CONSTRUCTION AND CHARACTERIZATION OF A 1,3-PROPANEDIOL OPERON

The genes for the production of 1,3-propanediol (1,3-PD) in Klebsiella pneumoniae, dhaB, which encodes glycerol dehydratase, and dhaT, which encodes 1,3-PD oxidoreductase, are naturally under the control of two different promoters and are transcribed in different directions. Thes e genes were reconfigured into an operon containing dhaB followed by d haT under the control of a single promoter, The operon contains unique restriction sites to facilitate replacement of the promoter and other modifications. In a fed-batch cofermentation of glycerol and glucose, Escherichia coli containing the operon consumed 9.3 g of glycerol per liter and produced 6.3 g of 1,3-PD per liter, The fermentation had tw o distinct phases, In the first phase, significant cell growth occurre d and the products were mainly 1,3-PD and acetate. In the second phase , very little growth occurred and the main products were 1,3-PD and py ruvate. The first enzyme in the 1,3-PD pathway, glycerol dehydratase, requires coenzyme B-12, which must be provided in E. coli fermentation s, However, the amount of coenzyme B-12 needed was quite small, with 1 0 nM sufficient for good 1,3-PD production in batch cofermentations. 1 ,3-PD is a useful intermediate in the production of polyesters, The 1, 3-PD operon was designed so that it can be readily modified for expres sion in other prokaryotic hosts; therefore, it is useful for metabolic engineering of 1,3-PD pathways from glycerol and other substrates suc h as glucose.