Divergent evolution of extreme production of variant plant monounsaturated fatty acids

Abstract

Metabolic extremes provide opportunities to understand enzymatic and metabolic plasticity and biotechnological tools for novel biomaterial production. We discovered that seed oils of many Thunbergia species contain up to 92% of the unusual monounsaturated petroselinic acid (18:1Δ6), one of the highest reported levels for a single fatty acid in plants. Supporting the biosynthetic origin of petroselinic acid, we identified a Δ6-stearoyl-acyl carrier protein (18:0-ACP) desaturase from Thunbergia laurifolia, closely related to a previously identified Δ6-palmitoyl-ACP desaturase that produces sapienic acid (16:1Δ6)-rich oils in Thunbergia alata seeds. Guided by a T. laurifolia desaturase crystal structure obtained in this study, enzyme mutagenesis identified key amino acids for functional divergence of Δ6 desaturases from the archetypal Δ9-18:0-ACP desaturase and mutations that result in nonnative enzyme regiospecificity. Furthermore, we demonstrate the utility of the T. laurifolia desaturase for the production of unusual monounsaturated fatty acids in engineered plant and bacterial hosts. Through stepwise metabolic engineering, we provide evidence that divergent evolution of extreme petroselinic acid and sapienic acid production arises from biosynthetic and metabolic functional specialization and enhanced expression of specific enzymes to accommodate metabolism of atypical substrates.

Keywords: biotechnology; chemical diversity; desaturase; fatty acid; vegetable oil.

Fig. 1.

Identification of petroselinic acid as the predominant seed FA of multiple Thunbergia species. (A) Flowers and seeds of T. laurifolia. (B) Gas chromatogram of isopropyl esters of FAs from the total lipid extract of T. laurifolia seeds with triheptadecanoin (17:0 TAG) added as an internal standard. (C) Mass spectral confirmation of petroselinic acid in T. laurifolia seeds analyzed as dimethyl disulfide derivatives of the methyl ester. (D) Comparison of the FA composition of TAG (orange) and PC (blue) fractions of T. laurifolia seed lipids. (E) FA composition of the total lipid of T. alata (blue), T. fragrans (orange), T. grandiflora (silver), T. coccinea (yellow), T. natalensis (brown), and T. lutea (green) seeds. Results of FA analyses in D and E are the average obtained from three to five independent biological replicates ± SD.

Fig. 2.

Phylogeny and in vitro activities of Δ6 desaturases of T. alata and T. laurifolia. (A) Unrooted phylogenetic tree showing the close relationship of the two T. laurifolia desaturases with the T. alata Δ6-16:0-ACP desaturase. (B) Multiple alignment with asterisks showing the conservative residues between different desaturases in A. Green shading indicates 100% amino acid sequence identify; blue shading shows the consensus match; and white identicates mismatch from the consensus. These residues were targeted for mutagenesis to understand their contributions to the variant activity of the Thunbergia desaturases (see Fig. 4). (C and D) Comparison of activities and regiospecificities of Δ6 desaturases from T. laurifolia and T. alata with 16:0- and 18:0-acyl-ACP substrates. Values are averages obtained from three independent enzyme assays ± SD. (E) Mass spectrum of dimethyl disulfide derivatives of FA methyl ester of products from assay of T. laurifolia desaturase with 17:0-ACP to confirm production of Δ6 unsaturated product. Species, desaturases, and gene identifiers or GenBank accession numbers for acyl-ACP desaturases shown in A: AtAAD1: A. thaliana AAD1 AT5G16240; AtAAD2: A. thaliana AAD2 AT3G02610; AtAAD3: A. thaliana AAD3 AT5G16230; AtAAD4: A. thaliana AAD4 AT3G02620; AtAAD5: A. thaliana AAD5 AT3G02630; AtAAD6: A. thaliana AAD6 AT1G43800; Cs C16 DES4: C. sativum Δ4-16:0-ACP desaturase, P32063.1; Hh C16 DES4: H. helix Δ4-16:0-ACP desaturase, AAY46941.1; Rc C18 DES9-1: R. communis Δ9-18:0-ACP desaturase, NP_001310674.1; Rc C18 DES9-2: R. communis Δ9-18:0-ACP desaturase, NP_001310638.1; Du C16 DES9: Dolichandra unguis-cati Δ9-16:0-ACP desaturase, AAC05293.1; Si C18 DES9-1: Sesamum indicum Δ9-18:0-ACP desaturase, XP_011074783.1; Si C18 DES9-2: S. indicum Δ9-18:0-ACP desaturase, XP_011091536.1; Ta C18 DES9-1: T. alata Δ9-18:0-ACP desaturase, AAA61559.1; Ta C18 DES9-2: T. alata Δ9-18:0-ACP desaturase, AAA61560.1; As C16 DES9: A. syriaca Δ9-16:0-ACP desaturase, AAC49719.1; Ta C16 DES6: T. alata Δ6-16:0-ACP desaturase, Q41510.1; Tl C18 DES6-1: T. laurifolia Δ6-18:0-ACP desaturase, OL757550; Tl C18 DES6-2: T. laurifolia Δ6-18:0-ACP desaturase, OL757551; Tl C18 DES9: T. laurifolia Δ9-18:0-ACP desaturase, ON393910.

Fig. 3.

Structure-guided mutagenesis of T. laurifolia Δ6-18:0-ACP desaturase for altered regiospecificity. (A–F) In vitro activity of T. laurifolia Δ6-18:0-ACP desaturase mutants that are designed based on amino acid sequence differences with T. alata Δ6-16:0-ACP desaturase and canonical Δ9-18:0-ACP desaturases. The relative production of Δ6 and Δ9 desaturation products is shown. (G) Structure of the T. laurifolia Δ6-18:0-ACP desaturase dimer determined at 2.0-Å resolution. (H) T. laurifolia Δ6-18:0-ACP desaturase crystal structure with the key amino acids labeled, (I) T. laurifolia Δ6-18:0-ACP desaturase structure in the surface model shows the K257 and Q275 at the bottom and top for the substrate pocket opening. (J) In vitro activity of K257M and (K) G183A/F184Y/K257M Δ6-18:0-ACP desaturase mutants. Data in A–F, J, and K are the averages ± SD from three independent assays (*P < 0.05, **P < 0.01, Student’s t test). nd, not detected. Confirmation of Δ10 desaturation product is shown in SI Appendix, Fig. S8.

Fig. 4.

Metabolic engineering for production of oils containing petroselinic acid and other Δ6 FA desaturase-derived FAs in camelina seeds by expression of genes from T. laurifolia or T. alata. (A) Relative amounts of Δ6 desaturase-derived monounsaturated FAs in the total FAs from homozygous T₃ camelina seeds of top-producing line engineered for expression of the T. laurifolia Δ6-18:0-ACP desaturase alone (Tl Δ6 Des) or coexpression with a T. laurifolia FatA cDNA (Tl Δ6 Des + TlFatA). Also shown are relative amounts of Δ6 desaturase-derived monounsaturated FAs in the total FAs of T₂ camelina DsRed⁺ seeds from top producing line engineered for coexpression with a T. laurifolia Δ6 desaturase, FatA and LPAT2 cDNAs with FATTY ACID ELONGASE1 FAE1 RNAi (Tl Δ6 Des + TlFatA + TlLPAT2 + FAE1 RNAi). n.d., not detected. (B) Relative amounts of Δ6 desaturase-derived monounsaturated FAs in the total FAs from homozygous T₃ camelina seeds of top-producing line engineered for expression of the T. alata Δ6-16:0-ACP desaturase alone (Ta Δ6 Des) or in a FA ELONGASE1 FAE1 RNAi background (Ta Δ6 Des + FAE1 RNAi). Data in A and B are the averages of measurements of seeds from three to five independent plants for each line ± SD. Proposed biosynthetic pathways for the production of Δ6 desaturase-derived monounsaturated FAs and mass spectral confirmation of the major products from transgenic expression of T. laurifolia and T. alata genes are also shown. Double-bond positions were determined by GC-MS analysis of dimethyl disulfide derivatives of FA isopropyl esters. n.d., not detected. (C) Comparison of Δ6 desaturase-derived monounsaturated FA production in T₂ DsRed⁺ camelina seeds expressing the T. laurifolia Δ6-18:0-ACP desaturase alone (Tl Δ6-18:0-ACP Des) or coexpressed with the Arabidopsis FatA-1 (+AtFatA-1), Arabidopsis FatA-2 (+AtFatA-2), or M. charantia FatA (+McFatA).

Fig. 5.

Expression analysis of genes for KAS I, KAS II, and FatA in developing T. alata and T. laurifolia seeds. (A–C) Results averages from three independent qPCR experiments ± SD (**P < 0.01, Student’s t test).