Engineering a new-to-nature cascade for phosphate-dependent formate to formaldehyde conversion in vitro and in vivo

Abstract

Formate can be envisioned at the core of a carbon-neutral bioeconomy, where it is produced from CO2 by (electro-)chemical means and converted into value-added products by enzymatic cascades or engineered microbes. A key step in expanding synthetic formate assimilation is its thermodynamically challenging reduction to formaldehyde. Here, we develop a two-enzyme route in which formate is activated to formyl phosphate and subsequently reduced to formaldehyde. Exploiting the promiscuity of acetate kinase and N-acetyl-γ-glutamyl phosphate reductase, we demonstrate this phosphate (Pi)-based route in vitro and in vivo. We further engineer a formyl phosphate reductase variant with improved formyl phosphate conversion in vivo by suppressing cross-talk with native metabolism and interface the Pi route with a recently developed formaldehyde assimilation pathway to enable C2 compound formation from formate as the sole carbon source in Escherichia coli. The Pi route therefore offers a potent tool in expanding the landscape of synthetic formate assimilation.

Identification of 2-Hydroxyacyl-CoA Synthases with High Acyloin Condensation Activity for Orthogonal One-Carbon Bioconversion

“…HACS variants for kinetic characterization were ordered from Twist Bioscience (South San Francisco, USA) as gene fragments with BsaI overhangs and were onboarded to the GFP drop-out construct pET28a_GFP using Golden Gate construction as described previously …”

“…Glycolyl-CoA was quantified by LC–MS/MS as described previously . Peak integration was performed in Agilent MassHunter Quantitative Analysis (QQQ) v10.0 and raw data were quantified against a glycolyl-CoA standard.…”

“…To address these issues, researchers developed different synthetic metabolic pathways, which are built upon newly discovered or engineered C1–C1 coupling enzymes, such as formolase (FLS) and associated variants − and 2-hydroxyacyl-CoA synthase (HACS). , However, because these enzymes often exploit a natural substrate promiscuity, many of them suffer from suboptimal kinetic parameters, even after extensive engineering efforts . For example, the engineered FLS that catalyzes C–C coupling of two or three formaldehyde molecules into glycolaldehyde or dihydroxyacetone (Figure ) demonstrated a turnover frequency ( k cat ) of only up to 1.5 s –1 and apparent Michaelis–Menten constants ( K M ) for formaldehyde exceeding 100 mM, even after several rounds of directed evolution. − , Considering the high toxicity of formaldehyde, , these enzymes are not suitable for use not only in actively growing cultures but also in resting cells. Efforts to substantially improve affinity of FLS toward formaldehyde ( K M = 18 mM) led to an extremely poor turnover ( k cat = 0.1 s –1 ), which requires high cell density (protein loading) to demonstrate product synthesis at the desired rate …”

Dual effects of endogenous formaldehyde on the organism and drugs for its removal

“…Thus, formate participates in the tricarboxylic acid cycle and is a necessary intermediate metabolite in single-carbon metabolism, sustaining basic metabolism processes (Kim et al, 2020;Nattermann et al, 2023).…”

Automated in vivo enzyme engineering accelerates biocatalyst optimization

“…When exploring new-to-nature enzymatic reactions (e.g., formyl-phosphate reductase 86 or glycolyl-CoA carboxylase 87 ), it can be useful to expand the solution space of mutations which can be screened. In this situation, directed evolution becomes a useful tool as it increases genetic diversity within a sequence of interest, provided that a high-throughput per experiment can be achieved.…”