Elevation of the Yields of Very Long Chain Polyunsaturated Fatty Acids via Minimal Codon Optimization of Two Key Biosynthetic Enzymes

Eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17) and Docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19) are nutritionally beneficial to human health. Transgenic production of EPA and DHA in oilseed crops by transferring genes originating from lower eukaryotes, such as microalgae and fungi, has been attempted in recent years. However, the low yield of EPA and DHA produced in these transgenic crops is a major hurdle for the commercialization of these transgenics. Many factors can negatively affect transgene expression, leading to a low level of converted fatty acid products. Among these the codon bias between the transgene donor and the host crop is one of the major contributing factors. Therefore, we carried out codon optimization of a fatty acid delta-6 desaturase gene PinD6 from the fungus Phytophthora infestans, and a delta-9 elongase gene, IgASE1 from the microalga Isochrysis galbana for expression in Saccharomyces cerevisiae and Arabidopsis respectively. These are the two key genes encoding enzymes for driving the first catalytic steps in the Δ6 desaturation/Δ6 elongation and the Δ9 elongation/Δ8 desaturation pathways for EPA/DHA biosynthesis. Hence expression levels of these two genes are important in determining the final yield of EPA/DHA. Via PCR-based mutagenesis we optimized the least preferred codons within the first 16 codons at their N-termini, as well as the most biased CGC codons (coding for arginine) within the entire sequences of both genes. An expression study showed that transgenic Arabidopsis plants harbouring the codon-optimized IgASE1 contained 64% more elongated fatty acid products than plants expressing the native IgASE1 sequence, whilst Saccharomyces cerevisiae expressing the codon optimized PinD6 yielded 20 times more desaturated products than yeast expressing wild-type (WT) PinD6. Thus the codon optimization strategy we developed here offers a simple, effective and low-cost alternative to whole gene synthesis for high expression of foreign genes in yeast and Arabidopsis.

The human body can synthesize small amounts of EPA and DHA mainly via the classic Δ6 desaturation/Δ6 elongation pathway by consumption of the essential fatty acids LA and ALA The effect of rare codon clusters on heterologous protein expression in E. coli has been reported [16,28]. For example, Kane [16] noted that the presence of individual AGG/AGA Arg , CUA Leu , AUA Ile , CGA Arg or CCC Pro codons in the DNA sequence could cause translational problems in E. coli. If these codons form a cluster it can significantly reduce both the quantity and quality of the synthesized protein. Kim and Lee [20] also reported that clusters of rare codons at the 5'-end of a gene could lead to significantly reduced levels of heterologous gene expression in the host.
Codon usage has been studied in a number of different organisms. Initial studies on codon optimization were carried out in bacteria (Escherichia coli) where the heterologous production of mammalian proteins was tested, resulting in great improvement of gene expression levels. For example, the very first study was carried out by Itakura et al in 1977 who reported production of the first functional human polypeptide in E. coli by utilizing a codon-optimized 14-codon-long DNA molecule encoding human somatostatin. Expression of the codon-optimized DNA fragment yielded 1-to 40-fold more polypeptide than the non-optimized DNA molecule [29]. Later, Kink et al [30] successfully expressed the Ca 2+ -binding protein calmodulin from Paramecium calmodulin, again in E. coli, by changing four TAA codons to optimized CAA codons in its encoding gene. This optimization resulted in around 170 times more calmodulin production in E. coli than in Paramecium cells.
The effect of rare codons on expression of heterologous proteins in yeast has also been reported [27,31,32,33,34,35]. For example, Yadava et al [31] codon-optimized the F2 domain of the Plasmodium falciparum erythrocyte binding antigen (EBA-175), a strong candidate for a vaccine against malaria. Two synthetic genes were produced for its expression in E. coli and Pichia pastoris, with the aim of identifying the best heterologous host for high-level production of biologically active F2 domain of EBA-175 (EBA-F2). Their results showed that codon optimization significantly improved the expression of EBA-F2 in both systems compared to the native sequence. However, the protein produced in Pichia was superior in terms of its expression levels, solubility, and biological activity. Another example was the high efficiency expression of a β-1,3-1,4-glucanase from Bacillus licheniformis following codon optimization for Pichia expression. This led to a 10-fold increase in production of an active enzyme [34]. Thus, it is clear that codon optimization of target genes according to the codon bias of the host cell can frequently result in 10-to 50-fold increases of target protein production [27,33,35].
Here we report on codon optimization of two genes involved in VCL-PUFA biosynthesis for improved heterologous expression in the higher plant Arabidopsis and in yeast. The first gene is the Δ9 elongase gene, IgASE1 which was isolated from a unicellular marine microalgae I. galbana [7], and the second a Δ6 desaturase gene, PinD6, from P. infestan [36]. Through PCR-based site-directed mutagenesis we optimized the first 15 codons of their N termini, and also all arginine codons in the coding sequences, as arginine displayed the highest degree of codon-usage bias. These modified variants together with their wild-type (WT) counterparts were subsequently expressed in Arabidopsis (IgASE1) and yeast (PinD6), respectively. Our results show that codon optimization of both genes resulted in marked increases in the production of VLC-PUFAs in transgenic hosts compared to the expression of the native genes.

Mutagenesis and Expression of IgASE1 in Arabidopsis
Overlap extension PCR technology was employed according to Ho et al [37] and Qi et al [38], using primer pairs listed in Table 1 The translation start codon ATG is in bold, mutated bases are in bold and italic and restriction sites are underlined. DNA fragments were subsequently cloned into the pMD18-T vector (Takara) and sequenced to confirm the presence of the desired changes. These were used as templates to generate R 10,35op , R 10,84op , R 10,35,84op and N op +3R op IgASE1 using different combinations of primer pairs listed in Table 1. Individual DNA fragments were cloned in the pMD18-T vector and sequenced to verify changes as above.
For expression in Arabidopsis, the WT and all 8 mutated IgASE1 DNA fragments were transferred via BamHI and SacI restriction sites to the plant expression vector pCambia 2300EC that contains a plant expression cassette that utilizes the CaMV 35S promoter and Nos terminator sequences [39]. Agrobacterium tumefaciens strain GV3101 was used to transform the WT Arabidopsis via the floral dipping method [40]. Transformants were selected on kanamycin containing ½ MS nutrient medium as described previously and PCR was carried out to check the presence of the transgene [41]. Ten transgenic plants from each line were randomly selected and total fatty acids from leaves were extracted and subjected to gas liquid chromatography as previously described [42]. Three higher C20 fatty acid producing lines that harbour a single copy of the transgene (segregating 3:1 for kanamycin resistant to kanamycin sensitive plants in the T2 seedlings) were taken to the T 3 generation. Homozygous transgenic plants were isolated from these lines if they all survived on kanamycin-selective ½ MS agar plates. Again, total fatty acids were extracted and analysed from leaves of these plants.

Functional Characterization of PinD6 and Its Codon-Optimized Variants in Yeast
Individual fragments of the WT and mutated PinD6 were generated as for IgASE1 mutants, using primer pairs listed in Table 1. They were cloned into the pMD18-T vector and sequenced to confirm the desired changes. Individual fragments were digested with BamHI and HindIII and ligated into the corresponding restriction sites of the yeast expression vector pYES2 (Invitrogen), downstream of the GAL1 promoter, to generate pYES-WT-PinD6, pYES-N op PinD6, pYES 4R op -PinD6 and pYES-N op +4R op , respectively.
These were introduced into S. cerevisiae strain YPH500 (ura3-52, lys2-801 amber , ade2-101 ochre , trp1-Δ63, his3-Δ200, leu2-Δ1) (Stratagene) by the lithium acetate method and selected on minimal media without uracil [41]. Expression of these enzymes was induced by the addition of 2% (w/v) galactose to cultures grown in raffinose minimal liquid media as described previously [7]. After induction, the cultures were grown for a further 48 hours at 22°C in the same medium with or without individually exogenously supplied fatty acid substrates (LA and ALA, 250μM) and 1% Tergitol Type NP-40 (Sigma).

Fatty Acid Analysis
Total fatty acids were extracted from leaves of Arabidopsis and yeast cells and transmethylated with methanolic HCl according to Browse et al [42]. Fatty acid methyl esters (FAMEs) were analyzed by gas chromatography (GC) on a 25-m×0.25-mm fused silica CP-Wax 52CB capillary column (Chrompack UK Ltd, London, UK), using a split/splitless injector (230°C, split ratio 50:1) and a flame ionization detector (300°C). After an initial temperature of 170°C for 3 min, the column was temperature-programmed at 4°C min −1 to 220°C and then was held at 220°C for 15 min. Hydrogen carrier gas at an initial flow rate of 1ml min −1 was used. FAMEs were identified by comparing to retention time with known standards.

Codon Optimization of the Δ9 Elongase Gene IgASE1 from I. galbana for Expression in Arabidopsis
The Δ9 elongase is the first enzyme in the Δ9 elongation/Δ8 desaturation pathways; therefore, it plays a decisive role in the final yield of EPA/DHA. We first analyzed the codon preference of the microalgal Δ9 elongase gene, IgASE1 [7] for expression in Arabidopsis. We found that the three arginine-encoding CGC Arg codons at positions 10, 35 and 84 are the least preferred codons used by Arabidopsis having a predicted codon usage efficiency of only 20% (S1 Fig). This would be expected to lead to lower expression levels of the Δ9 elongase in this host plant and hence result in a reduced final yield of AA/EPA. After comparing the usage frequency of all six arginine-encoding synonymous codons by Arabidopsis we found the frequencies to be 100% for AGA, 57% for AGG, 49% for CGT, 34% for CGA, 26% for CGG, and 20% for CGC, respectively. Thus, we altered the 3 CGC codons to AGA codons individually in the first instance (single codon optimization). Next, we made two-site codon optimized combinations of R10 op +R35 op , R10 op +R84 op , R35 op +R84 op , and also triple codon optimization of R10 op +R35 op +R84 op (Fig 1A). In addition, we optimized 11 codons within the first 15 codons (N op ) at the N-terminus to their high usage synonymous codon equivalents where their predicted usage frequency by Arabidopsis is 100% ("ATG gcT ctT gcT aaT gaT gcT gga gaA AgA atT tgg gcT gct gtT" replacing "ATG GCC CTC GCA AAC GAC GCG GGA GAG CGC ATC TGG GCG GCT GTG", Fig 1B).
All 8 codon-optimized IgASE1 constructs and the WT sequence were cloned in the plant binary vector pCambia2300EC and transferred into WT Arabidopsis plants. Transgenic plants were selected on kanamycin-containing nutrient media agar plates. Total fatty acids from leaves of ten randomly selected mature plants carrying each construct were analyzed. IgASE1 catalyzes the conversion of LA to EDA (ω6 pathway) and ALA to ETrA (ω3 pathway) in yeast [7] and in Arabidopsis [10,43]. The conversion rate [conversion rate = product/(product+substrate)x100] of LA to EDA (ω6 pathway), and ALA to ETrA (ω3 pathway) was analyzed. We found that the majority of WT-IgASE1-expressing transgenic plants had less than 10% conversion rate for ω3 and 30% for ω6 substrate fatty acids and none of them achieved more than 15% fatty acid conversion rate for ω3 or 40% for the ω6 pathway (Fig 2). However, all the codon-optimized variants had improved expression levels of IgASE1, and as a result increased production of EDA and ETrA in transgenic Arabidopsis (Fig 2). For example, expressing N op -IgASE1 resulted in 6 out of the 10 transgenic lines converting 20% of ALA to ETrA in the ω3 pathway, and 7/10 transgenic lines converting 45% of LA to EDA in the ω6 pathway. Changing all 3 CGC Arg to AGA Arg codons also resulted in higher numbers of plants having fatty acid conversion rates of 20% (5/10 for the ω3) and 45% (4/10 for the ω6 pathway). Interestingly, optimizing 2 CGC and single CGC codons at different positions only increased the expression level of IgASE1 slightly (Fig 2). A few transgenic plant lines harboring all 3 CGC Arg -, or the N-terminus-optimized IgASE1 constructs are among the highest producers of the elongated C20 PUFAs (Table 2). For example, while the WT-IgASE1 expressing transgenics produced 5.1 mol% total fatty acids of EDA (LA to EDA conversion rate was 38.1%), plants containing N op -and 3R op -IgASE1 produced nearly twice as much EDA compared to WT-IgASE1 expressing plants at about 9.9 and 8.9 mol% total FAs, and the conversion rate for LA to EDA was 64.3% and 59.7%, respectively. These plants also produced ETrA at 13.8 and 12.4 mol% total FAs compared to only 8.2 mol% total FAs in WT-IgASE1 expressing plants (conversion rate of ALA to ETrA was 30.0% and 25.8% compared to 16.5% in WT) ( Table 2). That is an increase of 1.7-and 1.5-fold of ETrA in N op and 3R op respectively, compared to WT-IgASE1 expressing plants. The total C20 PUFAs (EDA+ETrA) accounted for 23.7 (N op -IgASE1 transgenics) and 21.3% mol (3R op -IgASE1 transgenics) compared to only 13.5% mol total FAs in WT-IgASE1 expressing plants, an increase of 64% and 61%, respectively ( Table 2).

Codon Optimization of P. infestans Δ6 desaturase Genes for Expression in Yeast
To test if the optimization methodology we developed for expression of IgASE1 in plant also applies to other genes involved in the biosynthesis of VLC-PUFAs in a different host we next optimized the fungal P. infestans Δ6 desaturase PinD6, for expression in yeast. Delta6 desaturase is involved in the first step in the Δ6 desaturation pathway for the biosynthesis of VLC-PU-FAs. It converts LA to GLA and ALA to SDA by adding a double bond at the Δ6 position of their hydrocarbon chains. Previously we reported that the activity of PinD6 was very low (with less than 5% substrate conversion rate) when expressed in yeast [36]. Codon usage analysis of The values given are expressed as mol % of total fatty acid methyl esters identified by gas-liquid chromatography (GC). Total fatty acids were extracted from rosette stage leaves of WT Arabidopsis (Col-0 ecotype), or transgenic Arabidopsis expressing the WT, N-terminal optimised (N op ), or all three CGC Arg (R 10,35,84op ) optimized IgASE1 variants. Three higher C20 fatty acid producing lines that harbour a single copy of the transgene were taken to T 3 generation and the homozygous plants were isolated. These were used for total fatty acid analysis. The % conversion for ω-6 fatty acids is calculated as: (EDA/ EDA+LA)x100. Likewise, the % conversion for ω-3 fatty acids is calculated as: (ETrA/ETrA+ALA)x100.
Each value represents the mean ± standard deviation from measurements of three plants. Different  PinD6 revealed that there are four arginine-encoding CGC codons at positions of R9, R276, R376 and R433, respectively (S2 Fig). These codons are predicted to be the least preferred by yeast with the lowest expression efficiency being 13%. As for the AGA codon preference for arginine in Arabidopsis, yeast also has the strongest preference for this codon where 100% expression efficiency is predicted to be achieved (Fig 3A). Because the highest yield of EDA and ETrA was achieved for codon optimized IgASE1 in which all 3 CGC codons were changed to AGA codons in Arabidopsis we decided to mutate all four CGC codons of PinD6 to AGA codons (4R op PinD6). Similarly, we also optimized the less preferred 14 codons within the first 16 codons at the N-terminus of PinD6 (ATG GTG GAC GGC CCC AAG ACC AAG CGC AAG ATC TCG TGG CAG GAG GTC) to their synonymous codons (ATG GTT GAT GGT CCA AAA ACT AAA AGA AAA ATT TCT TGG CAA GAA GTT), where their predicted codon usage frequency in yeast is 100%. In addition, we generated a third DNA fragment containing all 4 AGA arginine encoding codons, plus the N-terminal 14 optimized codons to obtain 4R op +N op PinD6 (Fig 3A and 3B).
The WT-PinD6 and its codon-optimised variants (4 arginine optimised, 4R op ; N-terminal 14 codon optimised, N op ; combined 4 arginine optimised plus the N-terminal 14 codon optimized, 4R op +N op ) were all cloned in the yeast expression vector pYES2 and transformed into yeast. The enzyme activities of the WT and the codon optimized PinD6 were monitored by feeding the transgenic yeast cells with the substrate fatty acids LA and ALA in the presence of 2% galactose to induce protein expression. Consistent with our previous data [36] it was found that the WT-PinD6 can convert LA and ALA to GLA and SDA, with each accounting for 0.8 mol% of total fatty acids with a conversion rate of 2.7% and 1.9% for LA and ALA respectively. However, expressing the N op -PinD6 in yeast yielded GLA and SDA at 9 mol% and 16.9 mol% of the total fatty acids respectively, and the conversion rate was 31.5% and 38.3% for LA and ALA (Fig 3C; Table 3). This represents a 12-fold increase for GLA and a 19-fold increase for SDA production compared to the WT-PinD6-expressing yeast. The 4R op -PinD6 transformed yeast cells contained the products GLA and SDA at 8.2 mol% and 14.6 mol% of the total fatty acids respectively and the conversion rate was 30.3% and 36.2% for LA and ALA (Fig 3C;  Table 3). The fold change was 10.2 for GLA and 18.2 for SDA which was slightly lower than that of N op PinD6. Thus, optimization of all 4 CGC arginine codons significantly improved the enzyme activity of PinD6.
To see if the N op and 4R op mutations had an additive effect with respect to PinD6 enzyme activity we next made a construct containing all these mutations. Analysis of the total fatty acid composition of the transgenic yeast cells showed that GLA and SDA accounted for 14.5 mol% and 19.0 mol% respectively and that the conversation rate was 36.5% for LA and 41% for ALA (Table 3; Fig 3C). This is equivalent to a 19.4-fold increase for GLA and a 23.8-fold increase for SDA in these transgenic yeast cells. This clearly demonstrates that the combined effect of N op and 4R op mutations on PinD6 enzyme activity was much higher than the two individually optimized enzymes.

Discussion
The biosynthesis of VLC-PUFAs in a higher plant was first reported in 2004 by Qi et al [10] who introduced the Δ9 elongation and Δ8 desaturation pathway into Arabidopsis, utilizing one fatty acid elongase and 2 desaturase genes isolated from lower eukaryotes. This was followed by another report where the researchers demonstrated the possibility of producing VLC-PUFAs in seeds of tobacco and linseed via the D6 desaturation pathway [11]. Since then various attempts have been carried out to engineer both pathways into oilseed crops and significant advances have been made [9,13,14]. However, there still remain several challenges that must be met before engineered oilseed crops can be introduced for field production. Amongst these the accumulation of adequate levels of VLC-PUFA is the main hurdle.
The expression of multiple codon-optimized transgenes derived from lower eukaryotic organisms involved in the biosynthesis of EPA and DHA has resulted in high levels of these fatty acids being achieved in higher plants [44,45,46]. However, in these examples the transgenes were synthesized with the majority of their codons being changed to match the preferred codons used by the respective hosts. A minimum of two desaturation steps and an elongation step are needed for the production of EPA. In the case of DHA a further round of elongation and desaturation is required. The typical length of a fatty acid elongase is just under 1 kb and that of a desaturase is~1.5 kb. Therefore,~4,000-6,500 bps of nucleotides for host production of EPA or DHA have to be synthesized. This is a very costly option that most research labs cannot afford. In addition, the specific nucleotide arrangement in a gene sequence is important for its function, thus too many codon changes may alter this dynamic and result in a less active enzyme. Therefore, we aimed to find a codon optimization strategy where minimal changes to the nucleotide sequence of the transgene are made, but with the outcome that high enzyme activity is achieved, for the production of VLC-PUFAs.
In this study, we optimized two key genes, a Δ9 elongase gene from the microalga I. galbana [7] and a Δ6 desaturase gene from the fungus P. infestans [36]. We chose these two genes because they catalyze the first steps in the Δ9 elongation/Δ8 desaturation and the Δ6 desaturation/Δ6 elongation pathways, respectively. Therefore, their activities will have significant impact on the subsequent steps in both pathways for the biosynthesis of VLC-PUFAs.
We first optimized the Δ9 elongase for expression in Arabidopsis by changing the least preferred Arg-encoding CGC codon (CGC Arg ) that has a predicted usage frequency of only 20% in Arabidopsis. There are 3 such codons in the IgASE1 ORF (S1 Fig). Through PCR-based sitedirected mutagenesis, we changed all three to AGA Arg codons which have a predicted usage frequency of 100% in Arabidopsis. As there were 3 CGC Arg codon sites we optimized each one of these individually as well as producing different combinations of them in order to find the site(s) that were responsible for low IgASE1 expression levels. We found that the expression level of the Δ9 elongase gene variants having just one CGC optimized codon (at either position +10, +35 and +84) or a pair of optimized codons (positions 10+35, 10+84, or 10+84) were only slightly higher than the WT Δ9 elongase control (Fig 2). However, when all these 3 CGC codons were mutated to AGA simultaneously the enzyme activity of this variant was much higher than that of the WT IgASE1 and the other single and double AGA Arg IgASE1 versions.
We also altered 11 out of the first 16 N-terminal codons to their frequently used Arabidopsis counterparts. Our data show that the N terminal codon-optimized IgASE1 had the highest enzyme activity compared to the WT-and all the AGA Arg -IgASE1 variants (Fig 2; Table 2). Conveniently, this construct is also the easiest to produce as only one round of PCR is required to incorporate all the changes in this region. Thus, the best approach to achieve high levels of expression with minimal effort for this Δ9 elongase gene in Arabidopsis is to optimize all 11 Nterminal codons-this strategy resulted in lines producing up to 64% more C20 PUFAs than the WT-IgASE1 (Fig 2).
Codon optimization studies have predominately been carried out in E. coli where genes originating from humans and a range of other organisms have been heterologously expressed. For example, Burgess-Brown et al [47] achieved a higher expression efficiency of 30 human genes by optimizing the most biased codons. Similarly by adding rare codon tRNAs in cell lines high expression efficiency of these genes was achieved. Therefore, the availability of rare tRNAs seems to be the limiting factor in reduced protein expression [47]. Two of the six arginine-encoding codons, AGG and AGA, are amongst the rarest codons used by E. coli, and many studies demonstrated that they frequently lead to no, or low, protein expression of foreign genes. Therefore various codon alteration strategies for improving the yields of proteins expressed in E. coli involve the alteration of these two arginine-encoding codons. Consistent with this, we also found that one of the arginine-encoding codons, CGC, in IgASE1 is the most biased codon used by Arabidopsis where only 20% usage frequency is predicted (S1 Fig). By changing the 3 CGC Arg codons in IgASE1 one-by-one to the favored Arabidopsis AGA Arg codon we found higher levels of C20 FA production correlated with increasing numbers of optimised AGA Arg codons. The positions of the CGC Arg did not seem to play a significant role in this effect (Fig 2). Thus it appears that the total number of CGC Arg codons, rather than their positions, contributes to lower IgASE1 activity (C20 FA yield) in transgenic Arabidopsis plants.
Changing all 11 out of the first 15 codons to their favorable Arabidopsis counterparts achieved even higher levels of C20 FAs than that obtained by changing all three CGC Arg codons to AGA Arg in transgenic Arabidopsis (Fig 2). Studies have shown that in E. coli if rare codons occur near the 5' end of an ORF, especially as clusters, the effect can be detrimental, resulting in very low to no expression of foreign genes [20,48,49,50,51,52]. By inserting the rarest codon AGA Arg at different positions near the 5' end of a gene Kim and Lee [20] reported that positioning at the +2 and +3 positions had the most negative effect. As the position of an AGA Arg codon moves further towards to the 3' end of the gene its effect becomes minimal. In IgASE1 the first arginine-encoding codon CGC is positioned at +10, however changing this codon to the favored AGA codon used by Arabidopsis resulted in only a very small enhancement in its activity (Fig 2). Therefore, the marked increase of enzyme activity achieved by optimizing all the 11 codons downstream of the ATG codon is very unlikely to be due to the mutation of CGC Arg at the +10 th amino acid position alone. There are also 3 low-usage alanine-encoding codons at the +2nd, +7th and +13th amino acid positions where only 37%, 33% and 33% usage frequencies are predicted to be used by Arabidopsis (S1 Fig). Therefore, we propose that these 3 alanine codons and the CGC Arg at the +10th position could form a rare codon cluster at the N-terminus of IgASE1 which could result in lower expression of WT-IgASE1 and hence lower EDA and ETrA yields in Arabidopsis (Fig 2; Table 2).
In order to further verify the utility of the codon optimization strategy taken for the I. galbana Δ9 elongase gene, we codon-optimized a Δ6 desaturase gene we isolated from P. infestans (PinD6) which exhibited very low enzyme activity when expressed in yeast [36]. Based on the results obtained from the IgASE1, we made 3 constructs containing: i) 14 optimized N-terminal codons, ii) 4 optimized highly biased CGC Arg codons, and iii) a combination of all these mutations. We found that the enzyme activities of both N op and 4R op variants were significantly elevated compared to the WT, with the N-terminal-optimised Δ6 desaturase having yields higher than that of the variant containing all 4 CGC Arg codons being optimized to AGA Arg . This was consistent with the result obtained from codon optimization and expression of IgASE1 in Arabidopsis, hence further confirming the usefulness of our codon optimization strategies. Interestingly we also found that when both the N op and 4R op mutations were combined the enzyme activity of PinD6 was dramatically increased (Table 3; Fig 3C). Therefore, the N op and 4R op have an additive effect for PinD6 enzyme activity. The yeast expression system has been used for verifying the functions of many eukaryotic genes. For example, most of the VLC-PUFA desaturase and elongase genes isolated from lower eukaryotic organisms were functionally characterized by exogenously expressing them in Baker's yeast. However, such genes have frequently shown very low [36,53] or sometimes no (Qi and Lazarus, unpublished) activity when expressed in yeast, making the verification of their enzyme activities difficult or impossible. The fact that the desaturated fatty acid products of PinD6 could be increased more than 10-fold by simply optimizing 14 of its N-terminal codons for tailored expression in yeast highlights the importance of codon bias to protein expression. Of course, the lower activity of this delta-6 desaturase in yeast made the verification of our codon optimization strategy more convincing. Importantly, this N-terminal codon optimization strategy could be extended to the functional characterization of heterologous fatty acid desaturase and elongase genes in yeast.
Through codon optimization of two key enzymes for VLC-PUFA biosynthesis we provide further evidence that different genetic codon preference exists between organisms leading to lower enzyme activities following their heterologous expression. Both the N-terminal codons and rare codons across the whole ORF contribute to the low enzyme activities found for the two enzymes studied. Thus the best codon optimization strategy, based on our findings, is to optimize the first 16 codons at the N-terminus as well as all the most biased codons in the entire ORF of a given gene. Future studies will be focused on codon optimization of other desaturase and elongase genes involved in EPA and DHA biosynthetic pathways in order to enhance the VLC-PUFAs content in transgenic oilseed crops. (Grant No. 009ZX08005-024B to BQ). We thank Dr. James Doughty, University of Bath, for assisting with editing the manuscript.