Expression of fusion proteins in S. cerevisiae yielded active enzymes
ScWS is most likely localized in the ER membrane, while MaFAR is a cytosolic protein. Even though coexpression of MaFAR with ScWS in Arabidopsis and Camelina resulted in high yields of wax esters in seed oil [15], colocalization of MaFAR with ScWS in the same membrane may increase wax ester production. Therefore, a ScWS-MaFAR fusion protein was generated by fusing MaFAR to the C-terminal end of ScWS. The ability of ScWS-MaFAR to produce wax esters was tested by heterologous expression in S. cerevisiae. Fatty alcohols and wax esters do not naturally accumulate in S. cerevisiae. Some cultures were therefore additionally supplemented with fatty alcohols to test WS activity independently of FAR activity. While as expected the coexpression of the single enzymes MaFAR/ScWS resulted in production of both fatty alcohols and wax esters, the ScWS-MaFAR fusion protein produced fatty alcohols and wax esters as well. These results show that the ScWS-MaFAR fusion protein has both FAR and WS activities (Additional file 1: Figure S1a).
AbWSD1 is a bifunctional WS/DGAT enzyme from A. baylyi ADP1, showing high preference for C18 substrates [13, 29, 30]. In this study, coexpression of AbWSD1 with MaFAR resulted in low amounts of wax esters in Arabidopsis seeds (Fig. 1, AbWSD1/MaFAR). Therefore, the first 20 amino acids of AbWSD1 were optimized for plant codon usage (PCOAbWSD1) as described previously [31], to increase its expression level in plant cells. In addition, a TMMmAWAT2-AbWSD1 fusion protein was generated by fusing two transmembrane domains (first 60 AA) of MmAWAT2 to the N-terminal end of AbWSD1, for a potential enhancement of enzymatic activity by targeting the enzyme to the ER membrane [32]. To test their WS activity, PCOAbWSD1 and TMMmAWAT2-AbWSD1 were expressed in S. cerevisiae. When feeding yeast cells with fatty alcohol, MmAWAT2 and PCOAbWSD1 only produced wax esters. The TMMmAWAT2-AbWSD1 fusion protein synthesized both wax esters and TAGs in S. cerevisiae, showing its activity as a bifunctional WS/DGAT enzyme (Additional file 1: Figure S1b).
Different enzyme combinations produced varying amounts of wax esters in seeds of Arabidopsis
To obtain high content of oleyl oleate from plant-derived wax esters, two bacterial WSs, AbWSD1 from A. baylyi and MaWS2 from M. aquaeolei, as well as the optimized AbWSD1 versions were coexpressed with MaFAR. In addition, the transgene for the ScWS-MaFAR fusion protein was either transferred as single copy, double copies or together with MaFAR to increase the abundance of wax ester forming enzymes and the supply of fatty alcohols. In total, seven enzyme combinations were expressed in seeds of Arabidopsis: ScWS-MaFAR, ScWS-MaFAR/ScWS-MaFAR, ScWS-MaFAR/MaFAR, MaFAR/AbWSD1, MaFAR/PCOAbWSD1, MaFAR/TMMmAWAT2-AbWSD1 and MaFAR/MaWS2 (Fig. 2).
After screening at least 30 heterozygous T2 lines for each combination, the best three performing lines of each combination were selected for quantification of wax esters and TAGs. The ScWS-MaFAR lines produced an average of 23 mg g seed−1 of wax esters, and the second copy of ScWS-MaFAR further increased the yield up to 35 mg g seed−1 (Fig. 1a). The ScWS-MaFAR/MaFAR coexpression led to the accumulation of wax esters up to 64 mg g seed−1, accounting for 31% of total neutral lipids (Fig. 1b). The MaFAR/MaWS2 combination produced wax esters up to 14 mg g seed−1, accounting for 6% of total neutral lipids (Fig. 1). However, very low yield of wax esters (4 mg g seed−1) was achieved by MaFAR/AbWSD1, while the MaFAR/PCOAbWSD1 and MaFAR/TMMmAWAT2-AbWSD1 combinations enabled to increase the yields of wax esters to 12 and 17 mg g seed−1, respectively (Fig. 1a). Overall, lower yields of wax esters were reached by the seven tested enzyme combinations in Arabidopsis seed oil, in comparison to MaFAR/ScWS lines obtained previously (Fig. 1) [15].
Different enzyme combinations showed diverse substrate specificities
The compositions of wax esters produced by the seven enzyme combinations were obviously diverse. The MaFAR/ScWS coexpression mainly incorporated 18:1-OH (40 mol%) and 20:1-FA (38 mol%) into wax esters (Fig. 3). Differently, the three combinations expressing ScWS-MaFAR fusion protein predominantly utilized 20:1-OH that accounted for 45–52 mol% of total fatty alcohol moieties, meanwhile a lower abundance of 18:1-OH (20–28 mol%) was observed (Fig. 3a). Furthermore, in comparison to MaFAR/ScWS, a lower level of 18:1-FA and a higher level of 20:1-FA were found in wax esters produced by the ScWS-MaFAR (Fig. 3b). AbWSD1 and MaWS2 were reported to have high preference for C18 substrates [29, 30, 33]. As expected, MaFAR/AbWSD1 and MaFAR/PCOAbWSD1 combinations showed similar substrate preference, incorporating high levels of 18:1-OH and 18:0-FA into wax esters. Interestingly, the MaFAR/TMMmAWAT2-AbWSD1 utilized higher levels of C18:1 substrates and lower levels of C20:1 substrates, compared with MaFAR/AbWSD1 and MaFAR/PCOAbWSD1 (Fig. 3). The MaFAR/MaWS2 combination predominantly incorporated C18:1 at fatty alcohol moiety (Fig. 3a) and utilized C18:0 acyl moiety for wax ester production (Fig. 3b).
Further analysis of the acquired data showed different specificities of the seven enzyme combinations regarding chain length and saturation degree of alcohol and acyl moieties (Fig. 3c–f). As in previous research, MaFAR/ScWS coexpression revealed a dominant utilization of C18 alcohols (around 60 mol%) for wax ester biosynthesis; however, the length of alcohol chain was obviously shifted from C18 to C20 by the ScWS-MaFAR fusion protein (15, Fig. 3c). Furthermore, the three enzyme combinations expressing ScWS-MaFAR fusion protein also showed a high incorporation of C20 acyl moieties (>50 mol%) into wax esters (Fig. 3d). The MaFAR/AbWSD1 and MaFAR/PCOAbWSD1 combinations predominantly use alcohols and acyl substrates in C18 chain length, the latter one showing higher selectivity for C18 chain length (Fig. 3c, d). Interestingly, MaFAR/TMMmAWAT2-AbWSD1 showed with >80 mol% incorporation of C18 alcohols, even higher preference than MaFAR/AbWSD1 for these substrates (Fig. 3c). The MaFAR/MaWS2 combination revealed the highest incorporation of C18 substrates into wax esters, with 90 mol% C18 alcohol moieties and >80 mol% C18 acyl moieties (Fig. 3c, d).
With regard to the saturation degree of alcohol and acyl moieties, the three combinations with ScWS-MaFAR incorporated around 80 mol% monoenoic alcohols, higher than the 60 mol% in MaFAR/ScWS (Fig. 3e), while similar levels of monoenoic acyl moieties (>50 mol%) were observed in both MaFAR/ScWS coexpression and ScWS-MaFAR lines (Fig. 3f). Comparatively, MaFAR/AbWSD1 and MaFAR/PCOAbWSD1 combinations showed lower specificity to monoenoic substrates, using around 50 mol% monounsaturated alcohols and 40 mol% monounsaturated acyl substrates. Instead, these two combinations preferred saturated and dienoic alcohols, as well as saturated acyl substrates for wax ester biosynthesis (Fig. 3e, f). Whereas, while MaFAR/TMMmAWAT2-AbWSD1 displayed similar preference to monoenoic substrates, it tends to use higher levels of unsaturated alcohols instead of saturated alcohols, compared with MaFAR/AbWSD1. Moreover, MaFAR/MaWS2 dominantly incorporated monoenoic substrates (>60 mol%) at the alcohol position and saturated ones (around 70 mol%) at the acyl position (Fig. 3e, f).
Specific production of oleyl oleate was achieved by expression of MaFAR/AbWSD1 in Arabidopsis fad2 fae1 double mutant
The molecular species of wax esters produced by MaFAR/AbWSD1 Arabidopsis seeds were measured by ESI–MS/MS. The MaFAR/AbWSD1 combination in Arabidopsis Col-0 background led to accumulation of 4 mg g seed−1 of wax esters (Fig. 1a) and 11 mol% 18:1/18:1 in all wax ester species, which was similar to MaFAR/ScWS (10 mol%) and higher than those of other previously studied enzyme combinations (Fig. 4a) [15]. The most abundant wax ester species 20:1/18:1 accumulated by MaFAR/AbWSD1 still accounted for 16 mol% of total wax esters, and additional accumulation of 20:1/20:1 (10 mol%), 20:1/18:2 (10 mol%) and 20:2/18:1 (7 mol%) were observed (Fig. 4a). Therefore, the substrate preference of MaFAR/AbWSD1 is suitable for the formation of oleyl oleate, but specific production of oleyl oleate to a high level was not reached by simply expressing enzymes with a higher substrate specificity.
It was shown that the profile of fatty acyl-CoAs for wax ester biosynthesis significantly influence molecular species of wax esters [14]. For specific accumulation of oleyl oleate, MaFAR/AbWSD1 was therefore also expressed in the Arabidopsis fad2 fae1 double mutant that is enriched in oleic acid in seed oil [34]. This led to accumulation of 5 mg g seed−1 of wax esters and up to 62 mol% 18:1/18:1 of total wax esters accumulated by expressing MaFAR/AbWSD1 in this high oleate background, resulting in a similar level of oleyl oleate that was reached by the previously studied enzyme combinations in the same background (Fig. 4b) [14, 15].
Crossing MaFAR/ScWS lines with a high oleate line led to high amount of oleyl oleate in seeds of Camelina
Among all tested enzyme combinations in previous and current studies, the MaFAR/ScWS combination led to limited accumulation of oleyl oleate (4.7 mol%), but the highest yield of wax esters (around 40 mg g seed−1) in seeds of Arabidopsis and Camelina (Fig. 1) [14, 15]. A high oleate (HO) Camelina line was kindly provided by Prof. Cahoon [24], which was generated via a RNAi approach, using FAD2/FAE1 RNAi sequences from Camelina and FAD3 RNAi sequence from Arabidopsis and contains a favorable fatty acid profile for the formation of oleyl oleate. Therefore, six individual MaFAR/ScWS Camelina lines with relatively high wax ester contents were crossed with the HO-line, resulting in six independent MaFAR/ScWS and High Oleic crosses (MaFAR/ScWS-HO). The yields of wax esters in seeds of these six MaFAR/ScWS-HO ranged from 13 to 40 mg g seed−1, accounting for 5–20% of total neutral lipids. Among them, two crosses (L4 and L5) resulted in the highest wax ester amounts exceeding 40 mg g seed−1 (Fig. 5).
All six MaFAR/ScWS-HO resulted in a much higher accumulation of 18:1/18:1 wax esters, compared with the original MaFAR/ScWS lines (Fig. 6) [15]. Importantly, the most abundant wax ester species is 18:1/18:1 for all produced MaFAR/ScWS-HO, with the range from 27% for L26 MaFAR/ScWS-HO to 34% for L4 and L9 MaFAR/ScWS-HO (Fig. 6a, c, f). Some individual lines of MaFAR/ScWS-HO even accumulated up to 45 mol% 18:1/18:1 of total wax esters (Additional file 5: Table S4). MaFAR/ScWS accumulated large amounts of very long-chain wax esters (C38–C40), with 17.7 mol% 18:1/20:1 and 10 mol% 20:1/20:1 in Camelina seed oil [15]. However, the levels of 18:1/20:1 decreased to 7 mol–10 mol%, and the levels of 20:1/20:1 decreased to 7 mol% in wax esters produced by MaFAR/ScWS-HO; in contrary, more wax esters with shorter chain length (C34–C36) were produced, with the levels of 18:1/16:0 increased to around 12 mol% (Fig. 6).
Overall, MaFAR/ScWS-HO led to an increased accumulation of 18:1/18:1 up to 34 mol% of total wax esters. Meanwhile, the total yields of wax esters in seeds of Camelina were not negatively affected by crossing. In addition, some seedlings of MaFAR/ScWS-HO were also delayed in germination and had white cotyledons, as previously observed for MaFAR/ScWS lines (Additional file 6: Figure S2).