/pdf/systems-metabolic-engineering-of-corynebacterium-glutamicum-hgoxy44c.pdf

Systems metabolic engineering of Corynebacterium glutamicum eliminates all by-products for selective and high-yield production of the platform chemical 5-aminovalerate.

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.

Fatty aldehydes (FALs) can be derived from fatty acids (FAs) and related compounds and are frequently used as flavors and fragrances. Although chemical methods have been conventionally used, their selective biotechnological production aiming at more efficient and eco‐friendly synthetic routes is in demand. α‐Dioxygenases (α‐DOXs) are heme‐dependent oxidative enzymes biologically involved in the initial step of plant FA α‐oxidation during which molecular oxygen is incorporated into the Cα‐position of a FA (Cn) to generate the intermediate FA hydroperoxide, which is subsequently converted into the shortened corresponding FAL (Cn‐1). α‐DOXs are promising biocatalysts for the flavor and fragrance industries, they do not require NAD(P)H as cofactors or redox partner proteins, and they have a broad substrate scope. Here, we highlight recent advances in the biocatalytic utilization of α‐DOXs with emphasis on newly discovered cyanobacterial α‐DOXs as well as analytical methods to measure α‐DOX activity in vitro and in vivo.

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/cbic.202100693

α‐Dioxygenases (α‐DOXs): Promising Biocatalysts for the Environmentally Friendly Production of Aroma Compounds

The compounds 5-aminovalerate and δ-valerolactam are important building blocks that can be used to synthesize bioplastics. The production of 5-aminovalerate and δ-valerolactam in microorganisms provides an ideal source that reduces the cost. To achieve efficient biobased co-production of 5-aminovalerate and δ-valerolactam in Escherichia coli, a single biotransformation step from L-lysine was constructed. Firstly, an equilibrium mixture was formed by L-lysine α-oxidase RaiP from Scomber japonicas. In addition, by adjusting the pH and H2O2 concentration, the titers of 5-aminovalerate and δ-valerolactam reached 10.24 g/L and 1.82 g/L from 40 g/L L-lysine HCl at pH 5.0 and 10 mM H2O2, respectively. With the optimized pH value, the δ-valerolactam titer was improved to 6.88 g/L at pH 9.0 with a molar yield of 0.35 mol/mol lysine. The ratio of 5AVA and δ-valerolactam was obviously affected by pH value. The ratio of 5AVA and δ-valerolactam could be obtained in the range of 5.63:1-0.58:1 at pH 5.0-9.0 from the equilibrium mixture. As a result, the simultaneous synthesis of 5-aminovalerate and δ-valerolactam from L-lysine in Escherichia coli is highly promising. To our knowledge, this result constitutes the highest δ-valerolactam titer reported by biological methods. In summary, a commercially implied bioprocess developed for the coproduction of 5-aminovalerate and δ-valerolactam using engineered Escherichia coli.

/pdf/coproduction-of-5-aminovalerate-and-d-valerolactam-for-the-fepim3krxz.pdf

Coproduction of 5-Aminovalerate and δ-Valerolactam for the Synthesis of Nylon 5 From L-Lysine in Escherichia coli

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.

/pdf/the-effect-of-heat-sterilization-on-key-filtration-1203yvmd.pdf

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

Nylon 5 and nylon 6,5 are recently explored as new commercial polyamides, of which the monomer includes δ-valerolactam. In this study, a novel catalytic activity of lysine 2-monooxygenase (DavB) was explored to produce δ-valerolactam from l-pipecolic acid (L-PA), functioning as oxidative decarboxylase on a cyclic compound. Recombinant Escherichia coli BS01 strain expressing DavB from Pseudomonas putida could synthesize δ-valerolactam from l-pipecolic acid with a concentration of 90.3 mg/L. Through the co-expression of recombinant apoptosis-inducing protein (rAIP) from Scomber japonicus, glucose dehydrogenase (GDH) from Bacillus subtilis, Δ1-piperideine-2-carboxylae reductase (DpkA) from P. putida and lysine permease (LysP) from E. coli with DavB, δ-valerolactam was produced with the highest concentration of 242 mg/L. α-Dioxygenases (αDox) from Oryza sativa could act as a similar catalyst on l-pipecolic acid. A novel δ-valerolactam synthesis pathway was constructed entirely via microbial conversion from feedstock lysine in this study. Our system has great potential in the development of a bio-nylon production process. • DavB performs as an oxidative decarboxylase on L-PA with substrate promiscuity. • Strain with rAIP, GDH, DpkA, LysP, and DavB coexpression could produce δ-valerolactam. • This is the first time to obtain valerolactam entirely via biosynthesis from lysine.

Metabolic engineering of Escherichia coli for polyamides monomer δ-valerolactam production from feedstock lysine.

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

Dan Zhou

Papers

Metabolic engineering of Escherichia coli for polyamides monomer δ-valerolactam production from feedstock lysine.

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