Membrane-Located Expression of Thioesterase From Acinetobacter baylyi Enhances Free Fatty Acid Production With Decreased Toxicity in Synechocystis sp. PCC6803

Abstract

It has been previously reported that photosynthetic production of extracellular free fatty acids (FFAs) in cyanobacteria was realized by thioesterases (TesA) mediated hydrolysis of fatty acyl-ACP in cytosol and excretion of the FFA outside of the cell. However, two major issues related to the genetically modified strains need to be addressed before the scale-up commercial application becomes possible: namely, the toxicity of FFAs, and the diversity of carbon lengths of fatty acids that could mimic the fossil fuel. To address those issues, we hypothesized that generating FFAs near membrane could facilitate rapid excretion of the FFA outside of the cell and thus decrease toxicity caused by intracellular FFAs in the cytosolic expression of thioesterase. To realize this, we localized a leaderless thioesterase (AcTesA) from Acinetobacter baylyi on the cytosolic side of the inner membrane of Synechocystis sp. PCC6803 using a membrane scaffolding system. The engineered strain with AcTesA on its membrane (mAcT) produced extracellular FFAs up to 171.9 ± 13.22 mg⋅L^-1 compared with 40.24 ± 10.94 and 1.904 ± 0.158 mg⋅L^-1 in the cytosol-expressed AcTesA (AcT) and wild-type (WT) strains, respectively. Moreover, the mAcT strain generated around 1.5 and 1.9 times less reactive oxygen species than AcT and WT, respectively. Approximately 78% of total FFAs were secreted with an average rate of 1 mg⋅L^-1⋅h^-1, which was higher than 0.44 mg⋅L^-1⋅h^-1 reported previously. In the case of mAcT strain, 60% of total secreted FFAs was monounsaturated (C18:1) which is the preferable biodiesel component. Therefore, the engineered mAcT strain shows enhanced FFAs production with less toxicity which is highly desirable for biodiesel production.

Keywords: Acinetobacter baylyi; Synechocystis sp. PCC6803; free fatty acids (FFAs); membrane scaffold; thioesterase.

FIGURE 1

Generation of fatty acid-producing mutant strains using synthetic protein scaffold on the membrane of Synechocystis sp. PCC6803. (A) Design of gene arrangement in WT, AcT and mAcT strain. The dashed line indicates the neutral sites for the insertion of target genes. In AcT, ‘AcTesA is the truncated thioesterase from Acinetobacter baylyi; Ptrc is the promoter; NSU and NSD are the upstream and downstream flanking regions of the slr1311, respectively; Km is the kanamycin resistance cassette. In mAcT, PcpcB is the promoter; NSU and NSD are the upstream and downstream flanking regions of the deleted gene, slr0168, respectively. In the –ssSec-lac-lgt-‘AcTesA- cassette, ssSec is the signaling sequence; lac is β-lactamase; lgt is phosphatidylglycerol: prolipoprotein diacylglycerol transferase, an inner membrane protein; flexible linker FL3 (red color and coil-shaped) is introduced between proteins to fuse β-lactamase to the N-terminus and ‘AcTesA to the C-terminus of Lgt. (B) Hypothetical presentation of the mechanism of membrane-localized ‘AcTesA in generating and facilitating the excretion of FFAs outside the cell. (C) PCR confirmation of the insertion in the AcT and mAcT mutant strains. Lane M is a marker. Lanes 1, 2, and 3 are bands of DNA amplified using primers NSUF (1311) and NSDR (1311) to confirm the –AcT-PcpcKm- insertion. In lanes 4–6, the primers NSUF (0168) and NSDR (0168) were used to amplify the –PcpcBssSecLacLgtAcTesAKm-cassette insertion to confirm the insertion with around 3.7 kb in size. WT is the band without target insertion around 1.9 and 1 kb with the primer pair NSUF/NSDR (0168) and NSUF/NSDR (1311), respectively. (D) Western blot analysis of fractionated protein from mAcT and total crude AcT. Here, M is the marker; PM, plasma membrane; TM, thylakoid membrane; WT, total crude protein from WT Synechocystis. MW of ‘AcTesA is approximately 20 kDa and Lgt-‘AcTesA fusion is approximately 50 kDa. (E) qRT-PCR analysis of expression of ‘AcTesA. Data shown here are the mean ± SE from biological triplicates with 4 technical replicates and “^∗” represents statistical significance as indicated by Student’s t-test with a maximum p-value of < 0.05.

FIGURE 2

Production and secretion of FFAs. Cultures were grown at 30°C in a BG-11 medium under continuous illumination of 25 μmol photons m^-2 s^-1 light. (A) Droplets of FFAs appeared on the culture medium of mAcT after 168 h. GC-MS-based analysis of (B) extracellular FFAs (FFAs) at 168 h, (C) extracellular FFAs (FFAs) at 360 h, (D) intracellular FFAs at 168h and (E) intracellular FFAs at 360 h from the culture of WT, AcT and mAcT set in a 250 mL flask. Data shown in (B–E) are the mean ± SE from biological triplicates, and ‘^∗’ represents statistical significance as indicated by Student’s t-test with a maximum p-value of <0.05.

FIGURE 3

Fatty acid profile of FFA-producing strain. Cultures were grown at 30°C in a BG-11 medium under continuous illumination of 25 μmol photons m^-2 s^-1 light. FFA compositions were analyzed by GC-MS. (A) Extracellular FFA profile at 168 h, (B) extracellular FFA profile at 360 h, (C) intracellular FFA profile at 168 h and (D) intracellular FFA profile at 360 h. Data shown here are the mean ± SE from biological triplicates.

FIGURE 4

Effects of CO₂ on extracellular FFA production. Cultures were grown at 30°C in a BG-11 medium under continuous illumination of 50 μmol photons m^-2 s^-1 light and bubbled with 1% CO₂-enriched air. Measurement of (A) extracellular FFAs at 168 h and (B) extracellular FFAs at 360 h. Profiles of (C) extracellular FFAs at 168 h and (D) extracellular FFAs at 360 h of WT, AcT and mAcT. (E) Extracellular FFA amount after normalization with dry cell weight (DCW) at two time points, 168 and 360 h. Data shown in (A–E) are the mean ± SE from biological triplicates, and ‘^∗’ represents statistical significance as indicated by Student’s t-test with a maximum p-value of < 0.05.

FIGURE 5

Effects of FFA production on cell survivability. (A) Growth pattern of fatty acid-producing strains. The culture was grown in 250 mL culture flask at 30°C in a BG-11 medium under continuous illumination of 25 μmol photons m^-2s^-1 light. OD was measured at 730 nm. Data shown here are the mean ± SE from biological triplicates. (B) Cellular toxicity and ROS generation in WT, AcT and mACT strains at 168 h. Data shown here are the mean ± SE from five biological replications along with four technical replications and ‘^∗’ represents statistical significance as indicated by Student’s t-test with a maximum p-value of < 0.05. (C) Percentage of damaged cells of WT, AcT and mAcT strains sorted by flow cytometry using SYTOX green nucleic acid stain. Data shown here are the mean ± SE from biological triplicates.

FIGURE 6

Effect of exogenously added C18:1 FFA and extracted media of mutant strain on WT cells. (A) Effects of exogenously added C18:1 fatty acid on the cell survivability of FFA-producing strain. Cells were grown either with or without C18:1 unsaturated fatty acid with different concentrations (70 mg⋅L^-1 to 300 mg⋅L^-1) in medium, and the damaged cell percentage was determined by FACS at different time points. (B) Role of membrane scaffold on cell survivability. The WT cells were grown in the absence (WT without MS media) or presence (WT in mAcT/AcT media) of media from mAcT/AcT strains (MS media) after 10 days of normal growth in BG-11 media. Data shown here are the mean ± SE from biological triplicates.