Establishing a pyrroloquinoline quinone-dependent pathway for efficient alcohol metabolism in Escherichia coli

• Applicant: Professor Dr.-Ing. Thomas Walther
• Subject Area: Biological Process Engineering ; Microbial Ecology and Applied Microbiology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 555503581

Ethylene glycol (EG) and methanol are promising liquid feedstocks for industrial bioproduction. Despite the efforts to enable the workhorse Escherichia coli to metabolize EG or methanol, engineered strains grow at rates significantly lower than for other species naturally capable of assimilating these alcohols. In aerobic EG- and methanol pathways, the alcohol is first oxidized into the corresponding aldehyde. In engineered E. coli, this step is carried by NAD-dependent alcohol dehydrogenase enzymes. But because such reactions are thermodynamically unfavorable, processing of the respective aldehyde (mainly through synthetic metabolic pathways) becomes very difficult due to their extremely low concentrations. In this proposal, this challenge is addressed by installing in E. coli a non-canonical pyrroloquinoline quinone (PQQ)-dependent system which can irreversibly oxidize the alcohol(s). The functioning of a PQQ-based alcohol oxidation system requires multiple components: a PQQ-dependent alcohol dehydrogenase enzyme, a PQQ biosynthesis route, and a co-factor regeneration pathway composed of cytochrome c-proteins and cytochrome c terminal oxidase. None of the components is present in E. coli. Crucially, the bacterium Pseudomonas putida possesses a PQQ-dependent alcohol dehydrogenase enzyme (PedE) shown to be active on EG or methanol. After providing a detailed characterization of the molecular components implicated in the PQQ recycling in P. putida, we shall focus on their expression and step-wise functional characterization in E. coli. Alcohol oxidation and PQQ regeneration take place in the periplasm. Hence, we shall start by engineering PedE and c-type cytochrome protein(s) by appending an appropriate signal peptide towards their efficient translocation into the periplasm of E. coli strains with reduced proteolysis, and expressing cytochrome c maturation system. After additionally expressing cytochrome c terminal oxidase activity, those components shall enable in vivo oxidation of EG to glycolaldehyde when the cultivation medium is supplemented with PQQ. We will eventually co-express the PQQ biosynthetic pathway together with the above-described elements to enable fully autonomous functioning of the PQQ-dependent alcohol oxidation system in E. coli. Initial glycolaldehyde production rates are expected to be rather low, reason for which we have conceived a glycolaldehyde-dependent growth-based assay. Once improved performance is attained, we shall establish the entire reaction sequence in prototrophic E. coli to demonstrate that EG can serve as the sole source of carbon and energy for biomass formation via the tartronic-SA pathway. We shall use adaptive laboratory evolution to improve pathway performance, and evolved strains shall be studied by DNA sequencing and proteome analyses. Finally, we aim to show by 13C-tracer analysis the assimilation of methanol by E. coli using the PQQ-dependent oxidation system and glycolaldehyde synthase.

• DFG Programme: Research Grants