METABOLIC ENGINEERING OF CULTURED TOBACCO CELLS

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Construction of a gene expression system in tobacco cultured cells (BY2) was studied. A 925 bp promoter fragment of a heat-shock protein Nicotiana tabacum gene (HSP18.2) of Arabidopsis thaliana showed clear heat-shock response of expression of the beta-glucuronidase (GUS) reporter gene in BY2 cells. Similar results were observed in a 500ml flask and 3-l jar fermentor.Isolation of strong promoters in BY2 cells was tried. cDNA clones of which mRNA level is high in log phase cells and copy number in genome is low were isolated. These clones showed high homology with F1-ATPase (mitochondria type), elongation factor 1-alpha, and a gene with unknown function of A.thaliana (clone 27), respectively. A5'-flanking region of clone 27 showed 6.2 times of promoter activity of the CaMV35S promoter in BY2 cells.Three cDNA clones which are expressed in stationary growth phase of BY2 cells were isolated by a differential screening. There clones showed high sequence homologies to alcohol dehydrogenase, pectin esterase and extensin. Promoters of these genes will be useful in gene expression in high cell density culture.