Co-expression of MaFAR with optimized AbWSD1 in seeds of A. thaliana

Even though the attempt of re-localization of AbWSD1 by trasmembrane fusion was an exercise in futility, it is still interesting to check whether the two optimized AbWSD1 enzymes would perform better regarding enzymatic activity and substrate specificity for wax ester biosynthesis. So, PCOAbWSD1 and TMMmAWAT2-AbWSD1 were seed-specific co-expressed with MaFAR in A. thaliana Col._0 background.

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Table 5.2.1 Numbers of T2 transgenic lines with MaFAR/AbWSD1, MaFAR/PCOAbWSD1 and MaFAR/TMMmAWAT2-AbWSD1 in Col._0 background, lines with MaFAR/AbWSD1 in fad2 fae1 dpuble mutant;

numbers of transgenic lines analyzed by TLC and GC-FID.

Construct Number of T2 lines TLC analysis GC-FID analysis

MaFAR/AbWSD1 50 30 3

MaFAR/PCOAbWSD1 10 6 3

MaFAR/TMMmAWAT2-AbWSD1 56 30 3

MaFAR/AbWSD1_fad2fae1 10 10 3

The numbers of T2 transgenic lines with MaFAR/AbWSD1, MaFAR/PCOAbWSD1 and MaFAR/TMMmAWAT2-AbWSD1 in Col._0 background, and the lines with MaFAR/AbWSD1 in the fad2 fae1 double mutant (MaFAR/AbWSD1_fad2fae1) are shown in Table 5.2.1. Three individual lines with strong wax ester signal on TLC were selected and their wax ester contents were measured by GC-FID (Figure 5.2.6).

Figure 5.2.6 Quantification of wax esters in seeds of A. thaliana Col._0 transformed with MaFAR/ScWS, MaFAR/AbWSD1, MaFAR/PCOAbWSD1 and MaFAR/TMMmAWAT2-AbWSD1, A. thaliana fad2 fae1 double mutant transformed with MaFAR/AbWSD1. (A) Absolute quantification of wax esters in mg g^-1 seeds. *means significantly different from MaFAR/AbWSD1, p<= 0.05; **means significantly different from MaFAR/AbWSD1, p

<= 0.01. (B) The relative quantification of total neutral lipids (WE, wax ester; TAG, triacylglycerol) in mass% are calculated according to the absolute quantification of each lipid class. The data shown is an average of three individual heterozygous T2 lines for each enzyme combination with two extraction replicates for each individual line.

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In comparison to the MaFAR/AbWSD1 combination, MaFAR/PCOAbWSD1 and MaFAR/TMMmAWAT2-AbWSD1 combinations enabled to increase the formation of wax esters up to 12 mg g^-1 and 17 mg g^-1 in the seeds of A. thaliana, respectively, which were three times than the wax esters produced by MaFAR/AbWSD1 (Figure 5.2.6 A); however, these were still much lower than that of MaFAR/ScWS (100 mg g^-1 seeds). The wax esters produced by MaFAR/PCOAbWSD1 and MaFAR/TMMmAWAT2-AbWSD1 accounted for 4% and 7% of total neutral lipids, much lower than the 43 mol% of MaFAR/ScWS (Figure 5.2.6 B).

Figure 5.2.7 Alcohol and acyl moieties of wax esters in seeds of A. thaliana Col._0 background transformed with MaFAR/ScWS, MaFAR/AbWSD1, MaFAR/PCOAbWSD1 and MaFAR/TMMmAWAT2-AbWSD1, A. thaliana fad2 fae1 double mutant background transformed with MaFAR/AbWSD1. (A) Relative abundance of alcohol moieties in mol%. (B) Relative abundance of acyl moieties in mol%. The data shown is an average of three individual heterozygous T2 lines for each enzyme combination with two extraction replicates for each individual line.

*means significantly different from MaFAR/ScWS, p<= 0.05; **means significantly different from MaFAR/ScWS, p<= 0.01.

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Interestingly, the compositions of wax esters produced by co-expression of MaFAR with AbWSD1, PCOAbWSD1 or TMMmAWAT2-AbWSD1 were observed to be different from those produced by the MaFAR/ScWS combination (Figure 5.2.7). With regard to the fatty alcohol moieties, the levels of 18:1-OH and 20:1-18:1-OH incorporated into the wax esters of MaFAR/AbWSD1 and MaFAR/PCOAbWSD1 were not significantly different from that of MaFAR/ScWS (Figure 5.2.7 A). Interestingly, in comparison to MaFAR/ScWS as well as MaFAR/AbWSD1, a significantly higher level of 18:1-OH (more than 50 mol%) and remarkably lower level of 20:1-OH were utilized by the MaFAR/TMMmAWAT2-AbWSD1 combination (Figure 5.2.7 A). When MaFAR/AbWSD1 was expressed in a high oleic background (fad2 fae1 double mutant), over 60 mol% of all fatty alcohols incorporated into wax esters was 18:1-OH.

In term of the fatty acyl moieties incorporated into wax esters, upon expression in Col._0 background, the MaFAR/AbWSD1, MaFAR/PCOAbWSD1 and MaFAR/TMMmAWAT2-AbWSD1 combinations incorporated no more than 10 mol% 20:1-FA into wax esters, significantly lower than MaFAR/ScWS utilizing 38 mol% 20:1-FA (Figure 5.2.7 B). Furthermore, in Col._0 background these three combinations utilized stearic acid (18:0-FA) as the predominant fatty acyl moiety for wax ester synthesis, which was significantly higher than that of MaFAR/ScWS. The MaFAR/AbWSD1 and MaFAR/PCOAbWSD1 combinations did not utilize much higher level of 18:1-FA in comparison to MaFAR/ScWS; however, significantly higher levels of 18:1-FA were used by MaFAR/TMMmAWAT2-AbWSD1. When MaFAR/AbWSD1 was expressed in the fad2 fae1 double mutant, the level of 18:1-FA in wax esters was as high as 50 mol%, followed by 17 mol% stearic acyl moiety (Figure 5.2.7 B).

With regard to the preference for substrate chain length, AbWSD1, PCOAbWSD1 and TMMmAWAT2-AbWSD1 showed less preference for longer-chain substrates (Figure 5.2.8 A and B). The MaFAR/PCOAbWSD1 and MaFAR/TMMmAWAT2-AbWSD1 combinations utilized much lower levels of C20 alcohols compared with MaFAR/ScWS. In comparison to MaFAR/AbWSD1 as well as MaFAR/ScWS, the MaFAR/TMMmAWAT2-AbWSD1 combination showed much higher preference for C18 alcohols (Figure 5.2.8 A). In fad2 fae1 double mutant, MaFAR/AbWSD1 incorporated over 90 mol% C18 alcohols into wax esters (Figure 5.2.8 A). In A. thaliana Col._0 background, AbWSD1 and its optimized enzymes utilized over 60 mol% C18 acyl substrates for wax ester synthesis, significantly higher than the 35 mol%

of ScWS. Moreover, these enzymes showed obvious less specificity to C20 acyl substrates compared with ScWS. In fad2 fae1 double mutant, AbWSD1 predominantly utilized C18 acyl substrates for wax ester production (Figure 5.2.8 B).

In A. thaliana Col._0 background, MaFAR/AbWSD1 and MaFAR/PCOAbWSD1 preferred to use much less monoenoic alcohols but more saturated alcohols compared with MaFAR/ScWS. While, MaFAR/TMMmAWAT2-AbWSD1 tend to incorporate more unsaturated alcohols instead of saturated alcohols compared with MaFAR/AbWSD1 (Figure 5.2.8 C). Similarly, significantly lower levels of monoenoic acyl substrates but higher level of saturated acyl substrates were utilized by AbWSD1, PCOAbWSD1 and TMMmAWAT2-AbWSD1 in comparison to ScWS (Figure 5.2.8 D). Additionally,

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AbWSD1 and PCOAbWSD1 utilized more dienoic acyl substrates than ScWS. In fad2 fae1 double mutant, MaFAR/AbWSD1 incorporated over 60 mol% monounsaturated alcohols and acyl substrates for wax ester production (Figure 5.2. 8 C and D).

Figure 5.2.8 Relative abundance of alcohol and acyl moieties of wax esters in seeds of A. thaliana Col._0 background transformed with MaFAR/ScWS, MaFAR/AbWSD1, MaFAR/PCOAbWSD1 and MaFAR/TMMmAWAT2-AbWSD1, A. thaliana fad2 fae1 double mutant background transformed with MaFAR/AbWSD1. (A) Alcohol moiety calculated by total carbon number. (B) Acyl moiety calculated by total carbon number. (C) Alcohol moiety calculated by desaturation degree. (D) Acyl moiety calculated by desaturation degree. The data is calculated according to the wax ester composition shown in Figure 5.2.7, and is an average of three individual heterozygous T2 lines for each enzyme combination with two extraction replicates for each individual line. *means significantly different from MaFAR/ScWS, p<= 0.05; **means significantly different from MaFAR/ScWS, p<= 0.01.

In summary, co-expression of AbWSD1 with MaFAR led to increased formation of 18:1/18:1, due to the preference of AbWSD1 for C18 acyl substrates. However, the activity of AbWSD1 in plant cells was low, so that the MaFAR/AbWSD1 combination resulted in low amounts of wax esters in seeds of A.

thaliana and C. sativa. Co-expression of MaFAR with optimized AbWSD1 increased the amount of wax esters, but the yield of these combinations were still lower than the MaFAR/ScWS combination. The transmembrane domains of MmAWAT2 affected the formation of molecular specificities of AbWSD1,

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so that TMMmAWAT2-AbWSD1 showed higher preference for C18 alcohol and acyl substrates compared with AbWSD1.