Efficient Expression And Directed Molecular Evolution Of Lysine Decarboxylase And Its Process Characteristics For Cadarverine Synthesis

Previous research in cadaverine production focused mainly on two lysine decarboxylases: Escherichia coli Cad A and Ldc C. However, their low acitivity and poor stability in higher p H condition make it very difficult produce cadaverine efficiently. This paper studied a novel lysine decarboxylase from Klebsiella oxytoca. Genetic engineering, protein engineering, fermentation engineering and enzyme engineering were comprehensivly applied to improve cadaverine production efficiency. Multi-tools in molecular biology were used to improve the heterologous expression of lysine decarboxylase gene at transcriptional level and translation level. The enzyme activity and stability in high p H environment was improved further by modifying target gene structure. The cadaverine synthesis process optimization was also conducted with the improved lysine decarboxylase from Klebsiella oxytoca. After these optimizations, the cadaverine systhesis efficiency was greatly improved. The main contents and the results are as the following:1. The lysine decarboxylase gene was amplified from the K. oxytoca genomic DNA, and cloned into the plasmid p UC18 to create p UC18-KOldc. Then p UC18-KOldc was transformed into E. coli K12 MG1655 to attain strain E. coli LN18. Codon and the promoter optimization were subsequently conducted. Codon optimization increased cadaverine production to 6.6 g/L, 50% higher than the control. With tac promoter to replace lac promoter, the recombinant strain can produce 7.6 g/L of cadaverine.2. Further improvement to the K. oxytoca lysine decarboxylase system was performed by randomly screening ribosome binding sequences in order to improve the translation of the lysine decarboxylase m RNA. E. coli LN1103 was identified as the highest cadaverine production strain from one thousand mutants, which was 34% higher than the starting strain E. coli LN24 and 2.3 fold of E. coli LN18. Error-prone PCR was also used to increase lysine decarboxylase activity and stability based on the high throughput screecing platform in alkli condition. From another one thousand mutants screening, E.coli LN3014 was the highest one to produce cadaverine, 20% higher than the starting strain E.coli LN1103 and 2.5 fold of E. coli LN18. Five top cadaverine producing strains from error-prone PCR improvement were chose to analysis the amino acids compositions and positions of the lysine decarboxylase. Though five top producers were respectively corresponding to the amino acid mutation alone, the mutant sites were randomly distributed across the lysine decarboxylase.3. When the p H increased from 6.0 to 8.0, the Vmax of the wide type lysine decarboxylase from E. coli LN18 decreased 60%, while the mutant one from E. coli LN3014 decreased only 7.0%. The Km of the wide type and the mutant one is almost the same. The relative activity from the E. coli LN3014 can keep 85% at the p H 8.0 for 36 h, while lysine decarboxylase Cad A from E.coli lost all activity at p H 8.0.4. The fermentation condition was optimized at a 10 L fermentor scale to improve the lysine decarbonxylase acitivty. Firstly, by orthogonal optimizing the medium, the lysine decarbonxylase activity of the E. coli LN3014 fermentation broth increased by 16% to 128 U/g in the shake flask. Batch fermentation and fed batch fermentation process were then optimized in 10 L fermentor. After the improvements, the lysine decarbonxylase activity and OD600 of E. coli LN3014 could attain 240 U/g and 82 respectively which was much higher than literatures reported.5. The biomass addition amounts, lysine concentration and co-enzyme amount were studied to optimize the catalytic process for cadaverine production. After these factors optimization, the cadaverine productivity could attain 887 mmol /(g-cell·h) without adjusting the system p H. Cells immobilization of E. coli LN3014 for cadaverine production was also studied. The enzyme activity recovery rate attained 45% when the polyvinyl alcohol concentration and the biomass amount was 5% and 3% respectively. Immobilized cells could be used 30 times repeatedly to produce cadaverine,and the yield from lysine to cardaverine was stable. The optimum p H of the lysine decarboxylase based on the immobilized cells was 6.0, higher than that of the free one. The lysine decarboxylase activity half-life of the immobilized cells and the free one were 30 days and 15 days at 4 oC condition respectively.