Multilevel Metabolic Engineering of Bacillus amyloliquefaciens for Production of the Platform Chemical Putrescine from Sustainable Biomass Hydrolysates

Putrescine is an important C4 platform chemical with extensive applications in bioplastics, pharmaceuticals, and agrochemicals. In this study, multilevel metabolic engineering of Bacillus amyloliquefaciens was performed to achieve the sustainable production of putrescine from biomass hydrolysates rich in glucose and xylose. First, the ornithine decarboxylase pathway was reconstructed in B. amyloliquefaciens by introducing an ornithine decarboxylase from Escherichia coli, resulting in the efficient transformation of ornithine to putrescine. The overall putrescine synthesis process was then recast into three modules including ornithine synthesis module, NADPH synthesis module, and ATP supply module. In the ornithine synthesis module, deletion of ornithine carbamoyltransferase gene argF and arginine repressor gene ahrC, and overexpression of N-acetylglutamate synthase gene argA significantly enhanced putrescine production. Using a cofactor engineering strategy, overexpression of glucose-6P dehydrogenase gene zwf and pyruvate kinase gene pyK proved optimal for putrescine production through NADPH synthesis and ATP supply modules, respectively. Finally, all beneficial genetic manipulations were combined in recombinant strain HZ/CFK Delta FC/pHY-argA, and its putrescine titer (5.51 g/L), productivity (0.11 g/(L h)) and yield (0.14 g/g) from xylose were much higher than that previously reported using xylose substrate. Using hydrolysates of Miscanthus floridulus, higher putrescine titer (6.76 g/L), productivity (0.14 g/ (L h)) and carbohydrate yield (0.17 g/g) were achieved. Thus, multilevel metabolic engineering strategies, including pathway reconstruction, modular engineering, and cofactor engineering, were effective for improving putrescine production. This study describes a proof of concept demonstration of multilevel metabolic engineering of B. amyloliquefaciens for putrescine production from sustainable biomass hydrolysates.