Poster: Metabolic engineering

Abs # 285: Towards increasing pantothenate through pathway engineering in Brassica napus

Presenter:	Chakauya, Ereck , ec259@cam.ac.uk
Authors	Chakauya, Ereck  (A)   MacDonald, Mary V.  (B)   Smith, Alison G.  (A)
Affiliations:	(A): Plant Science, University of Cambridge, CB2 3EA, UK (B): Biogemma UK, Cambridge Science Park, CB4 4GZ, UK
Web Site:	http://www.plantsci.cam.ac.uk/Smith/Index.html

Pantothenate (vitamin B₅) is the precursor of coenzyme A and acyl carrier protein (ACP). These are essential cofactors in biological reactions including Krebs cycle and production, transportation and release of energy from fatty acids. Plants and microorganisms synthesise the vitamin, but animals get it from their diet. The commercial product is produced by chemical synthesis for pharmaceuticals and feed additives. This is expensive and requires high optical resolution of racemic intermediates. Thus, other production routes have been proposed including plants and microorganisms. The E. coli pathway is well-studied consisting of four genes (panB, panC, panD and panE encoding KPHMT, PS, ADC and KPR). We have cloned all four genes and in order to test the possibility of using them to generate plants with increased levels of pantothenate. Transgenic plants were generated by cloning the genes in a binary vector under a CaMV35S promoter and transformation into Brassica napus. Several transgenic plants were recovered by kanamycin selection and then confirmed by PCR. Segregation analysis shows normal Mendelian inheritance and linkage segregation in selfed F₁ progeny. Co-segregation of pantothenate genes indicated 100% linkage with the NPTII gene. Further analysis of the T₁ lines by RT-PCR could detect the mRNA transcript in some lines suggesting that the genes are expressed in seedlings. The T-DNA copy number is being confirmed by Southern blotting. Further biochemical analysis of these transgenic plants including pantothenate levels and oil composition of the seeds is in progress. In addition, F₁ hybrid lines overexpressing different enzymes are being generated to see if there is an additive effect. The implication of this on increasing pantothenate is being investigated.