Knocking out the carboxyltransferase interactor 1 (CTI1) in Chlamydomonas boosted oil content by fivefold without affecting cell growth

Abstract

The first step in chloroplast de novo fatty acid synthesis is catalysed by acetyl-CoA carboxylase (ACCase). As the rate-limiting step for this pathway, ACCase is subject to both positive and negative regulation. In this study, we identify a Chlamydomonas homologue of the plant carboxyltransferase interactor 1 (CrCTI1) and show that this protein interacts with the Chlamydomonas α-carboxyltransferase (Crα-CT) subunit of the ACCase by yeast two-hybrid protein-protein interaction assay. Three independent CRISPR-Cas9 mediated knockout mutants for CrCTI1 each produced an 'enhanced oil' phenotype, accumulating 25% more total fatty acids and storing up to fivefold more triacylglycerols (TAGs) in lipid droplets. The TAG phenotype of the crcti1 mutants was not influenced by light but was affected by trophic growth conditions. By growing cells under heterotrophic conditions, we observed a crucial function of CrCTI1 in balancing lipid accumulation and cell growth. Mutating a previously mapped in vivo phosphorylation site (CrCTI1 Ser108 to either Ala or to Asp), did not affect the interaction with Crα-CT. However, mutating all six predicted phosphorylation sites within Crα-CT to create a phosphomimetic mutant reduced this pairwise interaction significantly. Comparative proteomic analyses of the crcti1 mutants and WT suggested a role for CrCTI1 in regulating carbon flux by coordinating carbon metabolism, antioxidant and fatty acid β-oxidation pathways, to enable cells to adapt to carbon availability. Taken together, this study identifies CrCTI1 as a negative regulator of fatty acid synthesis in algae and provides a new molecular brick for the genetic engineering of microalgae for biotechnology purposes.

Keywords: ROS; acetyl‐CoA carboxylase; carboxyltransferase interactor; cell growth; de novo fatty acid synthesis; peroxisome fatty acid β‐oxidation.

Figure 1

Chlamydomonas contains one orthologue of the Arabidopsis CTIs which interacts with the α‐CT subunit of ACCase. (a) Phylogenetic tree of CrCTI1 and three Arabidopsis CTI homologues constructed by comparing amino acid sequences. (b) Protein domain structures of A. thaliana CTI1 (AtCTI1), C. reinhardtii CTI1 (CrCTI1), A. thaliana α‐CT (Atα‐CT) and C. reinhardtii α‐CT (Crα‐CT). The location of putative phosphorylation sites was highlighted in red colour. (c) Prediction model of coiled‐coil interaction (highlighted by dot circle) between Crα‐CT (light green) and CrCTI1 (magenta) using AlphaFold2. (d) Yeast two‐hybrid (Y2H) assay showed that full‐length Crα‐CT interacts with the coiled‐coil domain of CrCTI1 in vitro. Empty vector and Crβ‐CT served as negative controls, the test between Crα‐CT and Crβ‐CT served as positive control. AD, pGADT7 (prey vector); BD, pGBKT7 (bait vector); SD, synthetic drop‐out medium supplements.

Figure 2

Generation and validation of knockout mutants for Chlamydomonas CrCTI1 using CRISPR‐Cas9. (a) The sgRNA target site on CrCTI1 gene. Black arrow boxes with numbers stand for exons, and the black lines represent the introns. (b) Genomic DNA (gDNA) PCR verification of cassette insertion in the crcti1 mutants. (c) Sanger sequencing results. (d) RT‐PCR analyses of CrCTI1 expression in WT and the three crcti1 mutants. RACK1, receptor for activated kinase C 1 was used as a reference gene. M, marker; PAM, protospacer adjacent motif; WT, wild type.

Figure 3

Chlamydomonas CrCTI1 mutants made more oil during mixotrophic growth without compromising photosynthesis yield and growth. (a) Total fatty acid content measured by GC–MS. FAME, fatty acid methyl ester. (b) TAG content was measured by thin‐layer chromatography (TLC). (c) Confocal fluorescence microscopy images of Chlamydomonas cells. Green, Bodipy fluorescence; red, chlorophyll autofluorescence. Images were present in high and low magnification. Scale bars = 10 μm. (d) Chlamydomonas cell growth curve. (e) PSII yield (Y(II)) measurement. Data are means of three biological replicates with standard deviation shown. *P < 0.05 and ***P < 0.001 (Student's t‐test).

Figure 4

Oil accumulation in cti1 mutants is affected by CO₂ level during photoautotrophic growth. (a) Total fatty acid content measured by GC–MS. (b) TAG content was measured by thin‐layer chromatography (TLC). (c) Cell growth curve cultured in photoautotrophic condition with additional 2% CO₂ supply in the air. (d) Cell growth curve cultured in photoautotrophic condition with ambient air. All experiments were performed in three biological replicates (± SD). *P < 0.05, **P < 0.01 and ***P < 0.001 (Student's t‐test). FAME, fatty acid methyl ester; MM, minimal medium.

Figure 5

Physiological characteristics of crcti1 mutants during heterotrophic growth. (a) Working flow of the experiments and the pictures of cell culture in flasks. Images were taken on the 3 days of pre‐culture or at the 4th day of sub‐culture in the dark. (b) Total fatty acids content measured by GC–MS. FAME, fatty acid methyl ester. (c) TAG content was measured by thin‐layer chromatography (TLC). (d) Confocal fluorescence microscopy images of Chlamydomonas cells. Green, Bodipy fluorescence; red, chlorophyll autofluorescence. Images were present in high and low magnification. Scale bars = 10 μm. (e) Chlamydomonas cell growth curve cultured in TAP medium in the dark. All experiments were performed in three biological replicates (± SD). *P < 0.05 and ***P < 0.001 (Student's t‐test).

Figure 6

Metabolic adaptations as a result of crcrti1 mutation under mixotrophic and photoautotrophic conditions. Fold‐changes of protein amounts between CrCTI1 mutants and wild‐type (average log₂FC between all three mutants, crcti1/WT) grouped according to pathways. N.Q. represent proteins not differently abundance with a P value ≤ 0.05. * represent two to more mutant lines with P‐value ≤ 0.01, and similar fold‐change values. ACH1, aconitate hydratase; ACLB1, ATP citrate lyase, subunit B; ACP2, acyl‐carrier protein 2; ACS2, acetyl‐CoA synthase 2; ACS3, acetyl‐CoA synthetase/ligase 3; ACX3, acyl‐CoA oxidase/dehydrogenase 3; ACX4, putative acyl‐CoA oxidase 4; ACX6, acyl‐CoA oxidase/dehydrogenase 6; BC1, biotin carboxylase (ACCase complex); BCC1, acetyl‐CoA biotin carboxyl carrier; CAT1, mono‐functional catalase; CIS1, citrate synthase, mitochondrial; CIS2, citrate synthase, glyoxysomal/microbody form; DLA1, dihydrolipoamide acetyltransferase; DLA2, dihydrolipoamide acetyltransferase; DLD2, dihydrolipoamide dehydrogenase 2; ECH3, enoyl‐CoA hydratase 1; ENR1, enoyl‐ACP reductase; FAB2, plastid acyl‐ACP desaturase; FBA1, fructose‐1,6‐bisphosphate aldolase; FBA2, fructose‐1,6‐bisphosphate aldolase; FBA3, fructose‐1,6‐bisphosphate aldolase, chloroplastic; FRK2, probable fructokinase‐2; GAPA1, glyceraldehyde‐3‐phosphate dehydrogenase a; GAPC, chloroplastic; GAPC1, glyceraldehyde 3‐phosphate dehydrogenase; GLB1, nitrogen regulatory protein PII; HAD1, 3‐hydroxyacyl‐ACP dehydratase; HBD1, 3‐hydroxybutyrate dehydrogenase; HXK1, hexokinase; IDH1, isocitrate dehydrogenase, NAD‐dependent; IDH2, isocitrate dehydrogenase, NAD‐dependent; KAR1, 3‐oxoacy‐ACP reductase; KAT1, 3‐oxoacyl CoA thiolase/acetyl‐CoA acyltransferase 1; LCS3, long‐chain acyl‐CoA synthetase; MAS1, malate synthase; MCT1, malonyl‐CoA:Acyl‐carrier‐protein transacylase; MDAR1, monodehydroascorbate reductase 1; MDH1, NAD‐dependent malate dehydrogenase 1, chloroplastic; MDH2, malate dehydrogenase 2; MDH3, NAD‐dependent malate dehydrogenase 3; MDH5, NADP‐malate dehydrogenase 5; MME6, NADP‐dependent malic enzyme 6; OGD1, 2‐oxoglutarate dehydrogenase, E1 subunit; OGD2, dihydrolipoamide succinyltransferase, oxoglutarate dehydrogenase E2 component; PCK1, phosphoenolpyruvate carboxykinase; PDC1, mitochondrial pyruvate dehydrogenase complex, E1 component, alpha subunit; PFK1, phosphofructokinase; PGK1, phosphoglycerate kinase, chloroplastic; PPD2, pyruvate phosphate dikinase; PRK1, phosphoribulokinase, chloroplastic; PYK1, pyruvate kinase 1; RBCS2, ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) small subunit 2, chloroplastic; RPE1, ribulose phosphate‐3‐epimerase, chloroplastic; SBE3, starch branching enzyme 3; SCLA1, succinyl‐CoA ligase alpha chain; SCLB1, succinate‐CoA ligase beta chain; SDH1, succinate dehydrogenase flavoprotein subunit; SDH2, iron–sulphur subunit of mitochondrial succinate dehydrogenase; TAL1, transaldolase; TAL2, putative transaldolase, plastid form; TRK1, transketolase, chloroplastic; αCT, Alpha‐carboxyltransferase (ACCase complex); βCT, beta‐carboxyltransferase (ACCase complex).

Figure 7

The highly abundant proteins in the enzymatic antioxidant defence pathway can effectively compensate for oxidative stress. (a) Intracellular ROS analysis by DCFH‐DA staining of WT and crcti1 mutants in mixotrophic and photoautotrophic condition (under air or supplemented with 2% CO₂). All experiments were performed in three biological replicates (± SD). *P < 0.05 (Student's t‐test). (b) Proteins detected by proteomics analysis. Fold‐changes of protein amounts between crcti1 mutants and wild‐type were calculated as log₂(crcti1/WT) and the significant differentially expressed proteins were displayed in the heat map. N.Q. represents protein not differently abundance with a P value ≤ 0.05. * represent two or more mutant lines with P‐value ≤ 0.01, and similar fold‐change values. APX, ascorbate peroxidase; Asc, ascorbate; CAT1, catalase; MDAR1, monodehydroascorbate reductase; MDHA, monodehydroascorbate; PRX, peroxiredoxin.