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

Abstract

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 oilproducing microalgae, including the diatom Phaeodactylum tricornutum, remain largely unknown.In P. tricornutum, a family of five genes (ptACSL1-ptACSL5) encodes LACS activities. 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.

Mitochondria and chloroplasts function in microalgae energy production

“…There are other examples of genetic manipulation of microalgae in order to modify metabolic pathways and/or mitochondrial functions to improve their productivity. One of the approaches used was the alteration of lipid metabolism using a multiplexed CRISPR/Cas9 to modify enzymes belonging to the long chain acyl-CoA synthetase (LACS) family ( Hao et al, 2022 ). Although the general role of LACS in other organisms is well known, little is known about how these enzymes regulate lipid metabolism in algae.…”

“…Although the general role of LACS in other organisms is well known, little is known about how these enzymes regulate lipid metabolism in algae. The study of Phaeodactylum tricornutum knockout/knockdown strains of LACS, particularly the mutants deficient in LACS3, which has a mitochondrial localization, showed a deficiency in the accumulation of triacylglycerol (TAG), as well as an alteration in the fatty acid profile ( Hao et al, 2022 ). This study demonstrated the importance of the proper function of mitochondria in the production of lipids in oleaginous microalgae.…”

UDP-glucose pyrophosphorylase as a target for regulating carbon flux distribution and antioxidant capacity in Phaeodactylum tricornutum

“…UGP1 and UGP2 enzyme assay. The enzymatic activities of UGP1 and UGP2 recombinant proteins were measured as described by Zhu et al 16 , and their kinetic constants were determined as described by Meng et al 24 and Hao et al 22 . Brie y, approximately 6 ng of puri ed protein was added to a 1 mL assay mixture composed of 100 mM Hepes (pH 7.5), 5 mM MgCl 2 , 1 mM UTP, and 0.08-1.6 mM Glc-1-P/Gal-1-P/Xyl-1-P/GlcNAc-1-P. After mixing thoroughly and incubating at 37°C for 20 min, the mixture was heated at 100°C for 5 min to terminate the reaction.…”

“…However, lipid accumulation caused by environmental stress is usually accompanied by reduced biomass, which is not suitable for industrial production [18][19][20] . Genetic engineering to increase lipid production focuses mainly on the regulation of enzymes in the lipid synthesis pathway, whereas the regulation of key nodes of other metabolic pathways to improve lipid synthesis has received little attention 21,22 . The biosynthesis of carbohydrates such as Chrl competes with lipid synthesis for the photosynthetic carbon source 3,14,23 .…”

Genetic engineering to enhance microalgal-based produced water treatment with emphasis on CRISPR/Cas9: A review

“…Frontiers in Bioengineering and Biotechnology frontiersin.org degradation could induce increased lipid yields in different microalgal species (Nguyen A. D. et al, 2020;Chang et al, 2020). Several other studies were also successful in implementing CRISPR/Cas9 to increase the production of different carotenoids in Dunaliella salina (Hu et al, 2021); and in Chlamydomonas reinhardtii (Baek et al, 2016), to improve the thermal tolerance of Tetraselmis suecica (Xu J. et al, 2020), and to investigate gene function in Phaeodactylum tricornutum (Hao et al, 2022;Llavero-Pasquina et al, 2022). Overall, CRISPR/ Cas9 has been successfully applied on many other algae species to improve their biomass productivities, tolerances to abiotic stressors, or increase lipid content, or that of other biomolecules and value-added products (Kumar et al, 2020;Jeon et al, 2021;Wang et al, 2021).…”