doron, L., N. Segal and M. Shapira "Transgene Expression in Microalgae-From Tools to Applications." Front Plant Sci 7: 505. (2016).100
[20] Effendi, S. S. W. and I. S. Ng "The prospective and potential of carbonic anhydrase for carbon dioxide sequestration: A critical review." Process Biochemistry 87: 55-65. (2019).132
[21] Eichler-Stahlberg, A., W. Weisheit, O. Ruecker and M. Heitzer "Strategies to facilitate transgene expression in Chlamydomonas reinhardtii." Planta 229(4): 873-883. (2009).133
[22] Galarza, J. I., J. A. Gimpel, V. Rojas, B. O. Arredondo-Vega and V. Henríquez "Over-accumulation of astaxanthin in Haematococcus pluvialis through chloroplast genetic engineering." Algal Research 31: 291-297. (2018).76
[23] Gordon, G. C., T. C. Korosh, J. C. Cameron, A. L. Markley, M. B. Begemann and B. F. Pfleger "CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002." Metabolic Engineering 38: 170-179. (2016).151
[24] Gutierrez, S. and K. J. Lauersen "Gene Delivery Technologies with Applications in Microalgal Genetic Engineering." Biology (Basel) 10(4). (2021).103
[25] Hopes, A., V. Nekrasov, S. Kamoun and T. Mock "Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana." Plant Methods 12(1): 49. (2016).136
[26] Hu, L., S. Feng, G. Liang, J. Du, A. Li and C. Niu "CRISPR/Cas9-induced beta-carotene hydroxylase mutation in Dunaliella salina CCAP19/18." AMB Express 11(1): 83. (2021).78
[27] Jain, S., S. Shukla, C. Yang, M. Zhang, Z. Fatma, M. Lingamaneni, S. Abesteh, S. T. Lane, X. Xiong, Y. Wang, C. M. Schroeder, P. R. Selvin and H. Zhao "TALEN outperforms Cas9 in editing heterochromatin target sites." Nature Communications 12(1): 606. (2021).101
[28] Jeon, S., H. G. Koh, J. M. Cho, N. K. Kang and Y. K. Chang "Enhancement of lipid production in Nannochloropsis salina by overexpression of endogenous NADP-dependent malic enzyme." Algal Research 54. (2021).73
[29] Jiang, W., A. J. Brueggeman, K. M. Horken, T. M. Plucinak and D. P. Weeks "Successful transient expression of Cas9 and single guide RNA genes in Chlamydomonas reinhardtii." Eukaryot Cell 13(11): 1465-1469. (2014).87
[30] Jinek, M., K. Chylinski, I. Fonfara, M. Hauer, J. A. Doudna and E. Charpentier "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Science 337(6096): 816-821. (2012).97
[31] Judé, S., S. Roger, E. Martel, P. Besson, S. Richard, P. Bougnoux, P. Champeroux and J.-Y. Le Guennec "Dietary long-chain omega-3 fatty acids of marine origin: A comparison of their protective effects on coronary heart disease and breast cancers." Progress in Biophysics and Molecular Biology 90(1): 299-325. (2006).114
[32] Kadono, T., N. Kira, K. Suzuki, O. Iwata, T. Ohama, S. Okada, T. Nishimura, M. Akakabe, M. Tsuda and M. Adachi "Effect of an Introduced Phytoene Synthase Gene Expression on Carotenoid Biosynthesis in the Marine Diatom Phaeodactylum tricornutum." Mar Drugs 13(8): 5334-5357. (2015).111
[33] Kao, P. H. and I. S. Ng "CRISPRi mediated phosphoenolpyruvate carboxylase regulation to enhance the production of lipid in Chlamydomonas reinhardtii." Bioresour Technol 245(Pt B): 1527-1537. (2017).13
[34] Kathiresan, S., A. Chandrashekar, G. A. Ravishankar and R. Sarada "Regulation of astaxanthin and its intermediates through cloning and genetic transformation of β-carotene ketolase in Haematococcus pluvialis." Journal of Biotechnology 196-197: 33-41. (2015).143
[35] Kim, J., K. S. Chang, S. Lee and E. Jin "Establishment of a Genome Editing Tool Using CRISPR-Cas9 in Chlorella vulgaris UTEX395." Int J Mol Sci 22(2). (2021).154
[36] Kim, J., S. Lee, K. Baek and E. Jin "Site-Specific Gene Knock-Out and On-Site Heterologous Gene Overexpression in Chlamydomonas reinhardtii via a CRISPR-Cas9-Mediated Knock-in Method." Front Plant Sci 11: 306. (2020).69
[37] Kindle, K. L. "High-frequency nuclear transformation of Chlamydomonas reinhardtii." Proc Natl Acad Sci U S A 87(3): 1228-1232. (1990).81
[38] Latowski, D., P. Kuczyńska and K. Strzałka "Xanthophyll cycle--a mechanism protecting plants against oxidative stress." Redox Rep 16(2): 78-90. (2011).109
[39] Lee, J.-W., M.-W. Lee, J.-S. Ha, D.-S. Kim, E. Jin, H.-G. Lee and H.-M. Oh "Development of a species-specific transformation system using the novel endogenous promoter calreticulin from oleaginous microalgae Ettlia sp." Scientific Reports 10(1): 13947. (2020).126
[40] Li, L., Z. Chen and Q. Huang "Exogenous γ-aminobutyric acid promotes biomass and astaxanthin production in Haematococcus pluvialis." Algal Research 52. (2020).15
[41] Li, Y., M. Horsman, B. Wang, N. Wu and C. Q. Lan "Effects of nitrogen sources on cell growth and lipid accumulation of green alga Neochloris oleoabundans." Appl Microbiol Biotechnol 81(4): 629-636. (2008).106
[42] Li, Z., T. Meng, X. Ling, J. Li, C. Zheng, Y. Shi, Z. Chen, Z. Li, Q. Li, Y. Lu and N. He "Overexpression of Malonyl-CoA: ACP Transacylase in Schizochytrium sp. to Improve Polyunsaturated Fatty Acid Production." J Agric Food Chem 66(21): 5382-5391. (2018).74
[43] Lin, J.-Y., W.-R. Lin and I.-S. Ng "CRISPRa/i with Adaptive Single Guide Assisted Regulation DNA (ASGARD) mediated control of Chlorella sorokiniana to enhance lipid and protein production." Biotechnology Journal n/a(n/a): 2100514. (2021).64
[44] Lin, J. Y., S. Sri Wahyu Effendi and I. S. Ng "Enhanced carbon capture and utilization (CCU) using heterologous carbonic anhydrase in Chlamydomonas reinhardtii for lutein and lipid production." Bioresour Technol 351: 127009. (2022).79
[45] Lin, J. Y., S. I. Tan, Y. C. Yi, C. C. Hsiang, C. H. Chang, C. Y. Chen, J. S. Chang and I. S. Ng "High-level production and extraction of C-phycocyanin from cyanobacteria Synechococcus sp. PCC7002 for antioxidation, antibacterial and lead adsorption." Environ Res 206: 112283. (2022).113
[46] Lin, J. Y., C. Xue, S. I. Tan and I. S. Ng "Pyridoxal kinase PdxY mediated carbon dioxide assimilation to enhance the biomass in Chlamydomonas reinhardtii CC-400." Bioresour Technol 322: 124530. (2021).14
[47] Lin, W.-R., Y.-C. Lai, P.-K. Sung, S.-I. Tan, C.-H. Chang, C.-Y. Chen, J.-S. Chang and I. S. Ng "Enhancing carbon capture and lipid accumulation by genetic carbonic anhydrase in microalgae." Journal of the Taiwan Institute of Chemical Engineers 93: 131-141. (2018).32
[48] Lin, W. R. and I. S. Ng "Development of CRISPR/Cas9 system in Chlorella vulgaris FSP-E to enhance lipid accumulation." Enzyme Microb Technol 133: 109458. (2020).68
[49] Liu, J., H. Gerken, J. Huang and F. Chen "Engineering of an endogenous phytoene desaturase gene as a dominant selectable marker for Chlamydomonas reinhardtii transformation and enhanced biosynthesis of carotenoids." Process Biochemistry 48(5): 788-795. (2013).144
[50] Liu, L., Y. Wang, Y. Zhang, X. Chen, P. Zhang and S. Ma "Development of a New Method for Genetic Transformation of the Green Alga Chlorella ellipsoidea." Molecular Biotechnology 54(2): 211-219. (2013).124
[51] Liu, Q., Y. Huang, R. Zhang, T. Cai and Y. Cai "Medical Application of<i> Spirulina platensis</i> Derived C-Phycocyanin." Evidence-Based Complementary and Alternative Medicine 2016: 7803846. (2016).112
[52] Mares-Perlman, J. A., A. E. Millen, T. L. Ficek and S. E. Hankinson "The body of evidence to support a protective role for lutein and zeaxanthin in delaying chronic disease. Overview." J Nutr 132(3): 518S-524S. (2002).83
[53] Mekonnen, D. W., U. I. Flügge and F. Ludewig "Gamma-aminobutyric acid depletion affects stomata closure and drought tolerance of Arabidopsis thaliana." Plant Sci 245: 25-34. (2016).157
[54] Memon, S. A., X. Hou, L. Wang and Y. Li "Promotive effect of 5-aminolevulinic acid on chlorophyll, antioxidative enzymes and photosynthesis of Pakchoi (Brassica campestris ssp. chinensis var. communis Tsen et Lee)." Acta Physiologiae Plantarum 31(1): 51-57. (2009).163
[55] Mojica, F. J., C. Díez-Villaseñor, J. García-Martínez and E. Soria "Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements." J Mol Evol 60(2): 174-182. (2005).92
[56] Molina-Márquez, A., M. Vila, J. Vigara, A. Borrero and R. León "The Bacterial Phytoene Desaturase-Encoding Gene (CRTI) is an Efficient Selectable Marker for the Genetic Transformation of Eukaryotic Microalgae." Metabolites 9(3). (2019).128
[57] Muñoz, C. F., L. de Jaeger, M. H. J. Sturme, K. Y. F. Lip, J. W. J. Olijslager, J. Springer, E. J. H. Wolbert, D. E. Martens, G. Eggink, R. A. Weusthuis and R. H. Wijffels "Improved DNA/protein delivery in microalgae – A simple and reliable method for the prediction of optimal electroporation settings." Algal Research 33: 448-455. (2018).102
[58] Murillo, A. G., S. Hu and M. L. Fernandez "Zeaxanthin: Metabolism, Properties, and Antioxidant Protection of Eyes, Heart, Liver, and Skin." Antioxidants (Basel) 8(9). (2019).110
[59] Naduthodi, M. I. S., P. Mohanraju, C. Sudfeld, S. D'Adamo, M. J. Barbosa and J. van der Oost "CRISPR-Cas ribonucleoprotein mediated homology-directed repair for efficient targeted genome editing in microalgae Nannochloropsis oceanica IMET1." Biotechnol Biofuels 12: 66. (2019).77
[60] Naeem, M. S., H. Warusawitharana, H. Liu, D. Liu, R. Ahmad, E. A. Waraich, L. Xu and W. Zhou "5-Aminolevulinic acid alleviates the salinity-induced changes in Brassica napus as revealed by the ultrastructural study of chloroplast." Plant Physiology and Biochemistry 57: 84-92. (2012).164
[61] Nguyen, H., H.-S. Kim and S. Jung "Altered tetrapyrrole metabolism and transcriptome during growth-promoting actions in rice plants treated with 5-aminolevulinic acid." Plant Growth Regulation 78(1): 133-144. (2016).165
[62] Niu, Y.-F., M.-H. Zhang, D.-W. Li, W.-D. Yang, J.-S. Liu, W.-B. Bai and H.-Y. Li "Improvement of Neutral Lipid and Polyunsaturated Fatty Acid Biosynthesis by Overexpressing a Type 2 Diacylglycerol Acyltransferase in Marine Diatom Phaeodactylum tricornutum." Marine Drugs 11(11): 4558-4569. (2013).146
[63] Nomura, T., K. Inoue, Y. Uehara-Yamaguchi, K. Yamada, O. Iwata, K. Suzuki and K. Mochida "Highly efficient transgene-free targeted mutagenesis and single-stranded oligodeoxynucleotide-mediated precise knock-in in the industrial microalga Euglena gracilis using Cas9 ribonucleoproteins." Plant Biotechnol J 17(11): 2032-2034. (2019).88
[64] Nymark, M., A. K. Sharma, T. Sparstad, A. M. Bones and P. Winge "A CRISPR/Cas9 system adapted for gene editing in marine algae." Sci Rep 6: 24951. (2016).135
[65] Oey, M., I. L. Ross, E. Stephens, J. Steinbeck, J. Wolf, K. A. Radzun, J. Kugler, A. K. Ringsmuth, O. Kruse and B. Hankamer "RNAi knock-down of LHCBM1, 2 and 3 increases photosynthetic H2 production efficiency of the green alga Chlamydomonas reinhardtii." PLoS One 8(4): e61375. (2013).86
[66] Onodera, J. and Y. Ohsumi "Autophagy is required for maintenance of amino acid levels and protein synthesis under nitrogen starvation." J Biol Chem 280(36): 31582-31586. (2005).82
[67] Parra, M., S. Stahl and H. Hellmann "Vitamin B(6) and Its Role in Cell Metabolism and Physiology." Cells 7(7). (2018).38
[68] Parupudi, P., C. Kethineni, P. B. Dhamole, S. Vemula, P. R. Allu, M. Botlagunta, S. Kokilagadda and S. R. Ronda "CO2 fixation and lipid production by microalgal species." Korean Journal of Chemical Engineering 33(2): 587-593. (2016).108
[69] Perez-Perez, M. E., I. Couso and J. L. Crespo "Carotenoid deficiency triggers autophagy in the model green alga Chlamydomonas reinhardtii." Autophagy 8(3): 376-388. (2012).26
[70] Perozeni, F., S. Cazzaniga, T. Baier, F. Zanoni, G. Zoccatelli, K. J. Lauersen, L. Wobbe and M. Ballottari "Turning a green alga red: engineering astaxanthin biosynthesis by intragenic pseudogene revival in Chlamydomonas reinhardtii." Plant Biotechnol J. (2020).21
[71] Ran, F. A., P. D. Hsu, J. Wright, V. Agarwala, D. A. Scott and F. Zhang "Genome engineering using the CRISPR-Cas9 system." Nature Protocols 8(11): 2281-2308. (2013).99
[72] Rathod, J. P., C. Vira, A. M. Lali and G. Prakash "Metabolic Engineering of Chlamydomonas reinhardtii for Enhanced beta-Carotene and Lutein Production." Appl Biochem Biotechnol 190(4): 1457-1469. (2020).16
[73] Recht, L., N. Töpfer, A. Batushansky, N. Sikron, Y. Gibon, A. Fait, Z. Nikoloski, S. Boussiba and A. Zarka "Metabolite profiling and integrative modeling reveal metabolic constraints for carbon partitioning under nitrogen starvation in the green algae Haematococcus pluvialis." J Biol Chem 289(44): 30387-30403. (2014).160
[74] Ren, Y., H. Sun, J. Deng, J. Huang and F. Chen "Carotenoid Production from Microalgae: Biosynthesis, Salinity Responses and Novel Biotechnologies." Mar Drugs 19(12). (2021).80
[75] Rengel, R., R. T. Smith, R. P. Haslam, O. Sayanova, M. Vila and R. León "Overexpression of acetyl-CoA synthetase (ACS) enhances the biosynthesis of neutral lipids and starch in the green microalga Chlamydomonas reinhardtii." Algal Research 31: 183-193. (2018).75
[76] Ritz, T., A. Damjanović, K. Schulten, J.-P. Zhang and Y. Koyama "Efficient light harvesting through carotenoids." Photosynthesis Research 66(1): 125-144. (2000).47
[77] Schroda, M., C. F. Beck and O. Vallon "Sequence elements within an HSP70 promoter counteract transcriptional transgene silencing in Chlamydomonas." Plant J 31(4): 445-455. (2002).121
[78] Seifikalhor, M., S. Aliniaeifard, F. Bernard, M. Seif, M. Latifi, B. Hassani, F. Didaran, M. Bosacchi, H. Rezadoost and T. Li "γ-Aminobutyric acid confers cadmium tolerance in maize plants by concerted regulation of polyamine metabolism and antioxidant defense systems." Scientific Reports 10(1): 3356. (2020).158
[79] Serif, M., G. Dubois, A. L. Finoux, M. A. Teste, D. Jallet and F. Daboussi "One-step generation of multiple gene knock-outs in the diatom Phaeodactylum tricornutum by DNA-free genome editing." Nat Commun 9(1): 3924. (2018).153
[80] Shin, Y. S., J. Jeong, T. H. T. Nguyen, J. Y. H. Kim, E. Jin and S. J. Sim "Targeted knockout of phospholipase A2 to increase lipid productivity in Chlamydomonas reinhardtii for biodiesel production." Bioresour Technol 271: 368-374. (2019).65
[81] Simon, D. P., N. Anila, K. Gayathri and R. Sarada "Heterologous expression of β-carotene hydroxylase in Dunaliella salina by Agrobacterium -mediated genetic transformation." Algal Research 18: 257-265. (2016).72
[82] Song, I., J. Kim, K. Baek, Y. Choi, B. Shin and E. Jin "The generation of metabolic changes for the production of high-purity zeaxanthin mediated by CRISPR-Cas9 in Chlamydomonas reinhardtii." Microb Cell Fact 19(1): 220. (2020).12
[83] Song, I., S. Kim, J. Kim, H. Oh, J. Jang, S. J. Jeong, K. Baek, W. S. Shin, S. J. Sim and E. Jin "Macular pigment-enriched oil production from genome-edited microalgae." Microb Cell Fact 21(1): 27. (2022).150
[84] Sproles, A. E., F. J. Fields, T. N. Smalley, C. H. Le, A. Badary and S. P. Mayfield "Recent advancements in the genetic engineering of microalgae." Algal Research 53: 102158. (2021).123
[85] Steinbrenner, J. and G. Sandmann "Transformation of the green alga Haematococcus pluvialis with a phytoene desaturase for accelerated astaxanthin biosynthesis." Appl Environ Microbiol 72(12): 7477-7484. (2006).142
[86] Sun, Z., Y. F. Chen and J. Du "Elevated CO2 improves lipid accumulation by increasing carbon metabolism in Chlorella sorokiniana." Plant Biotechnol J 14(2): 557-566. (2016).46
[87] Suttangkakul, A., A. Sirikhachornkit, P. Juntawong, W. Puangtame, T. Chomtong, S. Srifa, S. Sathitnaitham, W. Dumrongthawatchai, K. Jariyachawalid and S. Vuttipongchaikij "Evaluation of strategies for improving the transgene expression in an oleaginous microalga Scenedesmus acutus." BMC Biotechnology 19(1): 4. (2019).127
[88] Takahashi, K., Y. Ide, J. Hayakawa, Y. Yoshimitsu, I. Fukuhara, J. Abe, Y. Kasai and S. Harayama "Lipid productivity in TALEN-induced starchless mutants of the unicellular green alga Coccomyxa sp. strain Obi." Algal Research 32: 300-307. (2018).89
[89] Tan, S. I. and I. S. Ng "Design and optimization of bioreactor to boost carbon dioxide assimilation in RuBisCo-equipped Escherichia coli." Bioresour Technol 314: 123785. (2020).131
[90] Tokunaga, S., D. Morimoto, T. Koyama, Y. Kubo, M. Shiroi, K. Ohara, T. Higashine, Y. Mori, S. Nakagawa and S. Sawayama "Enhanced Lutein Production in Chlamydomonas reinhardtii by Overexpression of the Lycopene Epsilon Cyclase Gene." Appl Biochem Biotechnol 193(6): 1967-1978. (2021).5
[91] Tran, Q. G., K. Cho, U. Kim, J. H. Yun, D. H. Cho, J. Heo, S. B. Park, J. W. Kim, Y. J. Lee, R. Ramanan and H. S. Kim "Enhancement of beta-carotene production by regulating the autophagy-carotenoid biosynthesis seesaw in Chlamydomonas reinhardtii." Bioresour Technol 292: 121937. (2019).1
[92] Tzfira, T. and V. Citovsky "Agrobacterium-mediated genetic transformation of plants: biology and biotechnology." Current Opinion in Biotechnology 17(2): 147-154. (2006).115
[93] Ungerer, J. and H. B. Pakrasi "Cpf1 Is A Versatile Tool for CRISPR Genome Editing Across Diverse Species of Cyanobacteria." Scientific Reports 6(1): 39681. (2016).137
[94] Villion, M. and S. Moineau "The double-edged sword of CRISPR-Cas systems." Cell Research 23(1): 15-17. (2013).96
[95] Waghmare, A. G., M. K. Salve, J. G. LeBlanc and S. S. Arya "Concentration and characterization of microalgae proteins from Chlorella pyrenoidosa." Bioresources and Bioprocessing 3(1): 16. (2016).117
[96] Wang, C., Y. Li, J. Lu, X. Deng, H. Li and Z. Hu "Effect of overexpression of LPAAT and GPD1 on lipid synthesis and composition in green microalga Chlamydomonas reinhardtii." J Appl Phycol 30(3): 1711-1719. (2018).70
[97] Wang, Q., Y. Lu, Y. Xin, L. Wei, S. Huang and J. Xu "Genome editing of model oleaginous microalgae Nannochloropsis spp. by CRISPR/Cas9." The Plant Journal 88(6): 1071-1081. (2016).148
[98] Wei, L., Z. Jiang and B. Liu "A CRISPR/dCas9-based transcription activated system developed in marine microalga Nannochloropsis oceanica." Aquaculture 546: 737064. (2022).149
[99] Wendt, K. E., J. Ungerer, R. E. Cobb, H. Zhao and H. B. Pakrasi "CRISPR/Cas9 mediated targeted mutagenesis of the fast growing cyanobacterium Synechococcus elongatus UTEX 2973." Microbial Cell Factories 15(1): 115. (2016).138
[100] Wu, Y., X. Jin, W. Liao, L. Hu, M. M. Dawuda, X. Zhao, Z. Tang, T. Gong and J. Yu "5-Aminolevulinic Acid (ALA) Alleviated Salinity Stress in Cucumber Seedlings by Enhancing Chlorophyll Synthesis Pathway." Front Plant Sci 9: 635. (2018).50
[101] Xue, C. and I. S. Ng "Sustainable production of 4-aminobutyric acid (GABA) and cultivation of Chlorella sorokiniana and Chlorella vulgaris as circular economy." Bioresource Technology 343: 126089. (2022).162
[102] Xue, C., T.-H. Yu and I. S. Ng "Engineering pyridoxal kinase PdxY-integrated Escherichia coli strain and optimization for high-level 5-aminolevulinic acid production." Journal of the Taiwan Institute of Chemical Engineers 120: 49-58. (2021).33
[103] Yang, J., Y. Pan, C. Bowler, L. Zhang and H. Hu "Knockdown of phosphoenolpyruvate carboxykinase increases carbon flux to lipid synthesis in Phaeodactylum tricornutum." Algal Research 15: 50-58. (2016).147
[104] Yang, L., J. Chen, S. Qin, M. Zeng, Y. Jiang, L. Hu, P. Xiao, W. Hao, Z. Hu, A. Lei and J. Wang "Growth and lipid accumulation by different nutrients in the microalga Chlamydomonas reinhardtii." Biotechnol Biofuels 11: 40. (2018).34
[105] Yao, L., I. Cengic, J. Anfelt and E. P. Hudson "Multiple Gene Repression in Cyanobacteria Using CRISPRi." ACS Synthetic Biology 5(3): 207-212. (2016).152
[106] You, S. K., Y. J. Ko, S. K. Shin, D. H. Hwang, D. H. Kang, H. M. Park and S. O. Han "Enhanced CO2 fixation and lipid production of Chlorella vulgaris through the carbonic anhydrase complex." Bioresour Technol 318: 124072. (2020).49
[107] Yu, T.-H., S.-I. Tan, Y.-C. Yi, C. Xue, W.-W. Ting, J.-J. Chang and I. S. Ng "New insight into the codon usage and medium optimization toward stable and high-level 5-aminolevulinic acid production in Escherichia coli." Biochemical Engineering Journal 177. (2022).35
[108] Zhao, Y., X. Song, D. B. Zhong, L. Yu and X. Yu "γ-Aminobutyric acid (GABA) regulates lipid production and cadmium uptake by Monoraphidium sp. QLY-1 under cadmium stress." Bioresour Technol 297: 122500. (2020).159
[109] Zhu, Z., H. Cao, X. Li, J. Rong, X. Cao and J. Tian "A Carbon Fixation Enhanced Chlamydomonas reinhardtii Strain for Achieving the Double-Win Between Growth and Biofuel Production Under Non-stressed Conditions." Front Bioeng Biotechnol 8: 603513. (2020).48