Biotechnology for Improving Hydroxy Fatty Acids Production in Lesquerella (*Physaria fendleri*)

*Grace Chen and Kumiko Johnson*

#### **Abstract**

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.

**Keywords:** hydroxy fatty acid, ricinoleic acid, lesquerolic acid, triacylglycerol, *Physaria fendleri*, lesquerella, seed oil, genetic transformation

#### **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) [2–4] resulting in 18:1-PC (**Figure 1**). An oleate 12-hydroxylase (FAH12) [5–8] 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].

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,

#### **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.*

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 [17–20]. 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) [23–25] 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**) [28–30].

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 *Biotechnology for Improving Hydroxy Fatty Acids Production in Lesquerella (Physaria… DOI: http://dx.doi.org/10.5772/intechopen.109271*

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**).

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) [18–20, 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

#### **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.*

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 [32–34]. 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].

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.*


*Biotechnology for Improving Hydroxy Fatty Acids Production in Lesquerella (Physaria… DOI: http://dx.doi.org/10.5772/intechopen.109271*

*\*\*p < 0.01.*

*\*\*\*p < 0.001.*

## **Table 1.**

*Fatty acid composition (mole %) in T1 seeds expressing AtFAD3 RNAi + CsFAE1 RNAi.*



**Table 2.**

*Fatty acid composition (mole %) in T1 seeds expressing CsFAD2 RNAi + AtFAD3 RNAi + CsFAE1 RNAi.*

*Biotechnology for Improving Hydroxy Fatty Acids Production in Lesquerella (Physaria… DOI: http://dx.doi.org/10.5772/intechopen.109271*

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 *FAH12*s, *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 s*n*-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.

#### **5. Summary**

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.

### **Conflicts of interest**

The authors declare no conflict of interest.

### **Notes/thanks/other declarations**

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].

## **Author details**

Grace Chen\* and 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

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Biotechnology for Improving Hydroxy Fatty Acids Production in Lesquerella (Physaria… DOI: http://dx.doi.org/10.5772/intechopen.109271*

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Section 2
