Cloning of algal FA synthesis genes
Using degenerated primers (Table ) and normal RT-PCR, five genes were successfully cloned, verified and submitted to NCBI GenBank. The nucleotide sequences of cDNAs of 3-keto acyl-acyl carrier protein synthase gene (KAS), acyl-acyl carrier protein thioesterase (FATA), ω-3 fatty acid desaturase (FAD), ACP and malonyl-CoA:ACP transacylase (MCTK) have been deposited in the GenBank database under the accession numbers, HM560033, HM560034, HM560035, HM560036, and HM560037, respectively. Together with other two known FA synthesis genes, biotin carboxylase (BC) and stearoyl-ACP-desaturase (SAD), total seven genes were investigated in this study (Figure ).
Primers for real time RT-PCR in this study
Expression of FA synthesis related genes
Haematocccus cells grown under different stress conditions and were harvested for RNA isolation. cDNA synthesis and gene expression of FA synthesis related genes were explored using quantitative real-time RT-PCR. The results showed that the mRNA levels of most selected genes were significantly or very significantly up-regulated under all stress conditions, with all genes were up-regulated under Fe + AC and HT conditions, and some genes, such as FATA, SAD and FAD, were more sensitive to treatments (Figure ).
Figure 2 Gene expression detected by real time RT-PCR in control and stress treatment conditions. BC: Biotin carboxylase; ACP: Acyl carrier protein; MCTK: Malonyl-CoA:ACP transacylase; KAS: 3-ketoacyl- ACP synthase; FATA: Acyl-ACP thioesterase; SAD: Stearoyl-ACP-desaturase; (more ...)
BC, as a subunit of acetyl coenzyme A carboxylase biotin carboxylase, involves in catalyzing acetyl-CoA to malonyl-CoA, the first step of de novo FA synthesis (Figure ). It is intriguing that different conditions had various impacts on BC mRNA level (Figure ). For instance, no change of BC mRNA was observed under AC treatment, while 1,626 fold of up-regulation was detected under HT. The change was 4.8 fold under Fe treatment, however, the combined salinity treatment (Fe + AC) resulted in about 10 fold up-regulation, which was almost 2 fold of up-regulation under Fe. Under LT, BC mRNA level was increased up to 4.8 fold, which was far lower than that of HT (1,626), indicating that FA biosynthesis in H. pluvialis is more sensitive to high temperature than low temperature. Depletion of nitrogen (NL) also induced BC expression level at about 1.3 fold. Student's t test analysis showed that NL significantly (p <0.05), Fe, Fe + AC, LT, and HT treatments very significantly (p <0.01) increased BC gene expression, while AC alone treatment had no significant effect. Multiple comparison analysis indicated that the gene expression of BC induced by Fe, AC, and Fe + AC were significantly different between the single and combined salinity treatment (data not shown).
ACP is an important component in both FA and polyketide biosynthesis with the growing chain bound during synthesis as a thiol ester at the distal thiol of a 4'-phosphopantethiene moiety (Figure ). Similar to BC gene, ACP mRNA was shown to react differently under different conditions (Figure ). The most significant up-regulation of ACP gene expression was observed under HT with a 8.7 fold rise, while LT induced only 2.6 fold. Fe + AC salinity treatment induced 9 fold of ACP expression, while separately salinity treatment increased less, at 1 and 0.4 fold under Fe and AC, respectively. Obviously, the combined salinity condition (Fe + AC) had higher impacts on ACP gene expression than separately treatments (Fe, or AC), implying a synergistic effect of Fe and AC on FA synthesis in H. pluvialis. LT treatment induced ACP gene expression at 3.6 fold, only 37.6% for the effect of HT treatment, suggesting that ACP gene may also be less sensitive to LT. The other conditions did not induce ACP gene expression significantly.
The initiation of the FA elongation step, which extends the length of the growing acyl chain by two carbons, requires MCTK to transfer malonyl moiety from malonyl-CoA onto the acyl carrier protein (Figure ). Very high induction of MCTK gene expression (2,261 fold) was observed under HT and LT caused about 1 fold up-regulation, while NL and AC conditions did not cause significant change to its expression (Figure ). Fe treatment enhanced MCTK gene expression about 2 fold, and combined salinity Fe + AC treatment induced about 5.4 fold. Multiple comparison analysis indicated that there was significantly different between the single (Fe or AC alone) and combined salinity treatment (Fe + AC) (data not shown).
KAS catalyzes the initial condensing reaction in FA biosynthesis (Figure ). Consistent with BC, ACP, and MCTK, KAS gene expression was promoted by Fe + AC for about 3.5 fold, while Fe and AC separated treatments induced its gene expression as 81% and 42% of that of control, respectively (Figure ). Under NL treatment, 1.7 fold of KAS gene expression increase was also observed. HT and LT treatments up-regulated KAS mRNA levels at about 1.1 fold and 20% only, respectively.
FATA is the chain-length-determining enzyme in de novo biosynthesis of plant FAs (Figure ). FATA mRNA levels were up-regulated significantly or very significantly under all treatments in this study, with 2.0 (NL), 2.9 (Fe + AC combined), 3 (LT), 9.9 (Fe), 13.8 (HT), and 18.8 fold changes (AC), respectively (Figure ). Interestingly, different from BC, ACP, MCTK, and KAS, the fold change of FATA gene expression under Fe + AC combined treatment was less than that under Fe or AC treatment alone. This indicated that for FATA, Fe + AC combined treatment had no synergistic but antagonistic effect.
SAD functions to position a single double bond into an acyl-ACP substrate and is best represented by the ubiquitous Δ9 18:0-ACP desaturase (Figure ). Similar to FATA, SAD gene expression was significantly or very significantly induced by all treatments (Figure ). In details, HT induced SAD mRNA levels about 625.0 fold, while LT induced only for 2.5 fold as only 1/180 of that under HT. Fe or AC treatment alone enhanced SAD gene expression 2.4 and 1.4 fold, respectively, compared with 8.6 fold of up-regulation under combined treatment Fe + AC. NL also increased SAD mRNA level at about 2.4 fold.
The enzyme of FAD converts linoleic to alpha-linolenic acid (C18:3n3) (Figure ). It seemed that FAD was the most sensitive among all FA biosynthesis genes selected in this study, since each treatment highly increased FAD gene expression in our study (Figure ). HT treatment still had the highest enhance (5,947 fold), and NL had the lowest impact with 5.0 fold of up-regulation. Fe up regulated SAD gene for 35.5 fold and AC enhanced 17.9 fold of FAD mRNA level. Interestingly, Fe + AC combined treatment only caused 12.8 fold increase (p < 0.01), as only 36% and 71.5% of those under Fe or AC separately treatment, respectively. LT treatment increased FAD mRNA levels at 8.90 fold.
FA content and composition
With internal standard, our FA extraction efficiency was 87.5% (data now shown). The FA profiles under treatments were listed on Table . We detected 24 individual FAs in H. pluvialis under different treatments. The overall FA profile in H. pluvialis was similar under control and stress conditions, and palmitic, stearic, oleic, linoleic acids were the major components (Table ), among which linoleic acid (C18:2n6) had the highest content under most conditions in H. pluvialis. Under NL, AC and HT, the total FA (TFA) content was, 77.2, 71.39, and 72.04 mg g-1, respectively, which were considerably higher than that observed under control conditions (58.4) (Table ). Moreover, no significant differences were found under the other stress treatments. The percentage of saturated fatty acids (SFA) was significantly higher in cultures grown under NL (23.99%) and AC (31.06%) conditions compared to the control (20.2%).
Fatty acid profile (mg/g dry weight) in control (C) and stress conditions (NL, Fe, AC, Fe + AC, LT and HT)
The total monounsaturated fatty acid (MUFA) content showed no significant differences between control and stress conditions, except that contents of most MUFA were significantly or very significantly increased under NL (C15:1, C16:1, C20:1, and C22:1) and HT (C16:1, C18:1, C20:1, C22:1 and C24:1). Similarly, percentage of MUFA in total FA indicated no significant differences between control and stress conditions, except for a lower percentage in algae grown under AC condition (Table ). Regarding polyunsaturated fatty acids (PUFA), there were significant alternation (increase or reduction) under stress conditions compared to the control (Table ). For example, most PUFAs were significantly or very significantly increased under NL condition. This increase may be attributed to higher proportions of linoleic (C18:2n6), eicosapentaenoic acid (EPA, C20:5n3) and docosahexanoic acid (DHA, C22:6n3). Similar increase of EPA was observed under Fe + AC, LT and HT conditions.
Fatty acid methyl esters (FAME) quality for biodiesel
FAs are precursors for biodiesel production. According to recent studies [22
], the FA profile of microalgae has a significant influence on the fuel properties of biodiesel, such as cetane number (CN), iodine number (IN) and saponification number (SN). Our analysis indicated that conditions selected in this study had no significant impact on SN, IN and CN (Figure ).
The biodiesel quality of Haematococcus. a-c, SN, IN and CN values under different treatments, respectively. Error bars represent standard error (n = 4).
As shown in Figure , the SN changes were slight under different conditions with a range of 201.9-205.7 (Figure ), while little reduction of IN was observed under Fe, AC, Fe + AC and HT, and little increase under NL and LT around 120.5-121.5 (Figure ). The CN of the FAME under different conditions were calculated and compared (Figure ). The CN values under different conditions were relatively high, ranging from 45.9 to 51.6. However, little difference of CN values was observed under different conditions, with a minimum CN values under NL condition. Based on SN and IN analysis, the increase of MUFA and PUFA was the key to the reduction of CN under NL condition.
Correlations between gene expression and FA profile
We determined both expression of key FA biosynthesis genes and FA profile under different treatments. To study the relationship between gene expression and FA profile, Pearson Correlation analysis (SPSS13.0) was carried out and specific results were summarized in Table .
The correlations between gene expression and fatty acid synthesis (cofactors, Pearson Correlation in SPSS13
Based on the summary on Table , the correlations between different FAs and gene expression were different. ACP, KAS, and FATA shared close correlations with FAs, while the other did not. For instance, C12:0 had significant positive correlations with all selected genes (Table ). ACP gene expression shared negative correlations with C15:0, C17:1, C18:3n6, C18:2n6, C20:4n6, C20:5n3, and positive correlation with C18:1n9, C20:2, C20:1, C22:1n9, and C24:1. KAS gene expression had negative correlations with C15:0, C18:3n6 and C20:4n6, while it shared positive correlations with C18:3n3, C18:0, C20:2, C20:1, C22:1n9, and C24:1. FATA gene expression was observed negatively correlated with synthesis of C17:1, C18:3n6, C20:4n6 and C22:6n3, while it positively correlated with synthesis of C18:3n3, C18:0, C20:1, C20:0, C22:1n9 and C24:1. The correlations between other genes and FA synthesis were found not significant.