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Enhanced accumulation of oil through co-expression of fatty acid and ABC transporters in Chlamydomonas under standard growth conditions

Abstract

Background

Chloroplast and endoplasmic reticulum (ER)-localized fatty acid (FA) transporters have been reported to play important roles in oil (mainly triacylglycerols, TAG) biosynthesis. However, whether these FA transporters synergistically contribute to lipid accumulation, and their effect on lipid metabolism in microalgae are unknown.

Results

Here, we co-overexpressed two chloroplast-localized FA exporters (FAX1 and FAX2) and one ER-localized FA transporter (ABCA2) in Chlamydomonas. Under standard growth conditions, FAX1/FAX2/ABCA2 over-expression lines (OE) accumulated up to twofold more TAG than the parental strain UVM4, and the total amounts of major polyunsaturated FAs (PUFA) in TAG increased by 4.7-fold. In parallel, the total FA contents and major membrane lipids in FAX1/FAX2/ABCA2-OE also significantly increased compared with those in the control lines. Additionally, the total accumulation contribution ratio of PUFA, to total FA and TAG synthesis in FAX1/FAX2/ABCA2-OE, was 54% and 40% higher than that in UVM4, respectively. Consistently, the expression levels of genes directly involved in TAG synthesis, such as type-II diacylglycerol acyltransferases (DGTT1, DGTT3 and DGTT5), and phospholipid:diacylglycerol acyltransferase 1 (PDAT1), significantly increased, and the expression of PGD1 (MGDG-specific lipase) was upregulated in FAX1/FAX2/ABCA2-OE compared to UVM4.

Conclusion

These results indicate that the increased expression of FAX1/FAX2/ABCA2 has an additive effect on enhancing TAG, total FA and membrane lipid accumulation and accelerates the PUFA remobilization from membrane lipids to TAG by fine-tuning the key genes involved in lipid metabolism under standard growth conditions. Overall, FAX1/FAX2/ABCA2-OE shows better traits for lipid accumulation than the parental line and previously reported individual FA transporter-OE. Our study provides a potential useful strategy to increase the production of FA-derived energy-rich and value-added compounds in microalgae.

Background

Microalgae are a group of photosynthetic microorganisms, that can convert CO2 and light energy into high value-added products and energy-rich reserve compounds such as oils (mainly triacylglycerols, TAG) [1]. They are considered as one of the most promising platforms to produce biofuels. However, the oil content in most microalgal species is naturally low, since most microalgae accumulate large amounts of oils only when subjected to stress conditions (e.g., nutrient depletion and high light) [2, 3]. At present, the industrial production of microalgal biofuels is not economically viable. One solution is to increase the oil productivity by genetic engineering approaches, which require a comprehensive understanding of lipid metabolism [4].

The green microalga Chlamydomonas reinhardtii (hereafter referred to as Chlamydomonas) is an established algal model due to its fully sequenced genome, high transformation efficiency, and versatile molecular tools available for this organism. Chlamydomonas has also emerged as a model organism to study the molecular mechanism of oil accumulation [5, 6]. Over the past decade, lipid metabolism has been modified through main approaches, such as enhancing the TAG biosynthesis, manipulating transcription factors/regulators, blocking competing pathways, modulating the redox state and reducing lipid degradation. Among those approaches, much of the research effort has focused on engineering the key enzymes in the lipid biosynthetic pathway in Chlamydomonas [4, 7]. For example, the over-expression of plastidial LPAAT1 (lysophosphatidic acid acyltransferase 1) or endoplasmic reticulum (ER)-located LPAAT2 was reported to increase the oil content (> 20%) in Chlamydomonas under nitrogen (N)-depleted conditions [8, 9]. However, the effect of the LPAAT2 over-expression on the TAG synthesis in Chlamydomonas under standard growth conditions remains negligible [8]. Diacylglycerol acyltransferases (DGAT) catalyse the last acylation step of the lipid biosynthetic pathway and convert diacylglycerol to TAG [10]. Chlamydomonas has six DGAT isozymes, which are classified into two types: type-I DGTA (DGAT1) and type-II DGAT (DGTT1-5) [11]. It was reported that over-expression of DGTT4 can enhance the TAG accumulation under phosphorus deprivation [12], while the over-expression of three of the five other type-II DGATs in Chlamydomonas showed no effects on the intracellular TAG levels under standard growth or stress conditions [13]. These results suggest that the over-expression of the key genes can be a strategy to enhance the TAG biosynthesis under certain conditions but does not always work as expected. Moreover, most of the mutants generated through those strategies can accumulate quantities of oils only under stress conditions when the biomass is compromised [4, 14]. Therefore, more alternative approaches are required to promote oil production from microalgae at cost efficiency.

In plants and microalgae, TAG biosynthesis requires substantial fatty acids (FA) as building blocks, which are synthesized in plastids and subsequently transported to the endoplasmic reticulum (ER) for modification [15]. The FA transporters involved in cellular lipid transport were reported to play critical roles in the lipid metabolism in Chlamydomonas [16, 17]. At present, two chloroplast inner-membrane-located FA exporters (FAX1 and FAX2) have been identified in Chlamydomonas [17]. The over-expression of either FAX1 or FAX2 in the Chlamydomonas UVM4 strain increased the TAG accumulation (up to 38%) under standard (N-replete) growth conditions [17]. Recently, we identified one of the ATP-binding cassette (ABC) transporters of subfamily A in Chlamydomonas, ABCA2, which is located in the ER and involved in the FA transport. The over-expression lines (OE) of ABCA2 exhibited higher TAG levels under standard growth conditions in Chlamydomonas [16]. Therefore, manipulating the expression of FA transporter genes is another promising alternative strategy to accelerate the FA transport from the chloroplast to the ER for TAG biosynthesis. However, individual expression of FAX1, FAX2 or ABCA2 can only result in a limited increase in oil contents (< 50%) [16, 17], and whether FA transporters synergistically contribute to the lipid accumulation in microalgae was unknown.

To investigate the function and effect of coordinate expression of chloroplast- and ER-located FA transporters on lipid metabolism in microalgae, we co-expressed FAX1, FAX2, and ABCA2 in Chlamydomonas in this study. FAX1/FAX2/ABCA2-OE showed enhanced accumulation of TAG and increased total FA and major membrane lipids relative to the parental line and individual FA transporter-OE under standard growth conditions. Additionally, the total accumulation contribution ratio of major polyunsaturated FAs (PUFA) to total FA and TAG synthesis in FAX1/FAX2/ABCA2-OE, was higher than that in the parental strain UVM4, and these increased PUFA could be remobilized from membrane lipids to TAG in FAX1/FAX2/ABCA2-OE. Consistently, the expression of key genes in de novo TAG synthesis and membrane lipid-derived TAG synthesis was significantly upregulated in the FAX1/FAX2/ABCA2-OE cells. Overall, our results indicate that the simultaneously increased expression of FAX1, FAX2 and ABCA2 has an additive effect on enhancing the TAG, total FA and membrane lipid accumulation, and favours the integration of PUFA from membrane lipids to TAG. The FAX1/FAX2/ABCA2-OE generated in this study showed better traits for lipid accumulation than previously reported individual FA transporter-OE. Therefore, this study provides additional molecular tools to further promote lipid production in other oleaginous microalgae.

Results

Generation of FAX1/FAX2/ABCA2-OE of Chlamydomonas

As a strategy to further enhance the oil content, we aimed to over-express the two FAX proteins responsible for fatty acid (FA) export from chloroplasts, and ABCA2, which helps deliver exported FAs to the ER, where the TAG synthesis mainly occurs. The Chlamydomonas UVM4 strain was transformed by electroporation with the pChlamy4-FAX1/FAX2 plasmid, where the DNA sequences of FAX1 (Cre10.g421750) and FAX2 (Cre08.g366000) were tandemly fused and driven by the hybrid promoter (Hsp70A-RbcS2) (Fig. 1a). Transformants were selected on TAP/agar plates supplemented with 15 mg/L zeocin. Zeocin-resistant clones were screened by PCR to verify the integration of pChlamy4-FAX1/FAX2 using specific primers to amplify the full-length DNA sequences of FAX1 and FAX2. A band corresponding to the expected size of 2092 bp was detected in the positive transformants (Additional file 1: Fig. S1a), which confirms the integration of DNA sequences of FAX1 and FAX2 into the genome of Chlamydomonas. The PCR-positive clones (FAX1/FAX2-13 and -35) were chosen for further analysis by RT-PCR using specific oligonucleotides to amplify the full-length cDNA sequences of FAX1 and FAX2. The predicted size of 996 bp of FAX1 and FAX2 cDNA was detected in the FAX1/FAX2-13 and -35 clones but not in the parental strain UVM4 (Fig. 1b), which further confirmed that FAX1 and FAX2 were expressed in transformants.

Fig. 1
figure 1

Overexpression of FAX1/FAX2 and ABCA2 in Chlamydomonas. a Schematic representation of the transformation vectors for generation of FAX1/FAX2 (left panel), and FAX1/FAX2/ABCA2 (right panel) overexpressing strains. Pro, Hsp70A/RbcS2 promoter; 2A, FMDV 2A peptide; Ter, Chlamydomonas RbcS2 terminator. b, c Reverse-transcription PCR (RT-PCR) analysis of the expression of FAX1/FAX2, and ABCA2 in the transformants. PL, parental line (FAX1-FAX2-35). Housekeeping gene used is RACK1 (Cre06.g278222, receptor of activated protein kinase C 1 initially described as CBLP). The PCR products amplified with the primers (FAX1-F/FAX2-R and ABCA2-F1/ABCA2-R1) at 28 cycles were shown, respectively. This is a representative of three independent experiments. d Quantitative RT-PCR analysis of the expression level of ABCA2 in FAX1/FAX2/ABCA2 transformant. The qRT-PCR results were normalized by the level of RACK1 expression. PL, parental line (FAX1-FAX2-35). Error bars represent standard errors based on three biological replicates (independent cultures) with three technical replicates each. Asterisks indicate statistically significant changes compared to the parental line FAX1-FAX2-35 by paired-sample Student’s t test (**P ≤ 0.01)

To generate FAX1/FAX2/ABCA2-OE of Chlamydomonas, the FAX1/FAX2-35 clone was used as the parental strain, where FAX1/FAX2 was highly expressed. ER-located ABCA2-encoding cDNA (Cre14.g613950) was subcloned into the pOpt-Clover-Paro vector to make the pOpt-ABCA2-Paro plasmid (Fig. 1a). After the transformation, the transformants were screened on agar plates containing 15 mg/L zeocin and 20 mg/L paromomycin. The insertion of the pOpt-ABCA2-Paro construct into the genome of selected colonies was confirmed by PCR (Additional file 1: Fig. S1b, c). Furthermore, the results show that the expression levels of ABCA2 in three representative FAX1/FAX2/ABCA2-OE lines significantly increased compared with those in the untransformed parental line (Fig. 1c). For example, the expression level of ABCA2 increased by up to 3.4-fold in FAX1/FAX2/ABCA2-51 compared to its parental strain UVM4, as analysed by qRT-PCR (Fig. 1d).

FAX1/FAX2/ABCA2-OE exhibit increased TAG accumulation

Changes in oil content in the triple over-expressor (FAX1/FAX2/ABCA2-OE) with the single over-expressor for ABCA2-OE [16], double over-expressor (FAX1/FAX2-OE), and untransformed parental strain (UVM4) were analysed here using thin-layer chromatography (TLC) coupled to GC–MS analysis. The strains were grown side by side under identical growth conditions for each experiment. Under standard growth conditions, we observed that FAX1/FAX2-35 and ABCA2-2 showed 91% and 36% more TAG than UVM4, while FAX1/FAX2/ABCA2-OE exhibited up to twofold more TAG than UVM4. Compared to the FAX1/FAX2-35 strain, there was 56% more TAG in FAX1/FAX2/ABCA2-51 when ABCA2 was further over-expressed (Fig. 2a). Under nitrogen (N)-depleted conditions, all three FA transporter-OE also showed increased TAG content compared with UVM4 (Additional file 1: Fig. S2a). Interestingly, the fold change in the increase in TAG contents under standard growth conditions in FA transporter-OE was comparable or even higher than that under N-depleted conditions (Fig. 2a; Additional file 1: Fig. S2a). Among the three independent strains of FAX1/FAX2/ABCA2-OE, FAX1/FAX2/ABCA2-51 was selected for further study due to its higher expression level of ABCA2 (Fig. 1c) and higher TAG content than the other strains under standard growth and N-depleted conditions (Fig. 2a; Additional file 1: Fig. S2a).

Fig. 2
figure 2

Levels of TAG and profile of fatty acids content of TAG in the transformants at exponential phase (day 2) under standard growth conditions. a TAG content, b fatty acids content of TAG. The distinct letters labelled indicate the statistically significant difference by Tukey’s honestly significant difference (HSD) test

The analysis of the individual FA contents of TAG reveals that almost all monounsaturated FA and PUFA contents increased in all three FA transporter-OE compared with UVM4 (Fig. 2b). Furthermore, the results show that FAX1/FAX2-35 exhibited some differences from ABCA2-2 in the augmentation of FA contents in TAG. In FAX1/FAX2-35, the contents of 16:0, 16:3 (7,10,13) and 18:1 (9) increased compared with UVM4, but they did not change in ABCA2-2. Noticeably, all FA contents of TAG significantly increased in FAX1/FAX2/ABCA2-51 compared with individual FA transporter-OE and UVM4, except 18:4 (5,9,12,15), which was unchanged (Fig. 2b). Taken together, these results indicate that the increased expression of FAX1/FAX2 and ABCA2 individually enhanced the oil accumulation, and this effect was further enhanced by simultaneously expressing the three genes.

FAX1/FAX2/ABCA2 over-expression increased the total fatty acids

We further examined the influence of FAX1/FAX2/ABCA2 over-expression on the FA content under standard growth conditions. Compared with the untransformed parental line UVM4, the total FA contents significantly increased in FAX1/FAX2-35 and FAX1/FAX2/ABCA2-51 but were not different from ABCA2-2. However, when ABCA2 was over-expressed in FAX1/FAX2-35, there were 38% and 61% more total FA in FAX1/FAX2/ABCA2-51 than in the parental strains FAX1/FAX2-35 and UVM4, respectively (Fig. 3a). The increase of total FA contents was also found in other FAX1/FAX2/ABCA2-OE, such as FAX1/FAX2/ABCA2-14 and FAX1/FAX2/ABCA2-74 (Additional file 1: Fig. S3). The individual FA content analysis shows that the contents of major FA (e.g., 16:0, 16:4 and 18:3) increased in FAX1/FAX2/ABCA2-51 compared with the control strains (UVM4, FAX1/FAX2-35 and ABCA2-2) (Fig. 3b). However, the individual FA content did not greatly change in either FAX1/FAX2-OE or ABCA2-OE compared with UVM4. In FAX1/FAX2-35, only 18:3 (9,12,15) increased, and 16:4 (4, 7, 10, 13) decreased, but other FA contents did not significantly change. In ABCA2-2, 16:1 (9) increased, but 16:0 was reduced (Fig. 3b). Additionally, the effect of these FA transporters on FA content was explored under N-depleted conditions. All total FA contents significantly increased in FAX1/FAX2-35, ABCA2-2 and FAX1/FAX2/ABCA2-51 compared with UVM4 (Additional file 1: Fig. S2b). For example, the contents of saturated FAs (e.g., 16:0 and 18:0) more significantly increased in both FAX1/FAX2-35 and ABCA2-2 than in UVM4. Noticeably, all FA levels were increased in FAX1/FAX2/ABCA2-51 compared with the control strains under N-depleted conditions (Additional file 1: Fig. S2c). Therefore, our results indicate that the increased expression of FAX1/FAX2/ABCA2 is likely to balance different substrate preferences towards fatty acyls among these different FA transporters and positively contributes to the accumulation of saturated and unsaturated FAs.

Fig. 3
figure 3

Total and individual fatty acids content in the transformants of FAX1/FAX2 and ABCA2 at exponential phase (day 2) under standard growth conditions. a Total fatty acids content. b Profile of individual fatty acid content in total fatty acids (TFA). The distinct letters labelled indicate the statistically significant difference by Tukey’s HSD test

Major membrane lipid levels were also altered in FAX1/FAX2/ABCA2-OE

To determine whether the increases in TAG and total FA in FAX1/FAX2/ABCA2-OE are accompanied by changes in polar lipids, the major membrane glycerolipids were analysed, including MGDG (monogalactosyldiacylglycerol), DGDG (digalactosyldiacylglycerol) and DGTS (diacylglyceryltrimethylhomoserine). The results show that the total membrane lipid content increased by 61% in FAX1/FAX2/ABCA2-51 compared with the untransformed parental strain UVM4, while no changes were found in FAX1/FAX2-35 and ABCA2-2 (Fig. 4a). In FAX1/FAX2-35, the amount of the major thylakoid membrane lipid MGDG decreased by 48%, while the second most abundant thylakoid membrane lipid DGDG and the major extraplastidic lipid DGTS both prominently increased compared with UVM4 (Fig. 4b). These results are consistent with previous reports on these two genes (FAX1 and FAX2) individually expressed in Chlamydomonas [17]. In ABCA2-2, the amount of DGDG significantly increased, while the content of DGTS decreased, and that of MGDG did not change compared with UVM4 (Fig. 4b). However, when FAX1, FAX2 and ABCA2 were co-expressed, all major membrane lipid levels in FAX1/FAX2/ABCA2-OE increased compared with those in UVM4, except that the SQDG (sulfoquinovosyldiacylglycerol) amount was unaltered (Fig. 4b; Additional file 1: Fig. S4). For example, in FAX1/FAX2/ABCA2-51, the contents of MGDG, DGDG and DGTS were 48%, 188% and 34% higher than those in UVM4, respectively (Fig. 4b). Furthermore, the contents of unsaturated FAs [16:3 (4,7,10), 16:4 (4,7,10,13), 18:2 (9,12) and 18:3 (9,12,15)] of MGDG were significantly higher in FAX1/FAX2/ABCA2-51 than in UVM4 (Additional file 1: Fig. S5a); for example, both major FAs of MGDG [16:4 (4,7,10,13) and 18:3 (9,12,15)] increased by more than 60%. The saturated FA (e.g., 16:0) and almost all detected unsaturated FAs in DGDG, such as 16:2 (7,10), 16:3 (7,10,13), 18:2 (9,12), and 18:3 (9,12,15) notably increased (Additional file 1: Fig. S5b). In particular, the contents of the major FAs of DGDG [18:2 (9,12) and 18:3 (9,12,15)] increased by 177% and 128% in FAX1/FAX2/ABCA2-51 compared with UVM4, respectively. In the FA profile of DGTS, the contents of saturated FA 16:0 and almost all unsaturated FA also increased in FAX1/FAX2/ABCA2-51 compared with UVM4 (Additional file 1: Fig. S5c). For example, the major FA of DGTS, 18:3 (5,9,12), increased by 73%. These results indicate that the phenotype of higher lipid contents observed in FAX1/FAX2/ABCA2-OE is probably mainly due to an increase in the de novo biosynthesis of FA, and the higher TAG level in this strain may be accompanied by de novo-synthesized membrane lipids, including MGDG, DGDG and DGTS, under normal growth conditions.

Fig. 4
figure 4

Alterations of membrane glycerolipid levels in the transformants of FAX1/FAX2 and ABCA2 at exponential phase (day 2) under standard growth conditions. a Total membrane lipid content. b Profile of individual membrane component contents. The total membrane lipid content referred to the total amount of major galactolipids (MGDG, DGDG and SQDG) and phospholipids (PE, PG and PI), and betaine lipid DGTS. MGDG, monogalactosyldiacylglycerol; DGDG, digalactosyldiacylglycerol; DGTS, diacylglycerol-N,N,N-trimethylhomoserine; SQDG, sulfoquinovosyldiacylglycerol; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PI, phosphatidylinositol. The distinct letters labelled indicate the statistically significant difference by Tukey’s HSD test

Analysis of the contribution source to accumulate total FA, TAG and membrane lipids in FAX1/FAX2/ABCA2-OE

To further elucidate the major contributors to the lipid accumulation, the contribution ratio of TAG and membrane lipids (PL) to total FA accumulation increased in FAX1/FAX2/ABCA2-OE relative to the parental strain UVM4 were firstly examined. The results showed that the contribution ratio of PL to total FA accumulation in FAX1/FAX2/ABCA2-51 was sevenfold than that of TAG relative to UVM4 (Additional file 1: Fig. S6a), which indicates that PL is the major contributor to total FA accumulation in FAX1/FAX2/ABCA2-OE. Furthermore, the main contributors to PL accumulation were MGDG, DGDG and DGTS in FAX1/FAX2/ABCA2-51 with contribution ratios of 39%, 43% and 10% in relative to UVM4, respectively (Additional file 1: Fig. S6b). Therefore, the biosynthesis of these three membrane lipids was remarkably enhanced in the over-expressed FAX1/FAX2/ABCA2-51, especially the thylakoid membrane lipids MGDG and DGDG. We also found that the PUFA levels of total FA, TAG and the major PL were significantly higher in FAX1/FAX2/ABCA2-51 than in UVM4 (Additional file 1: Fig. S6c). Then, we analysed the contribution ratio of the major PUFA to total FA accumulation, and the results showed that the increased ratio of 18:3 (9,12,15) in FAX1/FAX2/ABCA2-51 was the most prominent, followed by 16:4 (4,7,10,13) relative to UVM4 (Additional file 1: Fig. S6d). The contribution ratios of the major FAs 18:3 (9,12,15) and 16:4 (4,7,10,13) to MGDG accumulation were more than 40%, and that of 18:3 (5,9,12) for DGTS was more than 50% in FAX1/FAX2/ABCA2-51 relative to UVM4 (Additional file 1: Fig. S6e). For DGDG accumulation, the contribution ratios of major FA 18:2 (9,12) and 18:3 (9,12,15) were also significantly higher in FAX1/FAX2/ABCA2-51 than in UVM4, although they were less than those of the major FAs in MGDG and DGDG (Additional file 1: Fig. S6e). Additionally, the total accumulation contributions of major PUFAs, e.g., 16:4 (4,7,10,13), 18:3 (9,12,15) and 18:3 (5,9,12), to the total FA and TAG accumulation in FAX1/FAX2/ABCA2-51 were 54% and 40% higher than UVM4, respectively (Additional file 1: Fig. S6d, f). Overall, the contents of PUFA, which serve as the major contributors to total FA, TAG and the major membrane lipids in FAX1/FAX2/ABCA2-OE, significantly increased when fatty acid and ABC transporters were co-expressed.

Expression levels of genes in the TAG biosynthesis and membrane lipid turnover in FAX1/FAX2/ABCA2-OE

To gain insights into the mechanisms of enhancing lipid accumulation when FAX1, FAX2, and ABCA2 were co-expressed, we first analysed the transcript levels of key enzymes for the TAG biosynthesis, diacylglycerol acyltransferases (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT), which are directly involved in the acylation of diacylglycerol and transacylation of phospholipids into TAG. The results showed that the expression of DGAT1 (type-I DGAT) was unaltered in the three FA transporter-OE. However, type-II DGAT showed differential expression levels in the three FA transporter-OE compared with UVM4 (Fig. 5a). In FAX1/FAX2-35, the expression of DGTT1, DGTT3 and DGTT4 was significantly upregulated (> 2.0-fold), while DGTT5 was slightly downregulated compared with UVM4 (Fig. 5a). In ABCA2-2, the expression of DGTT5 was upregulated by 1.8-fold, while that of DGTT1, DGTT2 and DGTT4 was slightly downregulated compared with UVM4 (Fig. 5a). When FAX1, FAX2, and ABCA2 were co-expressed, the expression levels of DGTT1, DGTT3 and DGTT5 were upregulated 2.0-fold, 1.3-fold and 3.2-fold than UVM4, respectively, compared with UVM4. However, the expression of DGTT2 and DGTT4 was downregulated in FAX1/FAX2/ABCA2-51 compared with UVM4 (Fig. 5a). Interestingly, the expression level of PDAT1 was upregulated in all three FA transporter-OE compared to UVM4 (Fig. 5b). Moreover, the expression level of MGDG-specific lipase (PGD1) was significantly upregulated (> twofold) in all three FA transporter-OE, especially that in FAX1/FAX2/ABCA2-51 (up to sevenfold) (Fig. 5c). The significantly upregulated expression of PDAT1 and PGD1 in FAX1/FAX2/ABCA2-OE, FAX1/FAX2- and ABCA2-OE revealed the vital roles of membrane lipid remodelling, especially MGDG, in the TAG biosynthesis. Overall, FAX1/FAX2/ABCA2-OE showed significantly higher expression of DGAT (DGTT1, DGTT3 and DGTT5), PDAT (PDAT1) and MGDG-specific lipase (PGD1) than UVM4, which was likely to account for the obvious accumulation of TAG in the FAX1/FAX2/ABCA2-OE strain.

Fig. 5
figure 5

qRT-PCR analysis of the expression of key genes in the TAG biosynthesis and membrane lipid turnover in the transformants at exponential phase (day 2) under standard growth conditions. The relative expression level of diacylglycerol acyltransferases (DGAT) (a), phospholipid:diacylglycerol acyltransferase 1 (PDAT1) (b), and plastid galactoglycerolipid degradation 1 (PGD1) (c). The housekeeping gene RACK1 (Cre06.g278222) was used as the internal standard. Values are the relative expression level compared with parental line UVM4. Error bars represent standard deviations based on three biological replicates (independent cultures) with three technical replicates each. Asterisks indicate statistically significant changes compared to the parental line UVM4 by paired-sample Student’s t test (*P ≤ 0.05; **P ≤ 0.01)

Physiological consequence of FAX1/FAX2/ABCA2-OE

The physiological effect on the cells was also examined, when FAX1, FAX2, and ABCA2 were co-expressed. The results showed that the cell growth rate of FAX1/FAX2/ABCA2-51 was significantly slower than that of the untransformed parental line UVM4, and the over-expression of either FAX1/FAX2 or ABCA2 only delayed growth in the stationary phase under standard growth conditions (Fig. 6a). The slow cell growth of the triple over-expressor is consistent with a lowered maximum quantum efficiency (Fv/Fm) of PS II compared with all control strains (UVM4, FAX1/FAX2-35 and ABCA2-2) (Fig. 6b). However, FAX1/FAX2/ABCA2-51 had bigger cell size and biomass productivity than all control strains (Fig. 6c, d). This result is consistent with a previous report that when FAX1 or ABCA2 homologous genes (AtFAX1 or AtABCA9) were over-expressed in Arabidopsis thaliana, the mutants showed an increase in dry weight compared with wild type [18, 19]. Our results suggested that the increased expression of FAX1/FAX2/ABCA2 contributes to the increase in cell size and biomass productivity of microalgae.

Fig. 6
figure 6

Phenotypic changes of the transformants under standard growth conditions. a Cell growth kinetics. b The maximum quantum efficiency (Fv/Fm) of photosystem II (PS II) c Averaged cell diameter. d Biomass productivity. In b and c, the cells were harvested at exponential phase (day 2) under standard growth conditions. The distinct letters labelled indicate the statistically significant difference by Tukey’s HSD test

Discussion

Chloroplast- and ER-localized FA transporters were reported to play important roles in TAG biosynthesis in Chlamydomonas reinhardtii [16, 17]. However, whether FA transporters synergistically contribute to lipid accumulation in microalgae is unknown. In this study, we found that under standard growth conditions, FAX1/FAX2/ABCA2-OE showed greatly more TAG and total amounts of major PUFA in TAG than the untransformed parental line (Fig. 2). Moreover, our results suggested that the contents of total FA and major membrane lipids also significantly increased in FAX1/FAX2/ABCA2-OE compared with the control strains (Figs. 3, 4). Additionally, the total accumulation contribution ratios of major PUFA to total FA and TAG synthesis in FAX1/FAX2/ABCA2-OE were both higher than UVM4 (Additional file 1: Fig. S6). Consistently, the expression of key genes in the TAG synthesis and major thylakoid membrane turnover was significantly upregulated in FAX1/FAX2/ABCA2-OE (Fig. 5). These results indicated that the simultaneously increased expression of FAX1, FAX2 and ABCA2 has an additive effect on enhancing the TAG and membrane lipid accumulation, and prominently accelerates the PUFA remobilization from membrane lipids to TAG by fine-tuning the key genes in lipid metabolism (Fig. 7).

Fig. 7
figure 7

Hypothetical model explaining adjustment of lipid metabolism under standard growth conditions when FAX1, FAX2 and ABCA2 were co-expressed in Chlamydomonas. ABCA2, ATP-binding cassette transporter subfamily A2; ACP, acyl carrier protein; DAG, diacylglycerol; DGTS, diacylglyceryltrimethylhomoserine; DGTTs, type II diacylglycerol acyltransferases; DGDG, digalactosyldiacylglycerol; ER, endoplasmic reticulum; FAX1/2, fatty acids exporters 1 and 2; FAS, fatty acid synthase; FFA, free fatty acids; G3P, glycerol-3-phosphate; LD, Lipid droplet; LPA, lysophosphatidic acid; MGDG, monogalactosyldiacylglycerol; PA, phosphatidic acid; PGD1, plastid galactoglycerolipid degradation 1; PDAT1, phospholipid:diacylglycerolacyltransferase 1; TAG, triacylglycerol. The 16:4n3 [16:4(4,7,10,13)]*1, 18:3n3 [18:3(9,12,15)] and 18:3n6 [18:3(5,9,12)]*2 derived from membrane lipids were highlighted in bold

In this study, ABCA2-OE (ABCA2-2) was generated by overexpressing of ABCA2 cDNA and characterized as we previously described [16]. To compare with the same single mutant (ABCA2-2), we generated FAX1/FAX2/ABCA2-OE using ABCA2 cDNA. Recently, it was reported that intron-containing transgenes mediate efficient recombinant gene expression in Chlamydomonas [20, 21]. We used the genomic DNA of FAX1/FAX2 for the generation of FAX1/FAX2/ABCA2-OE, considering that native regulatory elements (introns) might facilitate the expression of nuclear transgenes. It is worth testing whether the expression levels of FAX1/FAX2/ABCA2 can be enhanced using a synthetic intron spread strategy in the future.

The accumulation of TAG is usually accompanied by the degradation of the membrane lipids in microalgae [10, 22, 23]. Interestingly, our results showed that the content of total membrane lipids significantly increased in the FAX1/FAX2/ABCA2-OE strain compared with the untransformed parental strain, while no changes were observed in the FAX1/FAX2-OE and ABCA2-OE strains (Fig. 4a). In FAX1/FAX2-35, the major lipid component of the thylakoid membrane, such as MGDG, significantly decreased, while DGDG and the major extraplastidic lipids such as DGTS increased compared with the untransformed parental strain (Fig. 4b), which is in consistent with previous reports on these two genes (FAX1 or FAX2) being individually expressed in Chlamydomonas [17]. In ABCA2-2, the amount of DGDG significantly increased, while MGDG was not changed compared with the untransformed parental strain (Fig. 4b). However, when the expression of FAX1, FAX2, and ABCA2 was synergistically increased, all major membrane lipids in FAX1/FAX2/ABCA2-51 increased compared with the untransformed parental line and individual FA transporter-OE (Fig. 4b). The contribution ratio of polar lipids to the accumulation of total FA in FAX1/FAX2/ABCA2-51 was up to 65%, while that of TAG was only 9%, compared with the parental strain UVM4 (Additional file 1: Fig. S6a). Noticeably, the biosynthesis of membrane lipids was remarkably enhanced in FAX1/FAX2/ABCA2-51, especially the plastidial photosynthetic membrane lipids MGDG and DGDG (Fig. 4b). Subsequently, all ratios of bilayer-forming lipid DGDG to nonbilayer-forming lipid MGDG in three FA transporter-OE increased (> twofold) compared with UVM4 (calculated from Fig. 4b), which indicates that the elevated membrane adaptations are similar to heat-stressed Chlamydomonas and higher plants [24]. Therefore, head group exchanges of glycerolipids can also be a mechanism that Chlamydomonas employs to adapt the over-expression of these FA transporters.

In microalgae, acyl-ACPs are generated by FA synthesis and cleaved by thioesterases (TEs) to form free FAs in the chloroplast. Free FAs are exported from the plastid to the ER, and esterified to acyl-CoAs to form TAG [10, 25]. The key enzymes involved in fatty acid synthesis (e.g., ACCase) are feedback-inhibited by long-chain fatty acyl-ACPs, and the expression of endogenous acyl-ACP TEs can relieve this inhibitory mechanism in microalgae [26]. For example, the over-expression of endogenous TEs in the diatom Phaeodactylum tricornutum enhanced its total FA content by 72% [27]. Moreover, heterologous over-expression of TEs from Dunaliella tertiolecta increased both neutral lipid and total FA content in Chlamydomonas [26]. Our results showed that both TAG and total FA also increased in FAX1/FAX2/ABCA2-OE. One of the possible reasons may partially be due to the TE-related deregulation in lipid metabolism, which must be studied in the future studies.

In terms of polyunsaturation, either total fatty acyls or main membrane lipids, i.e., MGDG, DGDG and DGTS, exhibited prominently incremental abundances of polyunsaturated fatty acyls in FAX1/FAX2/ABCA2-51 (Additional file 1: Fig. S6c). Meanwhile, the PUFAs attached with more than two double bonds [e.g., 16:4 (4,7,10,13), 18:3 (5,9,12) and 18:3 (9,12,15)], prominently accumulated in FAX1/FAX2/ABCA2-51 relative to UVM4 (Fig. 2b). These PUFA mainly reside in membrane lipids, and their assembly involves elongation and desaturation, which also depend on carrier membrane lipids [28, 29]. The main contributor to MGDG augmentation was its primary fatty acids, 16:4 (4,7,10,13) and 18:3 (9,12,15), and their contribution ratios both increased (up to 44%) relative to UVM4 (Additional file 1: Fig. S6e), which indicates the vital role of the main molecule 18:3 (9,12,15)/16:4 (4,7,10,13) in the MGDG of FAX1/FAX2/ABCA2-OE. In the second most abundant photosynthetic galactolipid, DGDG, the contribution ratio of 18:3 (9,12,15) was 18% and that of 18:2 (9,12) was 13% in FAX1/FAX2/ABCA2-51 (Additional file 1: Fig. S6e). In parallel, the pivotal functional molecules of DGDG were 18:3 (9,12,15)/16:0 and 18:2 (9,12)/16:0. In contrast, the major FA of the ER-located betaine lipid DGTS is 18:3 (5,9,12) [30, 31], whose contribution ratio to DGTS formation was up to 56% in FAX1/FAX2/ABCA2-51 (Additional file 1: Fig. S6e). The key molecule in DGTS was considered to be 18:3 (5,9,12)/16:0, whose content was found to notably increase in FAX1/FAX2/ABCA2-51. In this study, we observed the co-accumulation of TAG and photosynthetic membrane lipids (mainly MGDG and DGDG) in FAX1/FAX2/ABCA2-OE, which might be a response to over-expression of three fatty acid transporters.

The conversion of diacylglycerol into TAG is the only committed final step to TAG synthesis in plants and microalgae, which is mainly catalysed by DGAT and PDAT [29]. In Chlamydomonas, there is only one characterized PDAT (PDAT1) [32], but six DGATs were found [13, 32]. PDAT1 is a multi-functional enzyme with proven galactolipase function that prefers membrane lipids, particularly de novo-synthesized MGDG, as substrates to synthesize TAG in Chlamydomonas [32, 33]. Recently, it was reported that the less-unsaturated-PE, which serves as a transient carbon reservoir, could be used by PDAT for oil biosynthesis in Nannochloropsis [34]. Therefore, the increased PE/PG in our study may also be used as a PDAT1 substrate to enhance the TAG biosynthesis in Chlamydomonas. The Chlamydomonas pgd1 null mutant reduced the turnover of de novo-synthesized MGDG, which decreased the TAG content [35]. In this study, significantly upregulated expression levels of PDAT1 and PGD1 in FAX1/FAX2/ABCA2-OE were found (Fig. 5b, c), which revealed their critical roles in membrane lipid remodelling, especially MGDG for TAG biosynthesis. The major FAs of MGDG, i.e., 16:4 (4,7,10,13) and 18:3 (9,12,15), are proposed to be hydrolysed by these two lipases (PDAT1 and PGD1). The fatty acyls released from MGDG may serve as the acyl pool for TAG assembly catalysed by type-II DGAT in FAX1/FAX2/ABCA2-OE (Fig. 7).

In microalgae, the substrates used for TAG synthesis can partially originate from the main polar lipid species, since the FA from the degraded membrane lipids can be recycled for TAG synthesis [3]. The total membrane lipid content increased (Fig. 4b), and two major PUFAs [16:4 (4,7,10,13) and 18:3 (9,12,15)] were synthesized more in the FAX1/FAX2/ABCA2-OE strain than in the parental strain UVM4 (Fig. 3b). These enriched PUFA in TAG are likely to originate from the turnover pathway of the newly synthesized photosynthetic membrane lipids instead of that of the nascent ones when the expression of three fatty acid transporters was simultaneously increased. Our results showed that the expression levels of key enzymes in the TAG synthesis, such as DGTT1, DGTT3, DGTT5, PDAT1 and PGD1, in the MGDG-specific degradation were upregulated in FAX1/FAX2/ABCA2-51 comparted with the parental strain UVM4 (Fig. 5). Therefore, the higher TAG content in FAX1/FAX2/ABCA2-OE could be due to the enhancement of de novo synthesis of TAG and remodelling of the de novo-synthesized MGDG. Based on the gene expression levels of the related enzymes in the TAG assembly, the major photosynthetic membrane lipid MGDG is likely to direct the newly formed 16:4 (4,7,10,13) and 18:3 (9,12,15) into the stored TAG in FAX1/FAX2/ABCA2-OE by upregulating lipases (e.g., PDAT1 and PGD1) with acyltransferases (e.g., type-II DGAT) (Fig. 7). This study may present a novel TAG assembly pathway when FA and ABC transporters are co-expressed in Chlamydomonas under standard growth conditions.

The FAX1 specificity in Arabidopsis thaliana is significantly higher for palmitic acid than for stearic and oleic acid [19]. We also found that over-expression of two different types of transporters has different effects on FA components. Under standard growth conditions, the total FA levels increased in FAX1/FAX2-OE, which was mainly due to the increase in 18:3 (9,12,15); however, the total FA in ABCA2-OE did not change (Fig. 3). ABCA2 homologous gene (ABCA9)-overexpressing plants also showed no significant differences in FA composition [18]. In this study, when ABCA2 was over-expressed in FAX1/FAX2-OE, there were 38% and 61% more total FA in FAX1/FAX2/ABCA2-51 than in the parental strains FAX1/FAX2-35 and UVM4, respectively. Moreover, almost all FA components increased in FAX1/FAX2/ABCA2-51 comparted with the untransformed parental line UVM4. In particular, the contents of 16:0, 16:4 (4,7,10,13) and 18:3 (9,12,15) significantly increased in FAX1/FAX2/ABCA2-51 (Fig. 3). These results indicated that these two types of FA transporters may have different FA preferences, and a simultaneously increased expression of these transporters can balance the differences and increase the FA content. Under N-depleted conditions, the TAG, total FA and most FA component contents increased in FAX1/FAX2/ABCA2-OE (Additional file 1: Fig. 2). These results also suggested that the increased expression of FAX1/FAX2/ABCA2 shows an additive effect on enhancing the total FA and FA component contents, in addition to the TAG and membrane lipid contents under both standard growth and N-depleted conditions.

The maximum quantum efficiency (Fv/Fm) of photosystem (PS) II was negatively correlated with lipid contents in microalgae [31, 36, 37]. Our results showed that both FAX1/FAX2-OE and ABCA2-OE showed no differences in Fv/Fm compared with the untransformed parental strain UVM4. However, when the expression of FAX1, FAX2 and ABCA2 was synergistically increased, FAX1/FAX2/ABCA2-OE exhibited lower Fv/Fm than the control lines. The relatively lower Fv/Fm can cause the higher lipid phenotype in FAX1/FAX2/ABCA2-OE. The effect of the FA distribution and acceleration of FA transport from the chloroplast to the ER may impact the photosynthesis metabolism and inhibit growth in Chlamydomonas. In the microalgae Synechococcus elongatus, PS II is more readily photodamaged when intracellular free FA overaccumulates [38]. Therefore, the synergetic over-expression of these FA transporters may affect the intracellular and extracellular free FA levels, which may reduce the PS II activity and retard cell growth. If so, the rescue of growth inhibition by the free FA exporter should also be considered in future studies. Although the cell growth rate of FAX1/FAX2/ABCA2-OE was adversely affected, the biomass productivity was increased (Fig. 6a, d). The higher biomass yields may be due to bigger cells and more lipid accumulation. The protein homologues of FAX also exist in other microalgae, such Chlorella variabilis, Chromochloris zofingiensis and Botryococcus braunii [17] (https://phytozome-next.jgi.doe.gov/). Therefore, the engineering strategy in this study may be applicable to other oleaginous microalgae to boost biofuel production.

Conclusion

In this study, FAX1/FAX2/ABCA2-OE were generated, which showed better traits for lipid accumulation than the parental line and previously reported individual FA transporter-OE. The co-expression of FAX1, FAX2 and ABCA2 enhanced the accumulation of TAG, total FA and membrane lipids under standard growth conditions. Moreover, we found that the increased expression of FAX1/FAX2/ABCA2 enhanced the lipid biosynthesis, and accelerated the PUFA remobilization from membrane lipids to TAG by fine-tuning the key genes in lipid metabolism. This study provided valuable insights into facilitating the production of oils in microalgae, which may promote the production of microalgal biofuels with cost efficiency in the future.

Methods

Strains and culture conditions

The Chlamydomonas UVM4 strain [39] provided by Prof. Ralph Bock (Max-Planck Institute for Molecular Plant Physiology, Germany) was used as the parental strain for over-expressor generation. ABCA2-OE was generated in our previous study [16]. Unless otherwise stated, cells were routinely cultivated mixotrophically at 25 °C in Tris–acetate phosphate (TAP) medium [40], with 120 rpm shaking and constant illumination at 100 µmol m−2 s−1 [41]. For nitrogen deprivation, exponentially grown cells in TAP medium were centrifuged at 500 g for 5 min, and cell pellets were washed twice with nitrogen-depleted media (TAP-N) before finally being re-suspended in TAP-N for starvation experiments. Cells concentration and growth were monitored with automated Algae Counter (Countstar BioMarine).

Construction of plasmids and transformation conditions

Genomic DNA of Chlamydomonas was isolated by the cetyltrimethyl ammonium bromide (CTAB) method as previously described [42]. The genomic DNA fragments coding for FAX1 (Cre10.g421750) and FAX2 (Cre08.g366000) were amplified using primers (FAX1-F and FAX1-R) and (FAX2-F and FAX2-R), respectively. The PCR reaction was carried out using the high fidelity KOD FX DNA Polymerase (Toyobo), according to the manufacturer’s instructions. The amplified DNA fragments of FAX1 and FAX2 were cloned as an EcoRI-XbaI fragment into the pChlamy_4 vector (Thermo Fisher) which contains the ble gene conferring zeocin resistance [41], generating pChlamy4-FAX1/FAX2 plasmid. The cDNA fragment of ABCA2 was amplified using pChlamy4-cABCA2 plasmid [16] as template with primers (ABCA2-F and ABCA2-R), and was digested and subcloned into the pOpt-Clover-Paro plasmid [43] as an EcoRI-NdeI fragment to generate the ABCA2 over-expression construct (pOpt-ABCA2-Paro). The sequences of all the primers used in this study are shown in Additional file 2: Table S1.

Before transformation, vectors were linearized with a single restriction enzyme SspI and purified using the MiniBEST DNA Fragment Purification Kit (Takara), according to the manufacturer’s protocols. Approximately 1.0 μg linearized plasmid DNA was used during each transformation experiment. The nuclear transformation was performed by electroporation as we previously described [41]. Briefly, Chlamydomonas cells were grown to approximately 1.5 × 106 cells/mL in TAP medium. The exponentially grown cells (2.5 × 107 cells) were harvested by centrifugation and suspended in 250 μL of TAP medium supplemented with 40 mM sucrose. The cell suspension was placed into a prechilled disposable electroporation cuvette with a 4-mm gap (Bio-Rad) for 5 min at 16 °C. Electroporation was performed by BioRad Gene Pulser Xcell with the following settings (voltage 500 V, Capacity 50 µF and Resistance 800 Ω). Electroporated cells were recovered with 5.0 mL of 40 mM sucrose in TAP medium for 16 h under the dim light (10 µmol m−2 s−1), and then plated on TAP agar plates containing single antibiotic (15 µg/mL zeocin) or dual antibiotics (15 µg/mL zeocin and 20 µg/mL paromomycin), and the plates were incubated under continuous light (50 µmol m−2 s−1) at 25 °C. Antibiotic-resistant colonies were visible after around 7 days.

PCR-based determination of gene-positive transformants

The stable integration of FAX1, FAX2 or ABCA2 in the transformants was determined by PCR analysis of cell lysates, as we previously described [44, 45]. In brief, exponentially grown cells (5.0 × 106) were resuspended in Tris–EDTA solution and incubated at 98 °C for 10 min. Aliquots (1.0 µL) of the supernatants from denatured cell lysates were then used as template for 20 µL PCR. For FAX1 and FAX2 gene positive screens, the primers (FAX1-F and FAX2-R) were used. For ABCA2 gene positive PCR screens, the primers ABCA2-Rev5/Rbcs2 Pro and ABCA2-Forw5/Strp-tag-R were used to N- and C-terminal of expression cassette, respectively. The sequences of all the primers used in this study are shown in Additional file 2: Table S1.

Nucleic acid extraction and expression level analysis

Total RNA was isolated as previously described [16, 41] using TRIzol reagent (Thermo Fisher), purified total RNA was treated with DNase I (Thermo Fisher) to remove the residual genomic DNA. For the first-strand cDNA was synthesized, 3.0 µg RNA was subjected to reverse transcription using PrimeScript 1st Strand cDNA Synthesis Kit (Takara), according to the manufacturer’s protocols. For RT-PCR, the PCR product of the housekeeping gene RACK1 (Cre06.g278222) was employed as a loading control using previously reported primers [46, 47]. To estimate gene expression levels, real-time PCR (qRT-PCR) was performed on Applied Biosystems 7500 using TB Green Premix Ex Taq (Takara). Relative gene expression was analysed by the 2−ΔΔCT method [48], using RACK1 as the reference gene. Primer sequences are listed in Additional file 1: Table S1.

Lipid extraction and analyses

Total lipids were extracted by modified Bligh and Dyer method with modifications as we previously described [31, 41]. Briefly, the cells cultivated under N-replete condition at logarithmic phase (60 million of cells) or N-depleted condition for 2 days (20 million of cells) were harvested, and 1.0 mL of 1.0 mM EDTA in 0.15 M acetic acid was added to resuspend the pellets. The mixture was vortexed for 10 min, after addition of 3.0 mL methanol:chloroform (2:1, v/v) mixture. Then, 1.0 mL of chloroform and 0.8 mL of 0.88% (w/v) KCl were added and vortexed, and then centrifuged at 3000 rpm for 5 min. The lower chloroform phase was recovered. Cells were then extracted again with hexane, and the supernatant was combined with the previous chloroform extracts. Extracted lipids were dried under a stream of nitrogen and then re-dissolved in chloroform for non-polar lipid or polar lipid analysis by thin-layer chromatography (TLC). The non-polar lipids (mainly TAG) were separated on TLC plates (Merck) by a solvent mixture of hexane-diethyl ether-acetic acid (85:15:1, v/v/v), and the polar lipids were separated on TLC plates using chloroform–methanol-acetic acid–water (25:4:0.7:0.3, v/v/v/v) as development solvent [11]. The lipids separated on the plates were visualized by spraying with 0.05% (m/v) primulin (Sigma-Aldrich) in acetone/water (80/20, v/v). The TAG or polar lipid containing silica was recovered, and transmethylated into fatty acid methyl esters (FAMEs). For total fatty acids analysis, the protocol of direct transmethylation of each lipid was used as previously described [49, 50]. Quantification of FAMEs was performed by gas chromatography with flame ionization detector (GC-FID) fitted with an Agilent DB-23 (60 m × 0.25 mm × 0.25 μm) column, the parameters of detections were previously described [51]. Analysis was carried out using Agilent Chemstation software, and glycerol triheptadecanoate (TAG 51:0, 17:0/17:0/17:0, Sigma-Aldrich) was used as an internal standard to determine fatty acid recovery for quantification. For each treatment, three biological replicates (independent cultures) with three technical replicates were performed.

Biomass determination

Biomass was determined by dry weight measurements as previously described [46, 52]. Briefly, the same concentration of cells (2.0 × 106 cells/mL) were inoculated and cultivated. Subsequently, 10.0 mL of culture medium containing cells at logarithmic phase was collected on the preweighed Whatman GF/C filters (47 mm diameter) and dried overnight at 80 °C in an incubator. The dried biomass was then gravimetrically measured.

Photosynthetic activity measurement

The maximal quantum conversion efficiency of PS II (Fv/Fm) of algal cells was measured by pulse amplitude-modulated (PAM) fluorometry (Water-PAM WALZ, Germany) as previously described [52, 53]. Briefly, the cells were dark-adapted for 10 min inside the spectrophotometer before the measurement of the maximal PS II quantum yield. Maximal PS II quantum yield was termed as Fv/Fm, where Fv represented the variation of chlorophyll fluorescence between maximal fluorescence (Fm) induced by saturating pulse and initial fluorescence (F0) (Fm−F0/Fm). F0 was recorded under a weak light (27 μmol m−2 s−1, peaking at 650 nm) and Fm was under a saturating pulse (0.8 s) of red light (4000 μmol m−2 s−1, peaking at 660 nm).

Statistical analysis

All the experiments in this study were performed at least three times. Cell samples were collected from three independent cultures (biological replicates) with three aliquots from the same culture (technical replicates) to obtain statistically data. The statistical significance of the differences was evaluated by Tukey’s honestly significant difference (HSD) test and Student’s t-test using SPSS 18.0.

Accession numbers

Sequence data of Chlamydomonas reinhardtii genes presented in this article can be found in Phytozome (https://phytozome-next.jgi.doe.gov/) with gene identifications as: FAX1 (Cre10.g421750), FAX2 (Cre08.g366000), ABCA2 (Cre14.g613950), PDAT1 (Cre02.g106400), PGD1 (Cre03.g193500), DGAT1 (Cre01.g045903), DGTT1 (Cre12.g557750), DGTT2 (Cre09.g386912), DGTT3 (Cre06.g299050), DGTT4 (Cre03.g205050), DGTT5 (Cre02.g079050) and RACK1 (Cre06.g278222).

Availability of data and materials

All data supporting the conclusions of this article are included within the article and in Additional files.

Abbreviations

ABC:

ATP-binding cassette

Chlamydomonas :

Chlamydomonas reinhardtii

DAG:

Diacylglycerol

FA:

Fatty acids

TAG:

Triacylglycerols

FAX:

Fatty acid exporters

PUFA:

Polyunsaturated fatty acids

ER:

Endoplasmic reticulum

DGAT:

Diacylglycerol acyltransferases

MGDG:

Monogalactosyldiacylglycerol

DGDG:

Digalactosyldiacylglycerol

DGTT:

Type II diacylglycerol acyltransferases

DGTS:

Diacylglyceryltrimethylhomoserine

PDAT1:

Phospholipid:diacylglycerol acyltransferase 1

PGD1:

Plastid galactoglycerolipid degradation 1

PS II:

Photosystem II

SQDG:

Sulfoquinovosyldiacylglycerol

PG:

Phosphatidylglycerol

PE:

Phosphatidylethanolamine

PI:

Phosphatidylinositol

RACK1:

Receptor of activated protein kinase C 1

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Acknowledgements

Thanks to Dr. Ralph Bock (Max-Planck institute of molecular plant physiology, Germany) for contributing UVM4 strain of Chlamydomonas reinhardtii. We also thank Dr. Xi Xie in Liaoning Ocean and Fisheries Science Research Institute for technical assistance.

Funding

This work was supported by the National Natural Science Foundation of China (31900221), Natural Science Foundation of Liaoning Province of China (2020-MS-102), and Fundamental Research Funds for the Central Universities (DUT21LK14).

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Contributions

FK designed and conceived the project. RC and FK made the constructs and generated the over-expression lines. FK and RC performed lipid analysis with ML and MY; FK carried out qRT-PCR with ML, HZ and HL. FK and RC carried out physiological analysis with SF, ML and XD; FK wrote the paper with comments from SX, CZ and ZC. All authors read and approved the final manuscript.

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Correspondence to Fantao Kong.

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The authors declare no competing interests.

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Supplementary Information

Additional file 1:

Fig. S1. PCR analysis of whole cell lysates from the FAX1/FAX2-OE and FAX1/FAX2/ABCA2-OE reveals that the transformants were stably transformed. (a) Colony PCR of FAX1/FAX2-OE using the primers (FAX1-F and FAX2-R) to amplify the full-length of DNA sequences of FAX1 and FAX2, and the predicted size of 2092 bp was detected. (b, c) Colony PCR of FAX1/FAX2/ABCA2-OE using the primers (ABCA2-Rev5/Rbcs2 Pro) and (ABCA2-Forw5/Strp-tag-R) to amplify the N- and C-terminal of the expression cassette, respectively. The predicted sizes of 484 bp and 381 bp were detected, respectively. M, DNA ladder. Fig. S2. Under nitrogen depleted condition (2 days), TAG content (a), total fatty acids content (b), and profiles of individual fatty acid content of total fatty acids (TFA) (c) in the transformants were analysed. The distinct letters labelled indicate the statistically significant difference by Tukey’s HSD test. Fig. S3. Total fatty acids content in the independent transformants of FAX1/FAX2/ABCA2 at exponential phase (day 2) under standard growth condition. The distinct letters labelled indicate the statistically significant difference by Tukey’s HSD test. Fig. S4. Alterations of membrane glycerolipid levels in the independent transformants of FAX1/FAX2/ABCA2 at exponential phase (day 2) under standard growth condition. (a) Total membrane lipid content. (b) Profile of individual membrane component contents. The total membrane lipid content referred to the total amount of major galactolipids (MGDG, DGDG and SQDG) and phospholipids (PE, PG and PI), and betaine lipid DGTS. MGDG, monogalactosyldiacylglycerol; DGDG, digalactosyldiacylglycerol; DGTS, diacylglycerol-N,N,N-trimethylhomoserine; SQDG, sulfoquinovosyldiacylglycerol; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PI, phosphatidylinositol. The distinct letters labelled indicate the statistically significant difference by Tukey’s HSD test. Fig. S5. Under standard growth condition at logarithmic phase, the profiles of individual fatty acids content of major thylakoid membrane MGDG (a), DGDG (b), and extraplastidic lipid DGTS (c) in the transformants were analysed. MGDG, monogalactosyldiacylglycerol; DGDG, digalactosyldiacylglycerol. The distinct letters labelled indicate the statistically significant difference by Tukey’s HSD test. Fig. S6. Analysis of the major contribution source for accumulation of TFA, TAG and membrane lipids in FAX1/FAX2/ABCA2-OE in relative to UVM4 under standard growth condition at logarithmic phase. (a) Contribution ratios of TAG and polar lipids (PL) to the accumulation of total fatty acids (TFA) in FAX1/FAX2/ABCA2-OE relative to UVM4, respectively. The contribution (%) is calculated as Con(%) = (cTAGOE –cTAGUVM4)/(cTFAOE-cTFAUVM4) ×100. cTAG means the content of TAG, and cTFA means the content of total fatty acids (b) Contribution ratios of MGDG, DGDG and DGTS to PL accumulation in FAX1/FAX2/ABCA2-OE relative to UVM4, respectively. The contribution (%) is calculated as Con(%) = (iPLOE – iPLUVM4)/(tPLOE-tPLUVM4) ×100. iPL means the content of individual polar lipids (MGDG, DGDG and DGTS), respectively, and tPL means the content of total polar lipids (c) Polyunsaturated FA levels in TFA, TAG, MGDG, DGDG, and DGTS in FAX1/FAX2/ABCA2-OE and UVM4, respectively. The polyunsaturated FA level (%) is calculated as (%) = cPUFA/cTFA×100. cPUFA means the content of total polyunsaturated FA, and cTFA total FA content. (d) Contribution ratios of the major PUFAs to TFA in FAX1/FAX2/ABCA2-OE relative to UVM4. The contribution (%) is calculated as Con(%) = (iPUFAOE –iPUFAUVM4)/(cTFAOE-cTFAUVM4) ×100. iPUFA mean the content of 16:4 (4,7,10,13), 18:2 (9,12), 18:3 (9,12,15), and 18:3 (5,9,12), respectively. cTFA means total FA content. (e) Contribution ratios of the major PUFA for MGDG, DGDG and DGTS accumulation in FAX1/FAX2/ABCA2-OE relative to UVM4, respectively. The contribution (%) is calculated as Con(%) = (iPUFAOE–iPUFAUVM4)/(iPLOE-iPLUVM4) ×100. iPUFA are annotated as above. iPL mean the content of MGDG, DGDG and DGTS, respectively. (f) Contribution ratios of the major PUFA to TAG in FAX1/FAX2/ABCA2-OE relative to UVM4. The contribution (%) is calculated as Con(%) = (mPUFAOE –mPUFAUVM4)/(cTAGOE-cTAGUVM4) ×100. mPUFA mean the content of 16:4 (4,7,10,13), 18:3 (9,12,15), and 18:3 (5,9,12), respectively. cTAG mean TAG contents. Asterisks indicate statistically significant changes compared to the parental line UVM4 by paired-sample Student’s t test (*P ≤ 0.05; **P ≤ 0.01).

Additional file 2:

Table S1. All primer sequences used in this study.

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Chen, R., Yang, M., Li, M. et al. Enhanced accumulation of oil through co-expression of fatty acid and ABC transporters in Chlamydomonas under standard growth conditions. Biotechnol Biofuels 15, 54 (2022). https://doi.org/10.1186/s13068-022-02154-6

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Keywords

  • Chlamydomonas reinhardtii
  • Genetic engineering
  • Fatty acid exporters
  • ABC transporter
  • Triacylglycerol
  • Polyunsaturated fatty acids