Biotransformation And Microbial Consortium Fermentation To Produce 1,5-Diaminopentane

1,5-diaminopentane,also known as cadaverine,is a biogenic diamine.Cadaverine has important industrial applications,it can polymerize with dicarboxylic acids and yield biobased polyamides,polyamide（PA）5×,with excellent properties compared with traditional polyamides,which is expected to substitute petroleum-based polyamides to solve problems like environmental pollution and resource shortage.The biosynthesis of cadaverine is mainly divided into bio-fermentation and bio-transformation,in which the main problems to be addressed are as follows:first is lysine decarboxylase as the key enzyme to synthesize cadaverine is low activity under alkaline conditions and stability;second is exogenous addition of pyridoxal 5′-phosphate（PLP）is needed to improve lysine decarboxylase activity in reaction system;third is higher concentration of intracellular cadaverine can induce closing of porins,which affects bacterial activity.In view of above problems,the works of this paper are as follows:First,the extracellular co-immobilization of lysine decarboxylase Cad A-EK and coenzyme PLP was studied.Barium alginate combined with polyethylenimine（PEI）to successfully prepare multifunctional carriers,one step purification and immobilization of Cad A-EK was realized under the function of Ba²⁺and His tag.PEI not only helps PLP immobilized on carriers,but also improves the stability of lysine decarboxylase.Finally,co-immobilization of enzyme and coenzyme was achieved.The catalytic efficiency,stability and kinetic parameters of immobilized enzymes were characterized,and the continuous flow microreactor catalyzing lysine to cadaverine was prepared.The results showed that the purification process for lysine decarboxylase was highly specific,immobilized enzymes exhibited preferable tolerance to high temperature,p H,organic solvent,high storage stability in 4℃and catalytic efficiency with the addition of PEI.Then,to solve the accumulation of cadaverine in cell,lysine/cadaverine antiporter（Cad B）was overexpressed,Cad A-EK and Cad B was combined with the help of Spy Tag/Spy Catcher to form transmembrane protein complex to efficiently transport in and out of lysine and cadaverine.As the insufficient supply of cofactor in E.coli,exogenous gene pdx ST introduced could synthesize PLP to improve catalytic efficiency of lysine decarboxylase,cofactor self-sufficient biocatalyst was realized.Results showed that the recombinant E.coli BL-ABST-Spy catalyzed>97%0.1mol/L lysine to cadaverine in 40 min without exogenous PLP addition.Besides,the optimal recombinant E.coli BL-ABST-Spy above constructed,was cocultured with engineering C.glutamicum cgl-FDK,which could utilize glycerol as sole carbon source to produce lysine,finally 1.16 g/L cadaverine was obtained.After fermentation optimization,the yield reached 2.3 g/L in 48 h under inoculation conditions,3:1（BL-ABST-Spy:cgl-FDK（v/v））,temperature 30℃and p H 6,the yield of cadaverine improved to 5.49 g/L with NH₄Cl as nitrogen source and C/N 3:2.Then under optimal conditions,the microbial consortium produced 9.3 g/L cadaverine in 126 h,0.074 g/（L·h）.