Short Chain Fatty Acid Biosynthesis in Microalgae Synechococcus sp. PCC 7942

Short chain fatty acids (SCFAs) are valued as a functional material in cosmetics. Cyanobacteria can accumulate SCFAs under some conditions, the related mechanism is unclear. Two potential genes Synpcc7942_0537 (fabB/F) and Synpcc7942_1455 (fabH) in Synechococcus sp. PCC 7942 have homology with fabB/F and fabH encoding β-ketoacyl ACP synthases (I/II/III) in plants. Therefore, effects of culture time and cerulenin on SCFAs accumulation, expression levels and functions of these two potential genes were studied. The results showed Synechococcus sp. PCC 7942 accumulated high SCFAs (C12 + C14) in early growth stage (day 4) and at 7.5g/L cerulenin concentration, reaching to 2.44% and 2.84% of the total fatty acids respectively, where fabB/F expression was down-regulated. Fatty acid composition analysis showed C14 increased by 65.19% and 130% respectively, when fabB/F and fabH were antisense expressed. C14 increased by 10.79% (fab(B/F)−) and 6.47% (fabH−) under mutation conditions, while C8 increased by six times in fab(B/F)− mutant strain. These results suggested fabB/F is involved in fatty acid elongation (C <18) and the elongation of cis-16:1 to cis-18:1 fatty acid in Synechococcus sp. PCC 7942, while fabH was involved in elongation of fatty acid synthesis, which were further confirmed in complementary experiments of E. coli. The research could provide the scientific basis for the breeding of SCFA-rich microalgae species.


Introduction
Short chain fatty acids (SCFAs) are usually defined as the carbon number of fatty acids between 6 and 14 [1]. Due to corresponding glycerides with extraordinary characteristics such as low viscosity, high extensibility, low freezing point, low surface tension, high transparency and oxidation stability, SCFAs have very broad application in cosmeceuticals, nutraceuticals, nutritional supplements, chemical industry, etc. [2]. Currently, main sources of SCFAs are tropical plants, such as coconut and palm trees [3,4]. However, people have to turn their attention to the potential oleaginous microorganisms because of the high cost of vegetable oils and the limitation of climate and land resources [5]. It is widely reported that bacteria, mold, yeast and microalgae are important oleaginous microorganisms [5], of which microalgae has received much attention for its unique advantages such as fast growth rate, high oil content, high photosynthetic efficiency, low land requirements and environmental protection [6].
There are abundant algae in the ocean, the unique ecological environment of which causes the special bioactive metabolites accumulation in the algae. These metabolites have a series of biological effects, such as moisturizing [7], bacteriostasis [8], anti-inflammatory [9], antivirus [10], inhibiting the growth of tumor cells [11], and resisting ultraviolet radiation [12]. It was found that a kind of ancient

Fatty Acid Compositions of Synechococcus sp. PCC7942 under Different Conditions
Microalgae synthesize and store different types of lipids in a single cell [32]. In contrast to the conventional fatty acids (FAs) composition of microalgae that contain long chain fatty acids (LCFAs) from C16 to C18, SCFAs (from C8 to C14) are generally classified to be unusual. Generally, the content of SCFAs in microalgae is relatively low under normal growth conditions. In order to better understand the synthesis mechanism of SCFAs in microalgae from a physiological perspective preliminarily, the fatty acid profiles of Synechococcus sp. PCC 7942 under different culture time periods was investigated. Table 1 shows the dynamic variations of fatty acids during the whole culture process. As shown in Table 1, the main fatty acid was C16 under different culture time periods, accounting for about 70%-80% of the total fatty acids, while little contents of LCFAs from C20 to C22 were detected. At the early growth stage (2 days-6 days), high content of SCFAs (C14 + C12) were observed, reaching highest content (2.44%) at day 4, among which C14:1 was 1.70% and C14:0 was 0.74% (Table 1). After day 8, contents of C14:0 and C14:1 did not change significantly, which were in the range of 0.3%-0.6% and 0.5%-0.9%, respectively, while C12 fatty acid could not be detected. These results suggested that the accumulation of SCFAs mainly occurred in the early growth stage (2 days-6 days), with C14 and C12 as the main composition. To our knowledge, this was the first report of SCFAs accumulation in the early growth stage in cyanobacteria. The fatty acid synthesis system of plants and bacteria belonged to the type II, where the KASs were related to the fatty acid carbon chain elongation and generally divided into KAS I (FabB), KAS II (FabF) and KAS III (FabH) [25,26]. It was reported that fatty acid synthase inhibitors have effects on the fatty acids carbon chain elongation in bacteria [34,35], among which cerulenin mainly played its role on KAS I and KAS II. In order to further understand the SCFAs synthesis in Synechococcus sp. PCC 7942, effects of different cerulenin concentrations on SCFAs accumulation were studied.
As shown in Table 2, the content of SCFAs (C12 + C14) accumulated as high as 2.84% of the total fatty acids under cerulenin concentration of 7.5 g/L, among which the content of C14 fatty acids increased from 1.58% (0 g/L) to 2.75% (7.5 g/L), increasing by 74.05% (p < 0.05), and C12 fatty acids increased from 0.04% to 0.09%, increasing by 125% (p < 0.05). At this time, C16 fatty acids decreased from 63.49% to 57.35%, decreasing by 9.67%, C18 fatty acids decreased from 18.44% to 13.7%, decreasing by 25.7%, respectively. Israel et al. [36] found that after 40 min of adding cerulenin into the medium with E. coli, 90% of oil synthesis and 25% of RNA and DNA synthesis were inhibited, but the protein synthesis was not affected. In addition, a previous study [37,38] on antibacterial drug targets showed that cerulenin has specific inhibition on KAS I and KAS II, which are responsible for the carbon chain elongation of C4-C16 fatty acids in bacteria. As shown in Table 2, the contents of longer chain fatty acids (C18 + C16) decreased and the contents of SCFAs (C12 + C14) increased, with the increase of cerulenin concentration. These results suggested that KAS I/II in Synechococcus sp. PCC 7942 might be inhibited, which resulted in the increase of SCFAs (C12 + C14). Based on the above results, it is assumed that the low expression levels of the related genes fabB/F (KAS I/II) in Synechococcus sp. PCC 7942 might inhibit the carbon chain elongation of fatty acids, leading to the sufficient accumulation of SCFAs at this stage. Therefore, the related genes expression levels under the above conditions were further investigated.

Genes Expression under Different Conditions in Synechococcus sp. PCC7942
Bioinformatic analysis showed that Synpcc7942_0537 and Synpcc7942_1455 in Synechococcus sp. PCC 7942 might have homology with fabB/F and fabH of plants and bacteria.
As mentioned above, the influences of culture time period and cerulenin concentration on the fatty acid compositions of microalgae were significant. In order to understand the relationship between genes expression level and fatty acids (FAs) carbon chain elongation, the influences of cerulenin concentration and culture time on transcriptional levels of the relative genes (fabB/F and fabH) were explored.
As shown in Figure 1a, the expression level of fabB/F was relatively low in the early growth stage (2 days-6 days), and reached the highest expression level at day 8, then decreased in the later period of culture time (10 days-16 days). The overall trend of expression level of fabB/F increased first and then decreased, which was consistent with the variations of fatty acid composition (Table 1), that is to say, the SCFAs mainly accumulated in early growth stage. The result indicated that fabB/F played an important role on the synthesis of SCFAs. On the other side, during the whole cultivation phase, the expression level of fabH was not high enough, and showed a trend of gradual decrease, only with relatively high expression level in the early growth stage (2 days-6 days) (Figure 1b). Inhibition of the expression of fabB/F will hinder the transformation of SCFAs (C12, C14) to relatively longer chain fatty acids (C16, C18), leading to SCFAs accumulation, which also verified the previous hypothesis that fabB/F is a key gene promoting the synthesis of SCFAs (C12, C14) to longer chain fatty acids (C16, C18). As shown in Figure 1c, under cerulenin concentration of 7.5 g/L, the expression level of fabB/F was relatively high at the early growth stage (2 days-8 days), then decreased significantly with the extension of culture time and reached the lowest expression level in the late growth stage (12 days-16 days). These results suggested that cerulenin could change the expression levels of fabB/F in Synechococcus sp. PCC 7942. Ter Beek et al. [39] also found that cerulenin could change the expression levels of fab (fatty acid biosynthesis) genes in fatty acid synthesis of Bacillus subtilis. The dynamic trend of fabB/F expression at 7.5 g/L of cerulenin was consistent with variation of SCFAs composition, compared with that in the absence of cerulenin or at low concentration of cerulenin ( Table 2). The expression level of fabH gene did not change significantly during the whole cultivation period under 7.5 g/L cerulenin concentration (Figure 1d), which indicated that cerulenin also has specific inhibition on KAS I and KAS II in Synechococcus sp. PCC 7942. However, high cerulenin concentration could cause the inhibition of the growth of Synechococcus sp. PCC 7942. When the cerulenin concentrations were between 0 g/L and 7.5 g/L, the OD730 value of the same culture time period decreased with the increase of the concentration of cerulenin. At 7.5 g/L cerulenin concentration, the OD730 value was the lowest, which was about one third of that at 0 g/L ( Figure S1, Supplementary Materials). Therefore, although there was no significant change during the whole cultivation period, the fabH expression level at 7.5 g/L cerulenin was much lower ( Figure 1d) compared with that in the absence of cerulenin ( Figure 1b). This is probably caused by the growth inhibition at 7.5 g/L cerulenin concentration ( Figure S1). So, the above dynamic expressions of two related genes in Synechococcus sp. PCC 7942 may be the result of both growth and gene inhibition.

Fatty Acid Compositions in fabB/F and fabH Sense/Antisense Expression Strains
It is reported that deletion of some important enzymes in the fatty acid synthesis pathway may lead to cell death [40]. Therefore, sense and antisense expression strains of fabB/F and fabH were constructed first to investigate the functions of the two genes in the process of fatty acids synthesis. Chen et al. [41] reported that inhibiting the expression of phosphoenol pyruvate carboxylase (PEPC) gene in rapeseed by antisense RNA led the oil content of transgenic rapeseed to 6.4%-18%, which was higher than that of the control group. Song et al. [42] successfully antisense expressed the As shown in Figure 1c, under cerulenin concentration of 7.5 g/L, the expression level of fabB/F was relatively high at the early growth stage (2 days-8 days), then decreased significantly with the extension of culture time and reached the lowest expression level in the late growth stage (12 days-16 days). These results suggested that cerulenin could change the expression levels of fabB/F in Synechococcus sp. PCC 7942. Ter Beek et al. [39] also found that cerulenin could change the expression levels of fab (fatty acid biosynthesis) genes in fatty acid synthesis of Bacillus subtilis. The dynamic trend of fabB/F expression at 7.5 g/L of cerulenin was consistent with variation of SCFAs composition, compared with that in the absence of cerulenin or at low concentration of cerulenin ( Table 2). The expression level of fabH gene did not change significantly during the whole cultivation period under 7.5 g/L cerulenin concentration (Figure 1d), which indicated that cerulenin also has specific inhibition on KAS I and KAS II in Synechococcus sp. PCC 7942. However, high cerulenin concentration could cause the inhibition of the growth of Synechococcus sp. PCC 7942. When the cerulenin concentrations were between 0 g/L and 7.5 g/L, the OD 730 value of the same culture time period decreased with the increase of the concentration of cerulenin. At 7.5 g/L cerulenin concentration, the OD 730 value was the lowest, which was about one third of that at 0 g/L ( Figure S1, Supplementary Materials). Therefore, although there was no significant change during the whole cultivation period, the fabH expression level at 7.5 g/L cerulenin was much lower (Figure 1d) compared with that in the absence of cerulenin (Figure 1b). This is probably caused by the growth inhibition at 7.5 g/L cerulenin concentration ( Figure S1). So, the above dynamic expressions of two related genes in Synechococcus sp. PCC 7942 may be the result of both growth and gene inhibition.

Fatty Acid Compositions in fabB/F and fabH Sense/Antisense Expression Strains
It is reported that deletion of some important enzymes in the fatty acid synthesis pathway may lead to cell death [40]. Therefore, sense and antisense expression strains of fabB/F and fabH were constructed first to investigate the functions of the two genes in the process of fatty acids synthesis. Chen et al. [41] reported that inhibiting the expression of phosphoenol pyruvate carboxylase (PEPC) gene in rapeseed by antisense RNA led the oil content of transgenic rapeseed to 6.4%-18%, which was higher than that of the control group. Song et al. [42] successfully antisense expressed the encoding gene (pcc) of PEPC in Synechococcus sp. PCC 7002, resulting in an oil content increase. It can be seen that antisense expression is very feasible in improving the oil content of microalgae.
As shown in Table 3, the SCFAs contents were not significantly changed in Synechococcus sp. PCC 7942 harboring sense plasmids pRL-Sense-fabB/F (Synpccw7942_0537) and pRL-Sense-fabH (Synpccw7942_1455) (The sense and antisense plasmids were all derived from pRL 489). Research in plants showed fabB/F was responsible for the longer chain fatty acids elongation, while fabH was related to the initiation of fatty acids synthesis and extending C2 to C4 [27]. The present study suggested that acyl-ACPs for longer chain fatty acids synthesis may enter the next step of fatty acid synthesis, which could not result in a massive accumulation of SCFAs (Table 3). It was also found that overexpression of KAS IV in a MCFA-producing strain of Dunaliella tertiolecta could allow MCFA accumulation [43], which lies in the control of a KAS enzyme to generate MCFA acyl-ACP substrates. We believed that enlargement of the MCFA acyl-ACP substrate pool by overexpressing KAS IV and concentration of MCFA acyl-ACP substrate pool caused by KAS enzyme all could lead to MCFAs accumulation [24]. Although the overexpression of KAS III gene in E. coli could change the fatty acid profile of E. coli, when C14:0 increased and C18:1 decreased [26], which were different from our results of Synechococcus sp. PCC 7942 with pRL-Sense-fabH (Table 3), indicating that the Synpccw7942_1455 (KAS III) gene had species difference. Furthermore, eukaryotic microalgae (Dunaliella tertiolecta) and bacteria/cyanobacteria are innately different, given that eukaryotic microalgae are capable to store free FAs in the form of SCFAs. The content of C18 of Synechococcus sp. PCC 7942 containing pRL-Antisense-fabB/F decreased from 18.44% of wild-type to 2.15% of antisense expression strain (Table 3), with a decrease of 88.34%. Meanwhile, the content of C14 in Synechococcus sp. PCC7942 containing pRL-Anti-fabB/F increased from 1.58% of the wild type to 2.61% of antisense expression strain (Table 3), with an increase of 65.19% (p < 0.05). This is because that the antisense gene fragment could inhibit the expression level of fabB/F. The expression levels of fabB/F in antisense expression of fabB/F strains were down-regulated (p < 0.05) as compared with that in the wild type strain during the whole growth period ( Figure S2, Supplementary Materials). In addition, as shown in Table 3, C16:2 in fabB/F antisense expression strain decreased from 0.27% to 0.13%, C18:2 decreased from 0.81% to 0.18%, C17 (17:0 and 17:1) fatty acids decreased from 1.3% to 0.87%. By referring to the studies of other microorganisms [3], fabB/F might be related to the extension of longer chain fatty acids. Further analysis showed that C16:1 of the antisense expression strain increased from 31.75% to 37.20% and C18:1 decreased from 15.84% to 1.25% (Table 3), which was consistent with the function of fabF for extending cis-16:1 into cis-18:1 mentioned in a previous report [44]. In E. coli, both KAS I (fabB) and KAS II (fabF) have the ability for longer chain elongation of saturated fatty acids. KAS I is one of the key enzymes involved in the de novo synthesis of unsaturated fatty acids, while KAS II only extends the cis-hexadecenoyl to cis-octadecanoyl and does not participate in de novo synthesis of unsaturated acyl [45]. Combined with the studies above, we speculated that fabB/F was involved in the synthesis of unsaturated fatty acids. Leonard et al. [46] have found that the gene with KAS II activity in Cuphea wrightii could regulate FA chain length regulation, and was co-expressed with specific thioesterases, leading the content of C10:0 and C12:0 accumulation. Besides, Pidkowich et al. [47] proved that KAS II had the function of extention 16:0-ACP to 18:0-ACP in plant. Our results suggested that Synpccw7942_0537 (fabB/F) also performed the function of fabF and fabB in microalgae.
The antisense expression fabH also caused the change of fatty acid composition. As Table 3 shows, the content of C18 of Synechococcus sp. PCC 7942 containing pRL-Antisese-fabH decreased from 18.44% of the wild-type strain to 5.47% of antisense expression strains, with a decrease of 70.34%. At the same time, the content of C14 increased from 1.58% of the wild type to 3.63% of the antisense expression strain (Table 3), with an increase of 130% (p < 0.05). In addition, C16:2, C17:0, C17:1 and C18:2 content of fabH antisense expression strain all decreased, when C16:2 decreased from 0.27% to 0.07%, C18:2 decreased from 0.81% to 0.13%, C17 (17:0 and 17:1) fatty acids decreased from 1.3% to 0.63%. It is generally believed that fabH was related to the initiation of fatty acid elongation and a rate-limiting step of fatty acid synthesis [30]. González-mellado et al. [48] found that KAS III in sunflower had a new substrate specificity, which could participate in the synthesis of C6 to C10 fatty acids. Our present results showed that the C18 fatty acid decreased with the increase of C14 fatty acid when the gene fabH was inhibited, suggesting that the Synpccw7942_1455 (fabH) in Synechococcus sp. PCC 7942 may only participate in the carbon chain extension (C < 18) (not the first condensation step in the fatty acid synthesis circle) and had acyl-ACP carbon chain substrate specificity. While antisense expression fabB/F and fabH are able to redirect FA synthesis from long to short chain lengths, the increases in yield are still not as high as those achieved in plants and some bacteria. It is thought that high content of SCFAs could be poisonous for microalgae growth. Therefore, SCFAs accumulation is not only governed by KAS, but also by the tolerance of SCFAs concentration in microalgae cells.

Fatty Acid Compositions of fabB/F and fabH Deletion Mutants
The functions of Synpccw7942_0537 (fabB/F) and Synpccw7942_1455 (fabH) were further explored by gene deletion mutants. Table 4 showed that C16 was the main fatty acid component in the fab(B/F) − mutant strain, accounting for about 60%-70% of the total fatty acid. The content of C8 increased by six times and C14 increased by 10.79%, while the content of C18 decreased by 9.87% (Table 4). In addition, the content of C15:0 fatty acid in the fab(B/F) − mutant strain was 0.48% of the total fatty acids, which was not detected in the wild type (Table 4). These results suggested that the mutation of fabB/F gene might block fatty acid elongation. It was reported that the homologous protein of FabB could not found in some bacteria, but the homologous protein of FabF (FabY) had the dual functions of KAS I and KAS II, but this is not common [40]. The above results suggested that fabB/F in Synechococcus sp. PCC 7942 might have the function of fatty acid carbon chain elongation (C < 18), which were consistent with the functions of fabB and fabF in E. coli, where they carried out the elongation steps in fatty acid synthesis. The fabB/F in Synechococcus sp. PCC 7942 was also involved in the de novo synthesis of the unsaturated fatty acids of the microalgae and forming cis-11-octadecenoyl ACP by extending palmitoyl-ACP.  Table 4 also showed that the content of C18 decreased from 18.23% in the wild type to 16.91% in fabH − mutant, with a decrease of 7.24%. Meanwhile, the content of C14 increased from 1.39% of the wild type to 1.48% of the mutant, with an increase of 6.47%, and 0.41% content of C15 was obtained (Table 4). These results indicated that the longer chain fatty acids decreased and shorter chain fatty acids increased when fabH was mutated. Previous studies showed that fabH was a rate-limiting step and also the first condensation step in the fatty acid synthesis circle in plants and bacteria [29,49]. The present results suggested that Synpccw7942_1455 (fabH) might only participate in the fatty acid carbon chain elongation (C < 18).
Nonetheless, the increment of SCFAs in mutants we observed was modest, far from our expectation. It was also reported that some enzyme had the activity of KAS III, with the domain similar to FabH, but whose molecular weight is larger than FabH, initializing fatty acid synthesis in Pseudomonas aeruginosa [46]. Therefore, it is assumed that fab(B/F) − and fabH − mutants were considered as strains deficient in fabB/F and fabH gene expression but not complete inactivation, or other proteins with similar activity executed corresponding functions (KAS III).

Complementation of E. coli
In order to further verify the functions of the related genes, we attempted to construct mutant strains of KAS I/II/III of E. coli and compared the fatty acid compositions of mutants with that of their complementation.
As shown in Table 5, the content of C12, C14 and C16 in fab(B/F/H) − mutants of E. coli BL21 all increased as compared with that of the wild type. These results were consistent with the results that KAS III could catalyze acetyl-CoA with malonyl-CoA to generate 4:0-ACP, KAS I could catalyze 4:0-ACP to generate 16:0-ACP and KAS II could catalyze 16:0-ACP to generate 18:0-ACP as well as regulate the ratio of 16:0-ACP/18:0-ACP [24]. Mutation of the genes might lead to changes in fatty acids. Synpccw7942_0537 (fabB/F) and Synpccw7942_1455 (fabH) from Synechococcus sp. PCC 7942 were used for E. coli mutants (fabB/F/H) − complementation. The results show that fatty acid compositions of mutants were basically restored to that of the wild type after complementation (Table 5). These suggested that the Synpccw7942_0537 (fabB/F) gene in Synechococcus sp. PCC 7942 has similar functions of fabB and fabF of E. coli and was related to the elongation of fatty acids. Synpccw7942_1455 (fabH) has similar function of fabH, which is also related to the elongation of fatty acid in Synechococcus sp. PCC 7942.

Fluorescence Localization of the Related Proteins
In order to describe the SCFAs synthesis pathway in microalgae, cellular localization of Synpccw7942_0537 (fabB/F) and Synpccw7942_1455 (fabH) by co-expressing green fluorescence protein gene and potential genes were carried out. Figure 2 shows that the fusion expressed proteins of eGFP-FabB/F and eGFP-FabH in Synechococcus sp. PCC 7942 were evenly dispersed on the cell membrane, indicating that the enzymes corresponding to fabB/F and fabH were located on the cell membrane. It is the first time to show the cell location of KASs.  Based on the above studies, a possible pathway for the SCFAs synthesis in Synechococcus sp. PCC 7942 is proposed. As shown in Figure 3, CO 2 enters the microalgal cell and finally forms malonyl-ACP through de novo fatty acids synthesis, then goes into the fatty acid synthesis pathway, where KAS I/II (fabB/F) and KAS III (fabH) located on the cell membrane could catalyze condensation reactions to accumulate different chain length fatty acids less than 18 carbon atoms. Previous research found that fatty acids synthetic system started from acetyl-CoA carboxylase (ACCase), catalyzing the conversion of acyl-CoA to malonyl CoA, which was thought to be a limiting step [50]. KAS III had also been proved to be a limiting step in microalgae fatty acid biosynthesis. The above two limiting steps may affect the fixation of CO 2 in microalgae fatty acid biosynthesis. In the whole photosynthetic CO 2 fixation process, to increase the lipid productivity, ACCase and KAS III are the best options available for operation. Overexpression of ACCase and KAS III genes has succeeded to regulate the lipid synthesis [29,51,52].
Cell growth in BG11 medium was set as the normal growth condition. Different cerulenin concentrations were set as follows: 0 g/L, 1 g/L, 2.5 g/L, 5 g/L and 7.5 g/L. Stock solution of cerulenin (100 mg/L, preserved in ethyl alcohol, −20 °C) was added to BG11 medium with the final concentrations at 0 day.

Microalgae Cultures
Synechococcus sp. PCC 7942 used in the study was provided by Prof. Dingji Shi (Shanghai Ocean University, Shanghai, China). Cells were preserved in the BG11 medium. The initial OD 730 was 0.1, the initial pH was 7.8-8.0. The cultivation of Synechococcus sp. PCC 7942 was bubbled under 27 ± 1 • C and 140 mmol m −2 s −1 in 1 L Erlenmeyer flask with 500 mL working volume of BG11 medium. BG11 medium consists of (1 L) 1.5 g NaNO 3 ; 0.03 g K 2 HPO 4 ; 0.075 g MgSO 4 ·2H 2 O; 0.036 g CaCl 2 ·2H 2 O; 0.006 g citric acid; 0.006 g ferric ammonium citrate; 0.001 g EDTA; 0.02 g Na 2 CO 3 and 1 mL micronutrient solution. The microelement solution consists of ( Cell growth in BG11 medium was set as the normal growth condition. Different cerulenin concentrations were set as follows: 0 g/L, 1 g/L, 2.5 g/L, 5 g/L and 7.5 g/L. Stock solution of cerulenin (100 mg/L, preserved in ethyl alcohol, −20 • C) was added to BG11 medium with the final concentrations at 0 day.

Genomic DNA Extraction and PCR Analysis
Genomic DNA of Synechococcus sp. PCC 7942 was isolated as described previously by Kuo et al. [30]. Genomic DNA of Synechococcus sp. PCC 7942 transformant was prepared from exponential growth phase cultures using a procedure based on cetyltrimethylammonium bromide (CTAB) method. PCR analysis of transformants was carried out with genomic DNA as a template using primers as shown in Table S1 (Supplementary Materials). PCR amplification was performed for 30 cycles of 95 • C for 1 min, 56 • C for 30 s, and 72 • C for 30 s, followed by 72 • C for 10 min.

Sense and Antisense Expression Vector Construction
Bioinformatics showed that two enzymes in Synechococcus sp. PCC 7942 were annotated with β-ketoacyl ACP synthase activity. Alignments were proved that the sequence of Synpccw7942_0537 (Gene ID: 3774775) is similar to fabB and fabF in plants and bacteria, while the sequence of Synpccw7942_1455 (Gene ID: 3773627) is similar to fabH in plants and bacteria. In this experiment, we labeled them as fabF/B (Synpccw7942_0537) and fabH (Synpccw7942_1455), respectively.

Deletion Mutant Construction
The plasmid pUC118 was constructed for gene deletion mutants of Synpcc7942_0537 (fabB/F) and Synpcc7942_1455 (fabH). DNA fragments upstream and downstream of Synpcc7942_0537 (fabB/F) and Synpcc7942_1455 (fabH) were amplified from Synechococcus sp. PCC 7942 genomic DNA using the primers in Table S1 (Supplementary Materials). Chloromycetin gene was amplified from pET28a. These upstream and downstream DNA fragments and chloromycetin gene were amplified by using overlap PCR to form fabB/F-cat and fabH-cat fragments, and inserted into pUC118, replacing the corresponding regions. The resulting plasmids (pUC118) contains homologous regions for gene deletion mutants of Synpcc7942_0537 (fabB/F) and Synpcc7942_1455 (fabH), and chloromycetin antibiotic resistance was used for selection. The deletion mutants were further confirmed by DNA sequence analysis ( Figure S3, Supplementary Materials).

Fusion Expression Vector of Green Fluorescence Protein Construction
The plasmid pRL489 was used for fusion expression vectors of green fluorescence protein gene and Synpcc7942_0537 (fabB/F) and Synpcc7942_1455 (fabH). DNA sequences of Synpcc7942_0537 (fabB/F) and Synpcc7942_1455 (fabH) were amplified from Synechococcus sp. PCC 7942 genomic DNA using the primers in Table S1 (Supplementary Materials). Enhanced green fluorescence protein gene (egfp) fragment was from plasmid preserved in our laboratory. Synpcc7942_0537 (fabB/F) and Synpcc7942_1455 (fabH) and egfp gene were amplified by using overlap PCR to form egfp-fabB/F and egfp-fabH, and inserted into the pRL489, resulting pRL-egfp-fabB/F and pRL-egfp-fabH. Kanamycin antibiotic resistance was used for selection.

Liquid Culture and Screening of Transgenic Microalgae
Monoalgal colonies screened under antibiotic were cultured in BG11 medium containing kanamycin sulfate. Initially, transgenic microalgae was cultured in BG11 medium containing 25 g/L of kanamycin, and then the concentration of kanamycin (50 g/L, 100 g/L, 150 g/L, 200 g/L, 250 g/L, and 300 g/L) was gradually increased. When the concentration of antibiotic was increased to 300 g/L, wild type strains completely died under the pressure of antibiotic.

RNA Extraction and cDNA Synthesis
Total RNA extraction was treated with Trizol reagent (Sangon Biotech, Shanghai, China). Microalgal cells were harvested at different times (2 day intervals) by centrifugation. Approximately, microalgal cells (0.1 g) were ground in liquid nitrogen to powder before adding 1 mL Trizol reagent. After centrifugation at 12,000 r.p.m. for 10 min at 48 • C, 200 mL chloroform was added and mixed thoroughly. The sample was centrifuged at 12,000 r.p.m. for 15 min, then 450 mL of the uppermost layer was transferred into a fresh tube. Isopropanol (450 mL) was added to precipitated RNA and then centrifuged at 12,000 r.p.m. for 15 min, washed with 1 mL ethanol (75%, v/v) twice, dissolved in 30 mL diethyl pyrocarbonate treated distilled water. M-MLV (Moloney Murine Leukemia Virus) reverse transcriptase (TaKaRa Biotech Co., Dalian, China) was used to synthesize the cDNA. Two fatty acid synthetase genes (fabF/B and fabH) were investigated in this study. Genome annotation identified putative components of fatty acid biosynthetic pathway including fabF/B and fabH. Primers were designed according to the highly conserved regions obtained from the alignment of public database and the deduced amino acid sequences. The primers used in the experiment were shown in Table S1 (Supplementary Materials). After amplification, the PCR products were purified and cloned into pMD-19T vector for sequencing.

Quantitative Real-Time PCR
Real-time PCR was carried out on CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) using SYBR Premix Ex TaqTMII (TaKaRa Biotech Co., Dalian, China). The reference gene was used as an internal standard, amplified with the rnpB-F/rnpB-R primers (Table S1, Supplementary Materials). Real-time PCR was conducted following the procedure: 95 • C for 30 s before performing 40 cycles of 95 • C for 5 s and 60 • C for 45 s, and a melting step at 60 • C-95 • C. PCR efficiency of each gene was calculated by relative standard curve using sequential dilutions of the cDNA. The 2 −∆∆CT method [53] was applied for the target gene expression calculation.

Lipid Extraction and Fatty Acid Analysis
Microalgal cells were harvested by centrifugation after cultivation. The total lipids were extracted according to the following method. Lyophilized microalgae powder (0.2 g) was pulverized in a mortar and extracted using 5 mL solvent mixture of chloroform:methanol (2:1, v/v). After shaking for 10 min, the samples were centrifuged (5804R, Eppendorf, Germany) at 10,000 r.p.m. for 10 min. The procedure was repeated three times to make sure the lipids were extracted completely. The solvent phase was transferred by pipette and evaporated in a water bath at 60 • C.
The fatty acids component of the lipids was analyzed by GC-linked mass spectrometry (GC-MS). Fatty acid methyl esters were obtained by acidic transesterification of the lipids. After reaction at 90 • C for 2 h, 2.5 mL hexane and 1 mL water were added to the sample, then vibrated gently and centrifuged. One milliliter of the organic upper phase was injected into an Auto System XL GC/Turbo Mass MS (Perkin Elmer, Germany) using a DB-5MS (5% phenyl)-methylpolysiloxane nonpolar column (30 m × 0.25 mm × 0.25 mm). At the beginning, the column temperature was kept at 60 • C for 4 min, then increased to 220 • C, and finally reached to 280 • C with a temperature gradient of 10 • C min, maintained for 10 min.

Conflicts of Interest:
The authors declare no conflict of interest.