CO2-limitation-inducible Green Recovery of fatty acids from cyanobacterial biomass

Abstract

Using genetically modified cyanobacterial strains, we engineered a Green Recovery strategy to convert membrane lipids into fatty acids for economical and environmentally sustainable biofuel production. The Green Recovery strategy utilizes lipolytic enzymes under the control of promoters induced by CO(2) limitation. Data indicate that strains of the cyanobacterium Synechocystis sp. PCC6803 engineered for Green Recovery underwent degradation of membrane diacylglycerols upon CO(2) limitation, leading to release of fatty acids into the culture medium. Recovered fatty acid yields of 36.1 × 10(-12) mg/cell were measured in one of the engineered strains (SD239). Green Recovery can be incorporated into previously constructed fatty-acid-secretion strains, enabling fatty acid recovery from the remaining cyanobacterial biomass that will be generated during fatty acid biofuel production in photobioreactors.

Fig. 1.

Genealogies of the Green Recovery constructions. Three lipolytic genes (fol, shl, and gpl) were inserted into SD100 (6803 wild type) and SD232 (an FFA-secretion 6803 strain), and controlled by two CO₂-limitation-inducible promoters (P_cmp and P_sbt). Detailed genetic information of the strains is described in Table S1.

Fig. 2.

The cultures of 6803 wild type, SD256 and SD257 in sealed flasks for four days for CO₂ limitation after being grown with vigorous aeration.

Fig. 3.

The fatty acid profiles of SD strains. All the cultures were grown to about 4 × 10⁸ cells/mL at 30 °C. For wild type, the columns show the fatty acid profile of total membrane lipids. For SD237, the columns show the released FFA profile by Green Recovery, which is similar to that of wild type with abundant unsaturated fatty acids. For SD232, the columns show the profile of secreted FFAs, which are highly saturated with significant amounts of C12∶0 and C14∶0. For SD239 before (Green Recovery), the columns show the profile of secreted FFAs before CO₂ limitation, which is similar to that of the FFA-secretion strain SD232. For SD239 after (Green Recovery), the columns show the profile of all the FFAs contributed by SD239 after CO₂ limitation, which is a mixture of secreted FFAs (e.g., C12∶0 and C14∶0) and released FFAs (e.g., C18∶2 and C18∶3).

Fig. 4.

FFA yields of SD256 and SD237 during Green Recovery. Ten CO₂-limiting flasks with 16-mL cultures were set in the same conditions on day zero for each strain. Every day, the whole cultures in duplicate flasks were extracted by hexane for FFA yields. The cell membrane damage was observed after SYTOX staining, and the permeable cell percentages are indicated above the columns.