Abstract
Carbon acquisition and utilization are fundamental processes required for the growth and metabolism of algae, eukaryotic organisms that play a crucial role in global carbon cycling and contribute to the primary productivity of aquatic ecosystems. Algal cells rely on carbonic anhydrases (CAs) to facilitate the acquisition of inorganic carbon (Ci) and its conversion into bicarbonate ions (HCO3−) for various cellular processes. Understanding the molecular mechanisms that underlie Ci regulation is vital for unraveling the adaptation of algae to different CO2 conditions and enhancing the production of algal biomass. In this study, we characterized mutant algae showing robust growth under low CO2 conditions but impaired growth in high CO2 environments. Our investigation revealed a significant reduction in intracellular Ci content in the mutant, suggesting a disruption in Ci regulatory mechanisms. Through molecular analysis, we identified the pKIWI502 gene homolog as one of the mutated loci, which played a critical role in Ci regulation and was responsible for the mutant phenotype. Introduction of the intact pKIWI502 gene into mutant algae resulted in the recovery of high-CO2 sensitivity and the restoration of intracellular Ci content. These findings highlight the possible role of the pKIWI502 homolog in pH homeostasis and Ci regulation, and provide insights into the molecular basis of the mutant phenotype. Furthermore, our study improves the understanding of algal physiology and offers potential strategies for enhancing algal biomass production and carbon capture technologies.
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Data availability
The data that support the findings of this study are available from the corresponding author (W-J.Jeong), upon reasonable request.
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Acknowledgements
This work was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIT) (NBC7322213), and by the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Programs (KGM5252322 and KGM1002311).
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11816_2023_866_MOESM1_ESM.jpg
Supplementary file1 Supplementary Fig. 1 Generation of pKIWI502 KO lines. a Nucleotide sequence of the pKIWI502 homolog in N. salina. Red letters in bold indicate the sequences of two target sites (Target 1 and Target 2) and that of the palindromic adjacent motif (PAM) used for editing the pKIWI502 homolog by CRISPR/Cas9. b Schematic diagram of the pNsCas9-sgRNA construct. c Growth performance of pNsCas9-sgRNA construct transformed lines in f/2N agar medium under high and low CO2 conditions. d Southern blot of pKIWI502 KO lines. e Northern blot of pKIWI502 KO lines. Numbers in red represent the KO lines used in this study. f PCR amplification of target 1 (exon 2) sequence. g Nucleotide sequences of target site 1 in pKIWI502 KO lines 1-19, 2-17 and 6-9 lines showing identical 3-bp deletions (top), and deduced amino acid sequences of the corresponding regions showing the deletion of tyrosine (Y) (bottom). h PCR amplification of target 2 (exon 3) sequence. i Schematic of the knock-in events in pKIWI502 KO lines 3-21, 6-15, and 13-3. Vector sequences were inserted at the target site, along with an additional 3-bp (ACC) or 4-bp (GGTT) sequence at the 5'-end (arrowhead on the left in line 13-3) or 3'-end (arrowhead on the right in line 6-15) of the cleaved site, respectively. Black capital letters in lines 3-21 and 13-3 indicate pKIWI502 sequence; red bold letters in lines 3-21, 6-15, and 13-3 indicate partial target-2 sequence; sky-blue letters indicate the U6 promoter sequence. Question marks (?) represent unidentified regions. (JPG 634 KB)
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Lim, JM., Jung, S., Min, S.R. et al. Isolation and characterization of high-CO2 sensitive Nannochloropsis salina mutant. Plant Biotechnol Rep 17, 677–686 (2023). https://doi.org/10.1007/s11816-023-00866-1
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DOI: https://doi.org/10.1007/s11816-023-00866-1