Triggering the stringent response enhances synthetic methanol utilization in Escherichia coli

• Published in: Metabolic engineering Vol. 61; no. C; pp. 1 - 10
• Main Authors: Bennett, R. Kyle, Agee, Alec, Gerald Har, Jie Ren, von Hagel, Bryan, Siu, Ka-Hei, Antoniewicz, Maciek R., Papoutsakis, Eleftherios T.
• Format: Journal Article
• Language: English
• Published: Belgium Elsevier Inc 01.09.2020; Elsevier
• Subjects: Escherichia coli; Methanol; ppGpp; Stringent response; Synthetic methylotrophy; Synthetic methylotrophy; ppGpp; Stringent response; Escherichia coli; Methanol
• ISSN: 1096-7176; 1096-7184
• DOI: 10.1016/j.ymben.2020.04.007

Synthetic methylotrophy aims to engineer methane and methanol utilization pathways in platform hosts like Escherichia coli for industrial bioprocessing of natural gas and biogas. While recent attempts to engineer synthetic methylotrophs have proved successful, autonomous methylotrophy, i.e. the ability to utilize methane or methanol as sole carbon and energy substrates, has not yet been realized. Here, we address an important limitation of autonomous methylotrophy in E. coli: the inability of the organism to synthesize several amino acids when grown on methanol. By activating the stringent/stress response via ppGpp overproduction, or DksA and RpoS overexpression, we demonstrate improved biosynthesis of proteinogenic amino acids via endogenous upregulation of amino acid synthesis pathway genes. Thus, we were able to achieve biosynthesis of several limiting amino acids from methanol-derived carbon, in contrast to the control methylotrophic E. coli strain. This study addresses a key limitation currently preventing autonomous methylotrophy in E. coli and possibly other synthetic methylotrophs and provides insight as to how this limitation can be alleviated via stringent/stress response activation. •Triggering the stringent response via ppGpp overproduction enhances methanol utilization in E. coli•Transcriptional upregulation caused by ppGpp overproduction allows synthesis of limiting amino acids from methanol carbon.•Overexpression of DksA and RpoS mimics the positive effects of ppGpp overproduction on methanol utilization in E. coli.