Analysis of the co-production capability of thraustochytrids to supply EPA and DHA
The unrooted tree evaluation was carried out with PKS from thraustochytrids. Though the fatty acid composition of various thraustochytrid genera differed due to the existence of impartial evolution, all PKS pathways are thought-about to be DHA/n-6 DPA sort21,26. Lastly, we chosen Schizochytrium sp., Aurantiochytrium sp., Thraustochytrium sp., and Hondaea sp. in several clades from the phylogenetic tree to guage the EPA and DHA co-production capability of the thraustochytrids (Fig. 1a). Thraustochytrium sp. can make the most of the ELO/DES pathway to supply VCL-PUFAs16. Nevertheless, the ELO/DES pathway of the opposite three strains was thought-about incomplete (Fig. 1b). Underneath the fermentation temperature of 28 °C, Schizochytrium sp., Aurantiochytrium sp., and Hondaea sp. may accumulate greater than 40% DHA, which confirmed glorious DHA synthesis potential, however DHA in Thraustochytrium sp. solely accounts for 23.45% of TFAs (Fig. 1c). Thraustochytrium sp. confirmed the strongest EPA synthesis potential among the many 4 strains, and the content material of EPA was as much as 5.65%, which was nearly 7 instances that of Schizochytrium sp. The EPA synthesis potential of Aurantiochytrium sp. and Hondaea sp. was additionally far inferior to that of Thraustochytrium sp.
a Unrooted phylogenetic tree of thraustochytrid genera based mostly on the amino acid sequences of the PKS area. b Schematic diagrams for 2 forms of VCL-PUFAs synthesis programs in thraustochytrids. VCL-PUFAs is synthesized solely by PKS pathway (e.g., Schizochytrium sp., Aurantiochytrium sp., and Hondaea sp.), or by each the PKS and EL/DE pathways (e.g., Thraustochytrium sp.). c, d DHA ratio (c) and EHA ratio (d) of the totally different thraustochytrid genera below 28 °C and 15 °C. Letters above the bars point out vital distinction (p ≤ 0.05), based mostly on one-way evaluation of variance (ANOVA) and Tukey’s truthfully vital distinction (HSD) check. Supply information are supplied as a Supply information file.
Low-temperature situations are thought-about to be extra conducive to the buildup of VCL-PUFAs27. To additional examine the potential of the 4 strains to co-produce DHA and EPA, we carried out fermentation at 15 °C. Surprisingly, the DHA synthesis potential of Thraustochytrium sp. was elevated, however its EPA content material was solely 57.2% of that at 28 °C. Correspondingly, the EPA accumulation capability of Schizochytrium sp. and Hondaea sp. was elevated, and the ultimate proportion of EPA in Schizochytrium sp. was elevated by 3.4 instances in contrast with that at 28 °C. Curiously, the co-production capability of EPA and DHA in Schizochytrium sp. and Hondaea sp. was principally the identical, whereas the VCL-PUFAs synthesis capability of Aurantiochytrium sp. was much less affected by temperature. Due to this fact, there may be an adjustable EPA synthesis pathway in Schizochytrium sp. and Hondaea sp. Furthermore, in contrast with Hondaea sp., Schizochytrium sp. has extra benefits in biomass and lipid accumulation; subsequently, we chosen Schizochytrium sp. for additional analysis (Supplementary Fig. S1).
Elucidating the EPA biosynthesis pathway in Schizochytrium sp
In Schizochytrium sp., identified remaining merchandise synthesized by the PKS pathway are DHA and n-6DPA, however the synthesis methodology of EPA has not been elucidated15,28. A number of genes associated to the fatty acid ELO/DES pathway are current within the Schizochytrium sp. genome29, and this end result was additionally present in Aurantiochytrium. Limacinum30 and H. fermentalgiana9. Nevertheless, no direct proof that Schizochytrium sp. has a whole ELO/DES pathway for EPA synthesis has been discovered. To verify the impact of the partial ELO/DES pathway current in Schizochytrium sp. on EPA accumulation, a protocol for exogenous addition of ELO/DES substrates was designed, which concerned the next 4 distinct addition schemes (2 g/L): solely C14:0, solely C16:0, C16:0, and C18:0, and linseed oil (Fig. 2a). On this pathway, particular oxygen-dependent desaturases and elongases catalyzed particular person desaturation and elongation steps to synthesize VCL-PUFAs from oleic acid (OA; C18:1 n-9), EPA and ARA because the precise manufacturing representatives31. In such situations, schemes 1, 2, and three elevated EPA synthesis by including C18:1 precursor fatty acids, whereas scheme 4 contained C18:1 downstream merchandise, which had been extra favorable for EPA synthesis for the ELO/DES pathway (Supplementary Fig. S2). For EPA, all fermentation schemes elevated barely, which was inconsistent with our assumption that EPA in scheme 4 was not clearly larger than that in different schemes (Fig. 2b). Moreover, not one of the schemes had any impact on DHA synthesis (Fig. 2c). As well as, all of the schemes didn’t have an effect on the expansion of Schizochytrium sp., which can be associated to the low quantity of fatty acids we added externally (Supplementary Fig. S3).
a Schematic diagram of the cardio pathways for VCL-PUFAs biosynthesis. The native EL/DE gene in Schizochytrium sp. is indicated with grey strong body, and the 4 addition schemes are marked with coloured dotted strains. MA myristic acid, PA palmitic acid, STA stearidonic acid, LA linoleic acid, ALA α-linolenic acid, EDA eicosadienoic acid, GLA γ-linolenic acid, STA stearidonic acid, ETrA eicosatrienoic acid, ETA eicosatetraenoic acid, DGLA dihomo-γ-linolenic acid, DTA docosatetraenoic acid. b, c EPA ratio (b) and DHA ratio (c) with totally different addition schemes in Schizochytrium sp. d Comparability of ELO/DES genes transcript stage between 28 °C and 15 °C of Schizochytrium sp. (24 h). Letters above the bars point out vital distinction (p ≤ 0.05), based mostly on one-way evaluation of variance (ANOVA) and Tukey’s truthfully vital distinction (HSD) check. e Comparability of DHA/n-6DPA ratio between the 2 temperatures. The statistical significances of the ultimate outcomes had been analyzed by t-test, ∗p < 0.05.
Moreover, we quantified the transcript ranges of ELO/DES genes at totally different fermentation temperatures by reverse transcription (RT)-PCR, for the reason that potential of Schizochytrium sp. for accumulating EPA was elevated by temperature management (Fig. 1d). In contrast with the transcript ranges at 28 °C, solely the transcript stage of △6 DES elevated by 22% at 15 °C. Nevertheless, the transcript ranges of △9 ELO, △8 DEO, and △12 DEO all decreased by totally different magnitudes (Fig. 2nd). Furthermore, the DHA/n-6 DPA ratio of Schizochytrium sp. reached 4.45 at 15 °C, whereas the proportion of DHA didn’t improve clearly. This indicated a lower of n-6 DPA (Fig. 2e). Earlier research have reported that DHA and n-6 DPA are attribute fatty acids of the PKS pathway in Schizochytrium sp., and the DHA/n-6 DPA ratio is from 2.6 to three.532,33. Primarily based on this, Schizochytrium sp. can have a regulatory mechanism that may launch EPA from the DHA/n-6DPA-type PKS pathway, however the present quantitative information are restricted18,24,25.
Figuring out a MetE-like area that regulates DHA/n-6 DPA sort PKS synthesis of EPA
To find out how temperature impacts fatty acid synthesis in Schizochytrium sp., we carried out comparative transcriptomics on cells grown below two development situations (28 °C and 15 °C). Amongst all differentially expressed genes, one gene (purple rectangle) with each AT and KS domains exhibited clearly transcriptional stage upregulation (Fig. 3a). This gene is called MetE-like, and it consists of the next 4 domains: AT area, KS (or KS/CLF) area, cobalamin-independent methionine synthase area (MetE), and flavoprotein area (CzcO) (Fig. 3b). In reality, the PKS group may be totally different in several organisms. As proven in Fig. 3c, in contrast to the Thraustochytrids-derived PKS system, the ER area exists independently in micro organism, whereas the AT area within the EPA-type PKS system additionally typically exists independently. Apart from, free purposeful domains are current in Psychroflexus torquis (ARA/EPA-type PKS), and Pseudoalteromonas sp. (EPA-type PKS), which signifies the impartial existence of purposeful domains within the PKS pathway doesn’t have an effect on the perform of the PKS system15.
a Volcano plot of the entire transcriptome. Blue and inexperienced dots signify proteins with clearly elevated and decreased ranges, respectively, grey dots signify genes with related transcript ranges below two development situations (MetE-like is circled in purple). b Useful domains included in MetE-like. c Construction of PKS pathway from microorganisms. DH Dehydratase, KR Ketoacyl-ACP reductase. d, e Most Chance phylogenetic evaluation was carried out with all AT (d) and KS (e) area sequences from Schizochytrium sp., Aurantiochytrium sp., a number of micro organism and MetE-like area.
To find out the perform of AT and KS domains situated on MetE-like, a most chance phylogenetic evaluation was carried out on all AT and KS area sequences from Schizochytrium sp., Aurantiochytrium sp., many micro organism, and MetE-like domains. The unrooted tree evaluation distinctly confirmed the three foremost well-supported clades, and AT area of MetE–like had excessive homology with the AT area of Pfa C from Aureispira marina, adopted by the AT domains of Pfa A and Pfa B from Photobacterium profundum (Fig. 3d). The PKS pathways of A. marina and P. profundum may synthesize ARA and EPA, respectively. Due to this fact, the MetE-like-AT could take part within the synthesis and launch of C20 fatty acids similar to the AT area of A. marina and P. profundium. Within the fatty acid synthesis pathway, thioesterases are required to launch free fatty acids from the intermediate metabolites of acyl service protein tether. Equally, the AT domains in PKS of Schizochytrium sp. and marine micro organism can catalyze the chain-release response of acyl-ACP to type free fatty acids22. And, Orikasa et al. reported that the Pfa B-AT area is the important thing enzyme in figuring out the ultimate product of EPA-type PKS and DHA-type PKS23. As well as, the KS area of MetE-like had larger homology with the KS area of Pfa C from micro organism (Fig. 3e). In reality, it has been hypothesized that the origin of PKS gene clusters in thraustochytrids is laterally transferred from marine micro organism34, which could clarify the similarity of MetE-like-KS with bacteria-KS. Tang et al.35 hypothesized that KS/CLF is the first determinant of polyketide chain size. Nevertheless, one other examine reported that the ultimate size of the carbon chain is set by each KSA and KSC (KS/CLF), and the KSA area catalyzes the condensation of C18 to C20. The ultimate elongation step in DHA synthesis is catalyzed by the KSC area in DHA synthase21. Due to this fact, the KS area of MetE-like could not catalyze the elongation from C20 to C22. Primarily based on this, MetE-like can have the perform of releasing EPA from the DHA/n-6 DPA-type PKS system.
Activating the expression of MetE-like for enhancing EPA biosynthesis
Methionine synthase (MetH) permits the conversion of 5-methyltetrahydrofolate and homocysteine to tetrahydrofolate and methionine (Met)36, respectively. It performs a central position within the C1 cycle (Fig. 4a). Nevertheless, the co-factor cobalamin is required for the catalytic exercise of MetH37. In contrast to MetH, the MetE area in MetE-like can exchange MetH to perform as MetH within the absence of cobalamin38. Sequence similarity searches recognized this putative MetE sequence as MetE-like. Nevertheless, it has low homology with different sources of MetE, which might have an effect on the perform and exercise of MetE-like-MetE (Fig. 4b). The C-terminus of MetE comprises conserved Zn2+-binding residues which might be required for catalytic exercise39. These residues, specifically His233, Cys235, Glu267, and Cys330 in MetE-like, are conserved in all the obtained MetE sequences (black pentagram). Primarily based on this, Schizochytrium sp. can activate the expression of MetE-like for the perform of MetE in a cobalamin-free surroundings.
a Metabolic map of a portion of the C1 cycle centered round MetE and MetH, with enzyme abbreviations in black, metabolite abbreviations in blue, and arrows depicting enzyme-catalyzed reactions. Hcy Homocysteine, THF tetrahydrofolate, SAH S-adenosine homocysteine, SAM S-adenosyl methionine, 5-CH2-THF 5-CH2-tetrahydrofolate, 5,10-CH2-THF 5,10-CH2-tetrahydrofolate. b A number of sequence alignment of MetE protein sequences from eight algae for the area spanning the functionally essential conserved residues required for binding Zn2+ (indicated by a black star), numerical values on amino acids signify the place of every amino acid within the MetE-like-MetE area. c, d Time programs of EPA ratio (c) and DHA/n-6DPA (d) ratio between cobalamin+ and cobalamin− of Schizochytrium sp. e Comparability of PKS genes and MetE-like transcript stage between cobalamin+ and cobalamin− of Schizochytrium sp. (60 h). f, g, h Results of various addition schemes on EPA ratio (f), DHA/n-6DPA ratio (g), and MetE-like transcript stage (h) between cobalamin+ and cobalamin− of Schizochytrium sp. (60 h). Letters above the bars point out vital distinction (p ≤ 0.05), based mostly on one-way evaluation of variance (ANOVA) and Tukey’s truthfully vital distinction (HSD) check. The statistical significances of the ultimate outcomes had been analyzed by t-test, ∗p < 0.05.
To find out the consequences of the AT and KS domains of MetE-like on the PKS pathway, we activated the expression of MetE-like by eradicating cobalamin from the Schizochytrium sp. tradition medium. For the EPA ratio, appreciable variations had been noticed with cobalamin addition (cobalamin+) and with out cobalamin addition (cobalamin−) (Fig. 4c). Particularly, below cobalamin−, EPA proportion elevated with the rise in fermentation time and eventually reached 7.63%. In distinction, with cobalamin+, EPA proportion regularly decreased with the prolongation of the fermentation time and leading to only one.26% of EPA content material. Lastly, the EPA titer and EPA yield reached 335.9 mg/L and 15.2 mg/g cell dry weight, respectively, and had been 2.03- and three.89-folds that of Cobalamin+ (Supplementary Fig. S4). The DHA/n-6DPA ratio with cobalamin− remained round 3.60 and was trending upwards, however the DHA/n-6DPA ratio below the cobalamin+ situation fluctuated round 3.0 (Fig. 4d). To additional confirm the connection between PKS and MetE-like, we quantified the transcript ranges of the 4 associated domains by RT-PCR (Fig. 4e). Notably, MetE-like was the one gene with the elevated transcript stage in cobalamin−, which was 4.21-folds larger than that below the cobalamin+ situation. Intriguingly, below cobalamin−, orf A, orf B, and orf C transcript ranges had been lowered by 74.8%, 78.6%, and 85.4%, respectively. In accordance with the genomic data, Schizochytrium sp. has just one MetE area on MetE-like40. Because of the demand for Met, the transcription stage of MetE was clearly elevated; thus, the transcription ranges of the AT and KS purposeful domains of MetE-like had been additionally elevated. These outcomes help that MetE-like can regulate EPA synthesis through the PKS pathway.
Additional, to restrict MetE-like expression below the cobalamin− situation, Met was chosen because the MetE area inhibitor. Furthermore, Czco is a kind of flavoprotein, which might use derivatives of riboflavin as a prosthetic group to take part in electron switch41. Notably, including Met or riboflavin to Schizochytrium sp. didn’t have an effect on EPA accumulation below the cobalamin+ situation, and including Met and riboflavin concurrently barely decreased EPA accumulation (Fig. 4f). Underneath cobalamin−, including Met or including Met and riboflavin decreased the flexibility of EPA accumulation, which signifies that Met can alleviate the malnutrition attributable to the cobalamin− situation; nonetheless, including riboflavin alone has no impact. In cobalamin+, the DHA/n-6DPA ratio is principally secure below the three further situations, that means that the PKS system was secure. Nevertheless, in cobalamin−, the DHA/n-6DPA ratio was much like that of EPA, and Met may ameliorate the dysfunction of PKS system (Fig. 4g).
Additional, the MetE-like transcript ranges in Schizochytrium sp. below totally different fermentation situations had been examined. In cobalamin+, all inhibitor addition regimens prompted somewhat distinction (Fig. 4h). In distinction, below cobalamin−, the MetE-like transcript ranges had been lowered by 36.2%, 1.8%, and 49.5%, respectively. Which means the requirement for MetE perform in Schizochytrium sp. is the direct driving power for the rise of MetE-like transcription ranges below the cobalamin− situation. Collectively, by regulating MetE-like transcription ranges, the flexibility of Schizochytrium sp. to build up EPA and DHA/n-6DPA within the PKS pathway present corresponding adjustments; therefore, MetE-like can regulate the synthesis of EPA through the PKS pathway.
Exploring the lipid compound composition below the situation of MetE-like activation
To additional examine the impact of cobalamin on EPA accumulation in Schizochytrium sp., lipidomics evaluation was carried out to investigate the variations in lipid compound compositions throughout the late stage of Schizochytrium sp. fermentation below cobalamin+ and cobalamin− situations. Metabolites with related expression patterns are sometimes functionally associated, and a complete of fifty metabolites from the metabolic set had been chosen for the cluster evaluation (Fig. 5a). Amongst them, 25 lipids had been triglycerides (TGs). TG is the foremost storage type of fatty acids in Schizochytrium sp.15. As well as, fatty acids in lipid storage kinds equivalent to phosphatidylcholine (PC), diradylglycerols (DG), phosphatidylglycerol (PG), lysophosphatidylcholine (LPC), and dimethylphosphatidylethanolamine (dMePE) had been additionally clearly altered. DHA is a attribute fatty acid of Schizochytrium sp.34, and 25 lipid compounds confirmed the variability in DHA storage. Just a few lipids comprise EPA, and solely PC (16:1/20:5), TG (9:0/20:5/22:6), PG (20:5/22:6), and dMePE (20:5/22:6) majorly contribute to the variability in EPA storage. Within the cobalamin− situation, LPC (17:0), PG (16:0/17:0), PG (15:0/22:5), and TG (17:0/20:3/22:5) had been elevated, whereas TG (16:0/16:0/20:4), TG (16:0/14:3/22:6), TG (14:0/14:1/14:2), PG (18:0/16:0), and TG (18:4/16:0/22:6) had been decreased.
a Lipid compounds cluster evaluation plot. The colours within the determine signify the relative expression ranges of lipid compounds on this group of samples, the left facet is the dendrogram of lipid compound clustering, and the correct facet is the identify of the lipid compounds. b, c EPA-enriched TG profile (b) and EPA-enriched DG profile (c) in Schizochytrium sp.(60 h). d Comparability of fatty acid storage associated genes transcript stage between cobalamin+ and cobalamin− of Schizochytrium sp. (60 h).
Subsequent, we analyzed the variations among the many lipids registered in HMDB. Typically, fatty acids in Schizochytrium sp. are primarily composed of glycerolipids (GLs) and glycerophospholipids (GPs)42. GLs and GPs accounted for greater than 95% of the overall lipids in lipids registered in HMDB; nonetheless, below cobalamin−, GL proportion elevated by 9.9%, whereas GP proportion decreased by 13.4% (Supplementary Fig. S5). Amongst GLs, below cobalamin+, the proportion of TG to GLs was about 70%, whereas TG solely accounted for 54.8% of GLs below the cobalamin− situation; nonetheless, below cobalamin−, DG proportion elevated by 60% (Supplementary Fig. S6). PC is the principle storage type of GPs and accounts for greater than 90%; phosphatidylethanolamine (PE) is the second-largest storage type of GPs, however PE proportion elevated by 86.5% below the cobalamin− situation (Supplementary Fig. S7).
Moreover, we analyzed the variations in EPA content material among the many lipids registered in HMDB. In EPA-containing TG, its proportion (20:5/20:4/20:5) elevated and as much as 23.99% below the cobalamin− situation, which was about 6.8 instances the proportion below the cobalamin+ situation. A complete of 18 EPA-containing TGs had been registered in HMDB, of which 13 had been clearly elevated (Fig. 5b). Equally, a complete of three EPA-containing DGs had been registered in HMDB, of which the DG proportion (20:5/22:6) within the cobalamin− situation was 35.63%, whereas that within the cobalamin+ situation was 14.82%, however the proportions of the opposite two EPA-containing DGs had been much less (Fig. 5c). As well as, amongst GPs registered in HMDB, PE, glycerophosphates (PA), and PC grew to become the principle EPA-storage kinds. In cobalamin−, the proportion of PE (20:5/22:6) elevated from 2.12% to fifteen.53%, whereas the proportion of EPA in PCs has typically elevated, however the proportion of PA (20:5/22:6) has decreased (Supplementary Figs. S8–S10). Curiously, the co-storage type of EPA and DHA was most popular amongst diester lipids. Schizochytrium sp. tended to retailer EPA in GLs type below the cobalamin− situation.
To find out the adjustments that happen in fatty acid storage in Schizochytrium sp., we quantified the transcript ranges of glycerol-sn-3-phosphate acyl-transferase (GPAT), lysophosphatidate acyl-transferase (LPAAT), phosphatidic acid phosphatase (PAP), and diacylglycerol acyltransferase (DGAT) genes below the cobalamin+ and cobalamin− situations by RT-PCR. TGs are synthesized through the Kennedy pathway in Schizochytrium sp.15. In step one, glycerol-3-phosphate is transformed into lysophosphatidate (LPA) by the rate-limiting enzyme GPAT. Subsequently, LPAAT transfers an acyl moiety from coenzyme A (CoA) onto the sn-2 place of LPA, leading to PA. Then, PAP catalyzes the formation of DG from PA, and eventually, DGAT is catalyzed to switch an acyl group from CoA to DG to type TG. Though the general transcript ranges of associated genes decreased clearly, the transcript ranges of LPAAT2, PAP1, and DGAT2 elevated 0.80, 4.17, and 0.86 instances, respectively, which can be an essential motive for the distinction in fatty acid composition in Schizochytrium sp. (Fig. 5d).
Relieving the expansion stress to extend the height manufacturing of EPA by optimizing carbon sources
To discover the impact of cobalamin on the EPA manufacturing capability of Schizochytrium sp., a combined carbon supply of crude glycerol and glucose was used to alleviate the expansion stress on Schizochytrium sp. below the cobalamin− situation43,44,45. Right here, to discover the impact of cobalamin on the EPA manufacturing capability of Schizochytrium sp., a combined carbon supply of crude glycerol and glucose was used to alleviate the expansion stress on Schizochytrium sp. below the cobalamin− situation. Within the pre-experiment, we examined the adjustments attributable to the totally different ratio of carbon sources below the cobalamin− situation and the ultimate combined carbon supply of 20 g/L crude glycerol + 40 g/L glucose was chosen (Supplementary Fig. S11). As proven in Fig. 6a, below cobalamin−, the ultimate biomass and lipids through fed-batch fermentation had been far decrease than below the cobalamin+ situation, however the combined carbon supply was favorable for biomass and lipid accumulation, which lastly reached 75.13 g/L and 21.23 g/L, respectively. Nevertheless, combined carbon supply can’t basically resolve the expansion drawback attributable to cobalamin deficiency.
a Time programs of Biomass (or dry cell weight) and Lipid in fed-batch tradition. b EPA ratio, DHA ratio, and EPA manufacturing (mg/g dry cell weight) of Schizochytrium sp. (84 h) in fed-batch tradition. The statistical significances of the ultimate outcomes had been analyzed by t-test, *p < 0.05.
Unexpectedly, carbon supply optimization didn’t improve EPA proportion, and EPA proportion obtained utilizing the only real glucose and combined carbon supply reached 11.1% and 10.6%, respectively, whereas it was solely about 1% below the unique fermentation situations. No apparent distinction in DHA proportions among the many three fermentation situations was noticed; nonetheless, below cobalamin−, the usage of combined carbon sources improved DHA accumulation, and DHA accounted for 45.4%. As well as, research indicated that the DHA content material was enhanced when glycerol was used as carbon supply, and subsequent transcriptomic evaluation revealed that the glycolysis and amino acid metabolism had been tremendously upregulated46. Lastly, below cobalamin−, the height manufacturing of EPA obtained by utilizing combined carbon sources reached 29.83 (mg/g cell dry weight), which was 7.12-folds larger than that of the unique fermentation situations (Fig. 6b). Due to this fact, now we have established a believable methodology for the co-production of EPA and DHA in Schizochytrium sp. by fermentation engineering.
Conclusion
The Schizochytrium sp. was chosen because the potential EPA produce by analyzing the fatty composition of various thraustochytrids. We first recognized the MetE-like advanced that may modulate the PKS system to supply EPA. When the transcriptional stage of MetE-like was activated, the manufacturing of EPA may be elevated by 7.12-folds in comparison with that of the unique fermentation situation, ensuing a co-production of two.25 g/L EPA and 9.59 g/L DHA. The outcomes of the current examine present essential insights into the position of PKS system for producing fatty acids and report the flexibility of Schizochytrium sp. As a possible EPA and DHA co-producer (Desk 1).
