An in vivo selection system with tightly regulated gene expression enables directed evolution of highly efficient enzymes

Abstract

In vivo selection systems are powerful tools for directed evolution of enzymes. The selection pressure of the systems can be tuned by regulating the expression levels of the catalysts. In this work, we engineered a selection system for laboratory evolution of highly active enzymes by incorporating a translationally suppressing cis repressor as well as an inducible promoter to impart stringent and tunable selection pressure. We demonstrated the utility of our selection system by performing directed evolution experiments using TEM β-lactamase as the model enzyme. Five evolutionary rounds afforded a highly active variant exhibiting 440-fold improvement in catalytic efficiency. We also showed that, without the cis repressor, the selection system cannot provide sufficient selection pressure required for evolving highly efficient TEM β-lactamase. The selection system should be applicable for the exploration of catalytic perfection of a wide range of enzymes.

A growth selection system for the directed evolution of amine-forming or converting enzymes

“…A medium or low expression, on the contrary, ensures growth of cells bearing only the most active variants if a moderate or decent template activity already exists. Other strategies, such as inducible promoter, 5′-untranslated region 41 , or protein degradation tags 45 could also be used to fine-tune the expression levels of the enzyme.

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“…However, selection requires a direct link between the activity of the enzyme and the survival of the host, which is often highly specialized and difficult to be established for many synthetically useful enzymes. Pioneering studies in selection-based enzyme evolution often focused on enzymes conferring antibiotic resistance (e.g., β-lactamases) 40 , 41 targeting proteins linked to the expression of antibiotic resistance genes 42 or phage coat protein pIII (essential for phage propagation) 43 . Yet, it is difficult to apply this concept to synthetically useful enzymes.…”

High-throughput directed evolution: a golden era for protein science

“…OPEN ACCESS techniques owing to its ease of use (Figure 2B). epPCR is still widely employed and has been used successfully to engineer the properties of proteins, such as to increase aggregation-resistance [1], to improve enzyme activity [27], to evolve complex proteins useful for biotechnology [28], and to determine protein fitness landscapes using DMS [6,10]. Another often-used method for in vitro gene diversification is DNA shuffling (Box 1), wherein libraries are created by random fragmentation and recombination of homologous DNA sequences (Figure 2C) [29].…”

“…Another often-used method for in vitro gene diversification is DNA shuffling (Box 1), wherein libraries are created by random fragmentation and recombination of homologous DNA sequences (Figure 2C) [29]. Since its invention, DNA shuffling has been widely used and adapted to engineer a range of properties, including improved thermostability [30] or catalytic activity [27], and, most recently, to develop chemogenetic fluorescent reporters with tuneable fluorescent properties [31].…”

Growth‐Coupled Evolutionary Pressure Improving Epimerases for D‐Allulose Biosynthesis Using a Biosensor‐Assisted In Vivo Selection Platform

“…[ 13 ] Pioneering research on growth‐coupled evolution frequently concentrated on targeting proteins that directly impart antibiotic resistance (e.g., β‐lactamases), which are linked to the expression of antibiotic resistance genes. [ 14 ] Biosensors have emerged as promising alternative approaches to relieve throughput limitations in metabolic pathways and enzyme engineering. [ 15 ] Prominently, genetically encoded, transcription factor‐based biosensors can transmit the input signal (target metabolite concentration) into an easily detectable output (e.g., fluorescence, antibiotic resistance, or growth phenotype), offering a promising solution to accelerate the identification of target mutants.…”