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Hydroxy fatty acid (HFA) is a vital raw material for numerous industrial products, such as lubricants, plasticizers and surfactants. Castor oil is the current commercial source of HFA which contains 90% ricinoleic acid (18,1OH). Castor seeds contain the toxin ricin and hyperallergic 2S albumins; it is detrimental to castor oil production. Lesquerella is a potential industrial oilseed crop for a safe source of HFA, because lesquerella seeds contain a valuable HFA, lesquerolic acid (20,1OH), at 55–60% in seed oil. This chapter describes current progress on improving HFA production in lesquerella through metabolic engineering.
U.S. Department of Agriculture, Western Regional Research Center, Agricultural Research Service, Albany, CA, USA
Kumiko Johnson
U.S. Department of Agriculture, Western Regional Research Center, Agricultural Research Service, Albany, CA, USA
*Address all correspondence to: grace.chen@usda.gov
1. Introduction
Lesquerella seed oil (triacylglycerol, TAG) biosynthesis follows common de novo fatty acid (FA) biosynthesis in plastid (Figure 1). Once oleic acid (18,1) is synthesized and exported to cytosol, it is activated to 18:1-Coenzyme A (CoA) for endoplasmic reticulum (ER)-mediated fatty acid modification and TAG assembly [1]. The 18:1-CoA can be esterified directly into membrane lipid phosphatidylcholine (PC) in the ER by the forwarding reaction of lyso-PC acyltransferase (LPCAT) [2, 3, 4] resulting in 18:1-PC (Figure 1). An oleate 12-hydroxylase (FAH12) [5, 6, 7, 8] hydroxylates 18:1-PC to form 18:1OH-PC (Figure 1). Lesquerella PfFAH12, however, converts 18:1-PC to both 18:1OH-PC and linoleic acid (18,2)-PC, because PfFAH12 posesses bi-functional FAD2-related oleate Δ12 - hydroxylase: desaturase activities [8].
Figure 1.
Proposed pathways for TAG biosynthesis in lesquerella seed. Kennedy pathway is indicated by blue arrows. Acyl editing reactions are indicated by purple arrows. PC-derived DAG formation is indicated by a green arrow. Enzymes catalyzing these reactions are underlined. Fatty acid numerical symbols and abbreviations are described in introduction.
Through the reverse reaction of LPCAT (Figure 1), or phospholipase A (PLA2)–type activity [9], the 18:1OH can be removed from PC and transferred back to cytosol to be activated as 18:1OH-CoA. A lesquerella fatty acid condensing enzyme (PfKCS18) (also called KCS3 or FAE1) elongates 18:1OH-CoA to 20:1OH-CoA [10] (Figure 1). Rapid acylation of 18:1 and de-acylation of 18:1OH by LPCAT (or by PLA2), and together with elongation of 18:1OH-CoA by PfKCS18 leads to enrichment of 20:1OH-CoA in cytosol. FA desaturase 2 (FAD2) [11] and FA desaturase 3 (FAD3) [12] converts 18:1-PC to 18:2-PC and 18:2-PC to linolenic acids (18:3)-PC, respectively (Figure 1). The FAD3 is also resposible for converting 20-1OH to auricolic acid (20,2OH) [13, 14] in lesquerella seeds. A functional lesquerella PfFAD3–1 isoform is a key enzyme producing 18:3 and 20:2OH [15]. FA-CoA or FA-PC are assembled to TAG through multiple mechanisms [1, 2]. First, FA-CoAs are acylated to a glycerol-3-phosphate (G3P) backbone by enzymes in Kennedy pathway [16]. Glycerol-3-phosphate acyltransferase (GPAT) acylates FA-CoA to the sn-1 position of G3P backbone forming lysophosphatidic acid (LPA). LPA acyltransferase (LPAT) is responsible to add another FA-CoA to the sn-2 of LPA forming phosphatidic acid (PA). Through PA phosphatase (PAP), PA is changed to 1,2-sn-diacylglycerol (DAG). The DAG produced in Kennedy pathway is referred to de novo DAG. The last FA-CoA can be added to the sn-3 of DAG by 1,2-sn-diacylglycerol acyltransferase (DGAT), creating TAG. In lesquerella, almost all seed TAG having 20:1OH at the sn-1 and sn-3 positions, and the sn-2 positions are occupied by unsaturated FAs, ie., 18:1, 18:2 and 18:3 [17, 18, 19, 20]. The reason of lack of HFA at the sn-2 position of TAG could be due to the selectivity of lesquerella LPAT (PfLPAT2) for unsaturated FA [21], which is a typical characteristic for most plant LPAT2 [22]. Second, PC can be converted to DAG (PC-derived DAG). PC:DAG cholinephosphotransferase (PDCT) [23, 24, 25] is a major enzyme to produce PC-derived DGA through exchange of the head group between PC and DAG. Alternatively, PC-derived DGA can be produced by other enzymatic reactions catalyzed by CDP-choline: DAG cholinephosphotransferase (CPT) [26], or phospholipases (PLC, or PLD) [2, 27]. The conversion of PC into DAG also provides a mechanism to increase the amount of 18:1, 18:2, 18:3 in sn-2-TAG. Third, FA on the sn-2 PC can be transferred to the sn-3 position of DAG by phospholipid:DAG acyltransferase (PDAT) (Figure 1) [28, 29, 30].
To develop lesquerella that produces 18:1OH-rich seed oils like castor, we have over-expressed castor RcLPAT2 [21]. The resulted transgenic lesquerella seeds increase 18:1OH content at the sn-2 position of TAG from 2–17%, and consequently, oil accumulates more TAGs with all three sn positions occupied by HFA [20, 21]. RNA interference sequences targeting KCS18, FAD2 and FAD3 have been introduced to lesquerella. Seeds from the transgenic lines had increased 18:1OH up to 26.6% compared with that of 0.4–0.6% in wild type (WT) seeds. Our studies enhance our understanding of plant lipid metabolism.
2. Castor LPAT2 increases castor oil-like triacylglycerols in lesquerella seed
We have produced 17 transgenic lesquerella lines expressing RcLPAT2 under the control of a seed specific promoter [21]. Our results indicate that RcLPAT2 enables the incorporation of 18:1OH at sn-2 position of LPA which increases the accumulation of 18:1OH and also tri-HFA-TAGs in lesquerella (Figures 2 and 3).
Figure 2.
TAG species composition. Triplicates of 50-seed sample were measured for wild type (wt) and transgenic lines (line 3–1, line 4–5). The data represent averages of three replicates ± SE. Two-tailed Student’s t test. * p < 0.05; ** p < 0.01; *** p < 0.001. 0-HFA, 1-HFA, 2-HFA, and 3-HFA indicate TAG molecular with zero, one, two, or three HFAs, respectively.
Figure 3.
HFA content at the sn-2 position of TAG. Triplicates of 50-seed sample were measured for wild type (wt) and transgenic lines (line 3–1, line 4–5). The data represent averages of three replicates ± SE. Two-tailed Student’s t test. * p < 0.05; ** p < 0.01; *** p < 0.001. 18:1OH and 20:1OH represent ricinoleic acid and lesquerolic acid, respectively.
In transgenic lesquerella expressing RcLPAT2, we observed an increase in 0-, 1-, and 3-HFA-TAG levels and a reduction in 2-HFA-TAG species (Figure 2). Regiochemical analysis showed that sn-2 18:1OH increased 6–7-fold or 1.5-fold, respectively (Figure 3). Further analysis of regioisomer of the transgenic seed oil reveals that RcLPAT2 increased 3-HFA-TAG content by acylating mostly 18:1OH at the sn-2 position of 20:1OH-LPA forming tri-HFA-TAG (20,1OH at sn-1, 3 and 18:1OH at sn-2) [18, 19, 20, 31]. This indicates that RcLPAT2 allows for a more efficient acylation of 18:1OH than 20:1OH to the sn-2 position of TAG in vivo. We have demonstrated that castor LPAT2 increases 18:1OH level through exclusively acylating 18:1OH at the sn-2 position of tri-HFAs-TAGs in lesquerella. RcLPAT2 holds a valuable property for the engineering of a new castor oil-producing crop, such as lesquerella.
3. Ricinoleic acid content can be increased in lesquerella seeds through suppressing an elongase and fatty acid desaturases
To develop lesquerella that produces 18:1OH-rich seed oils like castor, silencing PfKCS18 would result in accumulation of 18:1OH; silencing FAD2 and FAD3 would block 18:1 flux to 18:2 and 18:3, respectively. To test the hypothesis, we generated transgenic lines expressing RNAi of camelina CsFAD2, CsFAD3, and Arabidopsis AtKCS18, which have 82–95% sequence homology with corresponding lesquerella genes [18]. RNAi constructs, CsFAD2 RNAi, CsFAD3 RNAi and AtFEA1 RNAi, are effective in silencing corresponding gene expression in camelina [32, 33, 34]. We therefore generated 16 transgenic lesquerella lines expressing AtFAD3 RNAi + CsFAE1 RNAi (2-dsRNA) (Table 1) [35], and 15 lines expressing CsFAD2 RNAi + AtFAD3 RNAi + CsFAE1 RNAi (Table 2) [35].
Three or four replicates of 30-seed sample were measured for wild-type and each transgenic line. All data are averages of measurements ±SD. Fatty acid legend: 18:1 is oleic; 18:2 is linoleic; 18:3 is linolenic; 18:1OH is ricinoleic; 20:1OH is lesquerolic; and 20:2OH is auricolic acid. a, total content of five common fatty acids: palmitic (16:0), palmitoleic (16:1), stearic (18:0), arachidic (20:0), and eicosenoic acids (20:1). Two-tailed Student’s t-test.
Three or four replicates of 30-seed sample were measured for wild-type and each transgenic line. All data are averages of three measurements ±SD. Fatty acid legend: 18:1 is oleic; 18:2 is linoleic; 18:3 is linolenic; 18:1OH is ricinoleic; 20:1OH is lesquerolic; and 20:2OH is auricolic acid. a, total content of five common fatty acids: palmitic (16:0), palmitoleic (16:1), stearic (18:0), arachidic (20:0), and eicosenoic acids (20:1). Two-tailed Student’s t-test.
As shown in Table 1, when the 2-dsRNAs (AtFAD3 RNAi and CsFAE1 RNAi) was introduced to lesquerella, we observed significant increases in 18:1OH from 0.6% of WT to 26.6% in line 1 (Table 1) and decreases in 20:1OH from 51.2% of WT to lowest 19% in line 1 (Table 1). 18:1 content was increased in 50% of transgenic population with the highest level of 32.1% in line 2 compared with 17% of WT (Table 1). Correlation analysis was performed to show the relationships between FA accumulation for 2-dsRNA group. Among the 16 T1 transgenic lesquerella lines, 15 lines shifted the accumulation of 18:3 to 18:2, showing a strong negative correlation between 18:2 and 18:3 (r = 0.93); 13 lines shifted 20:1OH to 18:1OH, which also displayed a strong negative correlation (r = −0.99). These results indicate that AtFAD3 RNAi and CsFAE1 RNAi are effective in silencing PfFDA3–1 and PfKCS18, respectively.
As shown in Table 2, 15 independent transgenic lines expressing the 3-dsRNAs, CsFAD2 RNAi + AtFAD3 RNAi + CsFAE1 RNAi were generated and their T1 seeds were analyzed for FA composition. Compared with 2-dsRNAs (Table 1), similar average contents in 18:2, 18:3 and 20:1 in lines expressing 3-dsRNA (Table 2) were observed. With the addition of CsFAD2 RNAi in the 3-dsRNA group, the average of 18:1 was higher at 27.8% (Table 2) compared with the average of 20.5% in the 2-dsRNA group (Table 1). Besides, less dynamic changes between average increase of 18:1OH and average decrease of total HFA were observed in the 3-dsRNA group, showing averages of 4.7% and 48.9%, respectively, (Table 2), compared with that of 7.7% and 53% in lines expressing 2-dsRNA, respectively (Table 1). FA composition in WT seeds (Tables 1 and 2) were similar to described [21, 36]. There was no change of growth phenotype for transgenic lesquerella expressing CsFAD2 RNAi, CsFAD3 RNAi and AtFEA1 RNAi.
We have demonstrated that high levels of 18:1OH can be achieved by blocking the elongation of 18:1OH to 20:1OH. Also, high levels of 18:1 and 18:2 were accumulated through suppression of desaturation steps. However, the accumulated 18:1 was not converted to 18:1OH and instead, 18:1 was largely channeled to seed TAG.
4. Bottlenecks and potential for production of a high 18:1OH-containing oil in lesquerella
Based on the data presented in Tables 1 and 2, significant amount of 18:1 was not utilized for 18:1OH production. One of the factors could be due to the bifunctional activity of PfFAH12 [8], which hydroxylates and desaturases 18:1 to produce 18:1OH and 18:2, respectively, thus diverting 18:1 flux to 18:2 production. Seeds contain 90% 18:1OH from castor [37] or 85% 20:1OH from Physaria lindheimeri [38, 39]. These species have strict FAH12s, RcFAH12 [7] and PlFAH12 [38]. Deleting PfFHA12 and at the same time introducing RcFAH12 or PlFAH12 in lesquerella should allow increased 18:1OH accumulation. On the other hand, the accumulation of 18:1 could also be due to a lesquerella LPAT that has substrate preference for 18:1-CoA, resulting in efficient incorporation of 18:1-CoA into TAG through Kennedy pathway. We already showed that castor RcLPAT2 increased 18:1OH in lesquerella [20, 21]. Besides, castor RcLPAT3B and RcLPATB also showed substrate preference to 18:1OH in Arabidopsis [40]. To enhance 18:1OH to a higher level in lesquerella, further engineering design should include knocking out a lesquerella PfLPAT2 and overexpressing RcLPAT2, RcLPATB, and RcLPAT3B. In addition to Kennedy pathway, some of the 18:1-PC could be converted by PDCT to 18:1-DAG for TAG assembly in lesquerella (Figure 1). Lesquerella seed TAGs contain about 21% 18:2 and 18:3 (Tables 1 and 2). There is strong evidence that seeds enriched with 18:2 or 18:3 may use the PC-derived pathway [2]. Therefore, it is likely that PC-derived DAGs are utilized in TAG assembly in lesquerella. To enhance flux from 18:1OH-PC to 18:1OH-DAG, it is advantageous to replace a lesquerella PfPDCT with a castor RcPDCT which was demonstrated effective in converting 18:1OH-PC to 18:1OH-DAG in Arabidopsis [25].
We observed increase in 18:1OH and decrease in 20:1OH in transgenic lesquerella seeds expressing CsFAE1 RNAi, which are expected. We, however, found that total HFAs are reduced (Tables 1 and 2). Considering lesquerella PfKCS18 is evolved to specifically elongate 18:1OH-CoA to 20:1OH-CoA [10] (Figure 1), it is possible that some enzymes in Kennedy pathway are co-evolved to adapt and utilize 20:1OH efficiently. Introducing additional enzymes with 18:1OH substrate selectivity may enhance total HFA level in lesquerella seeds. Although most plant GPATs select a wide range of acyl-CoA substrates [2, 41], castor RcGPAT9 was able to incorporate HFAs including 18:1OH at the sn-1 position of G3P, thus playing a critical role for sn-2 and sn-3 HFA acylation by LPAT and DGAT [42, 43]. Castor RcDGAT2 prefers 18:1OH to common FAs for esterifying 18:1OH to the sn-3 position of DAG [44, 45]. Thus future engineering design may target RcGPAT9 and RcDGAT2. Lesquerella seed transcriptome analysis reveals one PfGPAT9 and three PfDGATs [46]. Evaluation on substrate preference by these genes will provide insights for enzyme characteristics in HFA-rich species.
One of the engineering examples demonstrates the interplay between Kennedy pathway and PC-mediated pathway for acylating HFA into PfKCS18 was found to efficiently elongate 18:1OH to 20:1OH [47] in transgenic camelina expressing RcFAH12 [32]. The transgenic camelina seeds expressing both RcFAH12 with PfKCS18 increased HFA content to 21% compared with the background line expressing single RcFAH12 [47]. 18:1OH-PC generated by RcFAH12 in camelina may be subjected to β-oxidation [48], or represents a bottleneck [24], because camelina is not equipped with enzymes and pathways for channeling 18:1OH-PC into storage TAG. PfKCS18 may ease the 18:1OH flux from PC to cytosol by converting 18:1OH to 20:1OH, thus relieving the bottleneck and facilitating the incorporation of HFA into TAG by Kennedy pathway (Figure 1) [16].
FA at the sn-2 position of PC can be transferred to the sn-3 position of DAG, by PDAT [28, 49] (Figure 1). Castor RcPDAT1–2 (or RcPDAT1A) transfers 18:1OH from its PC to DAG for HFA-TAG synthesis [29, 30]. To further enhance 18:1OH accumulation in lesquerella TAGs, RcPDAT1–2 is another candidate target for genetic engineering.
Significant increases in 18:1OH content are achieved through over expressing RcLPAT2 and silencing FAD2, FAD3 and KCS18. Intriguingly, the accumulated 18:1 was not efficiently utilized to produce 18:1OH and instead, 18:1 was largely channeled to seed TAG. Future research efforts may focus on implementing genetic approach that targets not only enhancement of 18:1OH synthesis, but also on increased 18:1OH acylation to TAG. These genes include RcFAH12 or PlFAH12, RcGPAT9, RcLPAT2, RcDGTAT2, RcPDCT, RcLPAT1–2. Nevertheless, we have demonstrated that lesquerella can be engineered for large increases in 18:1OH levels from 0.4–0.5% in WT to a stable high level of 15–20% in transgenic seed oils.
Authors note that Figure 1 was revised from the original publication listed in ref. [36]; Figures 2 and 3 are cited from the original publication listed in ref. [21]; Tables 1 and 2 are cited from the original publication listed in ref. [36].
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Written By
Grace Chen and Kumiko Johnson
Reviewed: 01 December 2022Published: 28 February 2023
Open access peer-reviewed chapter
