A simple method for isolation and construction of markerless cyanobacterial mutants defective in acyl-acyl carrier protein synthetase

Abstract

Cyanobacterial mutants defective in acyl-acyl carrier protein synthetase (Aas) secrete free fatty acids (FFAs) into the external medium and hence have been used for the studies aimed at photosynthetic production of biofuels. While the wild-type strain of Synechocystis sp. PCC 6803 is highly sensitive to exogenously added linolenic acid, mutants defective in the aas gene are known to be resistant to the externally provided fatty acid. In this study, the wild-type Synechocystis cells were shown to be sensitive to lauric, oleic, and linoleic acids as well, and the resistance to these fatty acids was shown to be enhanced by inactivation of the aas gene. On the basis of these observations, we developed an efficient method to isolate aas-deficient mutants from cultures of Synechocystis cells by counter selection using linoleic acid or linolenic acid as the selective agent. A variety of aas mutations were found in about 70 % of the FFA-resistant mutants thus selected. Various aas mutants were isolated also from Synechococcus sp. PCC 7002, using lauric acid as a selective agent. Selection using FFAs was useful also for construction of markerless aas knockout mutants from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002. Thus, genetic engineering of FFA-producing cyanobacterial strains would be greatly facilitated by the use of the FFAs for counter selection.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-7850-8) contains supplementary material, which is available to authorized users.

Long-Chain Saturated Fatty Acids, Palmitic and Stearic Acids, Enhance the Repair of Photosystem II

“…The photoinhibition of PSII results in the inhibition of cell growth in plants, algae, and cyanobacteria [ 15 , 16 ]. In Synechocystis , 12:0 and 18:3 inhibit cell growth, and 16:0 and 18:0 accelerate it [ 4 , 13 ]. To investigate the effects of FFA on the photoinhibition of PSII, we exposed Synechocystis cells to strong light at 1500 μmol photons m −2 s −1 at 32 °C in the absence or presence of various FFA at the concentration of 0.1 mM.…”

“…PG molecule anchors D1 polypeptides and enhance PSII repair [ 19 , 20 ]. Since the mutant of AAS tolerated to 12:0 [ 13 ], the incorporation of MCFA into PG molecules might inhibit the de novo synthesis of thylakoid proteins that is required for the repair of PSII.…”

“…The accelerated photodamage by 18:3 was observed only under light ( Figure 2 ), indicating the action of 18:3 on photodamage depends on photosynthetic electron transport. The mutant of AAS was tolerant to 18:3 [ 12 , 13 ], suggesting that effect of 18:3 on the cyanobacteria appears after the activation of 18:3 to 18:3-ACP by AAS. Unsaturation of lipids increases membrane fluidity and permeability, which might inhibit the formation of proton motive force across the membrane.…”

“…Among FFA molecules, linoleic (18:2) and linolenic acids (18:3) severely inhibit photosynthetic electron transport and the cellular growth of cyanobacteria [ 4 , 12 ]. Further investigation of the effect of FFA on cellular growth reports that the sensitivity of cyanobacteria to extracellular FFA is improved in the aas mutant [ 12 , 13 ]. These results suggest that inhibitory effects of FFA on the cellular growth and photosynthesis occur when FFA are activated as acyl-ACP.…”

New Applications of Synthetic Biology Tools for Cyanobacterial Metabolic Engineering

“…Counter-selection based on sacB is not functional in Synechococcus PCC 7002 (Zhang and Song, 2018), possibly because sacB selection is sensitive to salt (Kunst and Rapoport, 1995) required for growth of this marine cyanobacterium. A few markerless gene deletion systems have been shown to work in multiple cyanobacterial strains, for example Kojima et al developed an efficient method for generating knockouts in Synechocystis PCC 6803 and Synechococcus PCC 7002 (Kojima et al, 2016). This system is based on knocking out the aas gene, an acyl-acyl carrier protein synthetase, and selecting the mutants by their free fatty acid tolerance.…”