A five carbon linear chain diamine, cadaverine (1,5-diaminopentane), is an important platform chemical having many applications in chemical industry. Bio-based production of cadaverine from renewable feedstock is a promising and sustainable alternative to the petroleum-based chemical synthesis. Here, we report development of a metabolically engineered strain of Escherichia coli that overproduces cadaverine in glucose mineral salts medium. First, cadaverine degradation and utilization pathways were inactivated. Next, L-lysine decarboxylase, which converts L-lysine directly to cadaverine, was amplified by plasmid-based overexpression of the cadA gene under the strong tac promoter. Furthermore, the L-lysine biosynthetic pool was increased by the overexpression of the dapA gene encoding dihydrodipicolinate synthase through the replacement of the native promoter with the strong trc promoter in the genome. The final engineered strain was able to produce 9.61 g L(-1) of cadaverine with a productivity of 0.32 g L(-1) h(-1) by fed-batch cultivation. The strategy reported here should be useful for the bio-based production of cadaverine from renewable resources.

Metabolic engineering of Escherichia coli for the production of cadaverine:A five carbon diamine.

Membrane processes can be integrated with fermentation for the selective separation of the products from the fermentation broth. Sterilization with saturated steam under pressure is the most widely used method; however, data concerning heat sterilization applicability to polymeric ultrafiltration (UF) membranes are scarcely available. In this study, the effect of the sterilization process on the filtration performance of a commercial polyvinylidene difluoride (PVDF) hollow fiber UF membrane was evaluated. Membrane modules were constructed and sterilized several times in an autoclave. Pure water flux tests were performed, to assess the effect of heat sterilization on the membrane’s pure water permeance. Dextran rejection tests were performed for the characterization of membrane typical pore size and its fouling propensity. Filtration performance was also assessed by conducting filtration tests with real fermentation broth. After repeated sterilization cycles, pure water permeance remained quite constant, varying between approx. 830 and 990 L·m−2·h−1·bar−1, while the molecular weight cut-off (MWCO) was estimated to be in the range of 31.5–98.0 kDa. Regarding fouling behavior, the trans-membrane pressure increase rate was stable and quite low (between 0.5 and 7.0 mbar/min). The results suggest that commercial PVDF UF membranes are a viable alternative to high-cost ceramic UF membranes for fermentation processes that require heat sterilization.

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The Effect of Heat Sterilization on Key Filtration Performance Parameters of a Commercial Polymeric (PVDF) Hollow-Fiber Ultrafiltration Membrane

Microbial Production of Diamines

Bio-based cadaverine, a crucial monomer for the production of bio-nylon 5X, can be synthesized during the bioconversion of l-lysine HCl relying on lysine decarboxylases. The rationally designed lysine decarboxylase ΔLdcEt3 has exhibited outstanding alkaline stability in pH 8.0 (half-life: 362 h); however, its catalytic activity still needs to be improved to meet the requirements of industrial cadaverine production. A novel ionozyme strategy can create a preferable reaction microenvironment, affect the intermediate formation, and/or improve the enzymatic stability and activity. Therefore, ionozymes ΔLdcEt3-[Emim]Cl and ΔLdcEt3-[Ch][Ser] have been successfully developed for efficient cadaverine production in this study. The results showed that the catalytic activities of ΔLdcEt3-[Emim]Cl and ΔLdcEt3-[Ch][Ser] improved 124.2 and 116.2%, respectively. Meanwhile, the catalytic efficiencies (kcat/Km) of ΔLdcEt3-[Emim]Cl and ΔLdcEt3-[Ch][Ser] were also increased by 1.5- and 1.2-fold, respectively. Particle size, circular dichroism, and Raman spectrum analyses showed that [Emim]Cl and [Ch][Ser] could affect short-range attractions related to the aggregation state and change the secondary structure. Protein surface analysis demonstrated that the addition of ionic liquids changed the hydrophobicity of ΔLdcEt3. In addition, isothermal titration calorimetry and molecular docking revealed that [Emim]Cl and [Ch][Ser] could promote the protein–ligand complexation during the enthalpy-driven l-lysine and ΔLdcEt3 binding process, which was confirmed by molecular dynamics. Therefore, ionozymes ΔLdcEt3-[Emim]Cl/[Ch][Ser] provide a novel possibility for high-level cadaverine production. 

Ionozymes for Efficient Synthesis of Cadaverine: Offering a Sustainable Way for Bio-nylon 5X Production

Nylon is a polyamide material with excellent performance used widely in the aviation and automobile industries, and other fields. Nylon monomers such as hexamethylene diamine and other monomers are in huge demand. Therefore, in order to expand the methods of nylon production, we tried to develop alternative bio-manufacturing processes which would make a positive contribution to the nylon industry. In this study, the engineered E. coli-overexpressing Lysine decarboxylases (LDCs) were used for the bioconversion of l-lysine to cadaverine. An integrated fermentation and microfiltration (MF) process for high-level cadaverine production by E. coli was established. Concentration was increased from 87 to 263.6 g/L cadaverine after six batch coupling with a productivity of 3.65 g/L-h. The cadaverine concentration was also increased significantly from 0.43 g cadaverine/g l-lysine to 0.88 g cadaverine/g l-lysine by repeated batch fermentation. These experimental results indicate that coupling the fermentation and membrane separation process could benefit the continuous production of cadaverine at high levels.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/elsc.202000099

Ge Hu

Papers

Coupling the fermentation and membrane separation process for polyamides monomer cadaverine production from feedstock lysine.