DNA scaffold assisted ectoine production in Escherichia coli

DNA scaffold that enhances the spatial proximity of enzymes and the local concentration of intermediates, is promising tools in optimizing heterologous metabolic pathways for target product biosynthesis. Here, we display the utility of a DNA scaffold system for the production of ectoine in E. coli MWZ003. Three fused enzymes EctA-ZFa, EctB-ZFb, and EctC-ZFc were firstly constructed by fusing enzymes of ectoine synthesis pathway with corresponding zinc finger domains. The copy number of the plasmid-expressing fusions was adapted by substitution of different replicons. Furthermore, a series of modifications were carried out on the DNA scaffold system through optimizing the spacer between enzyme binding sites, the binding direction of fusion enzymes, the repeating unit of DNA scaffolds, the stoichiometric ratio of enzyme binding sites, and the expression level of the rate-limiting enzyme. The optimized DNA scaffold system in the plasmid pFV30 involving use of pMB1 replicon, reverse binding, 11-bp spacer, 4 repeating units, stoichiometric ratio (1:2:2), and enhanced expression of EctB-ZFb increased the ectoine titer and yield, respectively, to 22.79 g/L and 0.65 g/g glucose with increase by 92% compared with that of the control strain. The post-translational strategy based on DNA scaffold was efficient in promoting heterologous synthesis of ectoine, which could also be used in combination with other genetic engineering tools.