Improved lipid production via fatty acid biosynthesis and free fatty acid recycling in engineered Synechocystis sp. PCC 6803

Abstract

BackgroundCyanobacteria are potential sources for third generation biofuels. Their capacity for biofuel production has been widely improved using metabolically engineered strains. In this study, we employed metabolic engineering design with target genes involved in selected processes including the fatty acid synthesis (a cassette of accD, accA, accC and accB encoding acetyl-CoA carboxylase, ACC), phospholipid hydrolysis (lipA encoding lipase A), alkane synthesis (aar encoding acyl-ACP reductase, AAR), and recycling of free fatty acid (FFA) (aas encoding acyl–acyl carrier protein synthetase, AAS) in the unicellular cyanobacterium Synechocystis sp. PCC 6803.ResultsTo enhance lipid production, engineered strains were successfully obtained including an aas-overexpressing strain (OXAas), an aas-overexpressing strain with aar knockout (OXAas/KOAar), and an accDACB-overexpressing strain with lipA knockout (OXAccDACB/KOLipA). All engineered strains grew slightly slower than wild-type (WT), as well as with reduced levels of intracellular pigment levels of chlorophyll a and carotenoids. A higher lipid content was noted in all the engineered strains compared to WT cells, especially in OXAas, with maximal content and production rate of 34.5% w/DCW and 41.4 mg/L/day, respectively, during growth phase at day 4. The OXAccDACB/KOLipA strain, with an impediment of phospholipid hydrolysis to FFA, also showed a similarly high content of total lipid of about 32.5% w/DCW but a lower production rate of 31.5 mg/L/day due to a reduced cell growth. The knockout interruptions generated, upon a downstream flow from intermediate fatty acyl-ACP, an induced unsaturated lipid production as observed in OXAas/KOAar and OXAccDACB/KOLipA strains with 5.4% and 3.1% w/DCW, respectively.ConclusionsAmong the three metabolically engineered Synechocystis strains, the OXAas with enhanced free fatty acid recycling had the highest efficiency to increase lipid production.

Synechocystis sp. PCC 6803 overexpressing genes involved in CBB cycle and free fatty acid cycling enhances the significant levels of intracellular lipids and secreted free fatty acids

“…5). Palmitic acid (C16:0) was the dominant FA in Synechocystis cells which is in agreement with previous studies in wild type 3,13,19 . It is interesting that myristic acid (C14:0) was also found in all constructed strains at day 10.…”

“…On the other hand, Farmer and Liao (2001) reported that Gro3P could be also converted to pyruvate intermediate which was sequentially utilized for isoprene and carotenoid synthesis. Moreover, in our previous work, Synechocystis cells with overexpressing aas gene significantly increased their intracellular lipid level under normal growth condition 3 . To develop more efficient strains in this study, we recombined the glpD and/or RuBisCo with aas overexpression which consequently generated OG, OAG and OAGR strains.…”

“…In this study, we highlight our results from genetically modified strains of cyanobacterium Synechocystis PCC 6803 with enhanced capacity on their lipid production via the synergistic integration of carbon fixation reaction (or CBB cycle) and FFA recycling. Our previous reports demonstrated the increased lipid levels in engineered Synechocystis by modifying genes directly associated with fatty acid and phospholipid synthesis, including plsX, plsC, accDBCA, and FFA recycling including aas using metabolic engineering approach 3,13 . We previously suggested that carbon source supplementation, particularly acetate addition, could induce the intracellular lipid content up to 39.1% w/DCW in PlsC_PlsX-overexpressing Synechocystis cells 13 (reviewed in Table 4).…”

“…PCC6803 (Synechocystis) strain was grown in BG 11 medium at a culture temperature of 28 °C with continuous illumination of approximately 50 µmol photons/m 2 /s. The aas-overexpressing Synechocystis (OXAas) strain was obtained in our previous study 3 , hereafter OA in this study. Other engineered strains were OG, OAG and OAGR strains, all of which were constructed in this study ( Table 1).…”

“…1, the pathway starts from acetyl-CoA, which is converted to malonyl-CoA catalysed by acetyl-CoA carboxylase (ACC encoded by acc gene), and involves multiple steps of fatty acid synthesis II (FAS II) to obtain an intermediate fatty acyl-ACP, which is mainly used as a substrate for phospholipid synthesis and minor alkane production 2 . A genetically modified Synechocystis 6803 strain in which alkane production is blocked by deleting aar, encoding an acyl-ACP reductase that catalyses the conversion of fatty acyl-ACP to alkane, and together with an overexpression of aas, a gene encoding acyl-acyl carrier protein synthetase (AAS), had enhanced lipid level of approximately 1.8-fold 3 . The FFA recycling occurs via the activity of AAS, encoded by aas, to recycle the degraded membrane lipids by lipase A, encoded by lipA, to intracellular FFAs and which is then reused as a precursor for fatty acyl-ACP production 4,5 .…”