Smith Richard

Juliette Jouhet

Chiara Gandini

Vladimir Nekrasova

Eric Marechal

Johnathan A Napier
email: johnathan.napier@rothamsted.ac.uk

Olga Sayanova
email: olga.sayanova@rothamsted.ac.uk

Richard Smith

Vladimir Nekrasov

J A N Wrote

Plastidial ACP ∆9-desaturase modulates EPA biosynthesis and TAG accumulation in Phaeodactylum tricornutum

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Main Text

Introduction

In recent years microalgae have attracted considerable attention as an alternative and sustainable platform to produce high value lipids. However, biotechnological improvement of algal strains requires further advances in our understanding of algal physiology and metabolic pathways, coupled with the refinement of molecular toolkits.

Heterokonts represent a major group of microalgae containing more than [START_REF] Arao | BIOSYNTHESIS OF POLYUNSATURATED LIPIDS IN THE DIATOM, PHAEODACTYLUM-TRICORNUTUM[END_REF].000 species and are major constituents of the collective biomass known as phytoplankton. Diatoms are thought to have derived from a secondary endosymbiotic event when a red alga was engulfed by a eukaryotic host cell. This led to the formation of secondary plastids surrounded by four membranes, corresponding to the exosymbiont endomembrane, the plasma membrane of the engulfed alga, and the two membranes of the primary plastids [START_REF] Prihoda | Chloroplast-mitochondria cross-talk in diatoms[END_REF]. It was demonstrated that in Heterokont species the outermost secondary chloroplast limiting membrane forms a continuum with the outer membrane of the endoplasmic reticulum (ER) [START_REF] Flori | Ultrastructure of the Periplastidial Compartment of the Diatom Phaeodactylum tricornutum[END_REF]. The chloroplast lipid profiles are characterized by the presence of four Recently, comprehensive studies of the glycerolipid content of two Phaeodactylum tricornutum ecotypes, Pt1 and Pt4, have demonstrated that the glycerolipid profiles are dominated by MGDG, SQDG, DGDG and phosphatidylcholine (PC) (~75% of the total content) while non-polar lipids, diacylglycerols (DAG) and triacylglycerol (TAG), represent ~ 3% of the total glycerolipids [START_REF] Abida | Membrane Glycerolipid Remodeling Triggered by Nitrogen and Phosphorus Starvation in Phaeodactylum tricornutum[END_REF][START_REF] Popko | Metabolome Analysis Reveals Betaine Lipids as Major Source for Triglyceride Formation, and the Accumulation of Sedoheptulose during Nitrogen-Starvation of Phaeodactylum tricornutum[END_REF]. In many Heterokonts species, thylakoid lipids contain the long chain polyunsaturate, eicosapentaenoic acid (EPA, 20:5n3) [START_REF] Abida | Membrane Glycerolipid Remodeling Triggered by Nitrogen and Phosphorus Starvation in Phaeodactylum tricornutum[END_REF][START_REF] Liang | Profiling of Polar Lipids in Marine Oleaginous Diatom Fistulifera solaris JPCC DA0580: Prediction of the Potential Mechanism for Eicosapentaenoic Acid-Incorporation into Triacylglycerol[END_REF]. EPA synthesis occurs in the ER [START_REF] Guschina | Lipids and lipid metabolism in eukaryotic algae[END_REF], implying the existence of unknown pathway(s) for its trafficking from the ER into the chloroplast. The current understanding of lipid metabolism and fatty acid synthesis in algae is based on genomic analysis and metabolic models derived from higher plants.

However, since higher plants neither contain EPA nor display the hallmarks of secondary endosymbionts, such parallels have their limitations. In terms of commonality, de novo FA synthesis in diatoms occurs in plastids and leads to formation of a C16-acyl carrier protein (ACP) by the fatty acid synthase of type II (FASII). In plants, the C16-ACP is then i) either retained in the plastids where it can be esterified by chloroplastic acyltransferase to glycerol-3-phosphate (G3P) for conversion into organellar membrane lipids (the so called "prokaryotic" pathway); ii) hydrolyzed from ACP by specific fatty acyl-ACP thioesterases (FATs) that release free fatty acids (FFAs) in the inner envelope of the chloroplast or iii) further elongated to C18-ACP. Alternatively, 16:0-and 18:0-ACPs can undergo further desaturation by the soluble ACP desaturases of the chloroplast stroma [START_REF] Shanklin | Desaturases: Emerging Models for Understanding Functional Diversification of Diiron-containing Enzymes[END_REF] and subsequently released by FATs. So far, no FATs similar to that of plants and bacteria have been identified in diatoms [START_REF] Gong | Characterization of a novel thioesterase (PtTE) from Phaeodactylum tricornutum[END_REF]. The formation of C16-18 FFA is considered the final step of de novo plastidial FA biosynthesis followed by the export of these fatty acids to the cytosol after being esterified to coenzyme A (CoA) by a longchain acyl-CoA synthase (LACS) located in the outer envelope of the plastid to form acyl-CoAs [START_REF] Schnurr | Fatty acid export from the chloroplast. Molecular characterization of a major plastidial acyl-coenzyme A synthetase from Arabidopsis[END_REF][START_REF] Li-Beisson | The lipid biochemistry of eukaryotic algae[END_REF]. These neosynthesized FAs may be further elongated or desaturated in the ER and used for glycerolipid synthesis (the so-called "eukaryotic pathway"). In plants, desaturation of 18:0 can be catalyzed either by the soluble stearoyl-ACP Δ9desaturase (SAD) of the chloroplast stroma [START_REF] Shanklin | Desaturases: Emerging Models for Understanding Functional Diversification of Diiron-containing Enzymes[END_REF] or by an extra-plastidic ER-bound acyl-CoA Δ9-desaturase (ADS) [START_REF] Fukuchi-Mizutani | Characterization of Delta 9 acyl-lipid desaturase homologues from Arabidopsis thaliana[END_REF], both generating 18:1n9, although SAD is the dominant pathway. Since only small amounts of C18-FAs could be detected in diatom chloroplast lipids, there is no direct evidence of 18:0-ACP synthesis in the plastids of diatoms, underlining another difference from plant FA biosynthesis. LC-PUFA biosynthesis is considered to start with desaturation of 18:1n9 by a Δ12-desaturase resulting in the production of linoleic acid (LA; 18:2n6) which is subsequently converted into α-linolenic (ALA; 18:3n3) by the action of Δ15-desaturase. Both LA and ALA are then converted into LC-PUFAs by the sequential desaturation/elongation reactions of that pathway.

The unicellular marine diatom P. tricornutum accumulates up to 35% of EPA and has been used as a model to study LC-PUFA biosynthesis due in part to a wellannotated genome sequence [START_REF] Bowler | The Phaeodactylum genome reveals the evolutionary history of diatom genomes[END_REF] and established transformation systems [START_REF] Apt | Stable nuclear transformation of the diatom Phaeodactylum tricornutum[END_REF]. In P.

tricornutum, the majority of EPA accumulates in polar lipids, especially in galactolipids such as MGDG and DGDG [START_REF] Abida | Membrane Glycerolipid Remodeling Triggered by Nitrogen and Phosphorus Starvation in Phaeodactylum tricornutum[END_REF][START_REF] Yongmanitchai | MOLECULAR-SPECIES OF TRIACYLGLYCEROLS FROM THE FRESH-WATER DIATOM, PHAEODACTYLUM-TRICORNUTUM[END_REF][START_REF] Arao | POSITIONAL DISTRIBUTION OF FATTY-ACIDS IN LIPIDS OF THE MARINE DIATOM PHAEODACTYLUM-TRICORNUTUM[END_REF]. Pulse chase experiments in P. tricornutum revealed that EPA can apparently be synthesized by a number of different routes with the predominant pathway proceeding via Δ6-desaturation of LA and ALA, and utilizing intermediates of both n6-and n3-pathways [START_REF] Arao | BIOSYNTHESIS OF POLYUNSATURATED LIPIDS IN THE DIATOM, PHAEODACTYLUM-TRICORNUTUM[END_REF]. Two main classes of FA desaturases involved in LC-PUFA biosynthesis have been previously identified in P. tricornutum and functionally characterized in yeast. Firstly, soluble enzymes, exemplified by PtFAD6, adding a double bond to an acyl-ACP substrate and secondly transmembrane enzymes (such as PtFAD2, PtD6 and PtD5), adding a double bond on acyl-glycerolipid susbtrates [START_REF] Domergue | Cloning and functional characterization of Phaeodactylum tricornutum front-end desaturases involved in eicosapentaenoic acid biosynthesis[END_REF][START_REF] Domergue | New insight into Phaeodactylum tricornutum fatty acid metabolism. Cloning and functional characterization of plastidial and microsomal Delta 12-fatty acid desaturases[END_REF]. Based on these observations, it was suggested that both Δ6-and Δ5desaturation and Δ6-elongation take place in the ER and newly synthesized EPA is then imported into the plastids for incorporation into galactolipids via an unknown route called the "omega pathway" [START_REF] Petroutsos | Evolution of galactoglycerolipid biosynthetic pathways -From cyanobacteria to primary plastids and from primary to secondary plastids[END_REF]. The understanding of EPA channelling is fundamental to manipulating LC-PUFA biosynthesis in diatoms.

To date, the mechanism of FA biosynthesis and export from the plastid in diatoms is still unknown. Since the 16:0 and 16:1n7 are the main FA synthesized in P.

tricornutum chloroplast (3) it is possible that the P. tricornutum acyl-ACP desaturase might utilize only 16:0-ACP substrate, acting specifically as a palmitoyl-ACP desaturase (PAD) rather than a stearoyl-ACP desaturase (SAD). Since 18:1n9 is considered to be a precursor of LC-PUFA synthesis in the ER and cannot be derived from 16:1n7, acyl-ACP-∆9 desaturase therefore represents an interesting target to modulate the accumulation of 18:0 and all downstream metabolites, including EPA. We overexpressed or disrupted the gene coding for the acyl-ACP-∆9 desaturase and analysed the impact on glycerolipid metabolism, revealing an unexpected effect of this soluble plastidial desaturase on EPA synthesis in the ER and also production of TAG. Our results indicate that the omega pathway relies on channelling processes occurring very early in plastidial FA desaturation and that this process can also be considered as a rate-limiting step in TAG synthesis.

Results

Generation of transgenic P. tricornutum overexpressing an acyl-ACP Δ9-

desaturase. Based on bioinformatic analysis only one homologue of the Arabidopsis soluble stearoyl-ACP Δ9-desaturase (SAD) gene, Phat3_J9316, can be found in the P.

tricornutum genome [START_REF] Bowler | The Phaeodactylum genome reveals the evolutionary history of diatom genomes[END_REF][START_REF] Dolch | Inventory of Fatty Acid Desaturases in the Pennate Diatom Phaeodactylum tricornutum[END_REF]. It contains predicted N-terminal bipartite targeting sequences with the conserved amino acid sequence motif "ASAFAP" surrounding the signal peptide cleavage site in diatoms {Kilian, 2005 #1643} (SI Appendix, Fig. S1A), consistent with a stromal location.

The native Phat3_J9316 acyl-ACP Δ9-desaturase sequence was chemically synthesized (Genscript, USA) and cloned into pPhos2 vector [START_REF] Hamilton | Metabolic engineering of Phaeodactylum tricornutum for the enhanced accumulation of omega-3 long chain polyunsaturated fatty acids[END_REF] under control of the EF2 promoter [START_REF] Seo | Development of a new constitutive expression system for the transformation of the diatom Phaeodactylum tricornutum[END_REF]. The resulted construct, pPhos2-PAD (SI Appendix, Fig. S1B) was used to transform P. tricornutum via particle bombardment. A total of 38 zeocin-resistant were screened by PCR for the presence of the transgene, out of which 20 were positive.

The fatty acid analysis of all PCR positive transformants revealed that 14 transgenic cell lines had elevated levels of 16:1n7 compared to that of wild type (WT) (SI Appendix, Fig. S2A). Analysis of transcript abundance of two representative lines (PtPAD#16 and PtPAD#32) with a clear chemotype of elevated 16:1n7, confirmed expression of the codon optimised gene and no change in endogenous gene expression (SI Appendix, Fig. S2B). Overexpression of the acyl-ACP Δ9-desaturase gene does not significantly change the total FA accumulation relative to WT, with an average total FA contents per cell of 3.2 nmol FA/million cells when harvested after 3 days of cultivation (SI Appendix, Fig. S2C).

Overexpression of acyl-ACP Δ9-desaturase in transgenic P. tricornutum results in reduced levels of EPA and alteration in lipid profiles. The fatty acid profiles of PAD#16 and PAD#32 were further analysed during the exponential (E) and stationary (S) phases of growth (Fig. 1A, SI Appendix, Fig. S3A). In accordance with previous observations [START_REF] Hamilton | Metabolic engineering of Phaeodactylum tricornutum for the enhanced accumulation of omega-3 long chain polyunsaturated fatty acids[END_REF], the major fatty acids in P. tricornutum Pt4 WT cells are palmitic acid (16:0), palmitoleic acid (16:1n7), and EPA. Transgenic lines overexpressing the acyl-ACP Δ9-desaturase accumulated significantly higher levels of 16:1n7 compared to that of WT at both growth stages. In the E phase the relative concentration of 16:1n7 is increased from 0.6 µg FA 10 6 cells ml -1 in the WT to 0.9-1.0 µg FA 10 6 cells ml -1 in the overexpression lines (1.5 fold, mean of both strains, p<0.05, Least Significant Different, LSD), (Fig. 1A) and remains elevated in both lines relative to the WT (1.4 -fold, p<0.05, LSD) at S stage (SI Appendix, Fig. S3A). In addition, the products of further desaturation is correlated with a reduction in the substrate 16:0 (0.52 -fold in the E phase and 0.65fold in the S phase, p<0.05, LSD).

Acyl-ACP Δ9-desaturase overexpression resulted in a significant decline of the FA intermediates involved in the proposed predominant route of LC-PUFA biosynthesis based on radiolabelling experiments (16): 18:1n9 →18:2n6 → 18:3n6→18:4n3. This was followed by the significant (~2-fold, p<0.05, LSD) reduction of the final products of the pathway, EPA (from 0.79 µg FA 10 6 cells ml -1 in the WT to an average of 0.36 µg FA 10 6 cells ml -1 in E growth phase (Fig. 1A) and from 0.5 to 0.25 to µg FA 10 6 cells ml -1 in S stage (SI Appendix, Fig. S3A) and DHA (from 0.08 to 0.04 µg FA 10 6 cells ml -1 in E growth and 0.04 to 0.02 µg FA 10 6 cells ml -1 in S growth stages). Together these data demonstrate an important role that that acyl-ACP Δ9-desaturase plays in channelling substrates at the very earliest stage of PUFA biosynthesis.

In order to determine if overexpression of the Δ9-desaturase gene could alter FA composition and abundance of specific lipid classes, a comprehensive glycerolipidome analysis was carried out. Total lipids were extracted from cells in both the E and S phases. Firstly, the detection and positional distribution of lipid species were characterised using direct infusion tandem mass spectrometry (MS2) with preferential loss analysis after separation by 1D and 2D TLC (3). Subsequently, lipid classes and species were quantified from total lipid extracts using HPLC-MS/MS.

The glycerolipid profile of WT P. tricornutum Pt4 cells in E phase were largely made up of plastidial membrane lipids, MGDG (36.4%), SQDG (16.4%) and DGDG (9%)

and extraplastidial lipid classes such as PC (10.7%), betaine lipid (BL)

diacylglycerylhydroxymethyltrimethyl-β-alanine (DGTA, 8.9%) and TAG (7.4%) (Fig. 1B).

In the WT cells grown to S phase, plastidial membrane lipids are degraded, resulting in a reduction in each class (MGDG, 0.39-fold; DGDG, 0.34-fold; SQDG 0.53 -fold; PG 0.06 -fold; relative to exponential phase) and TAG is increased 7-fold (SI Appendix, Fig. DGTA is a structural isomer of DGTS and the two molecules are difficult to differentiate by MS analysis alone. In this study, the presence of DGTA was confirmed by migration on 2D-TLC and M2S fragmentation (SI Appendix, Fig. S4). The presence and concentration of DGTA is similar to that reported in another P. tricornutum ecotype, Pt1

(3). We also confirmed the presence of EPA-acyl-SQDG, as reported previously, however this could not be quantified accurately by LC-MS/MS (3). The level of TAG in exponential phase of Pt4 was substantially higher (7.4%) than that reported in Pt1 (1-3%, (3), however it is important to note that the level of TAG is highly depended on growth conditions. Comparing the relative amounts of glycerolipids in acyl-ACP Δ9desaturase over-expressers (OE), significant increase of MGDG was observed in the E stage of cell growth (1.34-fold, p<0.05 LSD), correlated with decrease in SQDG (0.73 fold), DGDG (0.78 fold) and DAG (0.71 fold) relative to WT (p<0.05 LSD) (Fig. 1B).

There are no significant differences in proportions of lipid classes between WT and OE lines in the S phase (SI Appendix, Fig. S3B).

The sn-position of FAs on each glycerolipid was determined by preferential loss using MS and MS2 analysis as described in [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]. In agreement with previous observations, EPA is overrepresented in membrane lipids (( 3), ( 15); ( 25)). Considering chloroplast lipids, the major forms contain a C16 FA at the sn-2 position, suggesting that the prokaryotic pathway for the assembling of glycerolipids is very active in diatoms. The most abundant species in MGDG is 36:8, comprising of 20:5 and 16:3 (21%), whereas in DGDG dominant species are 20:5/16:2 (25.5%) and 20:5/16:1 (17.8%), and in PG, the major form is 20:5/16:1 (51%). (SI Appendix, Table S1). All these forms have a "prokaryotic" signature, a 20:5 at the sn-1 position and a C16 at the sn-2 position, supporting import of EPA into chloroplast from the ER, followed by plastidial acylation.

The overexpression of acyl-ACP Δ9-desaturase resulted in overall decline in all 20:5-containing species of chloroplast lipids (MGDG, DGDG, SQDG and PG) followed by an increase in C16-unsaturated containing species at both E and S stages of growth (SI Appendix, Tables S1-S4). We observed major reduction in the proportions of S1-4). Similar FA backbone as in TAG, 20:5/16:0 with 20:5

FA at the sn-1 position was also reduced in direct precursor DAG and also in PC, DGDG and PG. The most significant alterations were observed in the increased proportions of 16:1/16:1-and decrease in 20:5/16:0-containing TAG species, suggesting that all these lipids may act as substrates for TAG biosynthesis.

Generation of transgenic P. tricornutum with acyl ACP Δ9-desaturase knocked

out. To test the hypothesis that plastidial Δ9-desaturation of 16:0 within the chloroplast acts as a bottleneck for 16:0 export and further elongation and desaturation to EPA, the native Phat3_J9316 gene was targeted for disruption using CRISPR/Cas9 and two sgRNA guides to ensure gene disruption (Fig. 2A, SI Appendix, Fig. S5A). Cells were transformed using the previously described method [START_REF] Hamilton | Metabolic engineering of Phaeodactylum tricornutum for the enhanced accumulation of omega-3 long chain polyunsaturated fatty acids[END_REF] with the transformation efficiency ~ 1.6 colonies μg -1 plasmid. Among 33 colonies which were screened by PCR, two (∆pad#3 and ∆pad#7) had a lower MW band, indicating deletion of sequences within the target gene (Fig. 2B). The sgRNAs were designed to generate a 57 nt deletion within the first exon of the gene (SI Appendix, Fig. S5A). Cloning and subsequent sequencing of the PCR products revealed mosaicism within each KO lines (SI Appendix, Fig. S5B-5E), including the insertion of a 371 bp fragment of the transformation vector backbone.

The presence of bands >500 bp on the agarose gel in both lines indicates that larger insertions may also present in the cell population. The relative abundance of each deletion indicates that the deletion happened in multiple events after initial cell division.

There was no detection of unaltered DNA sequence between the gRNAs in either ∆pad#3 and ∆pad#7 lines, confirming a deletion in both these alleles. Consequently, the lines were analysed without further isolation of sub-clones. Both KO lines had significantly reduced levels of acyl-ACP Δ9-desaturase mRNA, most likely indicating increased post-transcriptional turnover of the mutated mRNA (Fig. 2C). tricornutum [START_REF] Arao | BIOSYNTHESIS OF POLYUNSATURATED LIPIDS IN THE DIATOM, PHAEODACTYLUM-TRICORNUTUM[END_REF]. The minor increase in proportions of DHA indicates that the pool size of EPA is not a bottleneck for synthesis; rather, as demonstrated by heterologous OtElo5 expression in P. tricornutum, accessibility of elongated products in the native strain is restrictive [START_REF] Hamilton | Metabolic engineering of Phaeodactylum tricornutum for the enhanced accumulation of omega-3 long chain polyunsaturated fatty acids[END_REF]. The residual 16:1n7 found in the KO line profile may be produced in the cytosol by a homologue of Arabodopsis ADS1 (26) acting on 16:0-CoA and imported back into the plastid. We have identified only one candidate Δ9-oleyl CoA desaturase, PtADS1 (Phatr3_J29797) based on the sequence homology. Increased expression of a putative PtADS1 desaturase in KO lines relative to the WT (~2.4 fold) supports this hypothesis (Fig. 2C).

Fatty acid and glycerolipid analysis of transgenic

Glycerolipidomic analysis was carried out to determine how disruption of the acyl-ACP Δ9-desaturase gene affected the distribution of glycerolipids and FA within each class. The WT profile, sampled in E phase, was largely similar to the previously outlined WT analysis, with the exception of a larger pool of TAG (24.5% vs 7.2% total lipid) which is likely due to slight differences in condition (synchronisation) of starting inoculum (Fig. 3B). Unlike the OE lines, KO of acyl-ACP Δ9-desaturase resulted in significant shift in the glycerolipid profile. KO lines had significant reduction in pools of MGDG (0.65-0.67-fold) and TAG (0.40-0.77-fold), and significant increases in SQDG Concerning changes in FA content, in chloroplast lipids in E phase of cell growth, EPA content increased significantly in SQDG (from 32.7 to 158.5 pmol FA 10 6 cells -1, ~5fold) and in PG (3-5-fold) (Table S9) whereas in MGDG it remains unaltered. In extraplastidial lipids the most increase was observed in DAG (~11-fold) followed by significant increase in PE (1.9-2.2-fold) and PC (1.5-2.5-fold). A sharp increase in C18

FAs was detected in SQDG and MGDG, followed by a more moderate uptick in PE, PC and DGTA. 16:1 levels were markedly reduced in all lipids. Interestingly, the most significant decrease in 16:1 was observed in DAG (26-72-fold), suggesting DAG pool is produced from neosynthesized FA. Similar pattern of FA accumulation was observed at S stage. In TAG, the most notable increase of EPA was observed in the stationary phase (~2-fold), followed by ~ 4-fold decrease in 16:1. Collectively, these observations support the hypothesis that the Phat3_J9316 acyl-ACP Δ9-desaturase acts as a competitor for 16:0 export and subsequent elongation/desaturation to EPA.

Heterologous characterization of the acyl-ACP Δ9-desaturase in Synechocystis.

For the functional definition of the Phat3_J9316 acyl-ACP Δ9-desaturase activity evaluated above, the cyanobacteria Synechocystis PCC6803 strain was used as a heterologous expression system. The Δ9-desaturase sequence Phat3_J9316 lacking predicted signal peptide was cloned into the pUR expression vector and expressed in Synechocystis to confirm its enzymatic activity (SI Appendix, Fig. S7). Synechocystis has a relatively simple fatty acid profile with 16:0, 16:1n7, 18:0, 18:1n9, 18:2n6, 18:3n6 and 18:3n3 as the major fatty acids, whereas 18:4n3 and 18:1n7 are found only in trace amounts (SI Appendix, Fig. S8A). The expression of the Phat3_J9316 acyl-ACP Δ9desaturase resulted in decreased levels of nearly all the major fatty acids except C18:0 and increased presence of C18 n7, the expected product of elongation of 16:1n7. To clarify the origin of 18:1n7, WT Synechocystis cultures were supplemented with 16:1n7 and grown until E phase before fatty acid analysis (SI Appendix, Fig. S8B). Exogenously supplied 16:1n7 only slightly increased the content of 16:1n7, but the levels of 18:1n7 were significantly increased, confirming the presence of a very efficient elongation of 16:1n7 to 18n7 in Synechocystis cells. Physiological studies. To explore whether the alterations of chloroplast ultrastructure have an impact on transgenic cells in different culture conditions, physiological studies were performed on the mutant and WT cells. Surprisingly, no differences were observed in specific growth rates and chlorophyll content between OE, KO and WT cells, suggesting that changes in thylakoid structure has no impact on the growth under normal conditions (SI Appendix, Fig. S10 andS11). To test the hypothesis that EPA acts as a sacrificial antioxidant molecule (to reduce ROX production under light stress), cellular growth of WT, OE PAD#16 and KO Δpad#3 strains was studied under light stress (SI Appendix, Fig. S12). Cultures were grown in 24 well plates under low light (20 µmol m -2 s -1 ) for 2 days, then transferred to high light (250 µmol m -2 s -1 ) and very high light (500 µmol m -2 s -1 ) or maintained at low light. The OE PAD#16 strain (with reduced EPA synthesis) showed no difference compared to WT, whereas growth was perturbed in KO Δpad#3 after 4 days.

Altered

Discussion

Our study confirms that in the marine diatom P. tricornutum Pt4 the main FAs synthesized de novo in the stroma of chloroplasts are 14:0-and 16:0-ACP [START_REF] Abida | Membrane Glycerolipid Remodeling Triggered by Nitrogen and Phosphorus Starvation in Phaeodactylum tricornutum[END_REF][START_REF] Popko | Metabolome Analysis Reveals Betaine Lipids as Major Source for Triglyceride Formation, and the Accumulation of Sedoheptulose during Nitrogen-Starvation of Phaeodactylum tricornutum[END_REF]). We identified one sequence in P. tricornutum genome, Phat3_J9316, containing both a predicted signal peptide and chloroplast transit peptide with a diatom-specific ASAFAP motif, and orthologous to the Arabidopsis Δ9-stearoyl-ACP desaturase (SAD) gene.

However, multiple lines of evidence support the classification of the Phat3_J9316 desaturase sequence as a Δ9 palmitoyl-ACP desaturase (PAD) as opposed to SAD.

Firstly, the absence of 18:0 in P. tricornutum plastid lipids suggests that a soluble Δ9acyl-ACP desaturase would act predominantly on 16:0-ACP, generating 16:1n7-ACP.

Overexpression of Phat3_J9316 leads to an increase in 16:1n7 correlated with reduced levels of 16:0 and 18:1n9, indicating that the enzyme has a strong preference to 16:0 as a substrate. In addition, we confirmed substrate specificity of Phat3_J9316 by functional characterization in the heterologous host Synechocystis providing evidence that increased accumulation of 18:1n7 in Phat3_J9316-overexpressing cells, is a result of 9-desaturase activity on 16:0 and the product of elongation of 16:1n7. In contrast, in P.

tricornutum KO mutants, where Phat3_J9316 is disrupted by CRISPR-Cas9 geneediting, there is a significant increase in levels of 16:0 consistent with the loss of PAD activity. Based on these results, we have assigned the function of palmitoyl-ACP 9desaturase (PAD) to Phat3_J9316.

Overexpression of this PAD gene in P. tricornutum leads to an overall reduction of the FA intermediates (SI Appendix, Fig. S13). in the presumed predominant LC-PUFA biosynthetic pathway starting from 18:1n9 ( 16) and 2-fold decline in EPA and DHA, indicating that PAD plays a key role in channelling processes from the very early stage of PUFA biosynthesis and the desaturation of 16:0 in plastid likely to be a "crossroad" for acyl flux through to 20:5. This is supported by the enhanced accumulation of 20:5 and concomitant reduction in 16:1 in stationary phase TAGs of PAD KO cells (Fig. 4). Δ9desaturation via PAD also regulates the levels of MGDG, the OE cells having higher MGDG levels while KO cells have lower, though both types of mutants displayed disturbed thylakoid membrane structures.

The inverse relationship between levels of 16:0/16:1 and EPA in OE and KO mutants, and the minor presence of C18 FA within chloroplast lipids, suggests that 16:0 is exported to the cytosol where it is converted into 16:0-CoA for further elongation and desaturation. This is in agreement with our previous observations that 20:5-CoA, 16:1-CoA and 16:0-CoA are the most abundant acyl-CoA species in P. tricornutum [START_REF] Hamilton | Metabolic engineering of Phaeodactylum tricornutum for the enhanced accumulation of omega-3 long chain polyunsaturated fatty acids[END_REF].

Recently, Dolch et al., 2017 demonstrated that Nannochloropsis gaditana saturated fatty acid elongase, Δ0-ELO1, could elongate palmitic acid [START_REF] Dolch | A Palmitic Acid Elongase Affects Eicosapentaenoic Acid and Plastidial Monogalactosyldiacylglycerol Levels in Nannochloropsis[END_REF]. N. gaditana Δ0-elo1 mutants exhibited a reduced EPA level and a specific decrease in MGDG. The study provided evidence that some of EPA used for MGDG production is biosynthesized by a channelled process initiated at the elongation step of palmitic acid by Δ0-ELO1, thus acting as a channelling enzyme for galactolipid production despite residing in the endoplasmic reticulum [START_REF] Dolch | A Palmitic Acid Elongase Affects Eicosapentaenoic Acid and Plastidial Monogalactosyldiacylglycerol Levels in Nannochloropsis[END_REF]. We identified two putative saturated ER fatty acid elongases, Phatr3_J16376 (Δ0-Elo1) and Phatr3_J49867 (Δ0-Elo02), homologues of N.

gaditana ER palmitic acid elongase Δ0-ELO1 ( 27) which could presumably elongate We identified only one putative Δ9-oleyl desaturase sequence, Phatr_28797, with an Nterminal sequence consistent with an ER localization. The present results suggest that acyl-CoA desaturase participates in desaturation at the Δ9 position of 18:0 in the ER.

The role of two putative Δ0-elongases and Δ9-oleyl desaturase in LC-PUFA biosynthesis is under investigation.

In higher plants, de novo plastidial 16:0-and 18:0-ACPs can enter two distinct routes of lipid synthesis, the "prokaryotic" or "eukaryotic". The "prokaryotic" or "eukaryotic" structure of plastid lipids is determined by the origin of phosphatidic acid (PA) and DAG. In plants, FA synthesized in the chloroplast are transferred onto glycerol-3-phosphate (G3P) by a plastidial acyl-G3P acyltransferase (ATS1). Further esterification by the plastid LPAAT, (ATS2), leads to the production of PA and DAG with C18 and C16 at position sn-1 and sn-2 respectively. Chloroplast desaturases acting on 16:0 produce a range of C16 unsaturated FA, found at the sn-2 position of MGDG and DGDG lipids. In the "eukaryotic" pathway, acyl-ACPs are hydrolysed by FAT to produce free FAs which are exported to the ER for the subsequent incorporation into membrane glycerolipids with C18 at sn-2 position before re-import to the chloroplast. The presence of a eukaryotic pathway involving recycling of diacylglycerol backbone coming from phospholipid for galactolipid synthesis, as described in higher plants, has not been demonstrated in Heterokonts. In P. tricornutum, EPA found in chloroplast lipids will have been synthesized in the ER by ER-located desaturases and elongases [START_REF] Domergue | Cloning and functional characterization of Phaeodactylum tricornutum front-end desaturases involved in eicosapentaenoic acid biosynthesis[END_REF]. Thus, EPA and other LC-PUFAs must also be re-imported to secondary plastids for incorporation into the plastidial glycolipids. However, the nature of precursors that are transported over the chloroplast-limiting membranes remains unknown.

In all analysed chloroplast lipids of WT and mutant cells EPA is exclusively found in the sn-1 position whereas C16 occupy mainly the sn-2 position, confirming the observation that the prokaryotic pathway for the synthesis of the glycerolipids is dominant in Heterokonts [START_REF] Abida | Membrane Glycerolipid Remodeling Triggered by Nitrogen and Phosphorus Starvation in Phaeodactylum tricornutum[END_REF][START_REF] Simionato | The Response of Nannochloropsis gaditana to Nitrogen Starvation Includes De Novo Biosynthesis of Triacylglycerols, a Decrease of Chloroplast Galactolipids, and Reorganization of the Photosynthetic Apparatus[END_REF][START_REF] Dolch | A Palmitic Acid Elongase Affects Eicosapentaenoic Acid and Plastidial Monogalactosyldiacylglycerol Levels in Nannochloropsis[END_REF]. This also suggests that the plastid LPAAT has a very high selectivity for C16 fatty acids as the acyl donors to generate sn-2-C16-PA giving rise to lipids with the prokaryotic signature. ER membrane glycerolipids of WT and both mutants, such as PC, DGTA and PE mostly have C18, C20 and C22 FA together with C16 species esterified at the sn-2 position indicating that either microsomal LPAAT has no selectivity for FA species or acyl-CoA:lysophospholipid acyltransferase (LPCAT) may be involved in ER membrane glycerolipid assembly.

In the different P. tricornutum cells used in this study, TAGs are always enriched in C16 fatty acids at their sn-2 position implying that the TAG synthesis may be similar to that described in Chlamydomonas reinhardtii where a chlorophyte-specific acyltransferase, CrLPAAT2, localized to the ER, prefers 16:0-CoA as the substrate for synthesis of glycerolipid intermediates for TAG assembly and/or transfer to the plastid [START_REF] Kim | Flux balance analysis of primary metabolism in the diatom Phaeodactylum tricornutum[END_REF]. Since C18 and C20 are hardly detected at the sn- [START_REF] Bates | The significance of different diacylgycerol synthesis pathways on plant oil composition and bioengineering[END_REF]. ER-synthesized EPA may be also imported into chloroplast and incorporated into glycolipids by the suggested "omega pathway". Newly formed EPA may be released from a phospholipid into the cytosolic acyl-CoA pool and then transported into the chloroplasts to be attached to MDGD at the sn-1 position by the first acyltransferase, ATS1 [START_REF] Petroutsos | Evolution of galactoglycerolipid biosynthetic pathways -From cyanobacteria to primary plastids and from primary to secondary plastids[END_REF].

Conclusions

The role of EPA in chloroplast lipids remains unknown. Based on the observation that the turnover of EPA-rich MGDG is higher under NPQ-inducing conditions, Dolch et al 2017 [START_REF] Dolch | A Palmitic Acid Elongase Affects Eicosapentaenoic Acid and Plastidial Monogalactosyldiacylglycerol Levels in Nannochloropsis[END_REF] suggested that EPA could act as a sacrificial molecule acting as supramolecular antioxidant capturing oxygen species during light stress. The results of our light stress experiments do not support this hypothesis as the growth rates in OE cells with less EPA/more MGDG were not inhibited relative to the WT by light stress.

However, the growth of KO cells containing more EPA /less MGDG was inhibited, probably indicating the presence of EPA in plastids is more important for membrane fluidity in cold adaptation. We propose that the PAD gene acts as a key factor in determining EPA levels to maintain phenotypic plasticity. In particular, the modulation of both MGDG and TAG levels through the presence/absence of 16:1n7 represents a simple "tag" by which different lipids can be sorted for further modification.

Materials and Methods

Strain and culture conditions. P. tricornutum Pt4 strain (UTEX 646) was grown in artificial sea water (Instant Ocean, Spectrum Brands) supplemented with F/2 nutrients.

Cultures were grown at 20°C under constant illumination (100 µmols m -2 s -1 , 4000K

White and 660nm LED lighting) and agitated continuously at 70 rpm. Growth was monitored by OD 750nm calibrated to cell density measured by an automated cell counter (Cellometer T4, Nexcelom). Lines were maintained on F/4 agar plates grown at 20°C under 50 µmols m -2 s -1 (3500K fluorescent tubes). biolistic transformation and screening was carried as described previously [START_REF] Hamilton | Metabolic engineering of Phaeodactylum tricornutum for the enhanced accumulation of omega-3 long chain polyunsaturated fatty acids[END_REF].

Construction of acyl-ACP Δ9-desaturase Overexpression

Generation of acyl-ACP Δ9-desaturase KO lines.

A universal knock-out CRISPR/Cas9 vector was constructed with a dual sgRNA design (33) using dual reporter gene selection system (designed by Mark Youles, personal communication).

Type IIS LOOP DNA assembly was used for Level 1 and 2 vector constructions, following the method described in Pollak et al., 2018 (34). The design of KO cassettes is explained in detail in SI Appendix, SI Materials and Methods. Transformation of P.

tricornutum Pt4 via biolistic transformation and screening was carried as described previously [START_REF] Hamilton | Metabolic engineering of Phaeodactylum tricornutum for the enhanced accumulation of omega-3 long chain polyunsaturated fatty acids[END_REF]. Expression is reported relative to the reference gene Aureochrome (Phatr3_J8113).

Cloning of acyl

Values presented are the average of three biological replicates, error bars represent SE.

Asterisks indicate significant difference relative to WT (p<0.05, LSD).

of 16 :

 16 1n7, following the route proposed by Domergue et al. (23), 16:1n7 → 16:2n4 → 16:3n4 → 16:4n1, and elongation to 18:1n7 also increased significantly (p<0.05, LSD) in E phase and remained elevated in S phase compared to the WT. An increase in 16:1n7

S3B) .

 . The glycerolipid profile of Pt4 contrasts with a recent analysis of Popko et al. (). Three main BLs have been identified in microalgae, DGTA, diacylglyceryl carboxyhydroxymethylcholine (DGCC) and DGTS.

20: 5 /

 5 16:3 form in MGDG (~ 4 -fold), 20:5/16:0 form in DGDG (~ 7-fold) and SQDG (4.8-6.8-fold) and 20-5/24:0 form in SQDG (12.6-fold) compared to WT. Extraplastidial polar lipids, PE, PC and DGTA, contain C16 FA together with C18, C20 and C22 FAs at sn-2 position, suggesting an unbiased FA species incorporation by microsomal acyl-CoA:lysophosphatidic acid acyltransferase (LPAAT) or the presence of acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) that may synthesize the precursor backbones involved in ER membrane glycerolipid assembly. Noteworthy, 20:5 is present at sn-2 position only in the eukaryotic 20/20:5 form and 22:6 is found only in 20:5/22:6 form (SI Appendix, TablesS1-S4). In PE, sn-1 position was mainly occupied by 20:5 acyl group. In the overexpresors, the greatest reduction was observed in 20:5/C18-and 20:5/LC-PUFA-forms of PC, DGTA and PE with major decline of 20:5/20:5 form in PC and DGTA (~8.4-5 and 6.3-4.3-fold respectively) and 20:5/22:6 in PC (9.9-fold). The major alteration observed was an increase in 16:1/16:1 and 16:1/16:2 forms in PC (5.7-8.0 and 8.0-11.4-fold respectively, pmol 10 6 cell -1 ) and DGTA (3.7-4.3 and 5.8-7.5-fold respectively, pmol 10 6 cell -1 ). More surprising was an increase in 20:5 -16:1 and 20:5 -16:2 forms in PE (5.0-6.0-and 6.0-7.4-fold respectively, pmol 10 6 cell -1 ) correlated with a decrease of these two major backbones in DGDG (~2 -3-fold), suggesting that 20:5/16:1 and 20:5/16:2 species could be potentially exported as DAG.Considering nonpolar lipids, the DAG pool is represented by 4 molecular species, comprising mainly of 16:1, 16:0 and 14:0 and 20:5 acyl groups, with 14:0 and 20:5 being found only at the sn-1 position. The DAG acyl composition (SI Appendix, TablesS1-S4) partially reflects that found in the main membrane lipids and we cannot rule out the recycling of the diacylglyceride backbone from membrane lipids. The major decline (~6fold) has been observed in 20:5/16:0 form found in most polar lipids followed by an increase in 16:1/16:1.Three DAG molecular species, 14:0/16:1, 16:1/16:1 and 20:5/16:0, are potential precursors of the TAG pool with sn-3 position occupied by 14:0 or C16 FA. In acyl-ACP Δ9-desaturase OE, TAG 20:5-containing species also declined followed by increase in 16:1 (SI Appendix, Tables

  P. tricornutum with disruption of endogenous acyl-ACP Δ9-desaturase reveals increases in EPA and major alterations in TAG. The fatty acid profiles of two disrupted/knock-out (KO) lines were analysed in the exponential and stationary phase of growth. KO of the acyl-ACP Δ9desaturase gene resulted in a significant reduction of 16:1n7 (94% reduction relative to WT) and, consequently, in a smaller pool of products of further desaturation (16:2n4,16:3n4,16:4n1) and elongation (18:1n7) in both E and S phases (Fig.3Aand SI Appendix, Fig.S6A). In contrast, the accumulation of 16:0, a substrate for acyl-ACP Δ9desaturase, increased ~1.45-fold followed by a significant increase in the levels of 18:1n9 vs WT, indicating independent desaturation of C18:0 by putative Δ9 acyl-CoA desaturase. Furthermore, the proportion of many intermediate FAs in extraplastidic PUFA synthesis pathway also significantly increased culminating in an elevated accumulation of EPA (~1.32-1.42-fold), increasing from 0.69 µg FA 10 6 cells -1 in the WT cultures, to 0.91-0.98 µg FA 10 6 cells -1 in the KO strains. Interestingly, the intermediates most affected by the KO (LA, ALA, 18:4n3, 20:4n3) are those highlighted by Arao and Yamada, 1994 to be involved in the dominant route of PUFA biosynthesis in P.

( 1 .

 1 34-1.40-fold), PG (2.64-4.31-fold), PE (2.30-2.70-fold), PC (1.99-3.01-fold) and DGTA (1.65-3.26-fold). Lipid classes analysis (Tables S6-S10) reveal the major increase in "TAG-diagnostic" 20:5/16:0 species in main chloroplast lipids, SQDG (4.15-4.85-fold), MGDG (14.1-15.7-fold), DGDG (4.17-5.19-fold) and PG (3.76-7.01-fold) correlated with significant decrease in 16:1-containing species and metabolic derivatives (16:2, 16:3 and 16:4) were undetectable. A dramatic increase in C18-FA was observed in 20:5containing lipids in MGDG and SQDG (~49-fold in 20:5/18:1 and 24-28-fold in 20:5/18:2 in MGDG; 3-5-fold increase in 20:5/18:2 in SQDG). The typical eukaryotic backbones, containing C18 FA and 20:4n3 at sn-2 position can come from the recycling of phospholipids such as PC, DGTA and PE similar to galactolipid synthesis in higher plants. Within the extraplastidial PLs there was a major increase in unsaturated C18, 20:5 and 22:6 containing forms. The 20:5/20:5 form was slightly elevated in PE (1.5fold), PC (1.8-fold) and DGTA (2-fold). Interestingly, an increase in 20:5/18:2 forms in PE, PC and DGTA was mirrored by similar changes in MGDG and SQDG, while 20:5/18:1 species concomitantly increased in PC, DGTA, MGDG and SQDG. This may imply that PE, PC and DGTA serve as precursors for galactolipids in P. tricornutum. The most significant changes in the DAG pool were increase in 20:5/16:0 form (21.65-22.92fold) and disappearance of 16:1/16:1 (non-detected in KO strains). Alterations in TAG reflected that of membrane lipids and a DAG pool with a noticeable increase in 18:1 and 20:5-containing forms. Significant increase is detected in 20:5/16:0/16:0 (1.68-3.64-fold) and 20:5/20:5/16:0 (4.31-7.35-fold) forms.

  Ultrastructure of Thylakoid membranes in transgenic P. tricornutum strains. Cross-sections of chloroplasts of E stage cells were analysed by transmission electron microscopy to access the impact of acyl-ACP Δ9-desaturase overexpression or inactivation on the ultrastructure of thylakoid membranes. WT chloroplasts contained several parallel thylakoid lamellae, each consisting of a stack of 3 membrane bilayers (SI Appendix, Fig.S9). Both mutants have disturbed thylakoid structure. The OE PAD#12 chloroplasts, containing more MGDG, has similar number of stacks, but they exhibit larger luminal space with increased packing of stacks and more extreme curvature of the stacks (red arrow) and less parallelism between membranes is observed. In KO Δpad#3 chloroplasts, containing less MGDG, thylakoids are distorted, less electron dense than that of WT (blue arrow) with expanded inter-space, reduced stroma space and curvature of the lamella (green arrow), and no obvious pyrenoid is present.

16: 0 -

 0 CoA to 18:0-CoA. This indicates that diatoms developed very different route of biosynthesis from plants, where the canonical conversion of 16:0 to 18:0 is carried out by plastidial a β-ketoacyl-ACP synthase II (KASII). Recently, Aslan et al., demonstrated that KO of KASII in tobacco resulted in increased accumulation of C16 FAs (28), suggesting that plants do not have an ability to utilize ER-located elongase for conversion of plastid 16:0 to 18:0. In the P. tricornutum genome a KASII orthologue has been annotated but not functionally characterized. Further modification of 18:0-CoA could be carried out by orthologues of the Arabidopsis ADS1, to produce 18:1Δ9-CoA.

  Cassette and Transformation. The PAD (Phatr3_J9316) gene was chemically synthesised (Genscript, USA) and codon optimised to remove conflicting restriction sites. The PAD gene was inserted into position 1 in the two-gene cassette transformation vector pPhOS2 (21) under the control of the EF2 promoter (22) generating pPhOS2_PAD construct. Construction of the transformation cassette pPhOS2_PAD is described in detail in SI Appendix, SI Materials and Methods. Transformation of P. tricornutum Pt4 via

Figure 1 .

 1 Figures and Tables

Figure 2 .

 2 Figure 2. Generation of acyl-ACP Δ9 desaturase KO strains by CRISPR/Cas9. (A) Schematic structure (important features) of the CRISPR/Cas9 target locus within the Δ9 desaturase gene. The positions of guide RNA complimentary binding sites in the first exon are indicated with black arrows. The approximate region of nucleotide deletion is shown in white. The grey arrows indicate the site of amplification for PCR genotyping and qPCR of cDNA. (B) PCR screen of native acyl-ACP Δ9 desaturase gene in KO lines (see SI Appendix, (Fig. S5) for position of primers and expected deletion). Single band shift indicates bi-allelic deletion. (C) Transcript abundance of acyl-ACP Δ9 desaturase (Phatr3_J9316) and putative Δ9 acyl-CoA desaturase, ADS (Phatr3_J28797) genes.

Figure 3 .

 3 Figure 3. FA and glycerolipid classes comparison in the WT and KO strains during the exponential stage of growth phase (3 days). (A) FA content. Values expressed in µg FA 10 6 cells -1 . (B) Glycerolipid classes. Values expressed in % of total lipids. Insert: Quantitative analysis of total glycerolipids (nmol of lipid 10 6 cells -1 ). Growth stage was determined by analysing cell density. Values presented are the average of three biological replicates, error bars represent SE. Asterisks indicate significant difference relative to WT (p<0.05, LSD).

Figure 4 .

 4 Figure 4. Proposed pathway for EPA biosynthesis and possible EPA import routes from the ER into the chloroplast of P. tricornutum. Red text indicates increased pool size of metabolite or activity of enzyme in KO cells. Blue text indicates reduced pool size or activity of enzyme. De novo synthesized 16:0-ACP enters prokaryotic pathway where it can be used in the plastid for the production of MGDG, DGDG, SQDG, and PG via a canonical prokaryotic pathway or desaturated by PAD to 16:1n7-ACP and further by

  An increase of 16:1 and concomitant reduction in EPA levels in TAG in OE cells may indicate that de novo FA synthesis contributes to TAG biosynthesis via the Kennedy pathway involving a diacylglycerol acyltransferase and de novo DAG and acyl-CoA synthesis. This may be supported by the observed decrease in 16:1 and increase in EPA levels in KO cells. Despite the significant changes to the FA profile, OE or KO of the PAD gene does not significantly change the total FA accumulation relative to WT, indicating no change in de novo FA synthesis. In addition, the DAG acyl composition does not fully reflect the composition of the main membrane lipids in support of a de novo synthesis and, in contrast to plants, does not indicate any substantial recycling of DAG moieties deriving from membrane glycerolipids

	hypothesis. Interestingly, MGDG and SQDG in KO cells contain increased levels of C18
	bound to sn-2 molecular species concomitant with an increase in these forms (16:1/18:1;
	20:5/18:1, 20:5/18:2) in PE, PC and DGTA suggesting a potential existence of a
	eukaryotic pathway with a recycling of a diacyl precursors coming from phospholipid for
	galactolipid synthesis, as described in higher plants and C. reinhardtii (31). Alternatively,
	ER located PA and DAG may serve as precursors of galactolipid and sulfolipid
	synthesis.
	2 position in thylakoid lipids in
	WT and OE cells, similar selectivity may operate in P. tricornutum, implying that ER-
	synthesized precursors (C16 bound to sn-2-PA) have diagnostic C16 acyl chains
	esterified at this position. A >20-fold increase in specific signature 20:5/16:0 form in DAG
	of KO cells followed by similar increase in this form in thylakoid lipids supports this

  One-way analysis of variance (ANOVA) was applied to data on specific growth, fatty acid, lipid class, and lipid species data. Data was transformed by natural log (quantitative data) or logit (relative %). Post-hoc comparison of the means was carried

	out using least significant difference (LSD) at 5%, 1% and 0.1% level of significance. A
	two-tailed Student's t-test was carried out in cases where only two strains are compared.
	Microsoft Excel was used for these analyses.

-ACP Δ9-desaturase (Phatr3_J9316) into Synechocystis expression vector and functional characterization in Synechocystis. To generate the Syn_PAD Statistics.

Acknowledgments

Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council (BBSRC), UK and this project was supported by grant BB/L002957/1. We would like to thank the following: Prof. Annegret Wilde for providing the pUR vector and Prof. Dario Leister for providing the Synechocystis PCC6803 GT WT strain. Dr Katrin Geisler for providing cloning modules (Ble, FcpB promoter, FcpC terminator). Dr. Mark Youles for providing technical advice and pICSL002215 vector (containing mRFP gene). Dr Bernado Pollack for providing Loop assembly vectors. Prof. Per Winge for providing pKS_diacas9 vector. Dr Chloe Economou for technical assistance. Dr Rebecca Lauder for bioimaging.

strain, the Synechocystis PCC6803 glucose-tolerant WT (Syn_WT, Himadri Pakrasi, Department of Biology, Washington University, St Louis, MO, USA) was transformed with the self-replicative vector pUR [START_REF] Wiegard | Biochemical analysis of three putative KaiC clock proteins from Synechocystis sp PCC 6803 suggests their functional divergence[END_REF] expressing the acyl-ACP Δ9-desaturase gene lacking the 5' putative signal peptide sequence (as predicted by SignalP online software). Generation of the vector, transformation and functional characterisation of the Phatr3_J9316 gene is described in detail in SI Appendix, SI Materials and Methods.

RNA Extraction and qRT-PCR.

For RNA extraction, 1-1.5x10 8 exponential phase cells were pelleted, flash frozen in liquid nitrogen and stored at -80°C. RNA extraction was carried out using the method described in Smith et al., 2018 (36). cDNA was synthesised and q-PCR analysis was carried out as described detail in SI Appendix, SI Materials and Methods.

Lipid analysis. Whole biomass FAME analysis was carried out following as previously reported ( 21), using of pentadecanoic acid and tricosanoic acid internal standards.

Further details are described in SI Appendix, SI Materials and Methods.

Glycerolipids were extracted following a method adapted from (29), further details are described in SI Appendix, SI Materials and Methods.

For positional analysis, lipids first were fractionated by 1D and 2D TLC (described in detail in SI Appendix, SI Materials and Methods), purified classes were then characterised by preferential loss analysis under low-energy collision-induced dissociation as described in previously described (3). After species characterisation, quantification of each species was carried out by LC-MS/MS 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].

Chlorophyll analysis. For chlorophyll analysis, 1.5-3x10 7 exponential phase cells were pelleted, washed in ammonium formate and frozen at -20°C. Chlorophyll was extracted with 2 ml of methanol and absorption was measured as outlined [START_REF] Ritchie | Universal chlorophyll equations for estimating chlorophylls a, b, c, and d and total chlorophylls in natural assemblages of photosynthetic organisms using acetone, methanol, or ethanol solvents[END_REF].

Transmission Electron Microscopy. TEM imaging was carried out by Rothamsted

Bioimaging (Harpenden). Processing of P. tricornutum cells for TEM imaging is described in detail in the SI Appendix, SI Materials and Methods.


Erwan Le Roux

Raluca Malacea

Eric Manoury
email: manoury@lcc-toulouse.fr

Rinaldo Poli

Luca Gonsalvi

Maurizio Peruzzini
email: mperuzzini@iccom.cnr.it

Highly efficient asymmetric hydrogenation of alkyl aryl ketones catalyzed by iridium complexes with chiral planar ferrocenyl phosphino-thioether ligands

Keywords: alkyl aryl ketones, asymmetric catalysis, ferrocene ligands, hydrogenation, iridium, P,S ligands

Iridium complexes of planar-chiral ferrocenyl phosphine-thioether ligands were tested in the hydrogenation of simple ketones. Conditions optimization led to a highly active catalytic system with turnover numbers up to 915 and turnover frequencies up to ca. 250 h -1 . Furthermore, very high enantioselectivities (up to >99%) together with complete conversions were obtained for the asymmetric hydrogenation of various acetophenones at 10 °C.

additional heteroatoms for secondary interactions of the substrate with the metal center, have long been a major goal [START_REF] Fehring | [END_REF][START_REF] Blaser | For recent reviews on asymmetric hydrogenation[END_REF] until Noyori et al. introduced new ruthenium-based [(diphosphine)RuCl2(diamine)] [4] complexes which proved outstanding in terms of both activities and enantioselectivities. Though many ruthenium-based catalytic systems have been developed during the last decade, [5] the use of other metals in this reaction have been rather rarely described. [6][7][8] Herein, we report a highly active and highly enantioselective catalytic system for the hydrogenation of various substituted acetophenones, based on Ir complexes of ferrocenyl phosphine-thioether ligands. We have recently developed syntheses of new chiral ferrocenyl P,S ligands in both racemic and enantiomerically pure form (R or S configuration), and briefly reported on their application to asymmetric catalysis, namely in palladiumcatalyzed allylic substitution. [9][START_REF] Mateus | For the synthesis of enantiomerically pure synthetic intermediates (R) or (S) (2diphenylthiophosphinoferrocenyl)-methanol[END_REF] We have also described iridium complexes 2-R (R = Ph, Et, tBu, see Scheme 1), obtained in high yields (85%-90%) and with complete diastereoselectivity via the addition of the bidentate ligands 1-R to [Ir(cod)Cl]2. [11] The coordination number of 2-R is controlled by the size of R (5-coordinate for the smaller Ph and Et, square planar 4-coordinate for the bulkier tBu), both in the solid state and in solution, while the absolute configuration at the coordinated sulphur atom is in turn controlled by the ferrocene planar chirality (only one diastereoisomer is obtained). [11a] By using the same procedure, we have now prepared the benzyl analogue (2-Bz) starting from ligand 1-Bz [9] and [Ir(cod)Cl]2. NMR data for 2-Bz are similar to those of 2-Et and 2-Ph. In particular, the 31 P signal (-9 ppm) is close to the corresponding signals of 2-Et and 2-Ph (respectively -3.1 ppm and -4.2 ppm) but not to the signal of 2-tBu (15.7 ppm). The 1 H NMR data (δ and JHH) of the two diastereotopic protons of the methylene group between the ferrocene moiety and the sulphur atom are also much more affected by coordination than in the case of 2-tBu. This suggests that the sulphur atom is coordinated to the iridium center and therefore that 2-Bz is pentacoordinated like 2-Et and 2-Ph.

In order to assess the efficiency of these complexes in ketone hydrogenation, the experimental conditions were first optimized for the hydrogenation of acetophenone as a model substrate with the racemic catalysts 2-R, followed by the asymmetric hydrogenation of various substituted alkyl aryl ketones with the enantiomerically pure complexes. The asymmetric hydrogenation of simple ketones is commonly carried out in alcoholic solvents, typically isopropanol, in the presence of a base. [4][5][6][7][8] Therefore, the hydrogenation of acetophenone was first studied in isopropanol (see table 1). In the absence of a base, 2-Et showed no catalytic activity (run 1). The addition of KI had no effect on the reaction rate [12] (run 2), whereas I2 had a positive but limited effect (run 3). [13] On the other hand, the addition of a strong base (NaOMe, KOtBu or KOH; 5 equivalents) efficiently promoted catalysis (runs 4-6). The weaker base NEt3 had no significant effect (run 7), contrary to former observations by Dahlenburg and Götz for related iridium-based catalytic systems. [8d] The iridium/substrate ratio could be decreased to 1/1500 while maintaining significant conversions after 5 h (61% corresponding to a turnover number of 915; run 8). The optimised conditions for 2-Et were finally applied to the other 2-R complexes. All proved to be efficient catalysts, with complete conversion after 5h in all cases (runs 9-11). These activities appear to compare quite favourably with those reported for other Ir complexes under similar conditions. [8] The reason for this difference may be related to a greater hemilability of these P,S ligands, [11b] relative to the other chelating ligands used so far.

The solvent influence on the catalytic activity has also been investigated (see table 2).

The runs in alcoholic solvents were stopped after 2h in order to better compare the catalytic activities. The catalytic activity depends on the alcohol nature (runs 12-16). In particular, the reactivity dropped dramatically in t-butanol (cf. runs 15 and 16 for the 2-Et catalyst), in contrast again with the system developed by Dahlenburg and Götz. [8d] This observation raised the question of the intervention of a transfer hydrogenation pathway, with the alcoholic solvent as the hydride source. However, the reaction could be efficiently carried out in toluene, which can not be a hydride source (runs 18 and 19), ruling out this mechanism under these conditions. Furthermore, the activity in isopropanol dropped dramatically when the reaction was carried out under reduced H2 pressure (2 bars, run 17). These two independent observations strongly support the hydrogenation mechanism in alcoholic solvents.

Acetophenone could also be hydrogenated efficiently in acetonitrile but not in dichloromethane or THF (runs 20-23). The replacement of NaOMe with KOtBu increased significantly the activity of 2-Et in toluene, probably because of the higher solubility of the latter base. In MeCN, where both bases are completely soluble, the observed activity is comparable.

The asymmetric hydrogenation using enantiomerically pure (S)-2-R has been studied under the optimised conditions (i.e. those of table 2, run14). In contrast with the catalytic activities, the enantioselectivities for the acetophenone hydrogenation strongly depend on the R substituent on sulphur, going from 43% with (S)-2-Ph to 77% with (S)-2-Bz (table 3, runs 24-27). For substituted acetophenones, enantioselectivities depend also strongly on the R substituent, especially for 2-substituted compounds where, with the more crowded complexes (S)-2-Ph and (S)-2-tBu, low enantioselectivities were observed (table 3, runs 29-3 and 4-substituted acetophenones (runs 36-51). For each substrate, complex (S)-2-Bz gives the best enantioselectivities (cf. runs 27, 31, 35, 39, 43, 47 and 51). The two best ligands in terms of enantioselectivity for the acetophenone hydrogenation (i.e. (S)-1-Et and (S)-1-Bz) were selected for further studies at lower temperature (10°C, see table 4). This resulted in much higher enantioselectivities (up to >99% for 4-fluoroacetophenone, table 4, runs 52-57), while the catalytic activities remained acceptably high. This outstanding increase is much greater than expected on the basis of a simple Arrhenius relationship for a mononuclear catalyst. It is therefore likely that several catalytically active species are involved. This hypothesis is in agreement with a preliminary investigation of the ee of the product as a function of enantiomeric purity of the catalyst, which shows negative non-linear effect. [14] This aspect of catalytic process is under further investigation.

In conclusion, we have shown that iridium complexes with planar chiral P,S ligands are effective catalysts for the asymmetric hydrogenation of various alkyl aryl ketones with high activities (TOF up to ca. 250 h -1 ) and ee's up to >99% with conversions up to >99%). To the best of our knowledge, these activities and enantioselectivities are the best ones reported so far for the iridium-catalyzed hydrogenation of ketones. Future work will focus on investigating the reaction mechanism and on expanding the substrate scope. Only four (S)-1-R ligands have been used in this study, but a variety of analogues can be easily synthesised, [9a] opening the way for searching the best complex for each substrate. Therefore, we hope that the fine tuning of the complex stereoelectronic properties, in particular by modification of the R group, will lead to a more efficient asymmetric hydrogenation of a large variety of simple ketones.

Experimental Section

General: All reactions were carried out under dry argon by using Schlenk glassware and vacuum line techniques. Commercial samples were used as received. Solvents were freshly distilled from standard drying agents. Synthesis of complex 2-Bz: Complex 2-Bz was synthesised following the published procedure [11a] from ligand 1-Bz. [9] 1 H NMR (500 MHz, CDCl3) δ(ppm): General procedure for asymmetric hydrogenation: in a glove box, a solution of 6.4 X 10 -3 mmol of catalyst, 3.2 X 10 -2 mmol of additives (5 equiv.) and 3.2 mmol of substrate (500 equiv.) in 2 mL of the desired solvent was transferred into a 5 mL glass ampoule which was then placed into a stainless steel autoclave. The reaction vessel was pressurized to the required H2 pressure and stirred with a magnetic bar for the desired time at controlled temperature (2 °C). The reaction was stopped by release of pressure and quenching of the solution with

CH2Cl2 at room temperature. The crude materials were obtained by evaporation of the solvent on rotavapor. The product was finally analysed by chiral GC (Supelco BETA DEX TM

225).

Table 1. Influence of the additives on the hydrogenation of acetophenone in isopropanol [a] run catalysts Additive Time (h) Conv.(%) [b] TOF(h -1 ) [c] 1 [a] Reaction conditions: racemic catalyst, 6.4 X 10 -3 mmol ; additive, 3.2 X 10 -2 mmol ; acetophenone, 3.2 mmol; (1/5/500) under 30 bars at 27°C in 2 mL of isopropanol. [b] Conversions determined by GC; 100% selectivity in (R/S)-1phenylethan-1-ol. [c] Global turnover frequency. [d] Amount of acetophenone increased to 9.6 mmol (S/C ratio = 1500). [e] The conversion was 91% after 2h.

2-Et

Table 2. Influence of the solvent and additives on the hydrogenation of acetophenone. [a] Run catalysts Additive Solvent Time (h) Conv.(%) [b] TOF(h -1 ) [c] 12 [a] Reaction conditions: racemic catalyst, 6.4 X 10 -3 mmol ; additive, 3.2 X 10 -2 mmol ; acetophenone, 3.2 mmol; (1/5/500) under 30 bars at room temperature in 2 mL of solvent. [b] Conversions determined by GC; 100% selectivity in (R/S)-1-phenylethan-1-ol. [c] Global turnover frequency. [d] Identical conditions as in [a] except P(H2) = 2bars.

Run

Catalyst X t (h) conv. (%) [b] ee (%) [b] [a] Reaction conditions: catalyst, 6.4 X 10 -3 mmol ; NaOMe, 3.2 X 10 -2 mmol; substrate, 3.2 mmol (1/5/500) at 10°C. [b] Conversion and ee determined by GC.

In each case, the absolute configuration configuration of product is S (assigned by comparison of rotation sign in literature data).

133. 9

 9 (d, JCP = 51.9 Hz, quat Ar); 131.7(d, JCP = 8.5 Hz, Ar); 130.6 (d, JCP = 3 Hz, Ar); 129.6 (s, Ar); 128.9 (s, Ar); 128.8 (s, Ar); 128.0 (s, Ar); 127.8 (d, JCP = 10.4 Hz, Ar); 127.1 (d, JCP = 8.9 Hz, Ar); 87.8 (d, JCP = 18.8 Hz, quat Cp); 72.5 (d, JCP = 7.0 Hz, subst Cp); 72.4 (s, subst Cp); 70.7 (d, JCP = 44.5 Hz, quat Cp); 70.5 (s, Cp); 68.8 (d, JCP = 4.6 Hz, subst Cp); 66.12 (s, CH/COD); 63.40 (d, CH/COD, JCP = 14.7 Hz); 40.13(d, JCP = 5.7 Hz, CH2Ph); 36.61 (d, JCP = 3 Hz, CH2/COD); 31.1 (s, CH2-Cp); 28.25 (s, CH2/COD). 31 P NMR (500 MHz, CDCl3) δ(ppm): -9.00. MS (DCI, NH3): m/z: 843 (M+H + , 2%); 807 (M-Cl -, 28 %).

  Scheme 1 Synthesis of complexes 2.

Table 3 .

 3 Asymmetric hydrogenation of alkylarylketones .[a]

	O		OH
	X	iPrOH, MeONa	X

Table 4 .

 4 Asymmetric hydrogenation of alkylarylketones .[a]

	O		OH
		30 bars H 2
	X	iPrOH, MeONa	X
	X=H, Me, F

Acknowledgements

We are grateful to the CNRS, the CNR, and the European Union (HYDROCHEM RTN, contract HPRN-CT-2002-00176) for funding.

Run

Catalyst X (position) t (h) conv. (%) [b] ee (%) [b]

24

(S)- [a] Reaction conditions: catalyst, 6.4 X 10 -3 mmol ; NaOMe, 3.2 X 10 -2 mmol; substrate, 3.2 mmol (1/5/500) at 27°C. [b] Conversion and ee determined by GC.

In each case, the absolute configuration configuration of product is S (assigned by comparison of rotation sign in literature data). Furthermore, very high enantioselectivities (up to >99%) together with complete conversions were obtained for the asymmetric hydrogenation of various acetophenones at 10 °C.


Dr Miguel Baya

Dr Jennifer Houghton

Jean-Claude Daran

Rinaldo Poli
email: poli@lcc-toulouse.fr

Dr J.-C Daran

Prof R Poli

Formation and Structure of a Sterically Protected Molybdenum Hydride Complex

Keywords: Molybdenum, Hydride ligands, Paramagnetic organometallic complexes, Oxidatively induced reductive elimination, EPR spectroscopy, X-ray crystallography

published or not. The documents may come

Sterically protected 15-electron molybdenum hydride

Formation and structure of a sterically protected molybdenum hydride complex with a 15-electron configuration: [(1,2,4-C5H2tBu3)Mo(PMe3)2H] + Miguel Baya, Jennifer Houghton, Jean-Claude Daran, Rinaldo Poli  Hydride complexes generally feature a closed shell configuration. They are implicated in a variety of catalytic cycles and are also intermediates of C-H oxidative addition processes. Open-shell, paramagnetic versions have not so far demonstrated broad utility, mainly because of their instability and multitude of decomposition pathways, including deprotonation, [1] disproportionation, [2] dihydrogen reductive elimination (for complexes containing at least two hydride ligands), [3] atom transfer, etc. [START_REF] Poli | Recent Advances in Hydride Chemistry[END_REF] Yet, open-shell hydride complexes appear to play a role in enzymatic processes such as hydrogenase and nitrogenase, [START_REF] Foerster | [END_REF][6][7][8] and their implication in a variety of electrocatalyzed transformation may be envisaged. Therefore, they are attracting renewed interest.

One-electron oxidation of compounds containing two 1-electron ligands, [M](X)(Y), may result in the reductive elimination of X-Y (oxidatively induced reductive elimination, OIRE). This has most clearly been demonstrated for dialkyl complexes, [M](R)2, to give the organic product R-R plus products originating from [M] + . [9][10][11][12] When X = Y = H, the process may lead to H2 evolution, since the oxidation of a polyhydride complex [M]Hn ix expected to favour the rearrangement to a nonclassical isomer, [M]Hn-2(H2) + . [START_REF] Poli | Recent Advances in Hydride Chemistry[END_REF]13,14] In fact, the oxidation of polyhydride complexes often results in dihydrogen evolution, [15] but the instability and the multitude of decomposition pathways of the intermediate oxidized polyhydride complexes often obscures the clean identification of OIRE. [START_REF] Poli | Recent Advances in Hydride Chemistry[END_REF] Indeed, the oxidation of complexes Cp*Ru(PPh3)H3 [13] and Os(PiPr3)2H6 [16] leads to hydrogen evolution, but this was shown to take place following proton transfer from the unstable oxidized complex to residual starting material, rather than from direct reductive elimination.

We have recently reported the first unambiguous case of H2 OIRE for complex Cp*Mo(dppe)H3 (dppe = Ph2PCH2CH2PPh2), thanks to the characterization of the solvent-stabilized product, [Cp*Mo(dppe)(S)H] + , by EPR (S = THF, CH2Cl2) [3] and electrochemistry (S = MeCN), [17] although the structure of the product could not be confirmed crystallographically. The oxidized trihydride complex, [Cp*Mo(dppe)H3] + , also decomposes simultaneously by deprotonation and disproportionation. All three pathways occur via the nonclassical intermediate [Cp*Mo(dppe)H(H2)] + , although theoretical calculations and circumstantial evidence indicates that the oxidized complex adopts a classical structure. [17] We have argued that both stronger electron donation and greater steric protection by the ligands stabilize paramagnetic hydride complexes by disfavouring, via different mechanisms, the various decomposition pathways. [START_REF] Poli | Recent Advances in Hydride Chemistry[END_REF] We have therefore turned our attention to a new Mo system, using the more donating PMe3 ligand in place of dppe and the more sterically protecting 1,2,4-C5H2tBu3 in place of Cp*. This has allowed us to isolate and structurally characterize, for the first time, the starting and end products of the H2 OIRE, [Cp tBu Mo(PMe3)2H3] + and [Cp tBu Mo(PMe3)2H] + (Cp tBu = 1,2,4-C5H2tBu3).

The starting compound, Cp tBu Mo(PMe3)2H3, was prepared by adapting the procedure previously employed for Cp*Mo-(dppe)H3, [18] from Cp tBu MoCl4 and LiAlH4 in the presence of PMe3. Its X-ray structure shows a very similar geometry to that of Cp*Mo(dppe)H3, [19] with the two PMe3 ligands symmetrically disposed in adjacent coordination positions (P-Mo-P angle of 93.71(2)°) and far away from the Cp tBu ligand, see Figure 1(a). [START_REF]The structures were solved by direct methods (SIR97) and refined by full matrix least-squares (SHELXL97). H atoms were treated with the riding model except for the hydrides which were fully refined with isotropic thermal parameters. Crystallographic data (excluding structure factors) have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC 619352 -619354. X-ray data[END_REF] When considering the Cp tBu ring as occupying a single coordination position at the ring centroid, it can best be described as a distorted trigonal prism.

<insert Figure 1 here> A preliminary electrochemical investigations shows a reversible one-electron oxidation process at E1/2 = -0.89 V in THF and -0.93 V in MeCN vs. ferrocene, suggesting that the oxidation product has a certain stability. The stoichiometric oxidation was accomplished by the use of Cp2Fe + PF6 -in THF, affording a product that was sufficiently stable to be isolated and crystallized. [START_REF]The structures were solved by direct methods (SIR97) and refined by full matrix least-squares (SHELXL97). H atoms were treated with the riding model except for the hydrides which were fully refined with isotropic thermal parameters. Crystallographic data (excluding structure factors) have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC 619352 -619354. X-ray data[END_REF] Its structure is closely related to that of the neutral precursor, see Figure 1(b). The Mo-Cp tBu (centroid) separation shortens upon oxidation, whereas the Mo-P bonds lengthen. Not much significance should be attributed to the parameters involving the imprecisely determined hydride positions, but the most notable change is an apparent decrease of the H2 … H3 contact (1.40 Å in the cation vs. 1.63 Å in the neutral complex), suggesting an increased H-H attraction in the oxidized complex. The complex, however, can still be described as a classical trihydride. In THF solution, the complex exhibits a rather broad EPR spectrum at ambient temperature, but sharpening occurs at lower temperatures allowing the identification of the expected quartet of triplet feature, due to coupling to three equivalent hydride ligands and to the P nuclei of two equivalent phosphine ligands, flanked by the 95 Although compound [Cp tBu Mo(PMe3)2H3]PF6 is quite stable as a crystallized solid and in THF solution at low temperatures, it slowly decomposed at T > 0°C, as indicated by a colour change from orange to green. Well formed green crystals were obtained by slow crystallization from THF/pentane at -20°C. The X-ray diffraction analysis revealed the identity of the compound as the H2 elimination product, [Cp tBu Mo(PMe3)2H] + PF6 -, see Figure 3. [START_REF]The structures were solved by direct methods (SIR97) and refined by full matrix least-squares (SHELXL97). H atoms were treated with the riding model except for the hydrides which were fully refined with isotropic thermal parameters. Crystallographic data (excluding structure factors) have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC 619352 -619354. X-ray data[END_REF] Its formation from [Cp tBu Mo(PMe3)2H3] + can be viewed as the collapse of the H2 and H3 atoms (Figure 1b) to yield a putative [Cp tBu Mo(PMe3)2-H(H2)] + PF6 -nonclassical intermediate, followed by H2 dissociation. Although, once again, structural parameters involving the hydride position may be imprecise, the quality of the data set allowed the identification of a single hydride ligand with a high level of confidence. Therefore, the product is a 15-electron complex of Mo III . When protected from air, the compound is stable over several months in the solid state and at least for several hours in THF solution at room temperature. This stability can be attributed to steric protection, since no additional electron density is available on the ligands for a -stabilization mechanism.

<insert Figure 2 here> Although the 15-electron configuration is quite common in the coordination chemistry of Mo III (associated without fail to a spin quartet state), it is extremely rare for organometallic compounds for reasons related to the lower cost of electron pairing in the presence of softer organic ligands. [START_REF] Poli | [END_REF]22] Two examples, which also enjoy high steric protection and adopt a spin quartet ground state, are [(C5Ph5)2Mo] + [23] and Mo(SPhMes2)3. [24] Open-shell hydride complexes are also extremely rare. An example with a 15-electron configuration is (Tp tBu,Me )CoH. [25] The spin configuration for complex [Cp tBu Mo(PMe3)2H] + could not be determined by magnetic methods, because of our inability to prepare a pure bulk sample (the compound crystallized together with other decomposition products, e.g. [Cp tBu Mo(PMe3)2H4] + obtained by proton transfer processes [START_REF]This compound has been isolated and fully characterized, including by X-ray crystallography[END_REF] ). However, we were able to confirm the spin quartet ground state by EPR spectroscopy. Unlike the spin doublet configuration, a spin quartet ground state is notoriously difficult to observe by EPR spectroscopy for 2 nd and 3 rd row transition metals with large zero-field splitting. According to theory, a slightly rhombically distorted tetragonal tensor should have gx and gy close to 4 and gz close to 2 for the spin-allowed ±1/2 transition. [START_REF] Muenck | [END_REF] In addition, a spin forbidden ±3/2 transition may also be accessible, yielding a weak feature at g ca. 6 for the z component. A rare example for Mo III is given by Mo(acac)3, for which a relatively broad spectrum can be observed between the liquid N2 and the liquid He temperatures. [28] Solutions of [Cp tBu Mo(PMe3)2H]PF6 in THF were EPR silent at room temperature and showed only a small resonance at the liquid nitrogen temperature, characteristic of residual [Cp tBu Mo(PMe3)2H3]PF6. The solid sample, however, exhibited the features shown in Figure 4 at liquid He temperature. The g tensor x and y components (±1/2 transition) are indeed visible at g = 3.74 and ca. 3.45, as well as a weak feature attributed to the ±3/2 transition (z component) at g = 5.33. The g = 3.74 peak appears to display a fine structure, possibly due to coupling to the two equivalent P nuclei. The gz component of the ±1/2 transition is not visible because it is overshadowed by stronger resonances of residual 17-electron trihydride at g = 2.009. <insert Figure 4 here> In summary, we have presented the first structural characterization of the starting and end products of an H2 oxidatively induced reductive elimination. The end product is a rare example of a 15-electron hydride complex.

Experimental Section

All operations were carried out under an argon atmosphere with Schlenk line and dry box techniques. Solvents were dehydrated by standard methods and distilled under dinitrogen. Instrumentation used was: cyclic voltammetry, EG&G 362 potentiostat; 1 H and 31 P NMR, Bruker AC200; EPR, Bruker ESP300; X-ray diffraction, Oxford-Diffraction XCALIBUR and Stoe IPDS. Compound Cp tBu MoCl4 was obtained from Cp tBu Mo(CO)3(CH3) and PhICl2, the carbonyl compound being obtained in turn from Mo(CO)6 and NaCp tBu , by adapting the literature procedure used for the Cp* analogue. [START_REF] Keogh | Synthetic Methods of Organometallic and Inorganic Chemistry[END_REF] Preparation of Cp tBu Mo(PMe3)2H3: To compound Cp tBu MoCl4 (1.42 g, 3.00 mmol) in THF (20 mL) was added PMe3 in THF (1 M, 8 mL, 8 mmol). After stirring for 30 min, a suspension of LiAlH4 (ca. 650 mg) in THF (40 mL) was carefully added, resulting in gas evolution. After stirring for 5 h, MeOH (ca. 6 mL) was added dropwise causing vigorous gas evolution. After an additional 1 h stirring the suspension was vacuum-dried. The residue was then extracted with Et2O (ca. 150 mL) and filtered through Celite. The final solution was vacuum-dried, and the pale yellow solid was washed with methanol and dried in vacuo. Yield: 727 mg (50 %). Anal. Calcd. for C23H50MoP2: C, 57.01; H, 10.40. Found: C, 56.48; H, 10.88. 1 H NMR (C6D6):  -5.20 (t, J = 51.0 Hz, 3 H, Mo-H), 1.37 (s, 9 H, t Bu), 1.49 (br, 18 H, P(CH3)3), 1.58 (s, 18 H, 2 x t Bu), 4.86 (s, 2 H, C5H2 t Bu3). 31 P{ 1 H} NMR (C6D6):  17.9 (s). A single crystal for the X-ray analysis was obtained by slow diffusion of a MeOH layer into a pentane solution at 5ºC.

Synthesis of [Cp tBu Mo(PMe3)2H3]PF6: A suspension of [Cp2Fe]PF6

(32 mg, 0.10 mmol) in THF (5 mL) was added dropwise to a cold solution (193 K) of Cp tBu Mo(PMe3)2H3 (53 mg, 0.11 mmol) in tetrahydrofuran (10 mL). The solution color immediately changed from pale yellow to dark blue and, within a few minutes, to orange. The reaction mixture was slowly warmed up to 253 K and then concentrated to ca. 1 mL. Addition of cold pentane (253 K, 10 mL) afforded a brown precipitate that was decanted and further washed with cold pentane (253 K, 3x10 mL) and finally vacuum-dried. Yield: 48 mg, 70 %. A single crystal for the X-ray analysis was obtained by diffusion of a pentane layer onto a THF solution at -80°C. EPR (THF, 193 K): g = 2.016 (triplet of quartets) aP = 36.2 G, aH = 11.4 G and aMo = 30.8 G.

Received: ((will be filled in by the editorial staff)) Published online on ((will be filled in by the editorial staff))

Figure 1 .

 1 Figure 1. ORTEP views of (a) Cp tBu Mo(PMe3)2H3 and (b) the cation in [Cp tBu Mo(PMe3)2H3]PF6. Ellipsoids are drawn at the 30 % probability level. All hydrogen atoms except the hydrides are not shown for clarity. Relevant parameters are listed in the order a/b (the parameters for the cation are averaged over two independent molecules in the asymmetric unit). Distances (Å): Mo-CNT, 2.0150(2)/2.002(2); Mo-P1, 2.3832(6)/2.473(2); Mo-P2, 2.3801(6)/2.473(2); Mo-H1, 1.58(3)/1.62(4); Mo-H2, 1.57(3)/1.54(4); Mo-H3, 1.58(3)/1.57(4); H2•••H3, 1.63/1.40. Relevant angles (°): CNT-Mo-P1, 130.62(2)/127.0(8); CNT-Mo-P2, 132.14(2)/127.4(3); P1-Mo-P2, 93.71(2)/100.3(1); CNT-Mo-H1, 106(1)/104(2); CNT-Mo-H2, 107(1)/108(2); CNT-Mo-H3, 114(1)/108(2). CNT is the Cp tBu ring centroid.

  Mo and 97 Mo isotope satellites. Satisfactory simulation, see Figure 2, provides the hyperfine coupling constants aP = 36.2 G, aH = 11.4 G and aMo = 30.8 G, which compare with those of the analogous but less thermally stable [Cp*Mo(dppe)H3] + complex (aP = 29.8 G; aH = 11.8 G). <insert Figure 2 here>

Figure 2 .

 2 Figure 2. EPR spectrum of compound [Cp tBu Mo(PMe3)2H3]PF6 in THF solution (T = 193 K).

Figure 3 .

 3 Figure 3. ORTEP view of the cation in [Cp tBu Mo(PMe3)2H]PF6. Ellipsoids are drawn at the 30 % probability level. All hydrogen atoms except the hydride are not shown for clarity. Distances (Å): Mo-CNT, 2.0096(3); Mo-P1, 2.4801(8); Mo-P2, 2.4685(9); Mo-H1, 1.83(3).40. Relevant angles (°): CNT-Mo-P1, 122.02(2); CNT-Mo-P2, 121.57(2); P1-Mo-P2, 109.55(3); CNT-Mo-H1, 125.2(9). CNT is the Cp tBu ring centroid.

Figure 4 .

 4 Figure 4. Liquid He, X-band EPR spectrum of compound [Cp tBu Mo(PMe3)2H]PF6 (polycrystalline solid sample).

  Figure 1

Figure 4

 4 Figure 4

We thank the European Commission through the HYDROCHEM program (contract HPRN-CT-2002-00176) for support of this work. MB thanks the Spanish Ministerio de Educación y Ciencia for a post-doctoral fellowship.

Sterically protected 15-electron molybdenum hydride

Miguel Baya, Jennifer Houghton, Jean-Claude Daran, Rinaldo Poli 