Microbial production of 1,3-propanediol in Escherichia coli: A model system for metabolic engineering

Metabolic engineering involves the use of recombinant DNA technology to construct or modify metabolic pathways in cells. The production of 1,3-propanediol (1,3-PD) in E. coli was chosen as a model system for metabolic engineering. The source of the 1,3-PD pathway genes was K. pneumoniae ATCC 25955. An enrichment for anaerobic glycerol and dihydroxyacetone (DHA) utilization, followed by screening for 1,3-PD producers resulted in the identification of E. coli AG1/pTC1 from a genomic library of K. pneumoniae. At least four genes of the dha regulon were transferred into pTC1. The yield of 1,3-PD from glycerol was 0.46 mol/mol.; Directions for further improvements of 1,3-PD production in E. coli were explored based on the stoichiometric/topological constraints and kinetic constraints. Several ideas from the above analyses were then implemented to complete the cycle between theoretical prediction and experimentation.; Cofermentation of glycerol and sugars was used as a physiological approach to overcome the topological/stoichiometric constraints. The yield of 1,3-PD by E. coli AG1/pTC1 was 0.63 mol/mol in a cofermentation of glycerol and glucose. Genetic manipulation was used to overcome the kinetic constraints. The genes of the dha regulon in cosmid pTC1 were subcloned into pBR322, and were also transferred into pBluescript II SK(+). The DHA kinase activity from the gene dhaK was further inactivated by an insertional mutation.; Cofermentations of glycerol and glucose by E. coli with new versions of pTC plasmids in 300 ml anaerobic flasks showed significant improvements in the yield of 1,3-PD from glycerol. A 5-l fed-batch cofermentation of glycerol and glucose by E. coli AG1/pTC9 gave yields of 1,3-PD ranging from 0.85 to 0.97 with different glucose to glycerol ratios. The highest productivity, 0.54 g/l/h, was observed with a glucose to glycerol ratio of 1.0 g/g. The final 1,3-PD concentration was 6.72 g/l with an overall productivity of 0.24 g/l/h by E. coli AG1/pTC9. The study of 1,3-PD production in E. coli demonstrates that metabolic engineering is a very powerful approach for process development in biotechnology.