Abstract
The integrative aspect on carbon fixation and lipid production is firstly implemented in cyanobacterium Synechocystis sp. PCC 6803 using metabolic engineering approach. Genes related to Calvin–Benson–Bassham (CBB) cycle including rbcLXS and glpD and free fatty acid recycling including aas encoding acyl-ACP synthetase were practically manipulated in single, double and triple overexpressions via single homologous recombination. The significantly increased growth rate and intracellular pigment contents were evident in glpD-overexpressing (OG) strain among all strains studied under normal growth condition. The triple aas_glpD_rbcLXS-overexpressing (OAGR) strain notably gave the highest contents of both intracellular lipids and extracellular free fatty acids (FFAs) of about 35.9 and 9.6% w/DCW, respectively, when compared to other strains at day 5 of cultivation. However, the highest intracellular lipid titer and production rate were observed in OA strain at day 5 (228.7 mg/L and 45.7 mg/L/day, respectively) and OG strain at day 10 (358.3 mg/L and 35.8 mg/L/day, respectively) due to their higher growth. For fatty acid (FA) compositions, the main saturated fatty acid of palmitic acid (C16:0) was dominantly found in both intracellular lipid and secreted FFAs fractions. Notably, intracellular FA proportion of myristic acid (C14:0) was induced in all engineered strains whereas the increase of stearic acid (C18:0) composition was found in extracellular FFAs fraction. Altogether, these overexpressing strains efficiently produced higher lipid production via homeostasis balance on both its lipid synthesis and FFAs secretion.
Highlights
Fatty acids and lipids, which are present at significant levels in cyanobacteria, may serve as crucial precursors for the production of renewable energy carriers, such as biofuel and biodiesel and they are mainly accumulated in terms of phospholipid membranes which are the composition of cell and thylakoid membranes[1]
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 and free fatty acids (FFAs) 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]
Summary
Fatty acids and lipids, which are present at significant levels in cyanobacteria, may serve as crucial precursors for the production of renewable energy carriers, such as biofuel and biodiesel and they are mainly accumulated in terms of phospholipid membranes which are the composition of cell and thylakoid membranes[1]. For lipid biosynthesis in the cyanobacterium Synechocystis PCC6803, as shown, 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]. Dihydroxyacetone phosphate (DHAP) which is one of intermediates in the CBB cycle can be converted to glycerol-3-phosphate (Gro3P) catalysed by glycerol-3-phosphate dehydrogenase (GPD encoded by glpD gene)[6], which is further partly used as a glycerol backbone for lipid molecules. The OAG strain contained double overexpression of aas_glpD which probably enhanced both glycerol backbone and fatty acyl-ACP substrate whereas triple-overexpression of aas_glpD_rbcLXS genes (OAGR) would synergistically induce RuBisCO function beside OAG. Our findings indicate that the apparent creation of genetically modified strains, in particular OAGR, was achieved to augment both intracellular lipid synthesis and free fatty acids secretion
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