Sucrose is a preferred feedstock for microbial fermentation due to its cheap price, high abundance in nature and its relatively environmentally friendly qualities. Sucrose utilisation in E. coli, however, is poorly understood, and most industrial E. coli strains cannot use sucrose. The aim of this study was to develop an efficient E. coli host that utilises sucrose as a carbon source for production of biochemicals, in particular isoprene. The major outcomes of this work are: (i) development of a detailed understanding of the molecular control of sucrose utilisation in E. coli W, (ii) development of molecular biology tool for integration of large DNA sequences onto the E. coli chromosome, (iii) successful transfer of MEP pathway genes from plant into E. coli for isoprene production, and (iv) identification of a negative regulation control for the MEP pathway.
E. coli W grows very fast on sucrose using the chromosomally encoded sucrose catabolism (csc) genes. The role of different csc genes in sucrose utilisation was examined. This work represents the first demonstration that the fructokinase gene (cscK) is not essential for sucrose utilisation and that the fructose moiety is not rate-limiting for growth on high sucrose concentrations but becomes rate-limiting when carbon is limited. Deletion of the csc repressor resulted in a strain which could grow efficiently on low sucrose and which had improved production of polyhydroxybutyrate (PHB). The resulting strain, WΔcscR, is a platform strain for production of industrial biochemicals from sucrose.
The planned metabolic engineering approach for developing a useful sucrose-utilising industrial strain relied on integration of very large DNA sequences. In order to facilitate the transfer of multiple genes into E. coli W, a series of knock-in/knock-out (KIKO) integration vectors was constructed. These vectors include large homologous ‘arms’ which are used to integrate DNA sequences at well-characterised loci via the lambda Red recombination system. The KIKO plasmids are a useful tool for efficient gene insertion in E. coli, providing the capacity to insert large and complex pathways as an effective alternative to plasmid-based methods. The approach allows for a more stable expression of exogenous proteins without the need for continuous antibiotic selection.
Isoprenoids are an interesting class of industrial biochemicals. However, E. coli native isoprenoid biosynthesis pathway flux is low and limits productivity. To increase pathway flux, reconstruction of a high flux plant pathway in E. coli was investigated. The KIKO vectors were used in integrate the eight MEP pathway genes onto the chromosome of the improved E coli W strain (WcscR). Improved production of isoprene, a C5 hydrocarbon with many industrial applications, was demonstrated using this approach. Furthermore, the modified strains were used to identify a previously-uncharacterised negative regulation control on the MEP pathway. The best titres achieved in this study were lower than what has been reported in other studies, but further improvements through optimised expression of the MEP pathway genes and other metabolic engineering strategies can be used to develop a platform for production of industrially relevant isoprenoids from sucrose.