摘要：All living organisms share similar reactions within their central metabolism to provide precursors for all essential building blocksand reducing power. To identify whether alternative metabolic routes of glycolysis can operate in E. coli, we complementarilyemployed in silico design, rational engineering, and adaptive laboratory evolution. First, we used a genome-scale model andidentified two potential pathways within the metabolic network of this organism replacing canonical Embden-meyerhof-Parnas(EmP) glycolysis to convert phosphosugars into organic acids. One of these glycolytic routes proceeds via methylglyoxal andthe other via serine biosynthesis and degradation. Then, we implemented both pathways in E. coli strains harboring defectiveEmP glycolysis. Surprisingly, the pathway via methylglyoxal seemed to immediately operate in a triosephosphate isomerasedeletion strain cultivated on glycerol. By contrast, in a phosphoglycerate kinase deletion strain, the overexpression ofmethylglyoxal synthase was necessary to restore growth of the strain. Furthermore, we engineered the “serine shunt” whichconverts 3-phosphoglycerate via serine biosynthesis and degradation to pyruvate, bypassing an enolase deletion. Finally, toexplore which of these alternatives would emerge by natural selection, we performed an adaptive laboratory evolution studyusing an enolase deletion strain. Our experiments suggest that the evolved mutants use the serine shunt. Our study reveals theflexible repurposing of metabolic pathways to create new metabolite links and rewire central metabolism.

摘要：Light-regulated gene expression systems allow controlling gene expression in space and time with high *** to previous synthetic light sensors that incorporate two-component systems which require localization at the plasma membrane,soluble one-component repression systems provide several advantageous ***,they are soluble and able to diffuse across the ***,they are smaller and of lower complexity,enabling less taxing expression and optimization of fewer ***,repression through steric hindrance is a widespread regulation mechanism that does not require specific interaction with host factors,potentially enabling implementation in different ***,we present the design of the synthetic promoter P_(EL)that in combination with the light-regulated dimer EL222 constitutes a onecomponent repression *** by previously engineered synthetic promoters and the Escherichia coli lacZYA promoter,we designed P_(EL)with two EL222 operators positioned to hinder RNA polymerase binding when EL222 is bound.P_(EL)is repressed by EL222 under conditions of white light with a light-regulated repression ratio of ***,alternating conditions of darkness and light in cycles as short as one hour showed that repression is *** design of the P_(EL)EL222 system herein presented could aid the design and implementation of analogous one-component optogenetic repression ***,we compare the P_(EL)-EL222 system with similar systems and suggest general improvements that could optimize and extend the functionality of EL222-based as well as other one-component repression systems.