Methanol Dehydrogenases as a Key Biocatalysts for Synthetic Methylotrophy

One-carbon (C1) chemicals are potential building blocks for cheap and sustainable re-sources such as methane, methanol, formaldehyde, formate, carbon monoxide, and more. These resources have the potential to be made into raw materials for various products used in our daily life or precursors for pharmaceuticals through biological and chemical processes. Among the soluble C1 substrates, methanol is regarded as a biorenewable platform feedstock because nearly all bioresources can be converted into methanol through syngas. Synthetic methylotrophy can be exploited to produce fuels and chemicals using methanol as a feedstock that integrates natural or artificial methanol assimilation pathways in platform microorganisms. In the methanol utilization in methylotrophy, methanol dehydrogenase (Mdh) is a primary enzyme that converts methanol to formaldehyde. The discovery of new Mdhs and engineering of present Mdhs have been attempted to develop synthetic methylotrophic bacteria. In this review, we describe Mdhs, including in terms of their enzyme properties and engineering for desired activity. In addition, we specifically focus on the application of various Mdhs for synthetic methylotrophy.

Improving the Methanol Tolerance of an Escherichia coli Methylotroph via Adaptive Laboratory Evolution Enhances Synthetic Methanol Utilization

There is great interest in developing synthetic methylotrophs that harbor methane and methanol utilization pathways in heterologous hosts such as Escherichia coli for industrial bioconversion of one-carbon compounds. While there are recent reports that describe the successful engineering of synthetic methylotrophs, additional efforts are required to achieve the robust methylotrophic phenotypes required for industrial realization. Here, we address an important issue of synthetic methylotrophy in E. coli: methanol toxicity. Both methanol, and its oxidation product, formaldehyde, are cytotoxic to cells. Methanol alters the fluidity and biological properties of cellular membranes while formaldehyde reacts readily with proteins and nucleic acids. Thus, efforts to enhance the methanol tolerance of synthetic methylotrophs are important. Here, adaptive laboratory evolution was performed to improve the methanol tolerance of several E. coli strains, both methylotrophic and non-methylotrophic. Serial batch passaging in rich medium containing toxic methanol concentrations yielded clones exhibiting improved methanol tolerance. In several cases, these evolved clones exhibited a > 50% improvement in growth rate and biomass yield in the presence of high methanol concentrations compared to the respective parental strains. Importantly, one evolved clone exhibited a two to threefold improvement in the methanol utilization phenotype, as determined via 13C-labeling, at non-toxic, industrially relevant methanol concentrations compared to the respective parental strain. Whole genome sequencing was performed to identify causative mutations contributing to methanol tolerance. Common mutations were identified in 30S ribosomal subunit proteins, which increased translational accuracy and provided insight into a novel methanol tolerance mechanism. This study addresses an important issue of synthetic methylotrophy in E. coli and provides insight as to how methanol toxicity can be alleviated via enhancing methanol tolerance. Coupled improvement of methanol tolerance and synthetic methanol utilization is an important advancement for the field of synthetic methylotrophy.

Methanol expression regulator 1 (Mxr1p) promotes xylulose 5-phosphate recycle via increaseing transketolase activity in Pichia pastoris

Background Methanol expression regulator 1 (Mxr1p) is a key transcription factor that plays a vital role in the methanol utilization pathway in Pichia pastoris ( P. pastoris ). Most genes referred to the methanol utilization pathway were regulated by Mxr1p. However, some genes did not show a significant difference between methanol and glycerol even though they play an important role in the methanol utilization pathway. So far, the regulation mechanism about these genes and the relationship with Mxr1p are still unknown. Results Methanol metabolic pathway analysis revealed that most of the methanol-induced genes were upregulated in transcriptional level when cultured in methanol. Whereas some genes like tkl1 (transketolase 1) did not show significant up-regulation in methanol even though it plays a very important role in Xu5P recycle, the reason is still not clear. To clarify this point, firstly, pull-down and MS experiments were performed. The result shows that Tkl1p protein combined with Mxr1p in vitro . Subsequently, this result was further confirmed by yeast two-hybrid in vivo , and the binding region mainly located from 150AA to 400AA. Moreover, Ser215 phosphorylation did not affect this interaction. In addition, Mxr1p-400AA integration into Δmxr1 could rescue cell growth in methanol. All the above results proved that Mxr1p played a post-translational role in the methanol utilization pathway and Mxr1p-400AA may achieved most of Mxr1p functions. Secondly, the function of Mxr1p-Tkl1p complex was expounded by detecting formaldehyde consumption and xylulose production in cell-free systems. Results showed that Mxr1p-Tkl1p mixture could promote formaldehyde consumption and xylulose production in vitro . Conclusion Mxr1p promotes methanol utilization via combining with Tkl1p to accelerate Xu5P recycle and this interaction was not affected by Ser215 phosphorylation.