Engineering ‘cell robots’ for parallel and highly sensitive screening of biomolecules under in vivo conditions

Abstract

Cells are capable of rapid replication and performing tasks adaptively and ultra-sensitively and can be considered as cheap “biological-robots”. Here we propose to engineer cells for screening biomolecules in parallel and with high sensitivity. Specifically, we place the biomolecule variants (library) on the bacterial phage M13. We then design cells to screen the library based on cell-phage interactions mediated by a specific intracellular signal change caused by the biomolecule of interest. For proof of concept, we used intracellular lysine concentration in E. coli as a signal to successfully screen variants of functional aspartate kinase III (AK-III) under in vivo conditions, a key enzyme in L-lysine biosynthesis which is strictly inhibited by L-lysine. Comparative studies with flow cytometry method failed to distinguish the wild-type from lysine resistance variants of AK-III, confirming a higher sensitivity of the method. It opens up a new and effective way of in vivo high-throughput screening for functional molecules and can be easily implemented at low costs.

Dynamic energy correlation analysis of E. coli aspartokinase III and alteration of allosteric regulation by manipulating energy transduction pathways

“…However, electrostatic interactions are the main source for non-bound energetic redistribution, while van de Waals interactions and H-bonded networks are both involved in energetic redistribution to some extent [17]. Residues with [18]. These results indicated that the mutation can change the energy transformation and then affect the allostery.…”

Robotics for enzyme technology: innovations and technological perspectives

“…Parallel and high-throughput screening was achieved based on cell-phage interactions. This cellphage screening system displayed high sensitivity in a highthroughput format with low cost (Song and Zeng 2017).…”

In vivo continuous evolution of metabolic pathways for chemical production

“…Under selection pressure, only the offspring with adaptive mutation can survive. These mutants can be used in biofuels productionModified natural mutagenesis with dnaQ proofreading element (PE) mutant library n -butanolFlaskContinuous genotype diversification is achieved by transfected PE mutantHuman intervention is required on every cycle of serial dilution.Limited to specific strain100-fold increase in survival rate in 2% n -butanol compared to wild type after 18 transfers.[125]Acetateeightfold increase in survival rate in 0.1% acetate compared to wild type after 12 transfers.PACE was performed to evolved the product protein aspartate kinase III, while l -lysine was used as a selection pressure for selection due to its inhibitory properties to aspartate kinase IIIModified natural mutagenesis with dnaQ926 l -lysineChemostatContinuous evolution without any human interventionLimited to specific strain which can be infected by bacteriophageAbsolute activity with 50 mM of lysine increase from 0.1 to 0.3; Less than 20% drop in relative activity when inhibited by 100 mM lysine[130]…”

“…The mutation rate can be further increased by mutators dnaQ926, umuC, umuD’ and recA730. Various proteins have been successfully evolved by this system [127–130].…”