Putrescine, a biogenic diamine, serves as an important component in various polyamides, medicine, and surfactants. One challenge for efficient putrescine production is to construct high-efficiency microbial cells. In this study, we designed the metabolic engineering strategies to significantly enhance the putrescine titer in Corynebacterium crenatum SYPA. First, an optimal synthetic pathway of putrescine comprising arginine decarboxylase (speA) and agmatinase (speB) genes from Escherichia coli was selected and introduced into C. crenatum. For efficient production of putrescine, the expression of agmatinase was optimized through a ribosome binding site regulation strategy. Next, the putrescine yield was furthermore increased by the knockout of snaA, speE, and cgmR to block putrescine degradation and overexpression of exporter CgmA. Additionally, the xylose utilization pathway was constructed for putrescine synthesis. Finally, the titer of putrescine was 41.5, 36.8, and 33.4 g/L from glucose, mixed sugar, and simulated wheat straw hydrolysates by fed-batch culture for 72 h, respectively. The yield was 0.18 g/g glucose, 0.15 g/g sugar, and 0.14 g/g sugar, respectively. To our knowledge, those results were the highest putrescine titers ever reported in the engineering of C. crenatum and Corynebacterium glutamicum. The engineering strains had the potential to produce putrescine from biomass hydrolysates.

The utilization of unnatural nicotinamide cofactors for reactions catalyzed by oxidoreductases has gained increasing interest. Totally synthetic nicotinamide cofactor biomimetics (NCBs) are cost-effective and convenient to synthesize. Thus, it has become increasingly important to develop enzymes that accept NCBs. Here, we have engineered SsGDH to favor a newly synthesized unnatural cofactor 3-carbamoyl-1-(4-carboxybenzyl) pyridin-1ium (BANA + ). Using in situ ligand minimization tool, sites 44 and 114 were identified as hotspots for mutagenesis. All the double mutants demonstrated 2.7-7.7-fold improvements in catalytic activity, and the best double mutant E44D/E114 L exhibited 10.6-fold increased catalytic efficiency toward BANA + . These results provide valuable information for the rational engineering of oxidoreductases with versatile NCBs-dependency, as well as the design of novel biomimetic cofactors.

Background Agmatine is a member of biogenic amines and is an important medicine which is widely used to regulate body balance and neuroprotective effects. At present, the industrial production of agmatine mainly depends on the chemical method, but it is often accompanied by problems including cumbersome processes, harsh reaction conditions, toxic substances production and heavy environmental pollution. Therefore, to tackle the above issues, arginine decarboxylase was overexpressed heterologously and rationally designed in Corynebacterium crenatum to produce agmatine from glucose by one-step fermentation. Results In this study, we report the development in the Generally Regarded as Safe (GRAS) l-arginine-overproducing C. crenatum for high-titer agmatine biosynthesis through overexpressing arginine decarboxylase based on metabolic engineering. Then, arginine decarboxylase was mutated to release feedback inhibition and improve catalytic activity. Subsequently, the specific enzyme activity and half-inhibitory concentration of I534D mutant were increased 35.7% and 48.1%, respectively. The agmatine production of the whole-cell bioconversion with AGM3 was increased by 19.3% than the AGM2. Finally, 45.26 g/L agmatine with the yield of 0.31 g/g glucose was achieved by one-step fermentation of the engineered C. crenatum with overexpression of speAI534D. Conclusions The engineered C. crenatum strain AGM3 in this work was proved as an efficient microbial cell factory for the industrial fermentative production of agmatine. Based on the insights from this work, further producing other valuable biochemicals derived from l-arginine by Corynebacterium crenatum is feasible.

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