Poster: Metabolic Engineering

Abs # 1012: Expression of E. coli L-aspartate-a-decarboxylase in plants

Presenter:	Fouad, Walid M, wfouad@ufl.edu
Authors	Fouad, Walid M (A)   Rathinasabapathi, Bala  (A)
Affiliations:	(A): University of Florida, Horticultural Sciences Department.

The prokaryotic L-aspartate-a-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 a and b subunits of 2.8 and 11 KDa, respectively. The active enzyme contains three each of a and b subunits and one unprocessed p peptide of 13.8 KDa. ADC decarboxylates L-aspartic acid generating b–alanine, an essential precursor for panthothenic acid. b–Alanine is also the precursor for an osmoprotectant b–alanine betaine, found in the plant family Plumbaginaceae. Our goal is to utilize E. coli panD gene to engineer b–alanine over-production in transgenic plants. The objective of the current study is to test whether E. coli panD expressed in an eukaryotic system will give rise to processed and active ADC enzyme. The E. coli panD gene was expressed in tobacco and arabidopsis under the control of the constitutive 35S-promoter. Several transgenic lines were recovered based on selection for kanamycin-resistance (Kan^r) coded by AAC-III gene. The panD insertion was confirmed by PCR screening of primary transformants. In some of the transgenic lines the single panD gene insertion was confirmed by Southern blot analysis and by co-segregation with the Kan^r phenotype. The transgenic lines showed varying levels of panD expression at the RNA level while the AAC-III expression was similar in most of the lines. This suggests a post-transcriptional control for the panD mRNA. The E coli panD gene was over-expressed in pET Blue-2 system and the ADC enzyme was purified for developing polyclonal antibodies. These antibodies will be used to study the processing of the ADC p peptide in transgenic plants.