Cellulosic Hydrocarbons Production By Engineering Dual Synthesis Pathways In Corynebacterium Glutamicum

Hydrocarbons are the main components of aviation fuel,which all come from non-renewable fossil fuels such as oil and coal.Biomanufacturing of aviation fuel from renewable resources is an important direction for the post-oil era and carbon neutral economy.Hydrocarbon-producing microorganisms have low hydrocarbons yield and require the consumption of large amounts of carbohydrate feedstock.Therefore,it is of great significance to use the most available lignocellulose feedstock for the production of hydrocarbons.Lignocellulose must be pretreated before effective enzymatic saccharification to produce fermentable monosaccharides,and hydrocarbon-producing microorganisms must have a strong tolerance to the inhibitors produced by pretreatment.Corynebacterium glutamicum is an important host strain for the production of hydrocarbons from lignocellulose feedstock due to its unique inhibitor tolerance and complete genetic operating system.In this study,the conventional industrial strain Corynebacterium glutamicum S9114 was used as the starting strain.Firstly,the AAR(acyl-acyl carrier protein ACP reductase)-ADO(aldehyde deformylating oxygenase)hyrdocarbons synthesis pathway from cyanobacteria was introduced into C.glutamicum and a hydrocarbon-producing strain was successfully constructed.After multi-level metabolic modification and optimization of the constructed hydrocarbons synthesis pathway,it was found that increasing the supply of cofactor NADPH could increase the hydrocarbons production by about 8.3%compared with the original strain.Next,in order to improve the hydrocarbons production of C.glutamicum,the pathway of fatty acid decarboxylation was constructed in C.glutamicum.The prominent metabolic characteristic of C.glutamicum is the lack of phosphatidate phosphatase(PAP)and diacylglyceryl acyltransferase(DGAT),which makes it impossible to synthesize microbial lipids such as triacylglycerol,and a large number of fatty acids accumulates in the extracellular space.In order to efficiently utilize fatty acids,the fatty acid decarboxylase OleTMc from Macrococcus caseolyticus was heterologously expressed in C.glutamicum to construct a pathway of directly decarboxylating free fatty acids to generate hydrocarbons.To avoid the damage of the transmembrane transport of hydrocarbons to the phospholipids and proteins of the cell membrane,the fatty acid decarboxylase OleTMC was secreted to the extracellular through the Sec pathway,and the yield of hydrocarbons was successfully increased to about 3 times of that when OleTMC was expressed intracellularly.Then the dual synthesis pathways,including the AAR-ADO pathway and the OleT secreting expression pathway.were integrated into one C.glutamicum strain to form an engineered C.glutamicum strain with dual synthesis pathways of hydrocarbons.The hydrocarbons production was increased to 3.3 folds of the engineered strain with AAR-ADO pathway and 6.7 folds of the engineered strain with the secreting expression OleT.Finally,by optimizing the fermentation conditions of the strain with dual synthesis pathways,the production of hydrocarbons was increased by 81.3%to 29.1 mg/L.The preliminary cellulosic hydrocarbons generation was carried out in the corn stover hydrolysate,and the yield reached 10.8 mg/L,which successfully realized the production of hydrocarbons from lignocellulose biomass and laid an important application foundation for the industrial production of cellulosic hydrocarbons.