Xiahui Hao

Wenchao Chen

Alberto Amato

Juliette Jouhet

Eric Maréchal

Daniel Moog

Hanhua Hu

Hu Jin

Lingjie You

Fenghong Huang

Mark Moosburner

Andrew E Allen

Yangmin Gong
email: gongyangmin@caas.cn

Multiplex 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

Keywords: Fatty acid, Long-chain acyl-CoA synthetase, Multiplexed CRISPR/Cas9, Phaeodactylum tricornutum, Triacylglycerol

Multiplex 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

Introduction

Fatty acids (FAs), a highly diverse class of molecules, have variable chain length and number of double bonds and are building blocks for all membrane lipids and storage lipids in the cells.

They are essential molecules in almost all organisms. Different from that in animals and fungi, which produce FAs primarily in the cytosol for lipid biosynthesis, in plants de novo synthesis of FA s takes place in the plastid via a repeated cycle of reactions with fatty acyl intermediates covalently attached to acyl carrier protein (ACP), providing 16:0-, 18:0-and 18:1-ACP as the final products [START_REF] Ohlrogge | Lipid biosynthesis[END_REF]. These fatty acyl-ACP thioesters can serve as substrates to plastid-localized acyltransferases and feed directly into the "prokaryotic" lipid synthesis pathway to produce phosphatidylglycerol (PG) and other thylakoid lipids such as sulfoquinovosyldiacylglycerol (SQDG), monogalactosyldiacylglycerol (MGDG), and digalactosyldiacylglycerol (DGDG) in the plastid [START_REF] Heinz | Similarities and differences in lipid metabolism of chloroplasts isolated from 18:3 and 16:3 plants[END_REF][START_REF] Jessen | Two activities of long-chain acyl-coenzyme Asynthetase are involved in lipid trafficking between the endoplasmic reticulum and the plastid in Arabidopsis[END_REF].

Alternatively, the current model proposes that a part of acyl-ACPs are hydrolyzed in the plastid stroma by the action of acyl-ACP thioesterase to release free FAs that are targeted for export from the plastid. Once activated into fatty acyl-CoAs, these acyl groups can enter the eukaryotic pathway in the endoplasmic reticulum (ER) for incorporation into extraplastid membrane lipids and storage lipids. In addition, plastid-derived FAs are also substrates for eukaryotic pathway to synthesize important lipid precursors, like phosphatidic acid (PA) and diacylglycerol (DAG), which are imported back to the plastid for thylakoid lipid biosynthesis. This two-pathway hypothesis for thylakoid lipid biosynthesis was originally postulated by Roughan and Slack [START_REF] Roughan | The role of chloroplasts and microsomal fractions in polar-lipid synthesis from [1-14 C]acetate by cell-free preparations from spinach (Spinacia oleracea) leaves[END_REF][START_REF] Roughan | Cellular organization of glycerolipid metabolism[END_REF], supported by specific acyl compositions of lipid molecular species derived from the plastid and ER pathways [START_REF] Heinz | Similarities and differences in lipid metabolism of chloroplasts isolated from 18:3 and 16:3 plants[END_REF], and later corroborated by genetic and biochemical analysis of several mutants in the model plant Arabidopsis thaliana [START_REF] Kunst | Altered regulation of lipid biosynthesis in a mutant of Arabidopsis deficient in chloroplast glycerol-3-phosphate acyltransferase activity[END_REF][START_REF] Wallis | Mutants of Arabidopsis reveal many roles for membrane lipids[END_REF][START_REF] Xu | Lipid trafficking between the endoplasmic reticulum and the plastid in Arabidopsis requires the extraplastidic TGD4 protein[END_REF]. In contrast to plants that contain the primary plastids with a two-membrane envelope, evidence is lacking to support such a similar pathway for phospholipid trafficking from the ER to chloroplasts to form galactolipids in photosynthetically active stramenopiles with a secondary plastid surrounded by four limiting membranes, such as the eustigmatophyte Nannochloropsis sp. and the diatom P. tricornutum. In these photosynthetic eukaryotes, MGDG and DGDG are characterized by a high content of 20:5, which is synthesized in the ER from the initial substrate of plastid-exported 16:0.

After synthesis in the ER, 20:5 needs to be imported into the plastid through the unknown "omega pathway" [START_REF] Petroutsos | Evolution of galactoglycerolipid biosynthetic pathwaysfrom cyanobacteria to primary plastids and from primary to secondary plastids[END_REF][START_REF] Dolch | Nitric oxide mediates nitrite-sensing and acclimation and triggers a remodeling of lipids[END_REF].

In plants, before becoming accessible to the "eukaryotic" lipid synthesis pathway, free FAs need to be activated by esterification with CoA to form acyl-CoA thioesters as they are chemically inert. The activation of free FAs to form fatty acyl-CoAs is catalyzed by long-chain acyl-CoA synthetases (LACSs,EC 6.2.1.3). This reaction occurs in two steps. First, the free FA reacts with ATP to generate an intermediate fatty acyl-AMP (acyl adenylate) with the release of pyrophosphate. In the second step, the enzyme bound acyl-AMP reacts with CoA to form acyl-CoA, with the release of AMP [START_REF] Groot | Fatty acid activation:specificity, localization, and function[END_REF]. In photosynthetic stramenopiles, LACS activity is likely to be not only required for activation of free FAs (16:0 and 16:1) exported from the plastid, but also involved in the downstream 16:0-to-20:5 route in which it provides activated FAs for FA elongases and acyltransferases.

LACS activities have been localized in various subcellular compartments including the chloroplast, cytoplasm, ER, peroxisome, mitochondria and oil bodies. The physiological function of LACS in cellular homeostasis, particularly in lipid metabolism, has been studied in detail in bacteria [START_REF] Black | Molecular and biochemical analyses of fatty acid transport, metabolism, and gene regulation in Escherichia coli[END_REF][START_REF] Black | Mutational analysis of a fatty acyl-coenzyme Asynthetase signature motif identifies seven amino acid residues that modulate fatty acid substrate specificity[END_REF], yeast [START_REF] Faergeman | The acyl-CoA synthetases encoded within Faa1 and Faa4 in Saccharomyces cerevisiae function as components of the fatty acid transport system linking import, activation, and intracellular utilization[END_REF][START_REF] Zou | Vectorial acylation in Saccharomyces cerevisiae. Fat1p and fatty acyl-CoA synthetase are interacting components of a fatty acid import complex[END_REF] and plants [START_REF] Lü | Arabidopsis CER8 encodes LONG-CHAIN ACYL-COA SYNTHETASE 1 (LACS1) that has overlapping functions with LACS2 in plant wax and cutinsynthesis[END_REF][START_REF] Weng | Organ fusion and defective cuticle function in a lacs1lacs2 doublemutant of Arabidopsis[END_REF][START_REF] Jessen | Combined activity of LACS1 and LACS4 is required for proper pollen coat formation in Arabidopsis[END_REF][START_REF] Jessen | Two activities of long-chain acyl-coenzyme Asynthetase are involved in lipid trafficking between the endoplasmic reticulum and the plastid in Arabidopsis[END_REF][START_REF] Zhao | Insertional mutant analysis reveals that long-chain acyl-CoA synthetase 1 (LACS1), but not LACS8, functionally overlaps with LACS9 in Arabidopsis seed oil biosynthesis[END_REF][START_REF] Zhao | Functional overlap of long-chain acyl-CoA synthetases in Arabidopsis[END_REF]. Like higher plants, plastids of photosynthetic microalgae are also the principle site for de novo FA synthesis while further desaturation and chain elongation occur in the ER and fatty acyl catabolism takes place in the mitochondria and/or peroxisome via -oxidation. We previously reported the identification of five LACS genes in the diatom Phaeodactylum tricornutum that can synthesize and accumulate large amounts of TAGs at stationary phase of growth and under stress conditions, particularly nitrogen starvation [START_REF] Guo | Identification and biochemical characterization of five long-chain acyl-coenzyme A synthetases from the diatom Phaeodactylum tricornutum[END_REF]. Although the LACS isozymes have been identified in Phaeodactylum, the functional role of individual LACS in lipid metabolism, especially their contribution to storage lipid biosynthesis, remains largely unknown. In the past ten years, development of genome-editing techniques, such as the mutagenesis with transcription activator-like effector nucleases (TALENs) and the CRISPR/Cas9 method [START_REF] Weyman | Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis[END_REF][START_REF] Daboussi | Genome engineering empowers the diatom Phaeodactylum tricornutum for biotechnology[END_REF][START_REF] Serif | One-step generation of multiple gene knock-outs inthe diatom Phaeodactylum tricornutumby DNA-freegenome editing[END_REF][START_REF] Moosburner | Multiplexed knockouts in the model diatom Phaeodactylum by episomal delivery of a selectable Cas9[END_REF], has allowed us to generate targeted and stable modification of the diatom genome to address the functions of key genes.

Considering acyl-CoA thioesters as key building blocks required for acyl-CoA-dependent TAG biosynthesis, we here used multiplexed CRISPR/Cas9 method to generate lacs single mutants and to assess the contribution of each ptLACS isozyme to lipid biosynthesis. In addition, we determined their subcellular localizations, protein expression patterns and enzyme activities. Our results advance the understanding on the functions of LACS isozymes in lipid metabolism in oilaccumulating unicellular microalgae.

Materials and methods

Strains and growth conditions

P. tricornutum strain Pt1 WT and derived mutants were grown in artificial seawater (ASW) media with F/2 nutrients [START_REF] Guillard | Culture of phytoplankton for feeding marine invertebrates[END_REF]) at 22 o C under white fluorescent lights (50 mol photons m -2 s -1 ) with a 12h:12 h light-dark cycle, and additional supplementation of 100 mg L -1 Zeocin was included for mutants. For time-course growth experiments in static culture, 200-mL algal cultures of were grown in batch mode for 12 d with an initial addition of inoculum to give a starting cell density of around 5×10 6 cells mL -1 . For growth experiments with aeration, duplicate 300-mL cultures were bubbled for 9 d with air supply sterilized by filtration through 0.22 m Millipore filters. Samples were taken daily for cell counting and measurement of Nile Red fluorescence and nitrate concentration. For the immunoblot experiment, algal cells were grown in N-replete F/2 medium to the exponential phase and then transferred to N-free medium for additional 72 h, or to N-replete medium bubbled with air, 1%, 4% and 10% CO2 enriched air for additional 6 h.

Plasmid construction

Plasmids for generation of Phaeodactylum ptACSL mutants using CRISPR/Cas9 method were constructed as described by [START_REF] Moosburner | Multiplexed knockouts in the model diatom Phaeodactylum by episomal delivery of a selectable Cas9[END_REF]. To construct eGFP fusion plasmids, full-length ptACSL sequences were cloned into the multiple cloning sites (MCSs) of the expression vector pPha-CG/ pPha-NG. For self-assembling split GFP constructs, we used the pPha-Dual vector that harbors two MCSs for gene cloning. Details are described in supporting information Methods S1. All the plasmids and primer sequences used in this study were listed in Table S1 andS2.

Generation of ptACSL1-5 knock out/down lines

For editing of each ptACSL gene, six sgRNAs were designed in the coding region, namely gRNA1 to gRNA6. Every three sites were designed to be assembled in a single vector carrying Cas9 and zeocin resistance cassette via Golden Gate cloning. Transformation of Phaeodactylum via bacterial-mediated conjugation and screening were carried out as described previously [START_REF] Moosburner | Multiplexed knockouts in the model diatom Phaeodactylum by episomal delivery of a selectable Cas9[END_REF]. For genotypic characterization, algal cell lysates served as the PCR templates and primers listed in Table S2 were used to amplify the various loci of interest. Cas9induced mutation for each ptACSL gene was analyzed by direct sequencing of PCR products containing the targeted sites. The Inference of CRISPR Edits tool (ICE, Synthego, https://ice.synthego.com) was used to genotype Phaeodactylum mutant lines. It offers the indel (insertion or deletion) type and the overall indel percentage (editing efficiency) by decoding of all the sequencing chromatograms with monoallelic mutation, homozygous and heterozygous biallelic mutations, which result in superimposed sequencing chromatograms.

In vitro LACS enzyme assay

Recombinant Phaeodactylum ptACSL proteins were produced from E. coli BL21 (DE3) pLysS and purified with Ni-NTA affinity column (Novagen). LACS enzyme activity was measured spectrophotometrically using an enzyme-coupled assay [START_REF] Horswill | Characterization of the propionyl-CoA synthetase (PrpE) enzyme of Salmonella enterica: residue Lys592 is required for propionyl-AMP synthesis[END_REF] Methods S1). Determination of kinetic constants of recombinant ptACSLs was described in Method S1.

Transformation of Phaeodactylum tricornutum

All the plasmids of GFP fusions were transformed into Phaeodactylum by microparticle bombardment using Bio-Rad Biolistic PDS-1000/He Particle Delivery System (Bio-Rad Laboratories, Hercules, CA) as previously described [START_REF] Zaslavskaia | Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes[END_REF]. Bacterial-mediated conjugation was used to introduce Cas9-ShBle:sgRNA episome into Phaeodactylum as described by [START_REF] Karas | Designer diatom episomes delivered by bacterial conjugation[END_REF]. Resistant colonies were selected on F/2 media supplemented with 100 g mL -1 zeocin.

Immunoblotting

Antibodies for ptACSL1 and ptACSL4 were generated as described previously [START_REF] Chen | Acetylome Profiling Reveals Extensive Lysine Acetylation of the Fatty Acid Metabolism Pathway in the Diatom Phaeodactylum tricornutum[END_REF].

The polyclonal antibodies that specifically recognize ptACSL2, ptACSL3 and ptACSL5 were raised by immunizing rabbits with purified full-length recombinant proteins. The expression of ptACSL1-5 was examined by immunoblotting as described in Methods S1.

Lipid extraction and analysis

Glycerolipids from Phaeodactylum WT and the ptACSL mutants were extracted as described by [START_REF] Gong | FFA, free fatty acid; OAA, oxaloacetate; TCA, tricarboxylic acid; ER, endoplasmic reticulum; FASII, fatty acid synthase II[END_REF]. The sn-position of FAs on individual glycerolipids and quantification of each molecular species were analyzed by preferential loss using MS and MS2 analysis and liquid chromatography-MS/MS, respectively, as previously described [START_REF] Jouhet | LC-MS/MS versus TLC plus GC methods: Consistency of glycerolipid and fatty acid profiles in microalgae and higher plant cells and effect of a nitrogen starvation[END_REF]. For detailed analysis of the change of TAG molecular species, TAGs were isolated by TLC from total lipids of WT and the ptACSL3 mutants grown at static (day 11) and air-bubbled (day 7) conditions, and LC-MS method was used to quantify each molecular species of TAGs as described by [START_REF] Xie | Ultrasoundassisted one-phase solvent extraction coupled with liquidchromatography-quadrupole time-offlight mass spectrometry for efficient profiling of egg yolk lipids[END_REF] (Methods S1).

RNA-seq analysis

WT and the ptACSL3 mutants were grown in static culture for 6 d and then transferred to the cultures bubbled with filtered air for additional 4 d. Cells were harvested at 2 d, 3 d, and 4 d for RNA-seq and samples under static culture for 6 d were used as control (0 d). Three biological replicates of WT and the ptACSL3 mutant and of each condition were processed for total RNA isolation using TRIzol reagent (Invitrogen, USA), and RNA sequencing were performed by the Illumina NovaSeq 6000 platform. RNA-seq reads were aligned to the Ensemble reference genome and gene model annotation files. The DESeq2 R package (1.20.0) was used to detect differential expressed genes (DEGs) between the WT and mutants. The DEGs were subjected to gene ontology (GO) enrichment analysis with a clusterProfiler R package (Methods S1).

Results

Substrate specificities of ptACSL15 determined by in vitro enzyme assay

The purified recombinant ptACSL proteins served as enzyme sources in LACS activity assays, using different free FAs as a substrate. The tested substrates are endogenous FAs and some are present in a high proportion in acyl lipids of Phaeodactylum. We used an enzyme-coupled method for measurement of LACS activity [START_REF] Horswill | Characterization of the propionyl-CoA synthetase (PrpE) enzyme of Salmonella enterica: residue Lys592 is required for propionyl-AMP synthesis[END_REF]. Substrate specificities and catalytic efficiencies of ptACSL15-catalyzed reactions were determined by varying concentrations of free FAs with fixed saturating concentrations of ATP and CoA. The plots of initial velocities versus free FA concentrations were fitted to the Michaelis-Menten equation to determine the Km, kcat, and kcat/Km values. As shown in Table 1, Km values for all five ptACSLs were in the range of 27451 M, and all five ptACSL enzymes exhibited LACS activity for oleic acid (18:1n-9) with kcat/Km values ranging from 1851 to 5912 M -1 s -1 . When supplied with different FA substrates, ptACSL1 proved to be the most active and have the highest catalytic efficiency (kcat/Km, the constant that better measures the specificity for the substrate), with EPA, with a value exceeding by 2 orders of magnitude that measured in presence of 16:0. Similarly, ptACSL4 also displayed a preference for EPA, with a kcat/Km value 5-and 184-fold higher than those obtained with the two other FAs tested.

Additionally, of the five LACS enzymes, ptACSL5 is only LACS showing no any activity for EPA.

The Km values determined for ptACSL5, which reflect the binding affinity of an enzyme for a substrate, indicate that ptACSL5 had the greatest affinity for 18:0 with Km value of 45 M. These data show that recombinant ptACSL proteins exhibit a difference in specificities towards free FAs, and ptACSL1 and ptACSL4 have the highest catalytic efficiency for EPA, the most abundant very long-chain polyunsaturated FA (VLC-PUFA) detected in Phaeodactylum.

Subcellular localization of LACS isozymes in Phaeodactylum

ptACSL1 was previously reported to be localized in the plastid [START_REF] Chen | Acetylome Profiling Reveals Extensive Lysine Acetylation of the Fatty Acid Metabolism Pathway in the Diatom Phaeodactylum tricornutum[END_REF]). To study its precise membrane location, we used the GFP self-assembling system [START_REF] Cabantous | Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein[END_REF] to determine the localization and orientation of ptACSL1. In this GFP reporter system, the last beta strand of GFP (GFP_S11) was attached to the C-terminus of ptACSL1 and the beta strands 1-10 (GFP_S1-10) were C-terminally fused either to a cER (chloroplast endoplasmic reticulum) marker to be expressed in the ER lumen or expressed as a cytosolically localized large GFP_S1-10 fragment.

These constructs were transformed into Phaeodactylum, and a cER-characteristic GFP fluorescence could be observed only when a fusion protein consisting of full-length sequence of ptACSL1 and GFP_S11 was simultaneously expressed with the large GFP_S1-10 fragment. In contrast, expression of the same ptACSL1 fusion protein with a cER-targeted PDI-GFP_S1-10 (PDI) fusion construct resulted in the absence of detectable fluorescence signal (Fig. 1). This indicates a localization of ptACSL1 in the cER or cER membrane (outermost membrane of the complex plastid) with a C-terminal orientation towards the cytoplasm. The hydrophobicity plot predicted a short transmembrane region at the N-terminus of ptACSL1, it is thus possibly localized in the cER membrane with the C-terminus facing the cytoplasm.

The attachment of ptACSL2 to GFP (ptACSL2:eGFP) caused GFP fluorescence to be located in the cytoplasm. In the transformants harboring ptACSL4:GFP fusion construct, GFP signal was also mainly dispersed throughout the cytoplasm. Fusion of ptACSL3 with GFP led to the mitochondria-characteristic fluorescence, indicating its location in the mitochondria that are in close proximity to the complex plastid. A peroxisomal targeting signal (PTS) type 1 (SKL), which is necessary for targeting and import of cytosolically expressed proteins into peroxisomes, was found to be located at the extreme C-terminus of the ptACSL5 isoform. For in vivo localization study we fused eGFP upstream of full-length sequence of ptACSL5 to ensure the PTS remained accessible, and this eGFP:ptACSL5 fusion construct resulted in a punctate GFP fluorescence pattern (Fig. 1), confirming its peroxisomal localization in transfected Phaeodactylum cells.

Immunoblot analyses of ptACSL accumulation in response to nitrogen deprivation and elevated CO2

Nitrogen-starved Phaeodactylum cells are capable of accumulating large amounts of TAGs, the major storage compounds for FAs. Lipid accumulation also increased in Phaeodactylum cells grown in seawater in response to elevated atmospheric CO2 concentration [START_REF] Jensen | Storage Compound accumulation in diatoms as response to elevated CO2 Concentration[END_REF]). We thus studied the expression of LACSs in response to nitrogen deprivation and increased CO2 supply.

Using ptACSL-specific antibody (Fig. S1), we detected the protein levels by Western blot in Phaeodactylum cells grown in F/2 media with nitrogen deprivation and supplied with different concentrations of CO2. As shown in Fig. 2a, the expression of ptACSL14 proteins was regulated by nitrogen deprivation while ptACSL5 showed a constant expression, suggesting differential expressions of these LACS isozymes upon nitrogen deprivation at protein levels . The expression of ptACSL1 and ptACSL3 was induced by nitrogen deprivation and their abundances peaked at 72 h. By contrast, the level of ptACSL2 was very high under nitrogen-replete condition but it declined during the time course of nitrogen deprivation. When cells were grown at atmospheric CO2 and at high CO2 (1%, 4% and 10%), the expression of ptACSL1, 2 and 4 was slightly suppressed at high CO2 compared to that at atmospheric CO2. Notably, the expression of ptACSL3 was upregulated at elevated CO2 and reached the highest level at 4% CO2 concentration followed by a decline at 10% CO2 concentration (Fig. 2b).

Multiplex CRISPR/Cas9 system induced mutation of single ptACSL gene

We used multiplex CRISPR/Cas9 genome editing system to generate single knockout (KO) mutants for each ptACSL gene [START_REF] Moosburner | Multiplexed knockouts in the model diatom Phaeodactylum by episomal delivery of a selectable Cas9[END_REF]. For creating the single gene mutants for all five ptACSL genes, we prepared ten Cas9-sgRNA episome-based constructs, each carrying three sgRNA expression cassettes to target three different sites within a single ptACSL gene (Fig. 3a). The final vectors expressing Cas9 and the sgRNA assay were transformed into Phaeodactylum, and PCR followed by Sanger sequencing was conducted to detect targeted mutations. Detailed analysis of total 246 sequenced sites from all five ptACSL genes showed that most colonies have a mixed genotype, which consists of wild-type (WT) sequence, nucleotide deletion and a very low percentage of nucleotide insertion. We compared the editing efficiencies of target mutations induced by different numbers of gRNAs, and found that mutation frequency of nucleotide insertion induced by double gRNAs was two-fold higher than that induced by a single gRNA. In double gRNAsinduced mutations, the frequencies of deletions with 1-10 bp in size was slightly higher than that of single gRNA-induced mutations while the frequencies of deletions with 10-30 bp in size were similar (Fig. 3b). Notably, we also detected homozygous biallelic mutations with large fragment deletions (158-230 bp) between two cleavage sites in some colonies. Homozygous biallelic mutations occurred in a varied frequency among different ptACSL genes, with the highest mutagenesis frequency of 30% in ptACSL4 and the lowest frequency of 2.5% in ptACSL1 (Fig. 3c).

We propagated these Phaeodactylum mutant lines in liquid media and then plated dilutions to obtain single colonies. Further sequencing was conducted to detect the targeted mutations, and it showed that the percentage of homozygous biallelic mutations was greatly increased. Finally, we confirmed the absence of wild-type gene using PCR with genomic DNA prepared from these sequenced single colonies. For this purpose, we designed a pair of primers with one being located in the region of fragmental deletion (between two active cleavage sites) while the other outside it and corresponding to the non-editing region (Figs 4a,4b). For instance, genomic PCR showed that the expected DNA bands could be observed in WT/control colonies, while there was no DNA band in the ptACSL1 or ptACSL3 KO mutants (Figs 4c,4d). Mutation of long fragment deletions was also verified using reverse transcription PCR (RT-PCR), indicating the complete lack of detectable expression of ptACSL genes (Figs 4e,4f). Using this method, we obtained the single KO mutants for ptACSL1, ptACSL2, ptACSL3, and ptACSL4, and knockdown mutants for ptACSL5 (Figs 4, S2). Therefore, creating long deletions with multiple gRNAs should be a more efficient approach to achieve targeted mutagenesis, and a rapid and low-cost PCR-based method could be used to screen positive KO colonies, not relying on expensive genotyping methods such as high-throughput next generation sequencing. Finally, we selected three independent mutant lines for each ptACSL single mutants for further phenotyping.

Growth and lipid content of ptACSL knockout mutants

To evaluate the influence of mutations on cell growth and lipid accumulation, we carried out time-course growth experiments using single mutants for each ptACSL gene under both static culture and air-bubbling conditions. During both 12 days of static cultivation and 9 days of growth with airbubbling, there was no difference in cell density, nitrate concentration in the media, and neutral lipid content revealed by Nile Red fluorescence intensity between WT and either ptACSL1, ptACSL2 or ptACSL4 mutants (Figs 5a, 5b, S3, S4). Interestingly, in static condition, there was a significant increase in neutral lipid content in the ptACSL3 mutants since day 7, at which nitrate concentration was reduced to as half as the initial concentration (Figs 5b,5c). The ptACSL3 mutants accumulated more TAGs compared with WT, which was revealed by a thin-layer chromatography (TLC) analysis of lipids from these algal samples (Fig. 5d). We further evaluated the growth of the mutants under lower or higher temperature, and found there was no difference in growth between WT and the ptACSL3 mutants at 15 o C in static culture (Fig. S5a). By contrast, WT cells grew slowly when the temperature was increased to 28 o C while the growth of the ptACSL3 mutants was almost arrested (Fig. S5b). However, when algal cultures were bubbled with air (low CO2, ~0.04% vol/vol CO2), cell densities of both ptACSL1 and ptACSL3 mutants were significantly reduced compared with WT (Fig. 5e), especially after 6 days of growth. Indeed, while nitrate in the F/2 media in WT was almost exhausted at day 6, nitrate concentration in the ptACSL3 mutants was reduced to the lowest level at day 8 (Fig. 5f). Therefore, neutral lipid content was significantly reduced in the ptACSL3 mutants after day 7 in comparison with WT, maybe because nitrogen starvation starts a bit later in ptACSL3 mutants. TLC also showed that the ptACSL3 mutants accumulated less TAGs, especially at day 7 (Figs 5g, 5h). In bubbled air condition, we observed a slower growth of the ptACSL5 knockdown mutants compared with WT from day 4 and nitrate was almost exhausted at day 5 for both WT and the mutants. However, the impairment of ptACSL5 did not result in a change of neutral lipid content, which was revealed by Nile red staining (Fig. S6).

ptACSL3 mutation affects TAG synthesis and fatty acid profiles of glycoglycerolipids when algal culture is bubbled with air FA s and lipids from WT and the ptACSL3 mutants were analyzed at the exponential (6 d) and stationary phases (9 d) of growth. When cells were bubbled with filtered air and harvested at dusk, total FA accumulation in the exponential phase was decreased from 738 nmol mg -1 of dry cell weight (dcw) in WT to 592 nmol mg -1 of dcw in the ptACSL3 mutants. In the stationary phase, total FA content declined from 1137 nmol mg -1 of dcw in WT to 811 nmol mg -1 of dcw in the ptACSL3 mutants. KO of ptACSL3 significantly reduced total FA accumulation relative to WT, indicating that de novo FA synthesis is inhibited in the ptACSL3 mutants. As the major FAs synthesized in the chloroplast, palmitic acid (16:0) and palmitoleic acid (16:1) showed significantly reduced accumulation. The level of 16:0 was reduced from 132 nmol mg -1 of dcw in WT to an average of 89 nmol mg -1 of dcw in the exponential phase, and from 331 to 189 nmol mg - 1 of dcw in the stationary phase. Similarly, the level of 16:1 was also decreased in the mutants at both growth phases (Fig. 6a). KO of ptACSL3 did not change the accumulation of other FA including the abundant EPA. Analysis of FA profiles revealed a significant decrease in the proportions of 16:0, 16:1 and 18:1 balanced by an increase in 16:2, 16:3 and 20:5 in the ptACSL3 mutants relative to WT at both growth phases (Fig. 6b).

Glycerolipidomic analysis was performed to determine how the inactivation of ptACSL3 influenced lipid class composition and the distribution of different molecular species within each lipid class. Compared to WT, disruption of the ptACSL3 gene led to a significant reduction of TAG (76%, p<0.0001), DAG (50%, p<0.01) and DGTA (46%, p<0.05) and an increase in every phospholipid, i.e. phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylethanolamine (PE) and phosphatidylcholine (PC) in the exponential phase (Fig. 7a). With the depletion of nitrogen, WT accumulated large amounts of TAGs in the stationary phase, accounting for 48% of total glycerolipids. However, the relative content of TAG was reduced to 25% in the ptACSL3 mutant (48% reduction, p<0.001) (Fig. 7a). In addition to TAG, the ptACSL3 mutants also showed lower DAG content (51% reduction at the exponential phase, p<0.01; 43% reduction at the stationary phase, p<0.01). The decrease in the proportions of DAG and TAG in the ptACSL3 mutants was compensated by a significant increase in PC (5-fold increase, p<0.0001) and three abundant thylakoid glycolipids (55% increase in SQDG, p<0.05; 47% increase in MGDG, p<0.0001; and 78% increase in DGDG, p<0.001) in the stationary phase (Fig. 7a).

Further quantification of molecular species showed that at both growth phases, both MGDG and DGDG in the ptACSL3 mutants had a significant decrease in the proportions of molecular species containing 16:0/16:0 and 16:1/16:0. In MGDG of the ptACSL3 mutants, 16:0/16:0 and 16:1/16:0 represent two species in which 16:0 is mainly enriched; 16:1 is distributed in seven molecular species, among which four species (14:0/16:1, 16:1/16:0, 16:1/16:2, and 16:1/18:1) showed significantly decreased levels while others (16:1/16:1, 16:1/16:3, and 20:5/16:1) remained unchanged at the stationary phase (Fig. 7b, Table S1). DGDG contains mainly nine molecular species and 16:0 is esterified in three species. Although the level of 20:5/16:0 was slightly increased in the ptACSL3 mutants, 16:0/16:0 and 16:1/16:0 that contained the highest proportion of 16:0 showed a pronounced decrease (Fig. 7c, Table S1). These results suggest that the most abundant galactoglycerolipids MGDG and DGDG in the ptACSL3 mutants had less 16:0 and 16:1, which is also supported by the analysis of FA composition of these two galactoglycerolipids (Figs S7, S8). As molecular species of MGDG and DGDG containing 16:0/16:0 and 16:1/16:0 in their DAG backbone are thought to be derived from the prokaryotic pathway, the impairment of ptACSL3 led to a pronounced alteration of FA profiles of chloroplast membrane lipids. In the plastid, 16:0 and 16:1 are released as acyl-ACPs from the FA synthase, and then transferred to glycerol backbone of galactoglycerolipids. This process does not require the activated form of acyl-CoA thioesters. Therefore, the reduction of 16:0 and 16:1 in two galactoglycerolipids in the ptACSL3 mutants reflects an inhibition of plastidic de novo FA synthesis. In the ptACSL3 mutants, MGDG contained more 16:2/16:3, 16:3/16:3, 20:5/16:2, 20:5/16:3, and 20:5/16:4, while DGDG had higher levels of 20:5/16:0, 20:5/16:1, 20:5/16:2, and 20:5/16:3 at the stationary phase (Figs 7b, 7c, Table S1). This change suggests that the chloroplast membrane lipids in the mutants contained more polyunsaturated FAs when compared with WT. More than ten FAs were found to be at the sn-2 position of PC, where 16:0 was enriched in two molecular species (PC16:1/16:0 and 20:5/16:0) and 18:1 was found in the other two species (PC16:1/18:1 and 18:2/18:1). The proportions of all these four molecular PC species and one species having two C18 acyl chains (18:2/18:2) were significantly/slightly reduced in the ptACSL3 mutants at both growth phases (Fig. 7d, Supporting information Dataset S1), which was compensated by an increase in 16:1/16:1 and 20:5-containing molecular species (20:5/16:1, 20:5/18:2, 20:5/18:3 and 20:5/22:6). Taken together, in air bubbled condition, the impairment of ptACSL3 led to a significant reduction of TAG accumulation, an increase in phospholipid content and a strong alteration in FA profiles of MGDG, DGDG and PC with reduced distribution of 16:0, 16:1 and 18:1 in these lipid classes.

To examine whether molecular species of TAG was affected in the ptACSL3 mutants, we used LC-MS to analyze TAG composition of the WT and mutants grown under static condition and in the bubbling of air. A heat map showing the variation of percentage abundance of 20 major TAG species as the effect of ptACSL3 disruption under both conditions revealed that the levels of three most abundant TAG species accounting for over 10% of total TAG species (48:1 (16:0/16:0/16:1), 48:2 (16:0/16:1/16:1) and 48:3 (16:1/16:1/16:1)) were not changed. When algal cultures were bubbled with filtered air, the levels of two molecular TAG species, including 50:1 (16:0/18:1/16:0) and 50:2 (16:1/18:1/16:0), and all 20:5/22:6-containing molecular species were decreased in the ptACSL3 mutants compared with WT (Fig. 8a). The decrease in percentage abundance of 18:1-and 20:5-containing TAG species in the ptACSL3 mutants was in accordance with the substrate selectivity of ptACSL3 isozyme towards these two FAs from in vitro enzyme assay (Table 1). In the static culture, the ptACSL3 mutants accumulated more TAGs than WT with no reduced growth rate. However, TAG profiling showed that there was also no difference in percentage abundance in three most abundant TAG species (48:1, 48:2 and 48:3) between the ptACSL3 mutants and WT, and the levels of only three molecular species accounting for less than 3% of total TAG species, TAG 50:3 (16:1/18:1/16:1), 50:4 (16:1/16:1/18:2) and 50:5 (16:1/16:1/18:3) were increased in the ptACSL3 mutants (Fig. 8b).

Impacts of the ptACSL3 KO on transcriptional change upon the transition of growth state

Because the ptACSL3 mutants showed different change of TAG accumulation under static culture (slow growth state) and in the culture bubbled with filtered air (rapid growth state), we induced after growth state transition. However, the expression of these genes was downregulated or remained unchanged (Fig. 9c). Moreover, the expression of genes encoding carnitine Oacyltransferase (CAT2, Phatr3_J48078), acyl-CoA dehydrogenase (MACAD1, J11014) catalyzing the first step in -oxidation, and 3-hydroxyacyl-CoA dehydrogenase (HAD2, Phatr3_J39681) was slightly downregulated in WT after transfer to the culture bubbled with air, but the degrees of downregulation were higher in the ptACSL3 mutants. These results indicate that disruption of ptACSL3 led to an inhibition of mitochondrial FA -oxidation upon transition from slow to rapid growth. As mitochondrial -oxidation generates acetyl-CoAs and reducing power and is essential for FA degradation and maintaining energy homeostasis in the cell, inhibition of mitochondrial oxidation might have a negative influence on FA synthesis under air-bubbling condition, which caused reduced FA content and TAG synthesis in the ptACSL3 mutants (Fig. 5h, Fig. 6a).

Effect of the ptACSL1/ptACSL5 mutation on lipid synthesis

As the expression of ptACSL1 was up-regulated during nitrogen deprivation, we analyzed lipid samples prepared from WT and the ptACSL1 mutants at the exponential phase (0 h) and deprived of nitrogen for 72 h (72 h). There was no difference in FA composition between WT and the ptACSL1 mutant, except a small decrease in 20:5 at exponential phase in the mutant (Fig. S9). This decrease could be explained by a global decrease in 20:5-containing TAG and polar lipid species (Dataset S1). At 72 h of nitrogen starvation, membrane lipids apart from DAG were more affected in the WT than in the ptACSL1 mutant, suggesting ptACSL1 might be involved in membrane lipid remodeling during nitrogen starvation.

The FA profiles and content of WT and the ptACSL5 mutants were analyzed in the exponential and stationary phase of growth (bubbled with air). It revealed a slight increase in total FA content of the ptACSL5 mutants at the stationary phase (WT, 766±55, and ptACSL5, 867±67 nmol mg -1 dcw), which was correlated by a slight increase in the levels of 16:1 and proportions of 16:1 in FA profiles at both phases (Fig. S10). Glycerolipidomic analysis showed that 16:1 was highly enriched in the sn-1 position of PC, and the levels of almost all 16:1-containing molecular species were increased in PC of the ptACSL5 mutants in the exponential phase at the expense of a reduction of 20:5containing species (Dataset S1). In the ptACSL5 mutants, 16:1-containing TAG species derived from prokaryotic pathway were increased, which was balanced by a slight decline of TAG 20:5containing species and TAG 16:0/16:0 species in both phases (Dataset S1). To further test whether peroxisomal ptACSL5 is involved in FA -oxidation, WT and the ptACSL5 mutants were maintained in N-free medium for 3 d to promote TAG accumulation. After that period, algal cultures were transferred to N-replete medium for additional 4 d to induce the turnover and hydrolysis of storage lipids. The ptACSL5 mutants still showed reduced growth rate compared with WT during both N starvation and resupply, but these algal strains displayed similar Nile red fluorescence (Fig. S11), indicating that storage lipid catabolism after a shift from N-starved to N-replete medium was not impaired in the mutants. Our results support that peroxisomal FA -oxidation is not correlated to storage lipid remobilization in Phaeodactylum but may be involved in the recycling of long-chain to very-long-chain FAs derived from polar lipids [START_REF] Jallet | Mitochondrial fatty acid -oxidation is required for storage-lipid catabolism in a marine diatom[END_REF]). To conclude, ptACSL5 might be involved in the balance of 16:0 versus 16:1 in acyl-CoA fluxes for membrane lipids (PC) and TAG synthesis, targeting a part of 16:0 into peroxisomal -oxidation to prevent it from further elongation to enter the 16:0-to-20:5 synthetic route.

Discussion

Phaeodactylum accumulates large amounts of TAGs and produces EPA up to 30% of total FAs with trace amounts of C18 FA intermediates, representing a particularly interesting model for studies on VLC-PUFA and lipid biosynthesis. Despite recent advances in understanding the main routes of lipid metabolism in Phaeodactylum [START_REF] Smith | Plastidial acyl carrier protein Δ9-desaturase modulates eicosapentaenoic acid biosynthesis and triacylglycerol accumulation in Phaeodactylum tricornutum[END_REF], little is known about FA activation, transport between different subcellular compartments, and flux governing the ultimate deposition of VLC-PUFA into galactoglycerolipids. Consistent with high proportions of palmitic acid (16:0), palmitoleic acid (16:1n-7) and EPA (20:5) in total FAs, palmitic, palmitoleic and EPA-CoA were detected as the most abundant species in acyl-CoA profiles of P. tricornutum cells [START_REF] Hamilton | Metabolic engineering of Phaeodactylum tricornutum for the enhanced accumulation of omega-3 long chain polyunsaturated fatty acids[END_REF]). These high levels of acyl-CoAs may potentially act as acyl donors or intermediates for VLC-PUFA synthesis and TAG assembly. P. tricornutum has at least five LACS isozymes catalyzing the formation of acyl-CoAs from free FAs and CoA. Recently, ptACSL4 was identified as one of lipid droplet-associated proteins in the proteome analysis [START_REF] Lupette | The architecture of lipid droplets in the diatom Phaeodactylum tricornutum[END_REF].

However, in our study the ptACSL4 mutants did not show any lipid-associated phenotype under the growth conditions. This finding is in contrast to its ortholog of the heterokont Microchloropsis gaditana. ptACSL4, also called PtACSBG (Phatr3_J45510), was recently identified as the LACS isoform of the bubblegum subfamily and shares five conserved ACSBG motifs with MgACSBG, the phylogenetically closely related LACS isoform of the bubblegum type in the eustigmatophytes M. gaditana [START_REF] Billey | Characterization of the Bubblegum acyl-CoA synthetase of Microchloropsis gaditana[END_REF]. In this oleaginous microalga, the MgACSBG mutant of point mutations led to an overall reduction of 16:1 in lipids and C20 acyls in the betaine lipid, supporting a critical role in the generation of 16:1-CoA and 18:3-CoA within the cell. We did not evaluate these two molecular species, but the data we obtained with tested acyl-CoA substrates,

showing that ptACSL4 was active exclusively with 18:1-CoA and 20:5-CoA, and inactive when provided with saturated acyl-CoAs (Table 1), are consistent with a specific function in the activation of unsaturated molecular species, similar to that reported in M. gaditana.

ptACSL1 is the only LACS isozyme located in the outermost of cER membrane with the Cterminus facing the cytoplasm (Fig. 1), indicating that it may be the most possible isozyme responsible for activating de novo-synthesized FAs exported from the plastid. During nitrogen starvation, membrane lipids seem to be protected from degradation or recycling in the ptACSL1 mutants compared to WT. In Arabidopsis, of the nine LACS isoforms only LACS9 was showed to reside in the plastid envelop and it might be the major LACS isoform involved in the export of plastidial FAs for TAG formation. Although the lacs9 null mutant had a 90% reduction of chloroplast LACS activity, the mutant did not exhibit any detectable phenotype. Further studies revealed that ER-localized LACS1 showed overlapping functions with LACS9 in TAG biosynthesis [START_REF] Zhao | Insertional mutant analysis reveals that long-chain acyl-CoA synthetase 1 (LACS1), but not LACS8, functionally overlaps with LACS9 in Arabidopsis seed oil biosynthesis[END_REF]). In our study, ptACSL1 displayed LACS activity towards all tested FAs and its disruption had no impact on the amounts of TAG (Fig. 5), which is mainly constituted of 16:0 and 16:1, suggesting one or more additional LACS isozymes may be involved in the activation of plastid-exported FAs and contribute to acyl-CoAs for storage TAG synthesis.

We identified the mitochondrial ptACSL3 as the LACS isozyme contributing to TAG synthesis.

The ptACSL3 mutants showed distinct TAG-associated phenotypes when cells were bubbled with air and in static culture (Figs 5d,5h). When algal cells are exposed to rapid changes in nutrient availability, temperature and light conditions, they must rapidly degrade or remodel storage and membrane lipids to ensure growth and survival. FA degradation thus plays an important role in the response to these changing conditions. Recently, [START_REF] Jallet | Mitochondrial fatty acid -oxidation is required for storage-lipid catabolism in a marine diatom[END_REF] confirmed the existence of a functional mitochondrial -oxidation pathway that plays a principle role in the liberation of energy from TAG-derived FAs in P. tricornutum. This pattern differs in the green alga Chlamydomonas that possesses the major reactions of FA -oxidation in peroxisomes [START_REF] Kong | Chlamydomonas carries out fatty acid -oxidation in ancestral peroxisomes using a bona fide acyl-CoA oxidase[END_REF]). In our study, when algal cultures were bubbled with filtered air, in rapidly growing cells mitochondrial FA oxidation plays a 'housekeeping' role in removing harmful free FAs which may be derived from the degradation of acyl-lipids. Disruption of ptACSL3 can result in the impairment of FA -oxidation that breaks the balance of keeping the amounts of cytosolic free FAs coming from phospholipids below the threshold and also led to a pronounced inhibition of plastidial de novo FA synthesis, which produces less FA acyls for TAG and chloroplast lipid synthesis and then reduced TAG content in the ptACSL3 mutants. By contrast, algal cells grown in static culture may not need a highly dynamic and active mitochondrial FA -oxidation to remove free FAs. Thus, we observed that a similar growth of the ptACSL3 mutants to WT under the static condition (Fig. 5a), which may ensure that the mutant could accumulate more TAGs through diverting a part of phospholipid derived FAs from mitochondrial -oxidation to the TAG synthesis pathway in the absence of ptACSL3.

In Phaeodactylum, free FAs may be derived from multiple routes: (1) de novo synthesis in the plastid, then hydrolysis by acyl-ACP thioesterase from ACP and finally export outside the plastid;

(2) turnover or recycling of membrane lipids (e.g. DGTA and PC) under some conditions such as nitrogen or carbon starvation; (3) TAG remobilization, possibly involving TAG lipases that remove acyl chains from glycerol backbone of TAG. Of the five LACSs, ptACSL1 is localized in the outermost of cER membrane and displayed the highest catalytic efficiency (kcat/Km) for 20:5 in our in vitro enzyme assay (Table 1). In addition, the ptACSL1 mutants grown in both the exponential phase (0 h) and N-deprived condition (72 h) showed slight decrease in the levels of 20:5-containing species in the plastid glycolipid of MGDG (Dataset S1), suggesting a possible role in transfer of ER-derived 20:5 to plastid membranes. ptACSL3 and ptACSL5 may play a role in generating acyl-CoAs that are channeled into mitochondria or peroxisome for -oxidation (Fig. 10). How ptACSL2 and ptACSL4 are involved in lipid metabolism in Phaeodactylum remains to be further elucidated.

Since the Phaeodactylum genome encodes multiple LACS isoforms, functional redundancy possibly exists within the lacs gene family. To distinguish the contributions of each ptACSL to lipid metabolism, generation of higher-order lacs KO mutants using multiplexed CRISPR/Cas9 method will provide more insights into the precise roles of each LACS and uncover the complex network of functions of all LACS isozymes in Phaeodactylum. vs 0d and 4d vs 0d of wild type and the ptACSL3 mutants. Data are presented in Table S2.

  performed mRNA-seq analysis to obtain mechanistic insight into the transcriptome response of Phaeodactylum to the shift of growth state. The global gene expression of Phaeodactylum as a function of time (i.e., over three time points, 2, 3, and 4 d) under the bubbling of air and static condition (0 d) was measured by mRNA-seq. A total of 5,622 genes, accounting for 45% of all the annotated genes in the Phaeodactylum genome (http://protists.ensembl.org/Phaeodactylum_tricornutum/Info/Index/), were aligned with at least one read (FPKM [Fragments Per Kilobase of transcript per Million mapped reads] value >10) in each biological sample (Supporting information Dataset S2). Based on the definition of differential gene expression, 746 genes were found to be significantly regulated at more than one time point in either WT or the mutant sample (Dataset S2). Growth state transition led to strong transcriptomic responses, which was revealed by the gradually increased numbers of differentially expressed genes (DEGs) during the growth in air-bubbling cultures. Compared with the static culture (0 d), 1937, 2543, and 3830 genes were significantly regulated (P<0.05) at 2 d, 3 d, and 4 d respectively in WT;while 2986, 3342, and 3913 genes were detected to be differentially expressed at 2 d, 3 d, and 4 d respectively in the ptACSL3 mutants. We used these DEG datasets to perform GO analysis with the Phaeodactylum genome as the reference, and found that the most obvious difference in the enriched GO terms between WT and the ptACSL3 mutant was observed at 2 d. In WT, these DEGs were mainly enriched for cell division and structural constituent of ribosome at 2 d; whereas in the ptACSL3 mutants the most over-represented GO term was "mitochondria" (Figs9a, 9b).In the mitochondria of most eukaryotes, both tricarboxylic acid cycle and FA -oxidation occur, which play a central role in the catabolism of organic fuel molecules such as glucose, FAs and amino acids. Mitochondria contain the full enzymatic machinery to oxidize FAs. After the conversion of FAs into CoA esters by a LACS enzyme, the acyl-CoA esters can enter the -oxidation spiral starting via the well-established cascade of four steps involving dehydrogenation, hydratation, dehydrogenation again and thiolytic cleavage of the acyl-CoA esters. We further determined if the expression patterns of genes associated with mitochondrial FA -oxidation were affected in the ptACSL3 mutants. In WT, genes encoding carnitine O-acyltransferase (CAT, Phatr3_J859), enoyl-CoA hydratase/3-hydroxyl-CoA dehydrogenase (HADA, Phatr3_J35240) and-ketoacyl-CoA thiolase (KCT3, Phatr3_J28068) known as the mitochondrial trifunctional protein -subunit, were

Figure 1 .

 1 Figure 1. In vivo localization of Phaeodactylum LACS isoforms. Self-assembly GFP system was used for analysis of ptACSL1 plastid localization. The (c)ER-characteristic fluorescence pattern could be observed when ptACSL1 fused to GFP_S11 was simultaneously expressed with a cytosolic GFP_S1-10. Expression of ptACSL1_S11 with the ER marker (PDI) fused to GFP_S1-10 led to no fluorescence. Expression of full-length ptACSL2 and ptACSL3 with C-terminally fused eGFP indicated the localization in plastid. Localization of ptACSL4:eGFP fusion led to indistinct results (plastid, plasma membrane and cytosol). eGFP with N-terminal fusion with ptACSL5 showed the fluorescence signal in the peroxisome. TL, transmitted light; PAF, plastid autofluorescence; eGFP, enhanced green fluorescence protein; PAF/eGFP, overlap of plastid and GFP fluorescence. Scale bar represents 10 m.

Figure 2 .

 2 Figure 2. Expression patterns of Phaeodactylum LACS family proteins. Lysates of Phaeodactylum cultures under nitrogen deprivation condition (A) and supplied with filtered air (low CO2, ~0.04%CO2) for 2 h (a2h) and 6 h (a6h) or high concentrations of CO2 (1%, 4% and 10%) for 2 h and 6 h (B) were analyzed by Western blotting using polyclonal antibodies against ptACSL1-5. Loading of

Figure 3 .

 3 Figure 3. Multiple targeted mutagenesis of ptACSL1-5 genes by CRISPR/Cas9 in Phaeodactylum. (A) Schematic depiction of the multiple CRISPR/Cas9 method for simultaneously targeting multiple sites in the Phaeodactylum genome. Each of the CRISPR-episome plasmids consists of three different gRNA expression cassettes driven by the U6 promoter, Cas9 protein, the ShBle resistance gene cassette, and the yeast-derived CEN6-ARSH4-HIS3 element conferring episomal replication in the diatom Phaeodactylum. The gRNAs are transcribed independently and direct Cas9 to multiple targets. (B) Frequencies of different editing events observed in ptACSL1-5 target regions induced by a single gRNA or double gRNAs using the multiple CRISPR/Cas9 system, which were calculated from 30 mutated sites (not including those of homozygous fragmental deletions between two targeted sites). (C) Comparison of mutation frequencies of large fragment deletions between two targeted sites occurred among five ptACSL genes.

Figure 4 .

 4 Figure 4. Genome editing of ptACSL1 and ptACSL3 using multiple CRISPR/Cas9 system led to the generation of fragmental deletions in Phaeodactylum. Schematic representations show the fragmental deletions between two targeted sites of the ptACSL1 (a) and ptACSL3 (b) mutants. A pair of primers was designed to detect the large fragment deletions induced by multiple CRISPR/Cas9 in the ptACSL1 (c) and ptACSL3 (d) mutants with PCR using genomic DNAs and total complementary DNAs (cDNAs) as templates. For DNA sequencing, PCR amplifications were carried out using primer pairs flanking the designed target sites, and the PCR products were sequenced directly using internal specific primers. Representative sequences of fragment deletions in the ptACSL1 (e) and ptACSL3 (f) mutants were aligned with that of wild type. The PAM region is shown in blue color letters, and the number at the end shown in red color letters indicates deleted (-) bases between two Cas9 cuts.

Figure 5 .

 5 Figure 5. Phenotype analyses of the ptACSL1 and ptACSL3 mutants. (a) Growth curves of the ptACSL1 and ptACSL3 mutants incubated in static cultures for 12 days. Cell densities were determined with a Z2 Coulter Counter. Results are the average of three biological replicates (independent lines) with error bars indicating standard deviations (n=3). (b) Nitrate concentrations in culture media for the ptACSL1 and ptACSL3 mutants incubated in static cultures. (c) Nile red fluorescence intensities of the ptACSL1 and ptACSL3 mutant cells incubated in static cultures for

Figure 6 .

 6 Figure 6. Comparative analysis of total fatty acid content (a) and fatty acid composition (b) (Mol of total fatty acids) of wild type and the ptACSL3 mutants grown at exponential and stationary phases.

Figure 7 .

 7 Figure 7. Compared analysis of the various glycerolipids (a) and lipid molecular species (b, MGDG; c, DGDG; d, PC) between wild type and the ptACSL3 mutants grown at the exponential and stationary phases. Values are means of three biological replicates. *Asterisk indicates significant difference between wild type and the mutant as determined by the Student's t-test. *, P<0.05, **P<0.01.

Figure 8 .

 8 Figure 8. The disruption of ptACSL3 affects molecular composition of TAG. LC-MS was used to quantify different molecular species of TAG in wild type and the ptACSL3 mutants in three biological replicates. A heat map shows the percentage abundance of 20 major TAG species in each biological replicate relative to the average percentage abundance of that species in wild type. Algal cultures were bubbled with filtered air and samples were harvested at day 7 (a), or cells were grown under static culture condition and harvested at day 9 (b) for lipid extraction and TAG profiling.

Figure 9 .

 9 Figure 9. Transcriptional change of the ptACSL3 mutants upon the shift from static culture to

Figure 10 .

 10 Figure10. Predicted LACS-mediated lipid metabolic pathways in Phaeodactylum. De novo fatty acid (FA) synthesis takes place in the plastid. 16:0-ACP is released from the fatty acid synthase, and then enters the prokaryotic pathway to be desaturated by soluble acyl-ACP desaturases to produce C16 FAs with one or more double bonds which are used for synthesis of MGDG, DGDG, SQDG and PG as chloroplast membrane lipids. The majority of 16:0 and 16:1 in the plastid is exported to the cytosol, converted to 16:0-CoA and 16:1-CoA and enters the Kennedy pathway for TAG biosynthesis. In the cytosol, 16:0-CoA can be elongated to 18:0-CoA and further desaturated to produce 18:1Δ9-CoA. This FA enters long-chain polyunsaturated FA synthesis pathway to produce 20:5 (EPA) and 22:6 via the combined n3 and n6 pathways. EPA can be imported back into the plastid via the unknown omega-3 pathway for the synthesis of chloroplast membrane lipids. Some free FAs, mainly 16:0 and 16:1 species, can be released from TAG hydrolysis, then activated by LACS enzymes and transported into mitochondria or peroxisomes for -oxidation.Enzymes are shown in red: PAD, palmitoyl-ACP Δ9-desaturase; FAD, fatty acid desaturases; Des6, plastidic soluble Δ6-desaturase; MGD, MGDG synthase; DGD, DGDG synthase; SQD2, SQDG synthase; GPAT, glycerol-3-phosphate acyltransferase; LPAAT, lysophosphatidic acid acyltransferase; PAP, phosphatidic acid phosphatase; DGAT, diacylglycerol acyltransferase. G3P, glycerol-3-phosphate; LPA, lysophosphatidic acid; PA, phosphatidic acid; DAG, diacylglycerols; TAG, triacylglycerols; MGDG, monogalactosyldiacylglycerol; SQDG, sulfoquinovosyldiacylglycerol; PC, phosphatidylcholine; DGTA, diacylglyceryl-hydroxymethyl-

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (41776175 and 31961133008 to YG) and the Agricultural Science and Technology Innovation Project of Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2013-OCRI) and in part by the 2021 CEA-CAS bilateral program (to A. Amato, EM, HH, and YG). A. Amato and EM were also supported by Agence Nationale de la Recherche (ANR-10-LABEX-04 GRAL Labex, Grenoble Alliance for Integrated Structural Cell Biology; ANR-11-BTBR-0008 Océanomics;

IDEX UGA CDP Glyco@Alps; Institut Carnot 3BCAR).

Authors'contributions

XH, WC, A. Amato, DM, HJ and LY performed experiments. JJ, EM, HH and FH analyzed data. MM and A. Allen contributed to the generation of ptACSL mutant lines by multiplexed CRISPR/Cas9 method. YG planned and designed the research and wrote the manuscript. XH and WC contributed equally.


Paméla Aoun

Ahmad Hammoud

Mayte A Martínez-Aguirre

Laurent Bouteiller

Matthieu Raynal
email: matthieu.raynal@sorbonne-universite.fr

Asymmetric hydroamination with far fewer chiral species than copper

Introduction

Although already identified in the sixties 1 on copolymers derived from optically active vinyl monomers, 2 the ability of a few chiral monomers (the "sergeants") to dictate their preferential conformation to a large fraction of achiral monomers (the "soldiers") was unambiguously revealed by Green and co-workers in 1989 during their thorough study of polyisocyanate copolymers. 3 Poly(alkyl isocyanates) adopt helical conformations in solution, which are particularly sensitive to weak chiral influences. 4,5 The "sergeants-andsoldiers" (S&S) effect was remarkable in this class of dynamic helical copolymers 6 since 4% of sergeants yielded a copolymer with an almost single-handed screw sense. This feature was later on observed for a large array of helical covalent polymers, 6,7 helical supramolecular polymers, 8,9 as a well as for discrete assemblies, 10,11 and thermodynamic description of this phenomenon was quantitatively assessed by means of statistical mechanical theory 12 and numerical models. 13 Implementation of the S&S effect in asymmetric catalysis requires a particular design in which the "soldier" bears a catalytic site and the "sergeant" is catalytically inactive but dictates the preferred handedness of the polymer main chain. 14 A key feature of this relatively new class of asymmetric catalysts is that catalytic centers are intrinsically achiral but experience the chiral environment provided by the helical backbone, which ultimately leads to stereodiscrimination in the transition states. In addition, it represents a unique, yet underexplored, strategy to decrease the amount of chiral inducers in catalytic processes. 15,16 Examples of S&S-type asymmetric catalysts have mainly been reported for helical covalent polymers, [START_REF] Reggelin | Proc. Natl. Acad. Sci[END_REF][START_REF] Takata | [END_REF] the most potent ones being based on the poly(quinoxaline-2,3-diyl) (PQX) scaffold. 14,19,20 PQX helical catalysts appended with phosphine ligands were found to provide excellent enantioselectivities in a range of palladium-catalysed reactions, whilst nitrogen-containing PQXs were employed in coppermediated 21,22 and organocatalytic processes. [23][24][25] PQX catalysts reported to date either consisted of copolymers embedding a large of fraction of chiral monomers (95%) [26][27][28][29][30][31][32] or on terpolymers containing an additional achiral monomer acting as "spacer" between the catalytic unit, [33][34][35][36] and for which, the "sergeants" are actually in excess relatively to the catalytic units. Alternatively, external guests [37][38][39][40] or solvents 41 can be added in order to induce and memorize a single handedness to helical catalysts but again their loading is large relatively to catalytic units. Transferring the chirality of the sergeant to a large number of catalytic centers is thus an ongoing challenge for S&S-type catalysts. Since 2013, 42 our group reported examples of S&S-type supramolecular helical catalysts, composed of phosphine- functionalized and enantiopure monomers, both based on the complementary benzene-1,3,5-tricarboxamide (BTA) scaffold. 43 These examples exploited the well-established S&S effect observed in BTA helical assemblies [44][45][46][47][48] and in related discotic monomers. 49 It was notably possible to perform the rhodiumcatalysed hydrogenation of dimethyl itaconate with a copolymer composed of one sergeant for two rhodium centers on average, thus representing the first example for which a substoichiometric amount of a chiral inducer was used relatively to the metal catalyst, without significant deterioration of the enantioselectivity. 50 The catalytic mixture between BTA P H (Chart 1a) and a sergeant (as represented in Chart 1b) was also evaluated in the copper-catalysed hydrosilylation of acetophenone derivatives, [51][52][53] but in that case, efficient S&S effect was observed only if a third co-monomer (a-BTA) was also present in the helical assemblies (Chart 1c). It was found that a-BTA both stabilize the assemblies and remove the conformational defects thus allowing efficient hydrosilylation of 4-nitroacetophenone in 90% enantiomeric excess (e.e.) at 200 K and as low as 0.25% of sergeant in the mixture. 54 Extending the scope of this highly efficient S&S effect requires to probe its validity with other soldier structures and under different reaction conditions. Herein, we report our efforts dedicated to the implementation of S&S-type BTA helical catalysts in the copper-catalysed hydroamination of styrene, a reaction that proved to be efficiently catalysed by conventional chiral diphosphine ligands. [55][56][57] Selection of the best-performing BTA ligand partner (the "soldier") and thorough screening of the experimental conditions afforded the amine product in 75% e.e. with as low as 0.51 mol% of sergeant in the catalytic mixture, i.e. one chiral molecule for 10 copper centers.

Results and discussion

Design and synthesis of BTA monomers. The asymmetric Cu-H promoted hydroamination of alkenes 55-57 is performed efficiently by a relatively narrow number of chiral diphosphine ligands, 58-83 with particularly strong effects exerted by substituents located on the aryl rings attached to the phosphorus atom. 84, 85 In addition to BTA ligands, BTA P H and BTA P Me , previously investigated in the copper-catalysed hydrosilylation of acetophenone derivatives, 51,52,54 it appears desirable to investigate more electron poor ligands as it may facilitate the hydrocupration step of the catalytic cycle. 85 Accordingly, BTA P p-F , BTA P p-CF3 and BTA P CF3 (Chart 1a) were prepared following previously established procedures and characterized by conventional techniques (ESI †). Initially screened co-assemblies were composed of one of these BTA . By comparison with the gmax value previously determined for homochiral assemblies of BTA P H (≈ 7.0× 10 -4 ), 54 it can be surmised a relatively high optical purity of the supramolecular helices for all mixtures. nd= not determined ligands and of (R)-BTA (resp. (S)-BTA) as the latter component has demonstrated its ability to intercalate into the helical assemblies of the BTA ligands 53 and induce a preferred left-(resp. right) handedness to the supramolecular co-assemblies (Chart 1b). 51 Characterization of the S&S-type co-assemblies. Prior to catalytic screening, we determined the composition, geometry and chiroptical properties of the BTA ligand homo-assemblies and of the S&S-type co-assemblies following our previously established analytical procedure. 1). The fraction of (R)-BTA monomers (sergeants) in the stacks, fs s , can be accurately determined, with similar results deduced independently from FT-IR and SANS analyses, and is found to be close to the fraction of (R)-BTA monomers initially introduced into the mixtures (compare fs 0 and fs s in Table 1). This means that (R)-BTA efficiently intercalates 53,89,90 into the stacks of the BTA ligands leading to the formation of long helical co-assemblies. Circular dichroism (CD) analyses (Figure S4a) revealed a negative band with a maximum around 300 nm for all S&S-type mixtures. This band, which belongs to the BTA ligand only (Figure S4b), is an induced CD (ICD) band 91 and thus reflects the chiral environment experienced by the ligand in the helical co-assemblies. By analogy with the BTA P H containing mixture, it can be inferred that all co-assemblies are lefthanded 51 and possess a relatively high optical purity (see g values, Table 1). 54 Overall, the combination of FT-IR, SANS and CD analyses indicates that the PAr2 moiety in this BTA ligand series has thus no significant influence on the structure of the helical co-assemblies. A subtle effect can be detected for the BTA P Me mixture which exhibits a lower g value as a probable result of a slightly lower amount of sergeants incorporated into the stacks, relatively to other co-assemblies. Likewise, the ICD band displayed by the BTA P CF3 mixture (Figure S4a) has a slightly different shape with two extrema at 285 and 305 nm instead of a single one at ca. 300 nm for all the other mixtures. This might be due to a subtle difference in the conformation adopted by the 3,5-bis-CF3-substituted PAr2 moiety. Finally, it is important to note that no significant change of the structure of the co-assemblies is expected upon copper binding, 52 thus coassemblies can be schematically represented as shown in Chart 1b. Although the precise coordination mode of the copper centers at the periphery of the helices cannot be ascertained, the formation of long and predominantly one-handed helical co-assemblies whatever the nature of the BTA ligand motivates their evaluation in catalysis.

Influence of BTA ligand in the copper-catalysed hydroamination of styrene. Initial conditions for the evaluation of the different S&Stype helical catalysts were selected as follows: i) a concentration in BTA ligand of 17 mM to ensure the presence of sufficiently long assemblies, ii) a fraction of sergeants of 50% to promote the formation of single-handed helices, iii) Amine-DM to minimize the detrimental direct reduction of the amine electrophile by Cu-H species, 60,92 and iv) a high concentration in dimethoxy(methyl)silane (DMMS) to increase the reaction rate 61 and provide a sufficient amount in N,N-dibenzyl-1phenylethanamine (DBA) whatever the ligand present in the S&S-type mixtures. The reactions were monitored by 1 H NMR The optical purity of DBA was determined by chiral HPLC analysis and configuration of the DBA enantiomers was established according to the literatu re. 55,61 Error bar according to integration of HPLC signals.

with 1,3,5-trimethoxybenzene as an internal standard and the results are plotted in Figure 1 (see Figures S5 to S9 for the NMR spectra). All helical catalysts promote rapid consumption of styrene and formation of DBA at the onset of the reaction followed by rapid plateauing of the reaction progress. This might be explained by rapid consumption of the reactants, including Amine-DM (ESI †), and possibly catalyst deactivation. 85 Whatever the ligand, styrene is only partly transformed into DBA: i) ethylbenzene, presumably formed by hydrolysis of the alkyl copper intermediate, 78 is detected in 10±5% in each mixtures (Figure S10) and ii) alternative hydrofunctionalization processes, such as styrene hydrosilylation, 93 constitute other potential side reactions. The nature of the BTA ligand strongly influences the outcome of the catalytic reaction (Table 2). CF3 substituted ligands, BTA P p-CF3 and BTA P CF3 , provided DBA with higher rates relatively to other ligands probably because the hydrocupration step is facilitated in these systems. All S&S-type helical catalysts furnish (R)-DBA as the major enantiomer, as expected from their same sense of rotation (vide supra), however with markedly different optical purities. Ligands with substituents at the meta positions of the phosphine aryl rings, BTA P Me and BTA P CF3 , give significantly higher enantioselectivities than the non-modified The entry was repeated 9 times, yielding the following e.e. values: 73%, 65%, 67%, 65%, 63%, 68%, 73%, 70% and 69%. Based on this repeatability assessment, a mean e.e. value of 68% can be deduced as well as an error bar of ±6% (equals to ±½ variance). Corresponding NMR yields are: 83%, 80%, 80%, 84%, 83%, 80%, 80%, 84% and 80% providing a mean yield of 82% with ±2% error bar (within the experimental error of NMR integration). (c) (S)-BTA was used instead of (R)-BTA.

ligand (BTA P H

) and substitution at the para position appears to be detrimental for the enantioselectivity. Modelling of transition states with chiral diphosphine Cu-H complexes have revealed the importance of through-bond and through-space interactions between the ligand and substrate for accelerating the hydrocupration step. 84,85 It is plausible that these interactions also affect the selectivity of the enantiodetermining step, as observed here. Undeniably, the helical catalyst composed of BTA P CF3 and (R)-BTA is the most efficient investigated system for the copper-catalysed hydroamination of styrene. Catalyst optimization. A range of parameters were screened to improve the catalytic performance of the helical catalyst based on the BTA P CF3 ligand (Tables S1.1 to S1.5). Parameters that led to significant improvement of the catalytic system relatively to initial conditions (entry 1) are shown in Table 3. The amount of DMMS can be decreased to 400 mol% with only a small erosion of the DBA yield (entry 2). Performing the reaction at 313 K leads to a greater DBA yield (82%) without dwindling of the enantioselectivity (entry 3). The reaction was repeated 9 times under these conditions providing fairly reproducible DBA yield (82±2 %) and e.e. (68±6 %). Screening of various copper sources (Table S1.2), copper/ligand ratios (Table S1.2), silanes (Table S1.3) and amine electrophiles (Table S1.4) does not lead to further improvement. Further increase of the temperature to 333 K neither proves to be beneficial (entry 4). It is interesting to note that the concentration in BTA P CF3 can be advantageously decreased down to 5 mM with no alteration of the catalytic performance (Table S1.5). Implementation of these modified conditions to BTA P Me -containing helical catalyst does not have a similar effect; low yields (ca. 20%) are obtained whatever the conditions (Table S2).

Finally, the yield in DBA is further improved by adding a larger excess of Amine-DM (entry 5), thus minimizing the extent of competing side reactions. When the reaction was conducted in presence of (S)-BTA instead of (R)-BTA, the opposite enantiomer of DBA is obtained with the same selectivity (entry 6), as expected since these S&S-type helical catalysts exhibit opposite handedness. A larger scale catalytic experiment (0.45 mmol) allowed the isolation of pure DBA in 60% yield and 64% e.e. (Figure S11).

Asymmetric hydroamination with low amount of sergeants.

Benefiting from the aforementioned screening of operating parameters, the catalytic hydroamination of styrene was performed with fractions of (S)-BTA (relatively to achiral monomers) ranging from 0.26% to 50%, which correspond to catalytic loadings (relatively to styrene) comprised between 0.05 mol% and 20 mol% (see Table S3 for the exact compositions). DBA isolated yields and e.e. values for reactions in presence and absence of a-BTA as the achiral additive 54 are plotted in Figure 2. In presence of 0.10 mol% of (S)-BTA, the mixture lacking a-BTA exhibits low yield (22%) and e.e. (10%). The low yield is likely the result of the limited solubility ‡ of this mixture at low (S)-BTA fractions whereas the low e.e. value is indicative of the absence of S&S effect. In sharp contrast, uniform NMR yields (80-99%) and isolated yields (55%-74%) are observed when the reactions are conducted in presence of a-BTA, whatever the amount of (S)-BTA. In addition, the effect of a-BTA on the enantioselectivity in DBA shows the anticipated hallmark of a high S&S effect: e.e. values around 80% are maintained for fraction of (S)-BTA as low as 2.5%. From there, the selectivity decreases smoothly to 42% for fs 0 = 0.26%. The magnitude of the S&S effect is remarkable relatively to previously reported helical catalysts, [33][34][35][36] but yet lower than that observed for the hydrosilylation of 4-nitroacetophenone with a related BTA system (optimal selectivity reached for fs 0 = 0.5%). 54 It is possible that a larger fraction of defects is present in the hydroamination catalyst as the result of a higher temperature required for efficient DBA formation. 48 Remarkably, the selectivity in DBA is also higher for the threecomponent system allowing to reach up to 81% e.e. versus 68±6% e.e. for the two-component one. Interestingly, an opposite effect was observed for the hydrosilylation reaction, i.e. sergeant-rich three-component mixture yielded lower selectivity. 54 This likely originates from a subtle change in the conformation of BTA P CF3 in presence of a-BTA as confirmed by different CD signatures of the three-and two-component BTA systems (Figure S12). These beneficial effects of the achiral additive allow DBA to be isolated in reasonable yield (55%) and 42% e.e. with as low as 0.05 mol% of (S)-BTA, i.e. one chiral molecule for 100 copper centers. Increasing the sergeant to copper ratio to 1/10 affords DBA in 64% yield and 75% e.e., i.e. better catalytic results than with a sergeant/copper ratio of 1 in absence of a-BTA. These results indicate that, in contrast to conventional organometallic catalytic systems, the optimal enantioselectivity of helical BTA catalysts can also be tuned by achiral additives that do not directly interact with coordination spheres of the metal centre. 94,95

Conclusion

Guiding rules are necessary to further exploit sergeants-andsoldiers-type helical systems in asymmetric catalysis. The Please do not adjust margins present work demonstrates that the recently disclosed ability of an achiral benzene-1,3,5-tricarboxamide additive, a-BTA, to dramatically increase the magnitude of the S&S effect in the Cu-H catalysed hydrosilylation 54 is also valid for the hydroamination reaction, even though more drastic conditions (313 K, excess of amine electrophile reagent) is required for this reaction to proceed efficiently. The combination of a-BTA and optimized reaction conditions affords the hydroamination product with reasonable yield (69%) and e.e. (81%). The effect of a-BTA is actually threefold since it proves to be beneficial not only for the magnitude of the S&S effect (close to optimal selectivity with as low as 0.51 mol% of sergeant) but also for the yield and enantioselectivity of the catalytic process. It motivates easily assembled multi-component BTA helical systems to be investigated in related Cu-H reactions [55][56][57] as well as different catalytic transformations.

Experimental

Sergeants-and-soldiers experiments with BTA P CF3 [9. were added to the tube and the mixture was briefly heated to reflux and stirred for 15 minutes at room temperature. Styrene (51 μL, 0.45 mmol, 100 mol%), and Amine-DM (292 mg, 0.81 mmol, 180 mol%) were added to the tube and the tube was flushed with argon for 10 seconds. The reaction mixture was heated to 40°C and DMMS was added (223 µL, 1.8 mmol, 400 mol%). After 2 hours, the reaction mixture was cooled down to room temperature and 2 mL of a saturated aqueous solution of Na2CO3 was added as well as ethyl acetate (1 mL). The phases were separated and the aqueous layer was extracted with ethyl acetate (2×1 mL). 1,3,5-trimethoxybenzene (75.7 mg, 0.45 mmol, 100 mol%) was added to the combined organic phases and the NMR yield was established after evaporation of the solvent. Acetonitrile was added to the crude and the insoluble material was discarded by filtration. The crude material was purified by flash column chromatography over silica gel, eluting with dichloromethane/ethyl acetate yielding (S)-DBA as a colourless oil (see isolated yields in Table S.3). The optical purity of DBA in each sergeants-and-soldiers mixture was determined by chiral HPLC. For additional experimental procedures, see the ESI †.

Chart 1

 1 (a) Chemical structures of the BTA monomers employed in this study. (b) Schematic representation of the S&S-type helical assemblies used in the catalytic screening. (c) Schematic representation of the S&S-type helical assemblies operating with low amount of sergeants (high S&S effect). (d) Initial conditions of the catalytic screening.

  (a) fs 0 = fraction of sergeant introduced into the solution= [(S)-BTA] 0 /([(S)-BTA ]0 + [BTA ligand] 0 ). (b) Molar fraction of BTA in stacks= ([BTA ligand] 0 +[(R)-BTA in stacks determined by FT-IR])/(total BTA concentration). (R)-BTA monomers that are not in the stacks form dimers (ESI † ). 86-88 (c) fs s = fraction of sergeant in stacks as determined by SANS (fs s -SANS) or by FT-IR (fs s -IR) analyses. fs s = ([(R)-BTA in stacks]/([(R)-BTA in stacks] + [BTA ligand] 0 ). (d) Kuhn anisotropy factor (g) was measured at the maximum of CD signals (close to 300 nm in all cases)

Figure 1

 1 Figure 1 Influence of the BTA ligand in the copper-catalysed hydroamination of styrene. (a) Conversion of styrene (a) and DBA formation (b) versus time. Inset: reaction points at the onset of the reaction.

( a )

 a Styrene conversion after 120 minutes. Error bar according to integration of the different styrene signals. (b) Initial rate corresponding to the derivative of the reaction points at the onset of the reaction. (c) NMR yield measured after 120 minutes, relatively to the internal standard. Error bar according to integration of DBA signals. (d)

  (a) NMR yield measured relatively to the internal standard. Error bar according to integration of DBA signals. (b)

Figure 2

 2 Figure 2 DBA isolated yield (left) and e.e. (right) for S&S-type catalytic experiments performed with BTA P CF3 and various amounts of (S)-BTA, in presence or absence of a-BTA. Inset: zoom on the region of low fs 0 values, catalytic loading in (S)-BTA is indicated for selected points. fs 0 = fraction of sergeant introduced into the solution= [(S)-BTA] 0 /([(S)-BTA] 0 + [BTA P CF3 ] 0 + [a-BTA] 0 ).

  6 mM], a-BTA [9.6 mM], and (S)-BTA: an oven-dried test tube was loaded with BTA P CF3 (43.4 mg, 45.0 μmol, 10.0 mol%), [Cu(OAc)2] (4.1 mg, 22.5 μmol, 5.0 mol%) and dry THF (500 μL). The solvent was removed under vacuum and the tube was kept under vacuum (10 -3 mbar) for 1 hour. (S)-BTA (0.23-90 μmol, 0.05-20.0 mol%), a-BTA (49.1 mg, 45.0 μmol, 10.0 mol%), and toluene (4000 μL)

Table 1

 1 Characterization of the helical co-assemblies for the S&S-type mixtures between (R)-BTA and the different BTA ligands. [BTA ligand]= 3.33 g.L -1 in toluene-d8 except for SANS analysis of BTA P H mixture (4.0 g.L -1 ).

	BTA ligand in the	fs 0	BTA in	fs s -SANS	fs s -IR	│g│ value at maximum
	S&S-type mixtures	(%) a	stacks (%) b	(%) c	(± 1%) c	(10 -4 ) d
	BTA P H	39	86±3	35±4	30	6.8
	BTA P Me	41	81±2	28±6	28	5.2
	BTA P p-F	41	88±2	nd	32	7.7
	BTA P p-CF3	44	86±3	nd	34	7.1
	BTA P CF3	47	86±3	31±6	39	6.5

51-53,87

Indeed, depending on their structure and on the conditions, BTAs can either selfassemble into stacks or into dimers, 86-88 but only the stacks can be expected to show strong S&S effects. Fourier-transform Infrared (FT-IR, Figures

S1-S2

) analyses indicated that all BTA ligands form long hydrogen-bonded stacks. ‡ Mixtures containing the different BTA ligands (3.33 g.L -1 ) and (R)-BTA (39%≤ fs 0 ≤ 47%, Table

1

) were analysed in toluene-d8, a suitable solvent for both assemblies' formation and catalysis. Small-Angle Neutron Scattering (SANS) analyses (Figure

S3

) are consistent with long (length> 27 nm i.e. DPn> 35) and onedimensional objects. (R)-BTA monomers are incorporated into the stacks of the BTA ligands as indicated by FT-IR and SANS analyses. Most of the BTA monomers in these S&S-type mixtures are actually present in the stacks (BTA in stacks≥ 81%, Table

Table 2

 2 Catalytic results in the copper-catalysed hydroamination of styrene for the different S&S-type mixtures.

	BTA ligand in the S&S-type mixtures	styrene conversion (± 5%) a	initial rate (10 -3 M.min -1 ) b	DBA yield (±4 %) c	DBA e.e. (±1 %) d
	BTA P H	75	3.8	17	29 (R)
	BTA P Me	50	4.7	22	58 (R)
	BTA P p-F	71	3.2	16	19 (R)
	BTA P p-CF3	94	12	36	7 (R)
	BTA P CF3	100	36	52	68 (R)

Table 3

 3 Catalyst optimization.

	Entry	DMMS (x mol%)	Amine-DM (x mol%)	Temperature (K)	DBA yield (± 4%) a	DBA e.e. (%)
	1	900	120	298	52	68 (R)
	2	400	120	298	42	69 (R)
	3	400	120	313	82	68±6 b (R)
	4	400	120	333	75	63 (R)
	5	400	180	313	93	69 (R)
	6	400	180	313	93	69 (S) c

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Acknowledgements

This work was supported by the French Agence Nationale de la Recherche (project ANR-17-CE07-0002 AbsoluCat, Ph.D. grant of A.H., PDRA grant of P.A.) and by the Consejo Nacional de Ciencia y Tecnologia (CONACYT, PDRA grant of M. M.-A.). Jacques Jestin (LLB, Saclay) and Omar Khaled (IPCM, Sorbonne Université) are acknowledged for assistance with SANS and chiral HPLC experiments, respectively. Lucrèce Matheron-Duriez and Gilles Clodic are acknowledged for the ESI analysis of catalytic samples.

Notes and references ‡ BTA ligand, BTA P CF3 , was not soluble on its own in toluene-d8 and thus only co-assemblies formed in presence of (R)-BTA have been characterized. Likewise, good solubility of this twocomponent mixture is obtained at the condition that sufficient amount of (S)-BTA (fs 0 ≥ 25%) is present in the mixture.

Author Contributions

Conflicts of interest

There are no conflicts to declare.