Dynamic control of the mevalonate pathway expression for improved zeaxanthin production in Escherichia coli and comparative proteome analysis.

Engineered heterologous multi-gene metabolic pathways often suffer from flux imbalance and toxic metabolites, as the production host typically lacks the regulatory mechanisms for the heterologous pathway. Here, we first coordinated the expression of all genes of the mevalonate (MEV) pathway from Saccharomyces cerevisiae using the tunable intergenic regions (TIGRs), and then dynamically regulated the TIGR-mediated MEV pathway to prevent the accumulation of toxic metabolites by using IPP/FPP-responsive promoter. After introduction of the dynamically controlled TIGR-mediated MEV pathway into Escherichia coli, the content and concentration of zeaxanthin in shaker flask cultures were 2.0- and 2.1-fold higher, respectively, than those of the strain harboring the statically controlled non-TIGR-mediated MEV pathway. The content and concentration of zeaxanthin in E. coli ZEAX (pZSPgadE-MevTTIGR-MevBTIGRIS-2) reached 722.46mg/L and 23.16mg/g dry cell weight (DCW), respectively, in 5.0L fed-batch fermentation. We also comparatively analyzed the proteomes between E. coli ZEAX and E. coli ZEAX (pZSPgadE-MevTTIGR-MevBTIGRIS-2) to understand the mechanism of zeaxanthin biosynthesis. The results of the comparative proteomes demonstrate that zeaxanthin overproduction may be associated with increased precursor availability, increased NADPH availability, increased ATP availability, oxidative stress response, and increased membrane storage capacity for zeaxanthin due to changes in both cellular shape and membrane composition.

Genome engineering Escherichia coli for L-DOPA overproduction from glucose.

Genome engineering has become a powerful tool for creating useful strains in research and industry. In this study, we applied singleplex and multiplex genome engineering approaches to construct an E. coli strain for the production of L-DOPA from glucose. We first used the singleplex genome engineering approach to create an L-DOPA-producing strain, E. coli DOPA-1, by deleting transcriptional regulators (tyrosine repressor tyrR and carbon storage regulator A csrA), altering glucose transport from the phosphotransferase system (PTS) to ATP-dependent uptake and the phosphorylation system overexpressing galactose permease gene (galP) and glucokinase gene (glk), knocking out glucose-6-phosphate dehydrogenase gene (zwf) and prephenate dehydratase and its leader peptide genes (pheLA) and integrating the fusion protein chimera of the downstream pathway of chorismate. Then, multiplex automated genome engineering (MAGE) based on 23 targets was used to further improve L-DOPA production. The resulting strain, E. coli DOPA-30N, produced 8.67 g/L of L-DOPA in 60 h in a 5 L fed-batch fermentation. This titer is the highest achieved in metabolically engineered E. coli having PHAH activity from glucose.