Multiplexed CRISPR/Cas9 editing of the long-chain acyl-CoA synthetase family in the diatom Phaeodactylum tricornutum reveals that mitochondrial ptACSL3 is involved in the synthesis of storage lipids

Hao, Xiahui; Chen, Wenchao; Amato, Alberto; Jouhet, Juliette; Maréchal, Eric; Moog, Daniel; Hu, Hanhua; Jin, Hu; You, Lingjie; Huang, Fenghong; Moosburner, Mark; Allen, Andrew E.; Gong, Yangmin

doi:10.1111/nph.17911

Title: Multiplexed CRISPR/Cas9 editing of the long-chain acyl-CoA synthetase family in the diatom Phaeodactylum tricornutum reveals that mitochondrial ptACSL3 is involved in the synthesis of storage lipids

Journal Article · Thu Dec 09 00:00:00 EST 2021 · New Phytologist

DOI:https://doi.org/10.1111/nph.17911· OSTI ID:1982920

Hao, Xiahui ^[1]; Chen, Wenchao ^[1];

^[2]; Jouhet, Juliette ^[2]; Maréchal, Eric ^[2]; Moog, Daniel ^[3];

^[4]; Jin, Hu ^[4]; You, Lingjie ^[1]; Huang, Fenghong ^[1];

^[5];

^[1]

Chinese Academy of Agricultural Sciences, Wuhan (China)
Univ. of Grenoble Alpes, Grenoble (France)
Philipps University of Marburg (Germany)
Chinese Academy of Sciences (CAS), Wuhan (China)
Univ. of California, San Diego, CA (United States). Scripps Inst. of Oceanography; J. Craig Venter Institute, Inc., La Jolla, CA (United States)

Long-chain acyl-CoA synthetases (LACS) play diverse and fundamentally important roles in lipid metabolism. While their functions have been well established in bacteria, yeast and plants, the mechanisms by which LACS isozymes regulate lipid metabolism in unicellular oil-producing microalgae, including the diatom Phaeodactylum tricornutum, remain largely unknown. In P. tricornutum, a family of five genes (ptACSL1–ptACSL5) encodes LACS activities. For this work, we generated single lacs knockout/knockdown mutants using multiplexed CRISPR/Cas9 method, and determined their substrate specificities towards different fatty acids (FAs) and subcellular localisations. ptACSL3 is localised in the mitochondria and its disruption led to compromised growth and reduced triacylglycerol (TAG) content when cells were bubbled with air. The ptACSL3 mutants showed altered FA profiles in two galactoglycerolipids and phosphatidylcholine (PC) with significantly reduced distribution of 16:0 and 16:1. ptACSL5 is localised in the peroxisome and its knockdown resulted in reduced growth rate and altered molecular species of PC and TAG, indicating a role in controlling the composition of acyl-CoAs for lipid synthesis. Our work demonstrates the potential of generating gene knockout mutants with the mutation of large fragment deletion using multiplexed CRISPR/Cas9 and provides insight into the functions of LACS isozymes in lipid metabolism in the oleaginous microalgae.

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Research Organization:: US Department of Energy (USDOE), Washington, DC (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER); National Science Foundation (NSF); Gordon and Betty Moore Foundation

OSTI ID:: 1982920

Journal Information:: New Phytologist, Vol. 233, Issue 4; ISSN 0028-646X

Publisher:: WileyCopyright Statement

Country of Publication:: United States

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
plant sciences
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multiplexed CRISPR/Cas9
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Title: Multiplexed CRISPR/Cas9 editing of the long-chain acyl-CoA synthetase family in the diatom Phaeodactylum tricornutum reveals that mitochondrial ptACSL3 is involved in the synthesis of storage lipids

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