Polyunsaturated fatty acid production by Yarrowia lipolytica employing designed myxobacterial PUFA synthases

Abstract

Long-chain polyunsaturated fatty acids (LC-PUFAs), particularly the omega-3 LC-PUFAs eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA), have been associated with beneficial health effects. Consequently, sustainable sources have to be developed to meet the increasing demand for these PUFAs. Here, we demonstrate the design and construction of artificial PUFA biosynthetic gene clusters (BGCs) encoding polyketide synthase-like PUFA synthases from myxobacteria adapted for the oleaginous yeast Yarrowia lipolytica. Genomic integration and heterologous expression of unmodified or hybrid PUFA BGCs yielded different yeast strains with specific LC-PUFA production profiles at promising yield and thus valuable for the biotechnological production of distinct PUFAs. Nutrient screening revealed a strong enhancement of PUFA production, when cells were phosphate limited. This represents, to the best of our knowledge, highest concentration of DHA (16.8 %) in total fatty acids among all published PUFA-producing Y. lipolytica strains.

Fig. 1

De novo polyunsaturated fatty acid (PUFA) biosynthesis in myxobacteria by iteratively acting, multifunctional PUFA synthases. a The starter unit acetyl-CoA (R = H) is consecutively elongated with the extender unit malonyl-CoA by several rounds of decarboxylative Claisen condensations, resulting in the extension of the fatty acyl chain by two carbons per cycle. After each round of elongation, the β-keto group is either fully reduced by ketoreduction, dehydration plus enoylreduction, or only reduced by ketoreduction and dehydration, giving rise to the trans double bond, which is then isomerized to synthesize an acyl chain bearing methylene-interrupted cis double bonds. After reaching its final length, the fatty acyl chain is presumably used for acylation of the 2-position of 1-acylglycerol-3-phosphate. KS, ketosynthase of Pfa2 and Pfa3; AT, acyltransferase of Pfa2 and Pfa3; ACP, acyl carrier protein of Pfa2; KR, ketoreductase of Pfa2; DH, dehydratase of Pfa2 and Pfa3; DH_I, dehydratase/isomerase of Pfa3; ER, enoylreductase of Pfa1; AGPAT, 1-acylglycerol-3-phosphate O-acyltransferase of Pfa3. b Structures of the main PUFAs produced by the myxobacteria Aetherobacter fasciculatus (SBSr002) and Minicystis rosea (SBNa008)

Fig. 2

Artificial pfa biosynthetic gene clusters encoding myxobacterial PUFA synthases for LC-PUFA production in Yarrowia lipolytica Po1h. a Synthetic pfa BGC (20.2 kb) version C1_V1 or C1_V2 comprising genes pfa1, pfa2, and pfa3 encoding the DPA/DHA-type PUFA synthase plus gene ppt encoding the 4′-phosphopantetheinyl transferase (PPTase) from Aetherobacter fasciculatus (SBSr002) adapted for the oleaginous yeast Yarrowia lipolytica. b Synthetic pfa BGC (21.1 kb) version C3 or C3_mod 5′ comprising genes pfa1, pfa2, and pfa3 encoding the AA/DTA-type PUFA synthase plus gene ppt encoding the PPTase from Minicystis rosea (SBNa008) adapted for Y. lipolytica. c LC-PUFAs produced by Y. lipolytica Po1h::Af4 (harboring synthetic pfa BGC version C1_V1), Y. lipolytica Po1h::Af7 (harboring synthetic pfa BGC version C1_V2), Y. lipolytica Po1h::Mr1 (harboring synthetic pfa BGC version C3), and Y. lipolytica Po1h::Mr2 (harboring synthetic pfa BGC version C3_mod 5′). Each coding sequence of all clusters is flanked by the strong hybrid hp4d promoter and the LIP2 terminator. Unique restriction enzyme sites are present at specific positions for pathway assembly and for interchangeability of inter- and intragenic regions. ER, enoylreductase; KS1+KS2, ketosynthases; AT1+AT2, acyltransferases; ACP, acyl carrier protein; KR, ketoreductase; DH1+DH4, polyketide synthase (PKS)-like dehydratases; CLF, chain length factor; DH2+DH3, FabA-like dehydratases/isomerases; AGPAT, 1-acylglycerol-3-phosphate O-acyltransferase. Cultivations were carried out in 10 mL YNBG medium+50 mM potassium phosphate buffer pH 6.8 at 28 °C and 200 rpm for 168 h. The indicated values are means and s.d. of three biological replicates, presented on a logarithmic scale. Source data are provided as a Source Data file. ETrA, eicosatrienoic acid; AA, arachidonic acid; DTrA, docosatrienoic acid; DTA, docosatetraenoic acid; DPA, docosapentaenoic acid; DHA, docosahexaenoic acid; TTA, tetracosatetraenoic acid

Fig. 3

LC-PUFAs produced by Yarrowia lipolytica Po1h using chimeric PUFA synthases encoded by artificial pfa biosynthetic gene clusters. a Hybrid pfa BGCs located on plasmid pHyb1, plasmid pHyb2a, and plasmid pHyb5a. b LC-PUFAs produced by Y. lipolytica Po1h::Hyb1, Y. lipolytica Po1h::Hyb2a, and Y. lipolytica Po1h::Hyb5a. c Hybrid pfa BGCs located on plasmid pHyb6, plasmid pHyb6b, and plasmid pHyb6b-H2270A. d LC-PUFAs produced by Y. lipolytica Po1h::Hyb6, Y. lipolytica Po1h::Hyb6b, and Y. lipolytica Po1h::Hyb6b-H2270. e Hybrid pfa BGCs located on plasmid pHyb8 and plasmid pHyb9. f LC-PUFAs produced by Y. lipolytica Po1h::Hyb8 and Y. lipolytica Po1h::Hyb9. g Hybrid pfa BGCs located on plasmid pHyb15. h LC-PUFAs produced by Y. lipolytica Po1h::Hyb15. Domains from the synthetic DPA/DHA-type pfa BGC are shown in blue; domains from the synthetic AA/DTA-type pfa BGC are shown in red. Cultivations were carried out in 10 mL YNBG medium+50 mM potassium phosphate buffer pH 6.8 at 28 °C and 200 rpm for 168 h. The indicated values are means and s.d. of three biological replicates. Source data are provided as a Source Data file. ETrA, eicosatrienoic acid; AA, arachidonic acid; ETA, eicosatetraenoic acid; EPA, eicosapentaenoic acid; DTrA, docosatrienoic acid; DTA, docosatetraenoic acid; DPA, docosapentaenoic acid; DHA, docosahexaenoic acid; TTA, tetracosatetraenoic acid; TPA, tetracosapentaenoic acid; THA, tetracosahexaenoic acid; THpA, putative tetracosaheptaenoic acid

Fig. 4

DHA production improvement in Y. lipolytica Po1h::Af4 based on an optimization of medium composition. Shake flask cultivations were carried out in triplicates. Performance parameters were determined after 200 h of cultivation. Graphs show means and s.d. Source data are provided as a Source Data file

Fig. 5

DHA production in a fed-batch process. The designed producer Y. lipolytica Po1h::Af4 was grown in minimal medium on either glucose or glycerol as sole carbon source. Fermentation was conducted at 28 °C, pH 5.5, and a dissolved oxygen level of 5% during the feed phase. Substrate feeding was initiated upon carbon depletion. Coefficients of variation (CVs) across biological replicates were below 5% for biomass, substrate, and citrate levels, and below 10% for PUFA and native fatty acid content. Source data are provided as a Source Data file