Engineered triacylglycerol accumulation

NoLDSP, a lipid droplet surface protein from the microalga Nannochloropsis oceanica, has functions partially analogous to plant oleosins25. Similar to oleosins, NoLDSP possesses a hydrophobic central region that likely mediates the anchoring on lipid droplets. To assess the impact of NoLDSP on AtWRI11–397-initiated triacylglycerol accumulation, we infiltrated leaves of N. benthamiana with Agrobacterium tumefaciens suspensions for transient production of AtWRI11–397 alone or in combination with NoLDSP (AtWRI11–397 + NoLDSP). In leaves producing AtWRI11–397 or AtWRI11–397 + NoLDSP, the triacylglycerol level was ~3-fold and 12-fold higher, respectively, than in control leaves without AtWRI11–397 (Fig. 1a). The results clearly demonstrated that the microalgal NoLDSP had no negative impact on triacylglycerol production and enhanced the accumulation of lipid droplets in infiltrated N. benthamiana leaves.

Fig. 1 Engineered patchoulol production in N. benthamiana leaves. Triacylglycerol (TAG) accumulation was initiated through expression of WRINKLED1 (AtWRI11–397) and further enhanced through co-expression of NoLDSP (a). Patchoulol production was engineered in the cytosol (b) and in the plastid (c) in the absence and presence of AtWRI11–397 and NoLDSP. To enhance FDP availability for patchoulol production, ElHMGR159–582 (cytosol), PbDXS (plastid) and AtFDPS (cytosol, plastid) were included in transient assays. The different construct combinations are indicated below each bar (black circle, was included; minus, was not included) and in the scheme next to each graph. Data were analyzed by Shapiro–Wilk, Welch–ANOVA (a P < 0.0006; b P < 0.0001; c P < 0.0001) and Brown–Forsythe ANOVA (a P < 0.0004; b P < 0.0001; c P < 0.0001) followed by t-tests (unpaired, two-tailed, Welch correction). Data are presented as individual biological replicates and bars representing average levels with SD (a N = 4; b N indicated below each bar; c N = 8). This experiment was replicated twice. Statistically significant differences are indicated by a–e based on t-tests (P < 0.05). Source Data are provided as a Source Data file. LD, lipid droplet Full size image

Sesquiterpenoid production in the cytosol and plastids

We then tested different engineering strategies for the production of sesquiterpenoids using patchoulol as a model compound. Like many other sesquiterpenoids, patchoulol is volatile and its engineered production in transgenic lines of N. tabacum resulted in significant losses from volatile emission15. In our study, losses by atmospheric terpenoid emission were not recorded as the engineering strategies were designed to sequester target terpenoids in lipid droplets in the plant biomass. Transient production of cytosolic Pogostemon cablin patchoulol synthase (cytosol:PcPAS) led to formation of a single low-level product, patchoulol, which was not detected in wild-type control plants (Fig. 1b). To enhance the precursor availability for sesquiterpenoid synthesis, a feedback-insensitive form of Euphorbia lathyris HMGR (ElHMGR159–582) and A. thaliana FDPS (cytosol:AtFDPS) were included in the transient assays. E. lathyris accumulates high levels of triterpenoids and their esters26, suggesting that its HMGR could be a robust enzyme for sesquiterpenoid production in N. benthamiana. The selection of the A. thaliana FDPS was based on its relatively high thermal stability27. The patchoulol content in N. benthamiana leaves producing ElHMGR159–582 + cytosol:AtFDPS + cytosol:PcPAS was ~5-fold higher than in leaves with cytosol:PcPAS which is consistent with enhanced precursor flux. Co-engineering of patchoulol and triacylglycerol synthesis impaired cytosolic terpenoid accumulation, independent of whether precursor availability was increased or not (Fig. 1b).

A previous study demonstrated that re-direction of PcPAS and avian FDPS to the plastid increased the (retained) patchoulol level in leaves of stable transgenic N. tabacum lines up to ~30 μg patchoulol g−1 fresh weight15. We modified this approach to further examine engineering strategies for the co-production of patchoulol and lipid droplets in N. benthamiana leaves. Targeting of patchoulol synthase to the plastids (plastid:PcPAS) led to accumulation of approximately 0.5 μg patchoulol g−1 fresh weight (Fig. 1c). To increase the precursor flux in the plastids, P. barbatus DXS (PbDXS) and plastid-targeted AtFDPS (plastid:AtFDPS) were combined with plastid:PcPAS in the assays. This strategy resulted in a 60-fold increase in the level of patchoulol (Fig. 1c). Synthetic lipid droplet accumulation impaired patchoulol production in leaves in the absence of PbDXS and plastid:AtFDPS, when precursor synthesis was not co-engineered (Fig. 1c). The negative impact of lipid droplet production on patchoulol synthesis was rescued when plastid:AtFDPS or PbDXS + plastid:AtFDPS were included in the assay. Leaves transiently producing PbDXS + plastid:AtFDPS + plastid:PcPAS + AtWRI11–397 + NoLDSP yielded the highest patchoulol level retained in leaves (up to ~45 μg patchoulol g−1 fresh weight), an average 90-fold and 1.5-fold higher compared to leaves producing plastid:PcPAS and PbDXS + plastid:AtFDPS + plastid:PcPAS, respectively.

Diterpenoid scaffold production in plastids and cytosol

Strategies for diterpenoid production in the N. benthamiana system were examined using the Abies grandis abietadiene synthase (AgABS) as diterpene synthase28,29. The bifunctional enzyme has class II and class I terpene synthase activity and catalyzes both the bicyclization of GGDP to a (+)-copalyl diphosphate intermediate and the subsequent secondary cyclization and further rearrangement. Transient production of the native plastidial A. grandis abietadiene synthase (plastid:AgABS) resulted in the accumulation of abietadiene (abieta-7,13-diene), levopimaradiene (abieta-8(14),12-diene), neoabietadiene (abieta-8(14),13(15)-diene) and, as minor product, palustradiene (abieta-8,13-diene) consistent with the previous findings30. These diterpenoids were not detected in wild-type control leaves of N. benthamiana. Sole production of plastid:AgABS yielded ~40 μg diterpenoids g−1 fresh weight (Fig. 2a). To enhance the production of diterpenoids, plastid:AgABS was co-produced in different combinations with PbDXS and a plastid GGDPS. GGDPSs are differentiated into three types (type I–III) according to their amino acid sequences around the first aspartate-rich motif. These three types differ in their mechanism of determining product chain-length31,32. Plant GGDPSs are type II enzymes that are regulated on gene expression, transcript, and protein level33,34,35. We hypothesized that inclusion of distantly related type I and type III GGDPSs or a cyanobacterial type II GGDPS may allow us to bypass potential regulatory steps limiting diterpenoid production in N. benthamiana. Six GGDPSs were selected: an archaeal GGDPS from Sulfolobus acidocaldarius (SaGGDPS, type I), a predicted archaeal GGDPS from Methanothermobacter thermautotrophicus (MtGGDPS, type I), a predicted cyanobacterial GGDPS from Tolypothrix sp. PCC 7601 (TsGGDPS, type II), two predicted plant GGDPSs from Euphorbia peplus (EpGGDPS1 and EpGGDPS2, type II), and one predicted GGDPS from the fungus Mortierella elongata AG77 (MeGGDPS, type III). SaGGDPS, MtGGDPS, and MeGGDPS share only 24%, 25 and 17% amino acid identities with EpGGDPS1, respectively, whereas TsGGDPS and EpGGDPS2 share 48 and 58% identities with EpGGDPS1, respectively. For transient assays in N. benthamiana, the coding sequences for the bacterial and fungal GGDPSs were codon-optimized (except for TsGGDPS) and modified to target the enzymes to the plastids, referred to as plastid:SaGGDPS, plastid:MtGGDPS, plastid:TsGGDPS and plastid:MeGGDPS. Co-production of PbDXS + plastid:AgABS or plastid:GGDPS + plastid:AgABS was insufficient to increase the diterpenoid content in N. benthamiana leaves more than 2-fold compared to the diterpenoid level in plastid:AgABS-producing leaves (Fig. 2a). In contrast, co-production of PbDXS + GGDPS + plastid:AgABS enhanced diterpenoid production up to 6.5-fold compared to leaves producing plastid:AgABS). Significant differences in diterpenoid yields were obtained depending on which GGDPS was included, apparently unrelated to a specific type of GGDPS (Fig. 2a). The highest diterpenoid levels were determined in N. benthamiana leaves co-producing PbDXS + plastid:AgABS with plastid:MtGGDPS (type I), plastid:TsGGDPS (type II), or EpGGDPS2 (type II), with similar yield between these combinations (Fig. 2a).

Fig. 2 Engineered diterpenoid production in N. benthamiana leaves. Production of AgABS led to accumulation of diterpenoids (abietadiene and its isomers). To enhance GGDP availability for diterpenoid production, ElHMGR159–582 (cytosol), PbDXS (plastid), and distinct GGDPSs (cytosol or plastid) were included in transient assays. The protein combinations are indicated below each bar (black circle, was included; minus, was not included) and in the scheme next to each graph. The production of diterpenoids was engineered in the plastid (a, b) and in the cytosol (c) in the absence and presence of AtWRI11–397 and NoLDSP. Data were analyzed by Shapiro–Wilk, Welch–ANOVA (a P < 0.0001; b P < 0.0001; c P < 0.0001) and Brown–Forsythe ANOVA (a P < 0.0001; b P < 0.0001; c P < 0.0001) followed by t-tests (unpaired, two-tailed, Welch correction). Data are presented as individual biological replicates and bars representing average levels with SD (N indicated below each bar). This experiment was replicated twice. Statistically significant differences are indicated by a-e based on t-tests (P < 0.05). Source Data are provided as a Source Data file. LD, lipid droplet Full size image

We further evaluated diterpenoid accumulation in the presence of lipid droplets. Co-production of plastid:AgABS + AtWRI11–397 had no significant impact on the diterpenoid level compared to control leaves producing plastid:AgABS, whereas in leaves producing plastid:AgABS + AtWRI11–397 + NoLDSP, the diterpenoid content was increased 2-fold (Fig. 2b). Similarly, co-production of plastid:MtGGDPS + plastid:AgABS + AtWRI11–397 + NoLDSP increased the diterpenoid level 2.5-fold compared to plastid:MtGGDPS + plastid:AgABS producing leaves. The results indicated that the increased abundance of lipid droplets was beneficial for the accumulation of diterpenoid products. Sequestration of the lipophilic diterpenoids into lipid droplets may have helped to circumvent negative feedback regulatory mechanisms and served as “pull force” in diterpenoid production. In fact, isolated lipid droplet fractions from leaves producing plastid:AgABS + AtWRI11–397 and plastid:AgABS + AtWRI11–397 + NoLDSP contained ~35-fold and 460-fold more diterpenoids, respectively, than control fractions from leaves with plastid:AgABS, consistent with the sequestration of diterpenoids in lipid droplets (Supplementary Fig. 1). Co-production of PbDXS and plastid:MtGGDPS together with plastid:AgABS yielded the highest diterpenoid level (Fig. 2b) independent of whether AtWRI11–397 was included for lipid droplet synthesis. In contrast, co-production of PbDXS + plastid:MtGGDPS + plastid:AgABS together with AtWRI11–397 + NoLDSP resulted in a significant reduction of the diterpenoid level (compared to leaves producing PbDXS + plastid:MtGGDPS + plastid:AgABS).

When A. grandis abietadiene synthase was targeted to the cytosol (cytosol:AgABS85–868), leaves accumulated ~0.2 μg diterpenoids g−1 fresh weight and addition of precursor pathway genes enhanced diterpenoid synthesis (Fig. 2c). Co-production of cytosol:AgABS85–868 together with ElHMGR159–582 and cytosolic M. thermautotrophicus GGDPS (cytosol:MtGGDPS) increased the diterpenoid yield more than 400-fold (relative to cytosol:AgABS85–868 containing leaves) and, thus, close to the highest diterpenoid yield achieved with plastid engineering approaches (Fig. 2b, c). Moreover, our data indicated an enhancing effect of lipid droplet accumulation on terpenoid production when cytosol:AgABS85–868 was co-produced with AtWRI11–397 or AtWRI11–397 + NoLDSP (Fig. 2c). Under these conditions, terpenoid production was increased up to approximately 3-fold which is consistent with diterpenoids being sequestered in lipid droplets. When ElHMGR159–582 + cytosol:MtGGDPS + cytosol:AgABS85–868 + AtWRI11–397 + NoLDSP were co-produced, no additive effects of lipid droplet engineering on terpenoid yield were detected (relative to ElHMGR159–582 + cytosol:MtGGDPS + cytosol:AgABS85–868) (Fig. 2c).

Triacylglycerol analysis of N. benthamiana leaves

To examine a potential impact of terpenoid engineering on triacylglycerol yield, the established approaches for low- or high-yield terpenoid synthesis combined with lipid droplet production were further tested. Four days after infiltration, the leaves were subjected to triacylglycerol analysis. Leaves co-engineered for lipid droplet and patchoulol production in the cytosol contained ~50% less triacylglycerol than leaves producing AtWRI11–397 + NoLDSP (Fig. 3a). A significant decrease in the triacylglycerol level was also detected when leaves were engineered for cytosol-targeted high-yield production of diterpenoids (compared to leaves producing AtWRI11–397 + NoLDSP) (Fig. 3b). When lipid droplet production was combined with a plastid-targeted approach for high-yield terpenoid synthesis, no negative impact on triacylglycerol accumulation was observed compared to control plants (Fig. 3a, b).

Fig. 3 Triacylglycerol yield in engineered N. benthamiana leaves. TAG accumulation was initiated through ectopic expression of WRINKLED1 (AtWRI11–397) and further enhanced through co-expression of NoLDSP. The impact of engineered patchoulol (a) and diterpenoid production (b) on TAG yield is depicted. The different construct combinations are indicated below each bar (black circle, was included; minus, was not included). Data were analyzed by Shapiro–Wilk, Welch–ANOVA (a P < 0.0001; b P < 0.0001) and Brown–Forsythe ANOVA (a P < 0.0001; b P < 0.0001) followed by t-tests (unpaired, two-tailed, Welch correction). Data are presented as individual biological replicates and bars representing average levels with SD (N indicated below each bar). This experiment was replicated twice. Statistically significant differences are indicated by a-e based on t-tests (P < 0.05). Source Data are provided as a Source Data file Full size image

Targeting diterpenoid production to lipid droplets

We next investigated whether lipid droplets in the cytosol can be used as platform to anchor biosynthetic pathways for the production of functionalized diterpenoids. The proof-of-concept experiments included modified A. grandis abietadiene synthase and Picea sitchensis cytochrome P450 (PsCYP720B4), previously reported to convert abietadiene and several isomers to the corresponding diterpene resin acids36. To target terpenoid synthesis to the lipid droplets, A. grandis abietadiene synthase lacking the N-terminal plastid targeting sequence (cytosol:AgABS85–868) and truncated PsCYP720B4 lacking the N-terminal membrane-binding domain (cytosol:PsCYP720B430–483) were produced as C-terminal and N-terminal NoLDSP-fusion protein, respectively. The NoLDSP-fusion proteins are here referred to as LD:AgABS85–868 and LD:PsCYP720B430–483. The construction of LD:AgABS85–868 as C-terminal NoLDSP-fusion protein was inspired by studies reporting on functional, C-terminal tagged diterpene synthases37,38. To re-target PsCYP720B4 to lipid droplets (LD:PsCYP720B430–483), the predicted N-terminal hydrophobic domain of native PsCYP720B4 was replaced by NoLDSP as a recent publication described that modifications or deletion of the membrane anchoring domain of CYP720B4 did not impair the enzyme’s activity38. Inclusion of CPRs has been shown to be crucial to drive metabolic fluxes in CYP-mediated production of high-value target compounds in non-native hosts and synthetic compartments39,40. In our experiments, Camptotheca acuminata CPR (cytosol:CaCPR70–708) was included as NoLDSP-fusion protein to co-localize the CaCPR and PsCYP720B4 activities on lipid droplets and facilitate the CYP-catalyzed production of functionalized terpenoids. As the C-terminus of CPRs is pivotal for catalytic activity and not suitable for modifications41,42, the predicted N-terminal hydrophobic domain of native CaCPR was replaced by NoLDSP to produce the fusion protein LD:CaCPR70–708.

To determine the localization in planta, the NoLDSP-fusion proteins were each produced as yellow fluorescent protein (YFP)-tagged proteins together with AtWRI11–397 for lipid droplet production. The YFP-signals in infiltrated leaves were subsequently compared to the signals obtained for YFP-tagged NoLDSP, which indicated that all three YFP-tagged NoLDSP-fusion proteins were targeted to the surface of the lipid droplets (Fig. 4). It is noteworthy that production of the YFP-tagged NoLDSP and NoLDSP-fusion proteins promoted clustering of small lipid droplets in planta and in isolated lipid droplet fractions, consistent with a previous report on ectopic production of A. thaliana OLEOSIN1 fused to green fluorescent protein43 (Fig. 4, Supplementary Fig. 1). As confirmed for NoLDSP, the clustering of small lipid droplets was independent of the presence or absence of the YFP-tag (Supplementary Fig. 2).

Fig. 4 Localization of heterologously expressed fluorescent-reporter tagged fusion proteins. N. benthamiana leaves producing yellow fluorescent protein (YFP)-tagged NoLDSP, LD:AgABS85–868, LD:PsCYP720B430–483, or LD:CaCPR70–708 were subjected to confocal laser scanning microscopy. Representative images are shown. The produced YFP-proteins are indicated in each line. Note that AtWRI11–397 was co-produced and leaf samples were stained with Nile red to visualize neutral lipids in lipid droplets. This experiment was replicated twice. Channels: YFP yellow fluorescent protein (scale bar 20 μm), NR Nile red (scale bar 20 μm), YFP NR, enlarged merge YFP and NR (scale bar 5 μm) Full size image

To compare different engineering approaches, the A. grandis abietadiene synthase was produced as plastid:AgABS (native), cytosol:AgABS85–868 or LD:AgABS85–868, each alone and combined with ER:PsCYP720B4 (native), cytosol:PsCYP720B430–483 or LD:PsCYP720B430–483 + LD:CaCPR70–708 (Fig. 5). Note that these assays also included either PbDXS + plastid:MtGGDPS or ElHMGR159–582 + cytosol:MtGGDPS to increase the precursor flux, and AtWRI11–397 to initiate lipid droplet accumulation. NoLDSP was included in those assays that lacked any NoLDSP-fusion proteins. Compared to the assays with plastid:AgABS, production of cytosol:AgABS85–868 and LD:AgABS85–868 resulted in similar diterpenoid yield. When native or modified A. grandis abietadiene synthase was co-produced with native or modified P. sitchensis PsCYP720B4, the leaves accumulated diterpene resin acids in free and glycosylated forms (Supplementary Figs. 3–5). The glycosyl modifications of the diterpenoid acids were consistent with those previously reported for engineered terpenoid products and are likely the result of intrinsic defense/detoxification mechanisms in N. benthamiana24,44,45. Incubation of such leaf extracts with Viscozyme® L resulted in the hydrolysis of the glycosylated diterpenoid acids to free diterpenoid resin acids which allowed determining the level of total diterpenoid acids produced in infiltrated leaves. To compare the different engineering strategies, the levels of both diterpenoids and total diterpenoid acids were quantified for each infiltrated leaf (Fig. 5). Co-production of plastid:AgABS with ER:PsCYP720B4, cytosol:PsCYP720B430–483 or LD:PsCYP720B430–483 decreased the diterpenoid level (compared to controls with plastid:AgABS) and resulted in the accumulation of diterpenoid acids, consistent with diterpenoids being converted to diterpenoid acids. The level of diterpenoid acids was ~4-fold and 3-fold higher in transient assays with plastid:AgABS including ER:PsCYP720B4 and plastid:AgABS + LD:PsCYP720B430–483 + LD:CaCPR70–708 compared to assays including cytosol:PsCYP720B430–483. The highest diterpenoid acid yield in transient assays with cytosol:AgABS85–868 was achieved in combination with ER:PsCYP720B4 which was ~2- and 3-fold higher than with cytosol:AgABS85–868 and LD:PsCYP720B430–483 + LD:CaCPR70–708, respectively (Fig. 5). In transient assays with LD:AgABS85–868, the diterpenoid acid level was 2-fold higher in assays with ER:PsCYP720B4 than in assays with either cytosol:PsCYP720B430–483 or LD:PsCYP720B430–483 + LD:CaCPR70–708 (Fig. 5).