An autoinducible trp‐T7 expression system for production of proteins and biochemicals in Escherichia coli

Abstract

Inducible expression systems can be applied to control the expression of proteins or biochemical pathways in cell factories. However, several of the established systems require the addition of expensive inducers, making them unfeasible for large‐scale production. Here, we establish a genome integrated trp‐T7 expression system where tryptophan can be used to control the induction of a gene or a metabolic pathway. We show that the initiation of gene expression from low‐ and high‐copy vectors can be tuned by varying the initial concentration of tryptophan or yeast extract, and that expression is tightly regulated and homogenous when compared with the commonly used lac‐T7 system. Finally, we apply the trp‐T7 expression system for the production of l‐serine, where we reach titers of 26 g/L in fed‐batch fermentation.

ASIA: An automated stress‐inducible adaptor for enhanced stress protein expression in engineered Escherichia coli

“…The orthogonality of T7 RNA polymerase (T7RNAP) with the T7 promoter is highly effective for initiating protein expression in Escherichia coli, and it is widely applicable across diverse organisms, including Bacillus subtilis, yeast, cyanobacteria-spanning from prokaryotic to eukaryotic systems (Wang et al, 2018), and even in cellfree systems recently (Wang et al, 2023). However, the substantial energy demands associated with T7 system (Landberg et al, 2020;Tan & Ng, 2020), particularly when expressing toxic and stressinducing proteins, have been known to hinder cell growth and lead to cell lysis during cultivation occasionally (Stargardt et al, 2020(Stargardt et al, , 2021Ting & Ng, 2023). On the other hand, the leakage expression of T7RNAP results in the rapid production of target proteins during the noninducing period and depletes resources for essential cellular growth.…”