Improvement of alka(e)ne production in Escherichia coli by the 3′-UTR engineering of acyl-ACP reductase

Abstract

The sustainable and efficient production of biofuels has generated considerable interest in the microbial synthesis of alka(e)nes, which are promising alternatives to fossil fuels. Acyl-ACP reductase (AAR) is a critical enzyme in the alka(e)ne biosynthetic pathway, and its poor solubility in Escherichia coli is a major bottleneck during the optimization of production yields. The approaches for enhancing protein solubility typically include the fusion of solubility tags at the N- or C-termini of target proteins, which can sometimes interfere with protein function or stability. The present study developed a novel strategy that leverages the regulatory potential of 3′-untranslated regions (3′-UTRs) by integrating the sequence coding thioredoxin (Trx), small ubiquitin-like modifier (SUMO), maltose-binding protein (MBP), or N-utilization substance protein A (NusA), into the 3′-UTR of the AAR gene. The strategy aimed to enhance the stability of AAR mRNA for improving the solubility and expression of AAR without altering its primary structure. The findings revealed that this strategy significantly enhanced the solubility and expression levels of AAR in Escherichia coli, which markedly increased alka(e)ne production. This method has potential widespread applications in metabolic engineering and synthetic biology. The study paves the way for the development of more efficient strategies aimed at producing biofuels, and highlights the untapped potential of the 3′-UTR engineering strategy.