An organic acid based counter selection system for cyanobacteria

Abstract

Cyanobacteria are valuable organisms for studying the physiology of photosynthesis and carbon fixation, as well as metabolic engineering for the production of fuels and chemicals. This work describes a novel counter selection method for the cyanobacterium Synechococcus sp. PCC 7002 based on organic acid toxicity. The organic acids acrylate, 3-hydroxypropionate, and propionate were shown to be inhibitory towards Synechococcus sp. PCC 7002 and other cyanobacteria at low concentrations. Inhibition was overcome by a loss of function mutation in the gene acsA, which is annotated as an acetyl-CoA ligase. Loss of AcsA function was used as a basis for an acrylate counter selection method. DNA fragments of interest were inserted into the acsA locus and strains harboring the insertion were isolated on selective medium containing acrylate. This methodology was also used to introduce DNA fragments into a pseudogene, glpK. Application of this method will allow for more advanced genetics and engineering studies in Synechococcus sp. PCC 7002 including the construction of markerless gene deletions and insertions. The acrylate counter-selection could be applied to other cyanobacterial species where AcsA activity confers acrylate sensitivity (e.g. Synechocystis sp. PCC 6803).

Figure 1. Growth of wild type and an adapted strain of PCC 7002 with acrylate.

(A) Growth of wild type PCC 7002 in unmodified medium (green diamonds) or 5 mM acrylate (red squares). (B) Growth of acrylate adapted cultures of PCC 7002 in unmodified medium (green diamonds) or 5 mM acrylate (red squares). Data points are the mean of biological triplicates and error bars represent the standard deviation.

Figure 2. Specific activity of AcsA towards the organic acids acetate, propionate, acrylate, and 3HP.

Figure 3. Homozygous mutants were obtained in both the acsA and glpK loci.

(A) Diagram of the primers used to screen for homozygous strains in the acsA locus. Primer set A amplifies from the up and down5' and 3' stream flanking regions of acsA and should be amplify 3.2 kb, 1.1 kb, and 2.6 kb in fragments for WT 7002, BPSyn_014, and BPSyn_015, respectively. Primer set B amplifies 1.2 kb from inside acsA to the region downstream of acsA. Primer set C amplifies 0.9 kb from the barcode (BC) region to downstream of acsA. Primer set D amplifies a 0.25 kb region internal to YFP. (B) 1% agarose gel showing colony PCR products amplified using the primer sets A, B, C, and D on WT PCC 7002, BPSyn_014, and BPSyn_015. Lane 1 contains a 2-log ladder from New England Biolabs. Lanes 2-5, 6-9, and 10-13 are primers sets A, B, C, and D amplifying WT PCC 7002, BPSyn_014, and BPSyn_015, respectively. (C) Diagram of the primers used to screen for homozygous strains in the glpK locus. Primer set E amplifies from the up and down stream5' and 3' flanking regions of glpK and should be amplify 2.7 kb, 4.8 kb, and 2.6 kb fragments from BPSyn_014, BPSyn_022, and BPSyn_027, respectively. Primer set F amplifies 0.72 kb from inside glpK to the region downstream of glpK. Primer set G amplifies a 2.3 kb region of the acsA cassette that was introduced into the glpK locus. (D) 1% agarose gel showing colony PCR products amplified using the primer sets E, F, G, and D on BPSyn_014, BPSyn_022, and BPSyn_027. Lane 1 contains a 2-log ladder from New England Biolabs. Lanes 2-5, 6-9, and 10-13 are primers sets E, F, G, and D amplifying BPSyn_014, BPSyn_022, and BPSyn_027, respectively.

Figure 4. Comparison of acsA and glpK mutant phenotypes.

(A) Growth of wild type PCC 7002 (green diamonds) and BPSyn_014 (blue squares) in Medium A⁺ supplemented with air. (B) Fluorescence measurements of strains BPSyn_015 and BPSyn_027, which contain YFP under the cpcB promoter in the acsA or glpK locus, respectively. Fluorescence is measured using arbitrary units (A.U.) at an excitation of 514 nm and an emission of 527 nm. Data points are the mean of biological triplicates and error bars represent the standard deviation.

Figure 5. Outline of counter selection protocol based on acrylate sensitivity.