Characterization and evolution of an activator-independent methanol dehydrogenase from Cupriavidus necator N-1

Abstract

Methanol utilization by methylotrophic or non-methylotrophic organisms is the first step toward methanol bioconversion to higher carbon-chain chemicals. Methanol oxidation using NAD-dependent methanol dehydrogenase (Mdh) is of particular interest because it uses NAD(+) as the electron carrier. To our knowledge, only a limited number of NAD-dependent Mdhs have been reported. The most studied is the Bacillus methanolicus Mdh, which exhibits low enzyme specificity to methanol and is dependent on an endogenous activator protein (ACT). In this work, we characterized and engineered a group III NAD-dependent alcohol dehydrogenase (Mdh2) from Cupriavidus necator N-1 (previously designated as Ralstonia eutropha). This enzyme is the first NAD-dependent Mdh characterized from a Gram-negative, mesophilic, non-methylotrophic organism with a significant activity towards methanol. Interestingly, unlike previously reported Mdhs, Mdh2 does not require activation by known activators such as B. methanolicus ACT and Escherichia coli Nudix hydrolase NudF, or putative native C. necator activators in the Nudix family under mesophilic conditions. This enzyme exhibited higher or comparable activity and affinity toward methanol relative to the B. methanolicus Mdh with or without ACT in a wide range of temperatures. Furthermore, using directed molecular evolution, we engineered a variant (CT4-1) of Mdh2 that showed a 6-fold higher K cat/K m for methanol and 10-fold lower K cat/K m for n-butanol. Thus, CT4-1 represents an NAD-dependent Mdh with much improved catalytic efficiency and specificity toward methanol compared with the existing NAD-dependent Mdhs with or without ACT activation.

Isolation and genomic characterization ofNovimethylophilus kurashikiensisgen. nov. sp. nov., a new lanthanide‐dependent methylotrophic species ofMethylophilaceae

“…Strain Ho1‐7 was identified as Cupriavidus necator (100% 16S rRNA gene identity with C. necator N‐1 T ), for which its methylotrophy was unknown (Makkar and Casida, ). Wu and colleagues () reported that C. necator N‐1 possessed a NAD‐dependent MDH to oxidize methanol, but it does not utilize methanol as a carbon source in the absence of La 3+ . However, the genome of C. necator N‐1 contains a putative xoxF gene (CNE_RS23020; CP002878; Poehlein, et al ., ), which indicates that C. necator N‐1 may be a La 3+ ‐dependent methylotroph.…”

Engineering Artificial Fusion Proteins for Enhanced Methanol Bioconversion

“…Consequently, their performance in platform microorganisms such as Escherichia coli is unsatisfactory . In addition to the activator‐dependent Mdhs, two activator‐independent Mdhs from Geobacillus stearothermophilus DSM 2334 and Cupriavidus necator N‐1 have been reported and characterized . Among all the reported Mdhs, Mdh from G. stearothermophilus DSM 2334 was proven to possess comparably higher V max and lower K M values towards methanol and work better in synthetic methylotrophs .…”

“…A key factor that affects the efficiency of methanol bioconversion is the balance between formaldehyde generation and consumption. Currently, all NAD‐dependent Mdhs suffer from low catalytic efficiency . The formaldehyde reduction activity of NAD‐dependent Mdh is at least 1000‐fold higher than methanol oxidation activity .…”

“…Currently,a ll NAD-dependent Mdhs suffer from low catalytic efficiency. [17,18] The formaldehyde reduction activity of NAD-dependentM dh is at least 1000-fold higher than methanolo xidationa ctivity. [17] Therefore, to drive the reaction to desired direction and prevent the toxicityo ff ormaldehyde to cells, the produced formaldehyde needs to be consumed efficiently.…”

Mixing and matching methylotrophic enzymes to design a novel methanol utilization pathway inE. coli

“…Similar results have also been reported in other model organisms such as Corynebacterium glutamicum, Pseudomonas putida and Saccharomyces cerevisiae (as reviewed recently by (Heux S. et al, 2018)). Improvements in methanol assimilation have been achieved using different strategies such as (i) optimizing the cultivation medium (Gonzalez et al, 2018), (ii) lowering the thermodynamic and kinetic constraints associated with NAD-dependent methanol oxidation (Roth et al, 2019; Wu et al, 2016), (iii) improving formaldehyde assimilation (Price et al, 2016; Woolston et al, 2018), (iv) increasing carbon fluxes through the autocatalytic cycle (Bennett et al, 2018), and (v) coupling the activity of the RuMP cycle to the growth of the host microorganism and then using adaptive laboratory evolution (Chen et al, 2018; He et al, 2018; Meyer et al, 2018). However, none of these synthetic strains are able to grow on methanol alone.…”