Overexpression of lipA or glpD_RuBisCO in the Synechocystis sp. PCC 6803 Mutant Lacking the Aas Gene Enhances Free Fatty-Acid Secretion and Intracellular Lipid Accumulation

Abstract

Although engineered cyanobacteria for the production of lipids and fatty acids (FAs) are intelligently used as sustainable biofuel resources, intracellularly overproduced FAs disturb cellular homeostasis and eventually generate lethal toxicity. In order to improve their production by enhancing FFAs secretion into a medium, we constructed three engineered Synechocystis 6803 strains including KA (a mutant lacking the aas gene), KAOL (KA overexpressing lipA, encoding lipase A in membrane lipid hydrolysis), and KAOGR (KA overexpressing quadruple glpD/rbcLXS, related to the CBB cycle). Certain contents of intracellular lipids and secreted FFAs of all engineered strains were higher than those of the wild type. Remarkably, the KAOL strain attained the highest level of secreted FFAs by about 21.9%w/DCW at day 5 of normal BG₁₁ cultivation, with a higher growth rate and shorter doubling time. TEM images provided crucial evidence on the morphological changes of the KAOL strain, which accumulated abundant droplets on regions of thylakoid membranes throughout the cell when compared with wild type. On the other hand, BG₁₁-N condition significantly induced contents of both intracellular lipids and secreted FFAs of the KAOL strain up to 37.2 and 24.5%w/DCW, respectively, within 5 days. Then, for the first time, we shone a spotlight onto the overexpression of lipA in the aas mutant of Synechocystis as another potential strategy to achieve higher FFAs secretion with sustainable growth.

Keywords: FFA secretion; Synechocystis sp. PCC 6803; acyl-ACP synthetase; lipase A; membrane lipid degradation.

Figure 1

Outline of intracellular lipid production and FFA secretion into cultured medium system in cyanobacteria. Key enzyme-encoding genes (represented in italic letters) associated with lipid and fatty acid synthesis, the Calvin–Benson–Bassham (CBB) cycle, polyhydroxybutyrate (PHB) synthesis, and free fatty acid (FFA) recycling, including accDACB, a multi-subunit acetyl-CoA carboxylase gene; lipA, a lipolytic enzyme-encoding gene; aas, acyl-ACP synthetase gene; plsX, plsY and plsC, putative phosphate acyl-transferases genes; and phaA, acetyl-CoA acetyltransferase gene; the RuBisCO gene cluster, including rbcLXS, encoding RuBisCO in respective order of large, chaperone and small subunits; glpD, glycerol-3-phosphate dehydrogenase gene. The abbreviated intermediates and products are: DHAP, dihydroxyacetone phosphate; fatty acyl-ACP, fatty acyl–acyl carrier protein; FFAs, free fatty acids; Gro3P, glycerol-3-phosphate; 3PG, 3-phosphoglycerate; PHB, polyhydroxybutyrate; RuBP, ribulose-1,5-bisphosphate. The thick arrows represent the overexpression (O) of indicated genes whereas the cross symbol represents knockout (K) of indicated gene.

Figure 2

Genomic maps of engineered Synechocystis PCC 6803 strains. The four modified strains are WTc (A), KA (B), KAOL (C), and KAOGR (D). The specific primers (Supplementary Materials, Table S1) were used to confirm the integration of each gene into Synechocystis genome. WTc is a wild-type control made by transforming both antibiotic chloramphenicol and kanamycin resistance cassettes (cm^r and km^r, respectively) into WT strain. KA was constructed by inserting a cm^r cassette to disrupt the aas gene. The integration of each strain was confirmed using PCR with genomic DNA from WT strain as the template. Lane M: GeneRuler DNA ladder (Fermentas). For (A) WTc strain; Lane 1: negative control using WTc as template (a–c), (a) Lanes 2–6: clone numbers 1 to 5 using UUSpsbA2 and Km_SR primers, (b) Lanes 2–6: clone numbers 1 to 5 using Cm_F and Km_SR primers, and (c) Lanes 2–6: clone numbers 1 to 5 using Km_FBamHI and DDSpsbA2 primers. For (B) KA strain; Lane 1: negative control using WTc as template (a,b), (a) Lanes 2–6: clone numbers 1 to 5 using Aas_F3 and aas_SR primers, and (b) Lanes 2–6: clone numbers 1 to 5 using USaas and DSaas primers. Only positive clones were selected for subsequent experiments; (A) WTc: clone numbers 2, 3, 5, (B) KA: clone numbers 1, 3, 4, 5, (C) KAOL: clone numbers 1–4, and (D) KAOGR: clone numbers 1–5.

Figure 3

The transcript levels (A) and relative transcript intensity ratios (B) of the accA, plsX, aas, lipA, rbcS, rbcL, glpD and 16S rRNA genes in Synechocystis PCC 6803 strains WTc, KA, KAOL and KAOGR. Oxygen evolution rate (C) and relative lipase activity (D) of all strains. Cells were grown in BG₁₁ medium and analyzed at day 5 of cultivation. The error bars represent standard deviations of means (mean ± S.D., n = 3). For (A), agarose gel (1.2%) of PCR product stained by RedSafe^TM nucleic acid staining solution (Intron Biotechnology Inc., Korea). For (D), control data (WTc) at 1.0 is derived from the lipase activity of 0.22 U/mg protein. For (C) and (D), the statistical differences of the results between WTc and engineered strain are indicated by an asterisk *, p < 0.05.

Figure 4

Growth curve (A), growth rate and doubling time (B), digital images of cell cultures (C,D), chlorophyll a content (E) and carotenoid content (F) of the Synechocystis PCC6803 wild-type control (WTc) and engineered KA strains. Cells were grown in BG₁₁ medium for 19 days. Error bars represent standard deviations of means (mean ± S.D., n = 3). For (C,D), images of 10-day cell culture flasks of WTc and KA were depicted. Floating white droplets in KA-cultured flask represent free fatty acids (FFAs) secreted to the medium (white arrow).

Figure 4

Growth curve (A), growth rate and doubling time (B), digital images of cell cultures (C,D), chlorophyll a content (E) and carotenoid content (F) of the Synechocystis PCC6803 wild-type control (WTc) and engineered KA strains. Cells were grown in BG₁₁ medium for 19 days. Error bars represent standard deviations of means (mean ± S.D., n = 3). For (C,D), images of 10-day cell culture flasks of WTc and KA were depicted. Floating white droplets in KA-cultured flask represent free fatty acids (FFAs) secreted to the medium (white arrow).

Figure 5

Contents (%w/DCW) of intracellular lipid (A) and extracellular FFAs (B) and total contents of intracellular lipid and extracellular FFAs (C) in Synechocystis PCC 6803 strains WTc, KA, KAOL and KAOGR. Cells were grown in BG₁₁ medium at the start of the experiment (day 0) and analysed at days 5 and day 10 of cultivation. Error bars represent standard deviations of means (mean ± S.D., n = 3). The statistical differences of the results between the WTc and engineered strains are indicated by an asterisk *, p < 0.05.

Figure 6

Fatty acid compositions (%) in extracellular fraction analysed by a GC instrument in Synechocystis PCC 6803 WTc and all engineered strains. Cells were grown in BG₁₁ medium for 10 days before being analysed. Error bars represent standard deviations of means (mean ± S.D., n = 3). The statistical differences of the results between the WTc and engineered strains are indicated by an asterisk *, p < 0.05.

Figure 7

Transmission electron micrographs (TEMs) of Synechocystis WTc (A) and KAOL (B) strains under normal BG₁₁ medium for 5 days of cultivation. In (A), WTc images are shown in 40,000× magnifications. In (B), KAOL images are shown in 50,000× magnifications. In (A), the white dots (white arrow) represent the examples of electron-transparent bodies. In (B), abundant droplet-like white dots were located on regions of thylakoid membranes inside KAOL cells.

Figure 8

Contents (%w/DCW) of PHB (A), intracellular lipid (B) and extracellular FFAs (C) in Synechocystis PCC 6803 WTc and KAOL strains under BG₁₁-N condition. For (A), cells were adapted for 5 days under BG₁₁ and BG₁₁-N conditions, respectively, before being analysed. For (B,C), cells were grown in BG₁₁ medium lacking NaNO₃ (BG₁₁-N) at 30 min, day 5 and day 10 of cultivations. Error bars represent standard deviations of means (mean ± S.D., n = 3). The statistical difference of the results between the WTc and KAOL strains is indicated by an asterisk at * p < 0.05.