Effects of Aas deficiency on cellular sensitivity to various FFAs
Figure 1a shows the effects of various FFAs on growth of the WT and the aas-deficient mutant strains of Synechocystis sp. PCC 6803. As previously reported, the WT Synechocystis cells grew fine in the presence of palmitic acid (16:0) and stearic acid (18:0) (Ruffing and Trahan 2014). The cells grew well also in the presence of myristic acid (14:0). In accordance with the previously published results (Sakamoto et al. 1998), the WT cells were sensitive to the C18 polyunsaturated FFAs, i.e., linoleic acid (18:2) and linolenic acid (18:3), while they could tolerate the presence of the monounsaturated FFA, oleic acid (18:1). The engineered aas-deficient mutant dAS11 was resistant not only to 18:3 as previously reported (von Berlepsch et al. 2012) but also to 18:2. The mutant was somewhat more tolerant to lauric acid (12:0) than WT.
Figure 1b shows the effects of FFAs on growth of the WT and the aas-deficient mutant strain of Synechococcus sp. PCC 7002, a model cyanobacterium that is thought to be useful for FFA production under the outdoor conditions because of its tolerance to strong irradiance and high salt concentrations (Reed and Stewart 1985; Ludwig and Bryant 2012). Similar to the results obtained with Synechocystis sp. PCC 6803, and in accordance with the previously reported results, the WT cells of Synechococcus sp. PCC 7002 grew well in the presence of 16:0 and 18:0 (Ruffing and Trahan 2014) but failed to grow in the presence of 18:2 and 18:3 (Sakamoto et al. 1998; Ruffing and Trahan 2014) (Fig. 1b). Synechococcus sp. PCC 7002 did not grow at all in the presence of 100 μM of 10:0, 12:0, and 14:0, but the engineered aas mutant dAS21 was resistant to these fatty acids. Unlike in Synechocystis sp. PCC 6803, deficiency of Aas did not confer the cells the resistance to the long-chain polyunsaturated fatty acids 18:2 and 18:3.
Isolation of aas mutants from WT cell population using FFAs as the selective agents
Although the WT cells of Synechocystis sp. PCC 6803 seemed to have completely died in the presence of 100 μM of 18:2 or 18:3 in 7 days, two and one colonies came up in the spot of undiluted cell suspension on the 18:2- and 18:3-containing agar plates, respectively, after 14 days of incubation. Transfer of the cells to new 18:2- and 18:3-containing media showed that they were resistant to both of the FFAs (not shown). PCR amplification and sequencing of the aas locus of these clones showed that they have mutations in the aas gene. These results indicated that aas-deficient mutants can be readily identified and isolated by using 18:2 or 18:3 as the selecting agent. For larger scale screening of naturally occurring mutations of the aas gene, WT Synechocystis cell suspension (OD730 = 3) was diluted to OD730 = 0.5 by BG11 and spread 100 μL of cell suspension onto agar plates containing 100 μM of 18:2 and 18:3, respectively. After 14 days of incubation, 12 and 54 colonies appeared on the 18:2- and 18:3-containing media, respectively. Cells from these colonies were subcultured for three passages on the FFA-containing media. DNA was subsequently purified from 10 and 17 of the lines obtained from the 18:2- and 18:3-containing media, respectively and subjected to PCR amplification and sequence analysis of the aas locus. Of the 27 strains examined, 17 strains carried a mutation in aas. There were 16 mutant alleles for aas, each carrying an indel or a base substitution that results in a frame shift, an amino acid substitution, or no amino acid substitution in the encoded protein (Fig. 2a).
The aas-deficient Synechococcus sp. PCC 7002 mutant failed to grow in the presence of 18:2 or 18:3, but it could grow in the presence of 10:0, 12:0, and 14:0, suggesting that these FFAs may be used as the selecting agents to identify and isolate aas-deficient mutants from the wild-type population of the cells. To determine whether 12:0 can be used for selection of aas-deficient mutants from the WT cultures of Synechococcus sp. PCC 7002, 100 μl of the cell suspension (OD730 = 0.5) was spread onto agar plates containing 100 μM of 12:0. Seventeen colonies obtained after 14 days of incubation were subcultured for three passages on the 12:0-containing medium. PCR amplification and sequence analysis of the aas locus revealed that 12 of the 17 strains carried a mutation in aas. There were eight mutant alleles for aas, each carrying an indel or a base substitution that would result in a frame shift or an amino acid substitution (Fig. 2b).
Markerless knockout of the aas gene using FFAs as the selective agents
Since FFAs were successfully used as selective agents for identification and isolation of the cells bearing spontaneous mutations in the aas gene, we further tried targeted inactivation of aas by counter selection, without using an antibiotic resistance marker. One of the Synechocystis aas mutants obtained by screening of 18:3-tolerant cells had a 97-base insertion in the aas ORF (Fig. 2a). A 2084-bp DNA fragment of the aas gene, carrying the 97-bp insertion, was amplified from the mutant by PCR using the primer pair a2/a6 (Fig. 3a) and used to transform WT Synechocystis sp. PCC 6803 cells to 18:3 resistance. Colonies from five independent transformant lines were isolated after three serial streak purifications on 18:3-containing medium, and all of them were shown to carry the 97-base insertion in the aas gene by PCR analysis (Fig. 3b) and nucleotide sequence analysis (not shown). Markerless knockout of the aas gene was attempted also in Synechococcus sp. PCC 7002, using 12:0 as the selective agent for counter selection. To this end, a 965-bp DNA fragment carrying the initiation codon of aas and 962 bases of its 5′-flanking sequence was amplified by PCR using the primers b1 and b3 (Fig. 3c). A 933-bp DNA fragment of the 3′-flanking sequence of aas, carrying the bases +3 to +935 with respect to the aas termination codon, was also amplified by PCR using the primers b8 and b9 (Fig. 3c). The two fragments were jointly cloned into the pGEM-Teasy plasmid to construct the plasmid p∆aas (Fig. 3c). The plasmid was used to transform WT cells of Synechococcus sp. PCC 7002 to 12:0 resistance, and colonies of five independent 12:0-resistant lines were streak purified. PCR analysis showed that the aas ORF had been deleted from the genome of four out of the five transformants (Fig. 3d). The remaining one transformant (Fig. 3d, #2) carried the wild-type aas gene, indicating that it had acquired the tolerance to 12:0 by a mutation(s) located elsewhere on the genome. Taken together, nine out of the ten FFA-tolerant transformants obtained from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 were defective in the aas gene. Although cyanobacteria have multiple copies of genomic DNA and are notorious for the difficulties in segregation of mutant and WT chromosomes, there was no sign of wild-type aas in the nine transformants (Fig. 3b, d). These results demonstrated that FFA-based counter selection applies strong enough selection pressure on cyanobacterial cells, allowing for inactivation of aas without using a drug resistance marker.