Functional Identification And Site-directed Mutagenesis Of Novel L-lysine Decarboxylase Genes For The Biosynthesis Of Pentanediamine From The Subtropical Soil Microorganisms

L-lysine decarboxylase?EC 4.1.1.18?is a highly specific enzyme that catalyzes the decarboxylation of L-lysine to pentanediamine with the cofactor of pyridoxal phosphate.It is also a key enzyme for the biosynthesis of pentanediamine.At present,the L-lysine decarboxylase enzyme activity is not high,and the stability is poor,which is the bottleneck problem of the inefficiency of the biosynthesis system.Therefore,it is of great practical significance and industrial application value to extract new and excellent L-lysine decarboxylase from environmental microorganisms.A metagenome library was constructed by directly extracting the metagenomic DNA of subtropical soil samples.Based on the functional screening strategy,a new L-lysine decarboxylase gene ldc1E?GenBank accession number:KX463450?was screened from the library.Sequence analysis,functional domain analysis and evolutionary relationship analysis indicated that Ldc1E belongs to type III pyridinium phosphate-dependent amino acid decarboxylase and aspartate transferase superfamily.Up to now,there is no report on the use of metagenomics technology to complete the research on the function of new L-lysine decarboxylase gene.The ldc1E gene was cloned and overexpressed in E.coli expression system.After purification by Ni-NTA and ultrafiltration desalination,the Ldc1E protein with higher purity was obtained.The enzymatic properties indicated that Ldc1E has ability to catalyze the L-lysine to pentanediamine;its optimum temperature is 40°C,the optimum pH is 6.5;K_m value of 1.08 mM,V_max value of 23.13?M/min,k_catat value of5.09/s,and the specific activity is 1.53 U/mg;the Ldc1E protein is stable to store at30°C for 1 h,the enzyme activity is maintained at about 80%;and Ldc1E is also stable to store at pH 6.5-7.0 for 24 h,the enzyme activity is maintained at about 80%;5 mM Mg²⁺and Ca²⁺have weak activation effect on Ldc1E;and Ldc1E has good resistance to chemical reagents EDTA and DMSO;in addition to the catalytic effect on L-lysine,it also has a certain catalytic effect on L-arginine and L-ornithine.The study results for the key amino acids binding to Ldc1E with cofactor and substrate indicated that Ldc1E has a certain conserved type in the spatial structure and active center.The enzymatic activities of mutants Y308A,E526D,V524A and L536A were significantly lower than the wild-type enzyme,indicating that Y308,E526,V524 and L536 had significant effects on the catalytic activity of the enzyme.The enzymatic activities of mutants K367A,K367R,S364A,H366A,E526A and W333A were completely lost,indicating that K367,S364,H366,E526 and W333 are key the binding sites for Ldc1E and play a crucial role in catalytic activity.We hypothesized that there is an interaction between the side chain of K367,S364,H366W333 and Y308 with the cofactor PLP.The mutation of these sites could change the spatial conformation,thus making Ldc1E cannot catalyze the substrate.There is an interaction between the side chains of E526,V524 and L536 with the substrate,and the mutations at these three sites lead to the instability of the substrate's spatial conformational at the active site,thereby affecting the efficiency of the Ldc1E catalytic the substrate.The strain L-2 with highly L-lysine decarboxylase activity was selected by pure culture technique.Morphological observations showed that the L-2 strain belonged to gram-negative rod-shaped bacteria with a size of about 2.1?m×0.75?m.The L-lysine decarboxylase L-2 strain was named Klebsiella quasipneumoniae subsp.similipneumoniae L-2 by 16S rDNA gene identification,physiological and biochemical identification,and sequence alignment at the genomic level.The L-lysine decarboxylase genes cad1A and ldc1C in Klebsiella quasipneumoniae subsp.similipneumoniae L-2 strain were cloned and overexpressed in E.coli expression system.The highly purified Cad1A and Ldc1C proteins were obtained after purification.Sequence analysis,domain analysis and evolutionary phylogenetic study indicated that the Cad1A belongs to the inducible enzyme,the Ldc1C belongs to the constitutive enzyme,which all have a typical?/?fold in structure,and belong to the type III pyridoxal-dependent decarboxylase and aspartate transferase superfamily members.The results of enzymatic properties study showed that both Cad1A and Ldc1C have ability to catalyze the L-lysine to pentamediamine,and the conversion ability of Cad1A is higher than that of Ldc1C.The optimum temperature is 40°C,and the optimum pH is 5.5 and 8.0;Cad1A has a K_m value of0.42 mM,V_max value of 36.27?M/min,k_cat value of 199.83/s,and the specific activity of 110.72 U/mg.Ldc1C has a K_m value of 0.69 mM,V_max value of 17.28?M/min,k_cat value 5.49/s,and the specific activity of 2.99 U/mg.Cad1A and Ldc1C are stable at 40°C.The Cad1A is stable under the condition of pH 6.0,and the Ldc1C is stable under neutral alkaline conditions;Cad1A is not dependent on metal ions,but 5 mM Mg²⁺and Ca²⁺have weak activation effect on Ldc1C,they have good tolerance to chemical reagents EDTA and DMSO.In summary,based on metagenomics techniques and pure culture techniques,series of new L-lysine decarboxylase genes were identified from subtropical soils,and their structures were studied,and their functions were analyzed.The results not only enriched the L-lysine decarboxylase resource pool,but also laid the foundation for the industrial application of the enzyme.At the same time,the study of the binding sites of Ldc1E with cofactor and substrate could provide the theoretical basis for molecular modification of L-lysine decarboxylase.