We have developed a genetic system we call "thermorecovery" that allows us to lyse cyanobacterial cultures and hydrolyze membrane lipids to release free fatty acids (FFAs), a biofuel precursor. The system uses thermostable lipases encoded by genes from thermophilic organisms that have been transferred into the cyanobacterial genome and can be synthesized by turning off CO(2) availability and subsequently activated by increasing the concentrated culture temperature. When synthesized in FFA-producing strains, the lipase Fnl from Fervidobacterium nodosum Rt17-B1 released the most FFA. Of the seven candidate lipases investigated, Fnl-synthesizing strains yielded 42.7±1.5 mg/l FFA at 47°C. We also determined that the optimal production conditions for SD338, the Synechocystis strain synthesizing Fnl, was to keep the cell concentrates at 46°C for two days after a one-day CO(2) limitation pretreatment of the culture. A 4-l continuous semi-batch production experiment with SD338 showed that daily harvested cultures (1l) released an average of 43.9±6.6 mg fatty acid and this productivity lasted for at least 20 days without significant decline. This improved thermorecovery process can be used in conjunction with other means to genetically engineer cyanobacteria to produce biofuels or biofuel precursors as the final step in recovery of membrane lipids.
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