Fatty acid production in genetically modified cyanobacteria

Abstract

To avoid costly biomass recovery in photosynthetic microbial biofuel production, we genetically modified cyanobacteria to produce and secrete fatty acids. Starting with introducing an acyl-acyl carrier protein thioesterase gene, we made six successive generations of genetic modifications of cyanobacterium Synechocystis sp. PCC6803 wild type (SD100). The fatty acid secretion yield was increased to 197 ± 14 mg/L of culture in one improved strain at a cell density of 1.0 × 10(9) cells/mL by adding codon-optimized thioesterase genes and weakening polar cell wall layers. Although these strains exhibited damaged cell membranes at low cell densities, they grew more rapidly at high cell densities in late exponential and stationary phase and exhibited less cell damage than cells in wild-type cultures. Our results suggest that fatty acid secreting cyanobacteria are a promising technology for renewable biofuel production.

Fig. 1.

The Synechocystis FAS pathways and modifications for FFA overproduction. The molecules and reactions in primary pathways toward FFA overproduction are indicated as bold text, whereas those in the competing pathways that uncouple the carbon flux from FFA overproduction are indicated as regular unbolded text. OPP, oxidative pentose phosphate; TCA, tricarboxylic acid; GA-3-P, glyceraldehyde-3-phosphate; 3-PGA, 3-phosphoglycerate; PEP, phosphoenolpyruvic acid; Ch FatB2, TE from C. hookeriana; Uc FatB2, TE from U. californica; Cc FatB1, TE from C. camphorum.

Fig. 2.

The genetic modifications in SD strains for FFA secretion. The genealogy of sequential modifications is shown on the left, and detailed modifications are shown on the right. As shown for SD215, P_nrsB is the nickel-inducible promoter that serves as the upstream flanking region (f1), whereas nrsCD is the downstream flanking region (f2). In SD216, f1 and f2 are the flanking regions for deletion of slr1609 (aas) and insertion of the ‘tesA cassette, of which, f1 contains the residual promoter of slr1609 (P_aas). In SD225, f1 and f2 are the flanking regions for deletion of slr1993 and slr1994; P_cpc and P_rbc are the promoters for the ACC genes (accB, accC, accD, and accA). In SD232, f1 and f2 are the flanking regions for deletion of sll1951; is an improved promoter from P_psbA2; Uc fatB1 is a TE gene from U. californica; and Ch fatB2 is a TE gene from C. hookeriana. In SD243, f1 and f2 are the flanking regions for deletion of slr2001 and slr2002. In SD249, f1 and f2 are the flanking regions for deletion of slr1710; Cc fatB1 is a TE gene from C. camphorum. In SD277, f1 and f2 are the flanking regions for deletion of slr2132; tesA137 is a ‘tesA gene with codon optimization.

Fig. 3.

Growth curves for SD strains. Cultures were grown at 30 °C in BG-11 medium and bubbled with 1% CO₂-enriched air. Cell density was transformed from culture optical density according to Fig. S5. The numbers pointed out by arrows are the damaged cell percentages in the SD232 and WT cultures at the specified times.

Fig. 4.

The secreted FFAs (white deposit) from an SD232 culture. Picture on the left shows an 800 mL culture of SD232 grown in an aeration flask for 4 d. Notice the secreted FFAs precipitated out of the culture medium as a granular ring on the flask wall above the aqueous phase. GC analysis indicates that the white precipitate contains about 60% water with half of the 40% dry weight being FFAs. Picture on the right shows microscopy of FFA secretion. (Scale bar: 10 µm.)