Cell-free synthesis system-assisted pathway bottleneck diagnosis and engineering in Bacillus subtilis

Metabolic engineering is a key technology for cell factories construction by rewiring cellular resources to achieve efficient production of target chemicals.However,the existence of bottlenecks in synthetic pathway can seriously affect production efficiency,which is also one of the core issues for metabolic engineers to solve.Therefore,developing an approach for diagnosing potential metabolic bottlenecks in a faster and simpler manner is of great significance to accelerate cell factories construction.The cell-free reaction system based on cell lysates can transfer metabolic reactions from in vivo to in vitro,providing a flexible access to directly change protein and metabolite variables,thus provides a potential solution for rapid identification of bottlenecks.Here,bottleneck diagnosis of the N-acetylneuraminic acid(NeuAc)biosynthesis pathway in industrially important chassis microorganism Bacillus subtilis was performed using cell-free synthesis system.Specifically,a highly efficient B.subtilis cell-free system for NeuAc de novo synthesis was firstly constructed,which had a 305-fold NeuAc synthesis rate than that in vivo and enabled fast pathway dynamics analysis.Next,through the addition of all potential key intermediates in combination with substrate glucose respectively,it was found that insufficient phosphoenolpyruvate supply was one of the NeuAc pathway bottlenecks.Rational in vivo metabolic engineering of NeuAc-producing B.subtilis was further performed to eliminate the bottleneck.By down-regulating the expression level of pyruvate kinase throughout the growth phase or only in the stationary phase using inhibitory Nterminal coding sequences(NCSs)and growth-dependent regulatory NCSs respectively,the maximal NeuAc titer increased 2.0-fold.Our study provides a rapid method for bottleneck diagnosis,which may help to accelerate the cycle of design,build,test and learn cycle for metabolic engineering.

THREE-STEP ITERATIONS FOR NONEXPANSIVE MAPPINGS AND INVERSE-STRONGLY MONOTONE MAPPINGS

This paper introduces a three-step iteration for finding a common element of the set of fixedpoints of a nonexpansive mapping and the set of solutions of the variational inequality for an inverse-strongly monotone mapping by viscosity approximation methods in a Hilbert space.The authors showthat the iterative sequence converges strongly to a common element of the two sets,which solves somevariational inequality.Subsequently,the authors consider the problem of finding a common fixed pointof a nonexpansive mapping and a strictly pseudo-contractive mapping and the problem of finding acommon element of the set of fixed points of a nonexpansive mapping and the set of zeros of an inverse-strongly monotone mapping.The results obtained in this paper extend and improve the correspondingresults announced by Nakajo,Takahashi,and Toyoda.