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.
Highlights
Acyl-acyl carrier protein synthetase (Aas), which is found in most organisms performing oxygenic photosynthesis, catalyzes esterification of free fatty acids (FFAs) to acyl carrier protein (ACP)
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
The toxic effect of these FFAs was not abolished by aas inactivation in Synechococcus sp
Summary
Acyl-acyl carrier protein synthetase (Aas), which is found in most organisms performing oxygenic photosynthesis, catalyzes esterification of free fatty acids (FFAs) to acyl carrier protein (ACP). Aas mediates incorporation of exogenously added FFAs into membrane lipids, but its natural substrate is thought to be the FFAs produced in the cell, because aas-deficient mutants secrete FFAs into the medium (Kaczmarzyk and Fulda 2010). Cyanobacterial cells produce FFAs by deacylation of the membrane lipids (Kaczmarzyk and Fulda 2010; Takatani et al 2015). Since deficiency of Aas results in destabilization of photosystem II and renders the cells sensitive to high light (>400 μE m−2 s−1), the Aas-mediated recycling of FFAs is deduced to be important for acclimation of cyanobacteria to highlight conditions (Takatani et al 2015). It is supposed that the concentration gradient of the FFA, which is maintained by 18:3-ACP formation on the cytoplasmic side of
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