Phosphoenolpyruvate Carboxylase Pathway Modulates The Metabolism Of Biodiesel Precursor In Synechococcus

Fossil fuels,an important energy resource,account for 85%of the world's energy demand.However,the excessive consumption of fossil fuels poses a great challenge for energy shortage and environmental pollution.Therefore,the development of renewable energy is currently a focus for the investigation of energy.Biofuels is a kind of clean energy which generated directly or indirectly by using animals,plants and microorganisms.Among them,biodiesel is expected to replace fossil fuels as a new type of transportation energy due to its safety,cleanliness,high combustion efficiency and low production cost.The raw lipid materials of biodiesel are mostly derived from animal and vegetable oils and fats,such as corn oil,soybean oil,animal fat,etc.Unfortunately,due to the slow growth of crops and high raw material costs,their promotion is limited.In recent years,single-cell cyanobacterial organisms with high photosynthetic efficiency,clear genetic background and rapid growth have gained increasingly attention.Synechococcus sp.PCC7002,a model organism of cyanobacterium,has been widely used for the study of photosynthetic systems,metabolic engineering,and biofuels.In this study,synechococcus sp.PCC7002 wild type was used as a model algae to knock out and overexpress the phosphoenolpyruvate carboxylase(PEPC)gene ppc in the central metabolic system to construct PCC7002(?)ppc::Sp and PCC7002-ppc~+strain,by measuring the biochemical indexes and the composition and content of fatty acid,accompanied with quantitatively analyzing the genes involved in the biosynthesis of triacylglycerol(TAG)and free fatty acids(FFAs),the regulation of the lipid synthesis in the cyanobacteria by the PEPC pathway was elucidated,and the main research details and results are as follows:1.The ppc knockout and overexpression cassette were constructed by PCR method.Subsequently,the linear DNA was transformed into PCC7002 by natural transformation and the PCC7002(?)ppc::Sp and PCC7002-ppc~+algae strains were successfully constructed by antibiotic screening and molecular verification.2.The cell growth,biomass accumulation,total carbohydrates,protein and lipids metabolic sinks were dynamically determined by spectrophotometry,dry cell weight method,anthrone colorimetry,BCA quantification and Bligh&Dyer method,respectively.The results showed that the knock-out of ppc significantly inhibited the growth of algal cells and the accumulation of biomass,and the content of glycogen and protein decreased significantly,while the lipids content remarkably increased and its synthesis became stabilized or even decreased in the later growth stage;over-expressed ppc gene had no significant effects on cell growth,biomass accumulation,protein and lipids content,but the glycogen synthesis and accumulation increased significantly.3.The composition and content of FFAs in algal cells were analyzed by gas chromatography-flame ionization detector(GC-FID).The results showed that all the strains contained C16:0,C16:1,C18:0,C18:1,C18:2,C18:3 and other FFAs,the first four accounted for 70-90%of the total FFAs,and the rest were polyunsaturated fatty acids(PUFAs),of which the percentage of C16:0 in the ppc engineered algae was significantly higher than that of the WT while C18:0 is lower than WT.Relative quantitative analysis showed that overexpression of ppc could remarkably increase the C16:0 content,but had no significant effect on the concentration of other FFAs,however,the synthesis and content of C16:0,C18:0 and C18:1 were observably increased after ppc knockout,and C16:1 and PUFAs did not change significantly.4.The differential expression of related genes involved in the synthesis of TAG and FFAs in the middle exponential phase of algal cells was quantitatively detected by qRT-PCR.The results showed that the expression levels of gps A,pls C and fab D genes were significantly up-regulated after ppc knock-out,but there was no significant change in the expression of acc A gene;conversely,the m RNA level of gps A,pls C and fab D was not significantly fluctuated when ppc gene was overexpressed while the acc A gene expression was significantly increased.In summary,this study demonstrated that the PEPC pathway has a certain regulatory effect on the lipid metabolism of cyanobacteria,and knock out ppc can significantly increase lipid synthesis and accumulation to provide a precursor molecule for biodiesel.Therefore,the ppc gene may be a potential target for biodiesel generation which use engineered cyanobacterial biomass as feedstocks,and provide a new theoretical basis and perspective for biofuel research.