Methanol-dependent Escherichia coli strains with a complete ribulose monophosphate cycle

Abstract

Methanol is a biotechnologically promising substitute for food and feed substrates since it can be produced renewably from electricity, water and CO2. Although progress has been made towards establishing Escherichia coli as a platform organism for methanol conversion via the energy efficient ribulose monophosphate (RuMP) cycle, engineering strains that rely solely on methanol as a carbon source remains challenging. Here, we apply flux balance analysis to comprehensively identify methanol-dependent strains with high potential for adaptive laboratory evolution. We further investigate two out of 1200 candidate strains, one with a deletion of fructose-1,6-bisphosphatase (fbp) and another with triosephosphate isomerase (tpiA) deleted. In contrast to previous reported methanol-dependent strains, both feature a complete RuMP cycle and incorporate methanol to a high degree, with up to 31 and 99% fractional incorporation into RuMP cycle metabolites. These strains represent ideal starting points for evolution towards a fully methylotrophic lifestyle.

Methanol Dehydrogenases as a Key Biocatalysts for Synthetic Methylotrophy

“…Many researchers are also trying to improve the methanol bioconversion efficiency of synthetic methylotrophy by searching for the NAD + -dependent Mdhs with better characteristics from different organisms via directed evolution (Table 3). The Mdhs from B. methanolicus (Müller et al, 2015;Witthoff et al, 2015;Dai et al, 2017;Meyer et al, 2018;Tuyishime et al, 2018;Hennig et al, 2020), B. stearothermophilus (Whitaker et al, 2017;Bennett et al, 2018;Tuyishime et al, 2018;Bennett et al, 2020;Rohlhill et al, 2020), and C. necator (Chen et al, 2018;Tuyishime et al, 2018;Woolston et al, 2018;Chen et al, 2020;Keller et al, 2020) have been used for synthetic methylotrophy in recent studies with E. coli as the most popular host (Müller et al, 2015;Whitaker et al, 2017;Bennett et al, 2018;Chen et al, 2018;Meyer et al, 2018;Woolston et al, 2018;Bennett et al, 2020;Chen et al, 2020;Keller et al, 2020;Rohlhill et al, 2020), besides C. glutamicum (Witthoff et al, 2015;Tuyishime et al, 2018;Hennig et al, 2020) and S. cerevisiae (Dai et al, 2017). In vitro system to mimic synthetic methylotrophy using scaffold system by enzyme assembly for enhancement of methanol utilization have been also attempt (Price et al, 2016).…”

“…Later, in 2018, Meyer et al performed reaction knockout (KO) analyses using FlexFlux based on the E. coli models iAF1260 and iML1515 containing additional reactions for NAD + -dependent Mdh, HPS and PHI (Meyer et al, 2018). As another example, Keller et al used cobra python for flux balance analysis (FBA) of the core metabolism of an E. coli model from BiGG (Keller et al, 2020).…”

“…However, so far, there are limitations in the engineering of native methylotrophs to produce heterologous products at high rates and titers due to the lack of genetic tools available. Recent advances in synthetic biology, integration of efficient methanol converting enzymes, genome engineering, and laboratory evolution are enabling the first steps toward the creation of synthetic methanol-utilizing microorganisms (Heux et al, 2018;Meyer et al, 2018;Bennett et al, 2020;Chen et al, 2020;Keller et al, 2020;Wang et al, 2020).…”

In-depth computational analysis of natural and artificial carbon fixation pathways

“…For the assimilation of these compounds, nature also has evolved specialized microorganisms with dedicated pathways like the Serine cycle or the Ribulose Monophosphate (RuMP) cycle that are thought to be more efficient than the traditional CBB cycle (Claassens et al, 2019). For biotechnological purposes, it was tried to implant these pathways into Escherichia coli with mixed success (He et al, 2018;Keller et al, 2020;. Additionally, multiple optimized artificial pathways for C1-assimilation have been proposed and their basic working principle could be shown (Chou et al, 2019;He et al, 2020;Siegel et al, 2015).…”

Sustainable biosynthesis of chemicals from methane and glycerol via reconstruction of multi‐carbon utilizing pathway in obligate methanotrophic bacteria

“…One possible solution to overcome this issue is to introduce methane utilizing pathway in fast‐growing bacteria like E. coli in which sugar substrate metabolism might supply reducing power for reduced products. Even though many efforts have been made to heterologously express methane monooxygenase, methanol dehydrogenase, formaldehyde dehydrogenase in a non‐native host, this approach faces engineering challenges for chemicals production due to the difficulties in operation of a methanotrophic lifestyle involving multiple‐enzyme expression levels, gene regulation and autocatalytic cycle constraints (West et al., 1992 , Woolston et al., 2018 , Kim et al., 2019 , Keller et al., 2020 ). To date, no heterologous host strain can replace methanotrophic bacteria to efficiently utilize methane as a carbon source for cell growth and production.…”