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Microalgae are widely recognized as a promising bioresource for producing renewable fuels and chemicals. Microalgal biorefinery has tremendous potential for incorporation into circular bioeconomy, including sustainability, cascading use, and waste reduction. In this study, genetic engineering was used to enhance the growth, lipid and lutein productivity of Chlamydomonas reinhardtii including strains of CC400, PY9, pCHS, and PG. Notably, CRISPRi mediated on phosphoenolpyruvate carboxylase (PEPC1) gene to down-regulate the branch pathway from glycolysis to partitioning more carbon flux to lipid was explored under meso-thermophilic condition. The best chassis PGi, which has overexpressed chaperone GroELS and applied CRISPRi resulting in the highest biomass of 2.56 g/L and also boosted the lipids and lutein with 893 and 23.5 mg/L, respectively at 35°C. Finally, all strains with CRISPRi exhibited higher transcriptional levels of the crucial genes from photosynthesis, starch, lipid and lutein metabolism, thus reaching a CO 2 assimilation of 1.087 g-CO 2 /g-DCW in mixotrophic condition., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors are grateful for the financial support received from the Ministry of Science and Technology (MOST 110-2221-E-006-030-MY3 and NSTC 111-2221-E-006-012-MY3) in Taiwan., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Carbon dioxide is the major greenhouse gas and regards as the critical issue of global warming and climate changes. The inconspicuous microalgae are responsible for 40% of carbon fixation among all photosynthetic plants along with a higher photosynthetic efficiency and convert the carbon into lipids, protein, pigments, and bioactive compounds. Genetic approach and metabolic engineering are applied to accelerate the growth rate and biomass of microalgae, hence achieve the mission of carbon neutrality. Meanwhile, CRISPR/Cas9 is efficiently to enhance the productivity of high-value compounds in microalgae for it is easier operation, more affordable and is able to regulate multiple genes simultaneously. The genetic engineering strategies provide the multidisciplinary concept to evolute and increase the CO 2 fixation rate through Calvin-Benson-Bassham cycle. Therefore, the technologies, bioinformatics tools, systematic engineering approaches for carbon neutrality and circular economy are summarized and leading one step closer to the decarbonization society in this review., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors are grateful for the financial support received from the Ministry of Science and Technology to I-Son Ng (MOST 111-2221-E-006-012-MY3 and MOST 110-2221-E-006-030-MY3) and to Tse-Min Lee (MOST 110-2311-B-110 -003, MOST 111-2311-B-110-002, MOST 111-2923-B-110 -001-MY2, and MOST 110-2622-E-110-016) in Taiwan., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Chlorella species are indispensable microalgae for biorefinery but are hardly in DNA manipulation due to the high guanine-cytosine (GC) contents of DNA. In this study, we established a new approach via 20 guanines for sgRNA design, which is annotated as "Adaptive Single Guide Assisted Regulation DNA (ASGARD)" and coupling with CRISPR interference associated dCas9 system to overcome the difficulties. At first, C. sorokiniana was predominate as its faster growth rate when compared to C. vulgaris and C. variabilis in the culture using Tris-acetate-phosphate (TAP) medium. Among all the genetic transformants, gene regulation via CRISPRa-VP64 (CRISPRa) enhanced the protein contents up to 60% (w/w) of dry cell weight, where the highest concentration was 570 mg L -1 . Meanwhile, CRISPRi-KRAB (CRISPRi) with ASGARD increased protein content to 65% and lipid formed in the range of 150-250 mg L -1 . From the transcriptome analysis, we deciphered 468 genes down-regulated and 313 genes up-regulated via CRISPRi, while less difference existed in CRISPRa. This novel design and technology reveal a high potential of gene-regulating approach to other species for the biorefinery and bio-industry., (© 2021 Wiley-VCH GmbH.)

[Display omitted] • The opportunity of heterologous carbonic anhydrase in CC-400 was evaluated. • This is the first attempt to cultivate CC-400 strain under autotrophic condition. • The carbon capture was achieved to 2.75 g-CO 2 /g-DCW in the optimal condition. • The biomass, lutein, and lipid were increased 2-, 4- and 8-folds in modified CC-400. • Transcriptional levels of major genes in the pathway decipher CA effect in CC-400. Chlamydomonas reinhardtii is a model microalga that has a higher growth rate and produces high levels of lutein and lipids, but biomass production is limited. Carbonic anhydrase (CA) converts atmospheric CO 2 to bicarbonate which is crucial for carbon‐concentrating mechanism (CCM) in microalgae and boosts cell density. Therefore, C. reinhardtii harboring the heterologous CA from Mesorhizobium loti (MlCA) and Sulfurihydrogenibium yellowstonense (SyCA) were explored to increase CO 2 capture and utilization (CCU) through different culture devices. Genetically modified C. reinhardtii was able to grow from mixotrophic to autotrophic conditions. Subsequently, biomass, lutein, and lipid were maximized to OD 680 of 4.56, 21.32 mg/L and 672 mg/L using photo-bioreactor (PBR) with 5% CO 2. Moreover, CO 2 assimilation rate was 2.748 g-CO 2 /g-DCW and 2.792 g-CO 2 /g-DCW under mixotrophic and autotrophic conditions, respectively. The biomass accumulation correlated with CA activity. In addition, the transcript levels of major genes in metabolic pathways of lutein and lipid were dramatically increased. [ABSTRACT FROM AUTHOR]