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Abstract
BackgroundIn engineered strains of Escherichia coli, bioconversion efficiency is determined by not only metabolic flux but also the turnover efficiency of relevant pathways. Methyl-d-erythritol 4-phosphate (MEP)-dependent carotenoid biosynthesis in E. coli requires efficient turnover of precursors and balanced flux among precursors, cofactors, and cellular energy. However, the imbalanced supply of glyceraldehyde 3-phosphate (G3P) and pyruvate precursors remains the major metabolic bottleneck. To address this problem, we manipulated various genetic targets related to the Entner–Doudoroff (ED)/pentose phosphate (PP) pathways. Systematic target modification was conducted to improve G3P and pyruvate use and rebalance the precursor and redox fluxes.ResultsCarotenoid production was improved to different degrees by modifying various targets in the Embden–Meyerhof–Parnas (EMP) and ED pathways, which directed metabolic flux from the EMP pathway towards the ED pathway. The improvements in yield were much greater when the MEP pathway was enhanced. The coordinated modification of ED and MEP pathway targets using gene expression enhancement and protein coupling strategies in the pgi deletion background further improved carotenoid synthesis. The fine-tuning of flux at the branch point between the ED and PP pathways was important for carotenoid biosynthesis. Deletion of pfkAB instead of pgi reduced the carotenoid yield. This suggested that anaplerotic flux of G3P and pyruvate might be necessary for carotenoid biosynthesis. Improved carotenoid yields were accompanied by increased biomass and decreased acetate overflow. Therefore, efficient use of G3P and pyruvate precursors resulted in a balance among carotenoid biosynthesis, cell growth, and by-product metabolism.ConclusionsAn efficient and balanced MEP-dependent carotenoid bioconversion strategy involving both the ED and PP pathways was implemented by the coordinated modification of diverse central metabolic pathway targets. In this strategy, enhancement of the ED pathway for efficient G3P and pyruvate turnover was crucial for carotenoid production. The anaplerotic role of the PP pathway was important to supply precursors for the ED pathway. A balanced metabolic flux distribution among precursor supply, NADPH generation, and by-product pathways was established.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0301-x) contains supplementary material, which is available to authorized users.
Highlights
In engineered strains of Escherichia coli, bioconversion efficiency is determined by metabolic flux and the turnover efficiency of relevant pathways
Deletion of pgi disrupted the recycling of F6P or glyceraldehyde 3-phosphate (G3P) to the oxidative PP pathway, resulting in decreased NADPH generation [19]
Among the genes involved in these steps, ytjC and pykFA have been identified as targets for modification to improve Methyl-d-erythritol 4-phosphate (MEP)-dependent biosynthesis
Summary
In engineered strains of Escherichia coli, bioconversion efficiency is determined by metabolic flux and the turnover efficiency of relevant pathways. The imbalanced supply of glyceraldehyde 3-phosphate (G3P) and pyruvate precursors remains the major metabolic bottleneck To address this problem, we manipulated various genetic targets related to the Entner–Doudoroff (ED)/pentose phosphate (PP) pathways. To construct an engineered strain with optimal phenotypes, the first steps are to enhance native pathways or introduce superior heterogeneous pathways for biosynthesis of target chemicals. Another strategy that can be more important and laborious is to seek and integrate separate genetic modification targets that improve productivity, even though the mechanisms of these targets are sometimes poorly understood. Bioconversion of carotenoids by engineered strain has been developed based on either of these two pathways [9, 10]
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