Poster: Metabolic engineering

Abs # 298: Processing of bacterial L-aspartate-α-decarboxylase in transgenic tobacco

Presenter:	Fouad, Walid , wfouad@ufl.edu
Authors	Rathinasabapathi, Bala  (A)   Fouad, Walid  (A)
Affiliations:	(A): University Of Florida

The prokaryotic L-aspartate-α-decarboxylase (ADC, EC 4.1.1.11 encoded by panD gene) is a pyruvoyl-dependent enzyme that undergoes an unusual self-processing. This processing gives rise to α and β subunits of 2.8 and 11 kDa, respectively. The active enzyme with MW of 55.4 KDa contains 3 each of α and β subunits and one unprocessed π peptide of 13.8 kDa. Our goal is to utilize E. coli panD gene to engineer β-alanine overproduction in transgenic plants. The objective of the current study is to test whether E. coli panD expressed in an eukaryote will give rise to processed and active ADC enzyme. The E. coli panD gene was overexpressed in tobacco under the control of the constitutive 35S-promoter. Several homozygous transgenic lines were recovered based on selection for kanamycin resistance coded by AAC-III gene. The transgenic lines showwed varying levels of panD expression at the RNA level. The E.coli panD gene was also overexpressed in pET Blue-2 expression system and the ADC protein was purified. Polyclonal antibodies raised against native ADC recognized ADC protein in immunoblot and immunoprecipitation experiments. When transgenic lines grown in a greenhouse were screened for ADC protein expression using immunoblots, the ADC polyclonal antibodies recognized a 13.8 kDa protein band corresponding to the unprocessed π peptide.