Metabolic Engineering Of Bacillus Amyloliquefaciens For ?-PGA Overproduction

Poly-?-glutamic acid(?-PGA)is an important natural occurring polymer.Due to its favorable features,it has been widely used in the fields of foods,cosmetics,medicines and materials,et al.B.amyloliquefaciens LL3 is a glutamic acid-independent ?-PGA producing strain and it has the potential to be used for industrial production.However,its ?-PGA production in the wild-type strain is relatively low.In this study,we aimed to improve the ?-PGA production by metabolic engineering strategy.The research strategies include three parts: modular pathway engineering method,pathway engineering for sucrose metabolism and pathway engineering for glutamic acid synthesis.Eight ?-PGA-synthesis-related metabolic pathways were engineered to improve ?-PGA production including pathways for ?-PGA degradation,cell respiration,extracellular protein and extracellular protease synthesis,microbial polysaccharides synthesis,small molecular by-products synthesis,autoinducer synthesis,glutamic acid precursor synthesis and ?-PGA synthesis.The final engineered NK-anti-rocG strain(with the deletion of epsA-O(responsible for extracellular polysaccharide synthesis),sac(responsible for levan synthesis),lps(responsible for lipopolysaccharide synthesis),pta(encoding phosphotransacetylase),pgdS(encoding poly-?-glutamate depolymerase),cwlO(encoding cell wall hydrolase),luxS(AI-2 synthetase)and the expression of anti-rocG sRNA)could produce 11.04 g/L ?-PGA in flask,which was 2.91-fold higher than that of the control strain.Its ?-PGA purity was also increased from 78.6% to 95.2%.The highest ?-PGA titer of the NK-anti-rocG strain was 20.3 g/L in a 5-L fed-batch cultivation.Two strategies were used to engineer the native sucrose metabolic pathway.The ?-PGA production was not increased after engineering its inducible sucrose metabolic pathway to the constitutive expression sucrose metabolic pathway.However,after replacing the native sucrose metabolic pathway to an energy-saving sucrose metabolic pathway,the finally obtained 3?-CES strain exhibited ?-PGA production increased about 38.5% compared with that of the control strain.We recruited new metabolic engineering strategies to improve B.amyloliquefaciens glutamic acid synthesis.After introducing an energy-saving glutamic synthesis pathway,?-PGA production of the NK-1(pHT315-gdh)strain increased 9.14% compared with that of the control strain.After introducing the metabolic toggle switch,the obtained NK-PO1(pHT01-xylR)strain ?-PGA production increased 66.2% compared with that of the control strain.