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Clostridium thermocellum is an anaerobic, thermophilic bacterium. C. thermocellum has garnered research interest due to its cellulolytic and ethanologenic abilities, being capable of directly converting a cellulosic substrate into ethanol by consolidated bioprocessing. This makes it useful in converting biomass into a usable energy source. The degradation of the cellulose is carried out in the bacterium by a large extracellular cellulase system called a cellulosome, which contains nearly 20 catalytic subunits. The cellulase system of the bacterium significantly differs from fungal cellulases due to its high activity on crystalline cellulose, being able to completely solubilize crystalline sources of cellulose, such as cotton. However, there are some shortfalls in applying the organism to practical applications due to it having low ethanol yield, at least partially due to branched fermentation pathways that produce acetate, formate, and lactate along with ethanol. There is also evidence of inhibition due to the presence of hydrogen and due to agitation. Some recent research has been directed to optimizing the ethanol-producing metabolic pathway in hopes of creating more efficient biomass conversion.
A genetically engineered strain for high-yield d-allulose and a construction method and application thereof
This invention relates to the fields of synthetic biology and metabolic engineering, and particularly to a genetically engineered strain producing high levels of D-allulose, its construction method, and its applications. The strain uses *Escherichia coli* as the starting strain and constructs a phosphorylation / epimerization / dephosphorylation synthetic pathway by introducing *AlsE* from *E. coli* and *CtA6PP* from *Clostridium thermocellum*. Competitive metabolic genes *pgm*, *zwf*, *pfkA*, and *rpiB* are knocked out to enhance carbon flux directing. Furthermore, the expression of *AlsE* and *CtA6PP* is precisely regulated at the transcriptional and translational levels through promoter engineering and RBS engineering to obtain the optimal expression combination. The engineered strain achieves a D-allulose yield of 5.26 g / L in in vitro fermentation and 33.05 g / L in a 5L fed-batch fermenter, exhibiting advantages of high yield, stability, and ease of scale-up, making it suitable for industrial production of D-allulose.
Owner:HENAN ZHONGDA HENGYUAN BIOTECH CO LTD