Modulation of the balance of fatty acid production and secretion is crucial for enhancement of growth and productivity of the engineered mutant of the cyanobacterium Synechococcus elongatus (original) (raw)
Related papers
Biotechnology for biofuels, 2017
Cyanobacterial mutants engineered for production of free fatty acids (FFAs) secrete the products to the medium and hence are thought to be useful for biofuel production. The dAS1T mutant constructed from Synechococcus elongatus PCC 7942 has indeed a large capacity of FFA production, which is comparable to that of triacylglycerol production in green algae, but the yield of secreted FFAs is low because the cells accumulate most of the FFAs intracellularly and eventually die of their toxicity. To increase the FFA productivity, enhancement of FFA secretion is required. Growth of dAS1T cells but not WT cells was inhibited in a liquid medium supplemented with 0.13 g L(-1) of palmitic acid. This suggested that when FFA accumulates in the medium, it would inhibit the release of FFA from the cell, leading to FFA accumulation in the cell to a toxic level. To remove FFAs from the medium during cultivation, an aqueous-organic two-phase culture system was developed. When the dAS1T culture was ov...
Plant and Cell Physiology, 2015
Most organisms capable of oxygenic photosynthesis have an aas gene encoding an acyl-acyl carrier protein synthetase (Aas), which activates free fatty acids (FFAs) via esterification to acyl carrier protein. Cyanobacterial aas mutants are often used for studies aimed at photosynthetic production of biofuels because the mutation leads to intracellular accumulation of FFAs and their secretion into the external medium, but the physiological significance of the production of FFAs and their recycling involving Aas has remained unclear. Using an aas-deficient mutant of Synechococcus elongatus strain PCC 7942, we show here that remodeling of membrane lipids is activated by high-intensity light and that the recycling of FFAs is essential for acclimation to highlight conditions. Unlike wild-type cells, the mutant cells could not increase their growth rate as the light intensity was increased from 50 to 400 mmol photons m-2 s-1 , and the highlight grown mutant cells accumulated FFAs and the lysolipids derived from all the four major classes of membrane lipids, revealing highlight induced lipid deacylation. The highlight grown mutant cells showed much lower PSII activity and Chl contents as compared with the wild-type cells or low-light-grown mutant cells. The loss of Aas accelerated photodamage of PSII but did not affect the repair process of PSII, indicating that PSII is destabilized in the mutant. Thus, Aas is essential for acclimation of the cyanobacterium to highlight conditions. The relevance of the present findings to biofuel production using cyanobacteria is discussed.
Photosynthesis Research, 2020
Sucrose, a compatible osmolyte in cyanobacteria, functions both as an energy reserve and as osmoprotectant. Sugars are the most common substrates used by microorganisms to produce hydrogen (H 2) by means of anaerobic dark fermentation. Cells of the unicellular, non-nitrogen fixing, freshwater cyanobacterium Synechococcus elongatus PCC7942 accumulate sucrose under salt stress. In the present work, we used this cyanobacterium and a genetically engineered strain of it (known as PAMCOD) to investigate the optimal conditions for (a) photosynthetic activity, (b) cell proliferation and (c) sucrose accumulation, which are necessary for H 2 production via anaerobic dark fermentation of the accumulated sucrose. PAMCOD (Deshnium et al. in Plant Mol Biol 29:897-902, 1995) contains the gene codA that codes for choline oxidase, the enzyme which converts choline to the zwitterion glycine betaine. Glycine betaine is a compatible osmolyte which increases the salt tolerance of Synechococcus elongatus PCC7942. Furthermore, glycine betaine maintains cell proliferation under salt stress and results in increased sucrose accumulation. In the present study, we examine the environmental factors, such as the NaCl concentration, the culture medium pH, and the carbon dioxide content of the air bubbled through it. At optimal conditions, sucrose accumulated in the cyanobacteria cells up to 13.5 mol per mole Chl a. Overall, genetically engineered Synechococcus elongatus PCC7942 produces sucrose in sufficient quantities such that it may be a viable alternative (a) to sucrose synthesis, and (b) to H 2 formation via anaerobic dark fermentation.
Biotechnology for Biofuels and Bioproducts
Background Based on known metabolic response to excess free fatty acid (FFA) products, cyanobacterium Synechocystis sp. PCC 6803 preferentially both recycles via FFA recycling process and secrets them into medium. Engineered cyanobacteria with well growth and highly secreted FFA capability are considered best resources for biofuel production and sustainable biotechnology. In this study, to achieve the higher FFA secretion goal, we successfully constructs Synechocystis sp. PCC 6803 mutants disrupting genes related to FFA recycling reaction (aas gene encoding acyl–acyl carrier protein synthetase), and surface layer protein (encoded by sll1951). Results Three Synechocystis sp. PCC 6803 engineered strains, including two single mutants lacking aas (KA) and sll1951 (KS), and one double mutant lacking both aas and sll1951 (KAS), significantly secreted FFAs higher than that of wild type (WT). Certain increase of secreted FFAs was noted when cells were exposed to nitrogen-deficient condition...
Applied microbiology and biotechnology, 2016
Cyanobacterial mutants defective in acyl-acyl carrier protein synthetase (Aas) secrete free fatty acids (FFAs) into the external medium and hence have been used for the studies aimed at photosynthetic production of biofuels. While the wild-type strain of Synechocystis sp. PCC 6803 is highly sensitive to exogenously added linolenic acid, mutants defective in the aas gene are known to be resistant to the externally provided fatty acid. In this study, the wild-type Synechocystis cells were shown to be sensitive to lauric, oleic, and linoleic acids as well, and the resistance to these fatty acids was shown to be enhanced by inactivation of the aas gene. On the basis of these observations, we developed an efficient method to isolate aas-deficient mutants from cultures of Synechocystis cells by counter selection using linoleic acid or linolenic acid as the selective agent. A variety of aas mutations were found in about 70 % of the FFA-resistant mutants thus selected. Various aas mutants w...
Biotechnology for Biofuels, 2019
Background: Cyanobacteria 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. Results: To 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. Conclusions: Among the three metabolically engineered Synechocystis strains, the OXAas with enhanced free fatty acid recycling had the highest efficiency to increase lipid production.
Synechococcus elongatus PCC 7942 as a Platform for Bioproduction of Omega-3 Fatty Acids
Life
Alpha-linolenic acid and stearidonic acid are precursors of omega-3 polyunsaturated fatty acids, essential nutrients in the human diet. The ability of cyanobacteria to directly convert atmospheric carbon dioxide into bio-based compounds makes them promising microbial chassis to sustainably produce omega-3 fatty acids. However, their potential in this area remains unexploited, mainly due to important gaps in our knowledge of fatty acid synthesis pathways. To gain insight into the cyanobacterial fatty acid biosynthesis pathways, we analyzed two enzymes involved in the elongation cycle, FabG and FabZ, in Synechococcus elongatus PCC 7942. Overexpression of these two enzymes led to an increase in C18 fatty acids, key intermediates in omega-3 fatty acid production. Nevertheless, coexpression of these enzymes with desaturases DesA and DesB from Synechococcus sp. PCC 7002 did not improve alpha-linolenic acid production, possibly due to their limited role in fatty acid synthesis. In any case...
Incorporation, fate and turnover of free fatty acids in cyanobacteria
FEMS Microbiology Reviews
Fatty acids are important molecules in bioenergetics and also in industry. The phylum Cyanobacteria consists of a group of prokaryotes that typically carry out oxygenic photosynthesis with water as an electron donor and use carbon dioxide as a carbon source to generate a range of biomolecules, including fatty acids. They are also able to import exogenous free fatty acids and direct them to biosynthetic pathways. Here, we review current knowledge on mechanisms and regulation of free fatty acid transport into cyanobacterial cells, their subsequent activation and use in the synthesis of fatty acid-containing biomolecules such as glycolipids and alka(e)nes, as well as recycling of free fatty acids derived from such molecules. This review also covers efforts in the engineering of such cyanobacterial fatty acid-associated pathways en route to optimized biofuel production.