Biosensor-Based Directed Evolution of Methanol Dehydrogenase from Lysinibacillus xylanilyticus

Abstract

Methanol dehydrogenase (Mdh), is a crucial enzyme for utilizing methane and methanol as carbon and energy sources in methylotrophy and synthetic methylotrophy. Engineering of Mdh, especially NAD-dependent Mdh, has thus been actively investigated to enhance methanol conversion. However, its poor catalytic activity and low methanol affinity limit its wider application. In this study, we applied a transcriptional factor-based biosensor for the direct evolution of Mdh from Lysinibacillus xylanilyticus (Lxmdh), which has a relatively high turnover rate and low KM value compared to other wild-type NAD-dependent Mdhs. A random mutant library of Lxmdh was constructed in Escherichia coli and was screened using formaldehyde-detectable biosensors by incubation with low methanol concentrations. Positive clones showing higher fluorescence were selected by fluorescence-activated cell sorting (FACS) system, and their catalytic activities toward methanol were evaluated. The successfully isolated mutants E396V, K318N, and K46E showed high activity, particularly at very low methanol concentrations. In kinetic analysis, mutant E396V, K318N, and K46E had superior methanol conversion efficiency, with 79-, 23-, and 3-fold improvements compared to the wild-type, respectively. These mutant enzymes could thus be useful for engineering synthetic methylotrophy and for enhancing methanol conversion to various useful products.

Phage-Assisted Continuous Evolution and Selection of Enzymes for Chemical Synthesis

“…Despite extensive use of ligand-dependent biosensors for detecting small molecules in cells [65][66][67] , their use in assays for directed evolution is rare [68][69][70] , and only one example of their use for continuous evolution systems has been reported 34 . In this study, we engineered multiple biosensors to detect diverse small molecule ligands and yield pIII or a reporter gene as an output.…”

Methanol Dehydrogenases as a Key Biocatalysts for Synthetic Methylotrophy

“…(such as C. necator (Wu et al, 2016)). In particular, their NAD +dependent Mdhs have been focused and reported for studies of recombinant E. coli as synthetic methylotrophs (Müller et al, 2015;Wu et al, 2016;Whitaker et al, 2017;Lee et al, 2020;Le et al, 2021). Three NAD + -dependent Mdhs have been found in B. methanolicus MGA3 (Mdh, Mdh2, and Mdh3).…”

“…In vitro studies suggest that ACT enhances the methanol affinity, oxidation rate, and catalytic activity of Mdhs; however, the detailed mechanism for activation is currently unclear (Hektor et al, 2002;Witthoff et al, 2015) and no effect has been shown in vivo in a synthetic methylotrophy (Müller et al, 2015) because detail research for the activator protein functions in native host has not been tested. To enable the assimilation of methanol as the carbon source in metabolic engineering, ACT-independent Mdhs and their mutants from C. necator (Wu et al, 2016;Chen et al, 2018) and L. xylanilyticus (Lee et al, 2020;Le et al, 2021) have been reported and introduced into E. coli for methanol assimilation. As best candidate for synthetic methylotrophy, NAD + -dependent Mdh that can perform its function under both aerobic and anaerobic conditions (Zhang et al, 2017).…”

“…For NAD + -dependent Mdhs, a metal ion is involved in cofactor binding which may influence enzymatic activity (Hektor et al, 2002). Several metal ions have been examined for the effects on the methanol oxidation activity of Mdhs, such as Fe 2+ , Mn 2+ , Zn 2+ , Cu 2+ , Co 2+ , Ni 2+ , or Mg 2+ ions (Sridhara et al, 1969;Arfman et al, 1991;Montella et al, 2005;Müller et al, 2015;Wu et al, 2016;Whitaker et al, 2017;Lee et al, 2020;Le et al, 2021). In general, the supplementation of Fe 2+ or Mn 2+ ions increase enzyme activity, and Mdh activity is inhibited by Cu 2+ , Co 2+ , or Zn 2+ (Sridhara et al, 1969;Montella et al, 2005).…”

“…In the methanol utilization in methylotrophy, one of the key steps is the oxidation of methanol to formaldehyde by oxidoreductase (Zhang et al, 2017), and methanol dehydrogenases (Mdhs) are the main enzymes as they catalyze the oxidation of methanol to form formaldehyde with two electrons and 2H + (Le et al, 2021) (Figure 1). There are three native pathways of formaldehyde assimilation, that have been discovered and biochemically described for growth support of microorganisms in methanol, as follows: the ribulose monophosphate (RuMP) cycle, serine pathway, and xylulose monophosphate (XuMP) cycle (Figure 1) (Zhang et al, 2017).…”