Development of a formaldehyde biosensor with application to synthetic methylotrophy

Abstract

Formaldehyde is a prevalent environmental toxin and a key intermediate in single carbon metabolism. The ability to monitor formaldehyde concentration is, therefore, of interest for both environmental monitoring and for metabolic engineering of native and synthetic methylotrophs, but current methods suffer from low sensitivity, complex workflows, or require expensive analytical equipment. Here we develop a formaldehyde biosensor based on the FrmR repressor protein and cognate promoter of Escherichia coli. Optimization of the native repressor binding site and regulatory architecture enabled detection at levels as low as 1 µM. We then used the sensor to benchmark the in vivo activity of several NAD-dependent methanol dehydrogenase (Mdh) variants, the rate-limiting enzyme that catalyzes the first step of methanol assimilation. In order to use this biosensor to distinguish individuals in a mixed population of Mdh variants, we developed a strategy to prevent cross-talk by using glutathione as a formaldehyde sink to minimize intercellular formaldehyde diffusion.Finally, we applied this biosensor to balance expression of mdh and the formaldehyde assimilation enzymes hps and phi in an engineered E. coli strain to minimize formaldehyde build-up while also reducing the burden of heterologous expression.This biosensor offers a quick and simple method for sensitively detecting formaldehyde, and has the potential to be used as the basis for directed evolution of Mdh and dynamic formaldehyde control strategies for establishing synthetic methylotrophy. pastoris convert methanol to formaldehyde using an FAD-linked alcohol oxidase (AOX) (Cregg, Madden, Barringer, Thill, & Stillman, 1989), and Gram-positive methylotrophs typified by Bacillus methanolicus perform the same conversion using an NAD-linked Mdh (Müller, Heggeset, Wendisch, Vorholt, & Brautaset, 2015).In all these organisms, formaldehyde acts as a branch point between further oxidization to CO 2 for energy conservation, and incorporation into biomass via the serine cycle, ribulose monophosphate (RuMP) pathway, or the xylulose-5-phosphate (Xu5P) pathway (Chistoserdova et al., 2009;Müller, Heggeset, et al., 2015;Yurimoto, Oku, & Sakai, 2011). These pathways are of growing interest in the field of metabolic engineering, where researchers seek to convert relatively cheap methanol feedstocks into higher value commodity chemicals with either native or "synthetic" methylotrophs (Kalyuzhnaya, Puri, & Lidstrom, 2015;Whitaker, Sandoval, Bennett, Fast, & Papoutsakis, 2015). Besides methylotrophs, formaldehyde is present at low levels in all organisms as a result of demethylation reactions (Kalasz, 2003). Because of its cytotoxicity, the intracellular formaldehyde concentration must be tightly controlled, which has led to the evolution of a variety of highly coordinated metabolic strategies for detoxifying formaldehyde (Yurimoto, Kato, & Sakai, 2005). The need to keep the concentration of formaldehyde low while supporting high flux places an even more stringent burden on methylotrop...

Improving formaldehyde consumption drives methanol assimilation in engineered E. coli

“…Beyond establishing Mdh as forward-driven, the KIE experiment revealed that, under conditions of sufficient Ru5P regeneration, Mdh activity limits methanol flux, even though the evolved C. necator Mdh variant used in this study is the most active variant known 17 , 29 . This suggests that future efforts to improve methanol assimilation should focus on improving Mdh kinetics.…”

Methanol-essential growth of Escherichia coli

“…Although enzymes that allow E. coli to consume methanol have recently been successfully implemented, all strains engineered so far depend on multi-carbon substrates and grow without methanol addition 26 , 28 , 31 , 33 – 35 , which renders the transition to growth on methanol as sole carbon and energy source a major hurdle. In order to metabolically link growth to the consumption of methanol, we evaluated methanol essentiality in silico using flux balance analysis (Fig.…”

Phage-Assisted Evolution of Bacillus methanolicus Methanol Dehydrogenase 2

“…Phage propagation rates were initially too low to support evolution in a continuous flow system, even when using formaldehyde-sensitized S1030 Δ frmA host cells that lack the full detoxification pathway needed to convert formaldehyde into formate. 37 Nevertheless, Mdh2-mediated gene III expression was sufficient to support phage-assisted noncontinuous evolution (PANCE) 4 (Figure 1B). The PANCE system uses iterative rounds of overnight phage propagation in discrete cultures of host cells and phage, instead of a continuous-flow lagoon (Figure 1A), allowing more stable monitoring, lower selection stringency, and maintenance of evolving phage populations since phage are only lost upon periodic dilution into fresh media with host cells.…”

“…We constructed a corresponding selection phage (SP) in which gene III in the M13 phage genome was replaced with the mdh2 gene from B. methanolicus MGA3. 33 To avoid cross-talk between infected cells producing differing amounts of formaldehyde, we added glutathione to the growth media, which we previously showed acts as an extracellular formaldehyde sink to prevent cell-to-cell formaldehyde diffusion 37 (Figure 1A).…”