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.

LanM Peptides – Unravelling the Binding Properties of the EF-Hand Loop Sequences Stripped from the Structural Corset

Since the discovery of the biological relevance of lanthanides (Lns) for methylotrophic bacteria in the last decade, the field has seen a steady rise in discoveries of bacteria using Lns. The major role of lanthanides here is in the active sites of enzymes: methanol dehydrogenases. Additionally, lanthanide binding proteins have also been identified. One such protein is lanmodulin (LanM) and, with a remarkable selectivity for Lns over Ca(II) and affinities in the picomolar range, it makes an attractive target to address challenges in lanthanide separation. Why LanM has such a high selectivity is currently not entirely understood, both the specific amino acid sequences of the EF-hand loops, together with cooperativity effects have been suggested. Consequently, we decided to remove the effect of cooperativity by focusing on the amino acid level. Thus, we synthesized all four 12-amino acid EF-Hand loop peptides of LanM using solid phase peptide synthesis and investigated their affinity for Lns (Eu(III), Tb(III)), the actinide Cm(III) and Ca(II). Using isothermal titration calorimetry and time resolved laser fluorescence spectroscopy combined with parallel factor analysis, we show that in the absence of cooperativity the short EF-Hand loop peptides have all similar affinities for lanthanides and that these are all in the micromolar range. Furthermore, calcium was shown not to bind to the peptides which was verified with circular dichroism spectroscopy. This technique also revealed that the peptides undergo a change to a more ordered state when lanthanides are added. These experimental observations were further supported by molecular dynamics simulations. Lastly, we put Eu(III) and Cm(III) in direct competition using TRLFS. Remarkably, a slightly higher affinity for the actinide, as was also observed for LanM, was found. Our results demonstrate that the picomolar affinities in LanM are largely an effect of pre-structuring in the full protein and therefore reduction of flexibility in combination with cooperative effects, and that all EF-Hand loops possess similar affinities when detached from the protein backbone, albeit still retaining the high selectivity for lanthanides and actinides over calcium.

Influence of Pink Pigmented Facultative methylotrophic bacteria (PPFM) as a foliar spraying on the growth and productivity of strawberry(Fragaria xananassa Duch.).

Pink pigmented facultative methylotrophic bacterium (PPFM) has a favorable impact on plant development and production, it is known as a biostimulator, biofertilizer and biocontroller. Here we investigate the effect of foliar spraying of PPFM, 10% methanol,30% methanol, and their combinations on the growth, fruit quality, and yield of two strawberry cultivars. PPFM was isolated from cotton leaves using imprinting technique. 16S rRNA sequence analysis identified it to be Methylobacterium radiotolerance. Its 16S rRNA sequence were deposited in the Gene Bank under accession number MT644122.1. Two field experiments were conducted during the 2017/2018 and 2018/2019 seasons to investigate the effect of foliar spraying of PPFM and methanol (10 and 30%) on the growth, fruit quality, and yield of two strawberry cultivars. The obtained results showed that, there were no significant differences in the most characteristics between the two cultivars except foliage fresh weight and early yield were higher in cv. Florid, however, Festival cv. recorded higher total yield /plant, anthocyanins and ascorbic acid content in both seasons. Spraying PPFM exhibited the highest values of chlorophyll, fresh weight, total yield and quality. Furthermore, PPFM combined with methanol 10% gave the highest values of leaf area, dry matter %, early yield and some fruit quality. Spraying cv. Florida with PPFM resulted in the best interactions for early yield. However, the best interaction for total yield and most fruit quality features was observed with Festival c.v. and spraying PPFM. It is reasonable to conclude that PPFM is the most effective treatment, increasing strawberry total yield/fed by 28.1 % in the 1st season and 27.91 % in the 2 nd season compared to the control.

Methylothon: a versatile course-based high school research experience in microbiology and bioinformatics-- with pink bacteria

ABSTRACTMethylothon is an inquiry-based high school learning module in microbial ecology, molecular biology, and bioinformatics that centers around pink-pigmented plant-associated methylotrophic bacteria. Here we present an overview of the module’s learning goals, describe course resources (available for public use on http://methylothon.com), and relate lessons learned from adapting Methylothon for remote learning during the pandemic in spring of 2021. The original in-person version of the module allows students to isolate their own strains of methylotrophic bacteria from plants they sample from the environment, to identify these using PCR, sequencing, and phylogenetic analysis, and to contribute their strains to original research in a university lab. The adapted version strengthens the focus on bioinformatics and increases its flexibility and accessibility by making the lab portion optional and adopting free web-based tools. Student feedback and graded assignments from Spring 2021 revealed that the lesson was especially effective at introducing the concepts of BLAST and phylogenetic trees, and that students valued and felt inspired by the opportunity to conduct hands-on work and to participate in community science.

Methane-derived carbon flows into host–virus networks at different trophic levels in soil

The concentration of atmospheric methane (CH4) continues to increase with microbial communities controlling soil–atmosphere fluxes. While there is substantial knowledge of the diversity and function of prokaryotes regulating CH4 production and consumption, their active interactions with viruses in soil have not been identified. Metagenomic sequencing of soil microbial communities enables identification of linkages between viruses and hosts. However, this does not determine if these represent current or historical interactions nor whether a virus or host are active. In this study, we identified active interactions between individual host and virus populations in situ by following the transfer of assimilated carbon. Using DNA stable-isotope probing combined with metagenomic analyses, we characterized CH4-fueled microbial networks in acidic and neutral pH soils, specifically primary and secondary utilizers, together with the recent transfer of CH4-derived carbon to viruses. A total of 63% of viral contigs from replicated soil incubations contained homologs of genes present in known methylotrophic bacteria. Genomic sequences of 13C-enriched viruses were represented in over one-third of spacers in CRISPR arrays of multiple closely related Methylocystis populations and revealed differences in their history of viral interaction. Viruses infecting nonmethanotrophic methylotrophs and heterotrophic predatory bacteria were also identified through the analysis of shared homologous genes, demonstrating that carbon is transferred to a diverse range of viruses associated with CH4-fueled microbial food networks.

Evaluation of Heterologous Biosynthetic Pathways for Methanol-Based 5-Aminovalerate Production by Thermophilic Bacillus methanolicus

The use of methanol as carbon source for biotechnological processes has recently attracted great interest due to its relatively low price, high abundance, high purity, and the fact that it is a non-food raw material. In this study, methanol-based production of 5-aminovalerate (5AVA) was established using recombinant Bacillus methanolicus strains. 5AVA is a building block of polyamides and a candidate to become the C5 platform chemical for the production of, among others, δ-valerolactam, 5-hydroxy-valerate, glutarate, and 1,5-pentanediol. In this study, we test five different 5AVA biosynthesis pathways, whereof two directly convert L-lysine to 5AVA and three use cadaverine as an intermediate. The conversion of L-lysine to 5AVA employs lysine 2-monooxygenase (DavB) and 5-aminovaleramidase (DavA), encoded by the well-known Pseudomonas putida cluster davBA, among others, or lysine α-oxidase (RaiP) in the presence of hydrogen peroxide. Cadaverine is converted either to γ-glutamine-cadaverine by glutamine synthetase (SpuI) or to 5-aminopentanal through activity of putrescine oxidase (Puo) or putrescine transaminase (PatA). Our efforts resulted in proof-of-concept 5AVA production from methanol at 50°C, enabled by two pathways out of the five tested with the highest titer of 0.02 g l–1. To our knowledge, this is the first report of 5AVA production from methanol in methylotrophic bacteria, and the recombinant strains and knowledge generated should represent a valuable basis for further improved 5AVA production from methanol.

Harnessing methylotrophs as a bacterial platform to reduce adverse effects of the use of the heavy lanthanide gadolinium in magnetic resonance imaging

Gadolinium is a key component of magnetic resonance imaging contrast agents that are critical tools for enhanced detection and diagnosis of tissue and vascular abnormalities. Untargeted post-injection deposition of gadolinium in vivo, and association with diseases like nephrogenic systemic fibrosis, has alerted regulatory agencies to re-evaluate their widespread use and generated calls for safer gadolinium-based contrast agents (GBCAs). Increasing anthropogenic gadolinium in surface water has also raised concerns of potential bioaccumulation in plants and animals. Methylotrophic bacteria can acquire, transport, store and use light lanthanides as part of a cofactor complex with pyrroloquinoline quinone (PQQ), an essential component of XoxF-type methanol dehydrogenases (MDHs), a critical enzyme for methylotrophic growth with methanol. We report robust gadolinium-dependent methanol growth of a genetic variant of Methylorubrum extorquens AM1, named evo-HLn, for evolved for heavy lanthanides. Genetic adaptation of evo-HLn resulted in increased xox1 promoter and XoxF MDH activities, transport and storage of Gd3+, and augmented biosynthesis of PQQ. Gadolinium-grown cells exhibited a shorter T1 relaxation time compared to cells with lanthanum or no lanthanide when analyzed by MRI. In addition, evo-HLn was able to grow on methanol using the GBCA Gd-DTPA as the sole gadolinium source, showing the potential of this strain for the development of novel GBCAs and gadolinium recovery from medical waste and/or wastewater.

EfgA is a conserved formaldehyde sensor that leads to bacterial growth arrest in response to elevated formaldehyde

Normal cellular processes give rise to toxic metabolites that cells must mitigate. Formaldehyde is a universal stressor and potent metabolic toxin that is generated in organisms from bacteria to humans. Methylotrophic bacteria such as Methylorubrum extorquens face an acute challenge due to their production of formaldehyde as an obligate central intermediate of single-carbon metabolism. Mechanisms to sense and respond to formaldehyde were speculated to exist in methylotrophs for decades but had never been discovered. Here, we identify a member of the DUF336 domain family, named efgA for enhanced formaldehyde growth, that plays an important role in endogenous formaldehyde stress response in M. extorquens PA1 and is found almost exclusively in methylotrophic taxa. Our experimental analyses reveal that EfgA is a formaldehyde sensor that rapidly arrests growth in response to elevated levels of formaldehyde. Heterologous expression of EfgA in Escherichia coli increases formaldehyde resistance, indicating that its interaction partners are widespread and conserved. EfgA represents the first example of a formaldehyde stress response system that does not involve enzymatic detoxification. Thus, EfgA comprises a unique stress response mechanism in bacteria, whereby a single protein directly senses elevated levels of a toxic intracellular metabolite and safeguards cells from potential damage.

Change in Abundance and Activity of Microbocenoses in the Area of Influence of a Large Landslide at the Bureya Reservoir

The results of field and experimental microbiological studies of water, soil, and rock samples in the influence zone of large landslide are presented. The landslide occurred in December 2018 and blocked the Bureya Reservoir from coast to coast. An artificial channel was created to restore the hydrological regime with the use of TNT (trinitrotoluene) and RDX (hexogen). A comparative analysis of the abundance of cultivated heterotrophic bacteria around the landslide body and in the artificial channel is carried out. The activity of microbial communities in relation to easily available (peptone, lactate, and starch) and difficult-to-mineralize humic compounds is also determined. With the use of spectrometry and gas chromatography, it is shown that an increase in the diversity of aromatic compounds in water is accompanied by an increase in the abundance of heterotrophic bacteria. A number of toxic substances, including methanol and methylated benzene derivatives, are found among the dominant components in the water. Its concentrations increased after the water drained through the landslide body and after imploding works. Many of the volatile organic compounds may have been products of microbial metabolism when water interacts with rocks. A hypothesis on the role of methanotrophic and methylotrophic bacteria in the genesis of methanol and toluene is discussed.