Synthesis Of Putrescine From L-arginine Using Recombinant Escherichia Coli Whole Cells

Putrescine,also known as 1,4-butylenediamine,is an aliphatic amine containing two amino groups.Widely used in the fields of medicine,agriculture and industry,it is one kind of small molecular compounds with high value.For example,putrescine plays an important role in the regulation of intestinal microorganisms,which has good fresh-keeping effect on postharvest crops,and could be used to produce high-quality industrial plastic nylon-4,6.Since the biosynthesis of putrescine is not yet mature,this study attempts to explore the biosynthesis of putrescine by whole-cell catalysis with L-arginine as the substrate and Escherichia coli as the host.The main results are shown as follows:(1)A strategy of using E.coli whole cells to catalyze the synthesis of putrescine from L-arginine in a dual-enzyme coupling method is proposed.L-arginine is first converted into agmatine under the catalysis of arginine decarboxylase(ADC),releasing a molecule of CO₂,and then agmatine is hydrolyzed to be urea and the product putrescine under the action of agmatine hydrolase(AUH).Five plasmids with different copy numbers were selected to ligate the double enzyme genes to five plasmids in different sequences,and the genes were recombined to construct ten engineered bacteria to balance the catalytic efficiency of the double enzyme reaction.Finally,the catalytic ability of each strain was compared,and the best strain E.coli BL21(DE3)/p ACYCDuet-spe B-spe A was obtained and named PUT4.(2)In the shaking flask fermentation stage,the induced expression conditions and the whole cell catalysis conditions were optimized respectively.The optimal induction conditions are as follows:the medium is SOC medium,the induction time is OD₆₀₀is 0.8,the concentration of inducer is 0.6 mM,the induction temperature is 20°C;the optimal whole-cell catalysis conditions are as follows:pH is 9.5,temperature is 45°C,rotation speed is 150 rpm,Mg²⁺concentration is 6 mM,substrate concentration is 20 mM.After optimization,the conversion rate increased by 17%and 10%,respectively.(3)Using PUT4 as the starting strain,the change of putrescine production over time in12 h was measured.The ADC was modified by molecular docking and the mutant D502A/D531A/D535A was constructed.The production of putrescine reached 17.1 mM(2.97g·L^-1),and the 12-hour space-time yield was 0.25 g·L^-1·h^-1.