Cloning And Expression Of L-Aspartate α-Decarboxylase Gene In C.Crenatum

A method of pantothenate acid and pantoate HPLC detection in transformation solution was built.The best condition for HPLC was showed as below:HPLC column Agilent Zorbax SB-Aq(5μm,4.6×250mm) was used and column temperature was 30℃.Mobile phase 0.02mol/L KH₂PO₄-CH₃CN(V:V=9:1)pH5.5 was adopted with flow rate of 1.0mL/min.Detecting wavelength was 200nm with reference wavelength of 600nm.And the injection volume was 20μL.To use this method,the retention time RSD of pantothenate acid and pantoate were 0.027%,0.040%respectively and peak area RSD were 0.29%,0.19%.Study of linear range showed:the linear equation of pantothenate acid was y=125393.43x+14429.43 with R²=0.9988 and linear range of 0.100g/L-2.000g/L;The linear equation of pantoate was y=1197.97x-113.86 with R²=0.9992 and linear range of 0.100g/L-5.000g/L. The recovery rate of these two substances were 87.22%～111.80%and 102.25%～91.32%respectively.L-aspartateα-decarboxylase was an important enzyme to produce β-Ala.The Corynebacterium crenatum panD gene,which encodes this enzyme,was cloned into plasmid pEKEx2 and electrotramsformed into the same strain to overexpress.The DNA sequencing result showed 99% homology with Corynebacterium glutamicum with 100%homology protein. Electroporation transformation method was improved and the best condition for transformation was 2.5kV,200Ω,25μF,5ms with incubation time of 3h and Kan concentration of 30μg/L,leading to a higher transformation efficiency of 1.81 cfuμg^-1DNA。panD of C.crenatum/pEKEx2-panD was overexpressed and lead to aβ-Ala production of 76.47mg/L.study of induction show inducer was needed in minimal medium,however little effection was found in complete medium.The best inducing temperature was 28℃.Best addition concentration of V_H was 10mg/L.L-Asp and pantoate were used by the engineering strain to produce pantothenate acid with a yield concentration of 36.20μmol/L.β-Ala production was increased to 398.57μmol/L when the pantothenate acid synthesization pathway was inhibited.