Natural Polyhydroxyalkanoates-An Overview of Bacterial Production Methods.

Polyhydroxyalkanoates (PHAs) are intracellular biopolymers that microorganisms use for energy and carbon storage. They are mechanically similar to petrochemical plastics when chemically extracted, but are completely biodegradable. While they have potential as a replacement for petrochemical plastics, their high production cost using traditional carbon sources remains a significant challenge. One potential solution is to modify heterotrophic PHA-producing strains to utilize alternative carbon sources. An alternative approach is to utilize methylotrophic or autotrophic strains. This article provides an overview of bacterial strains employed for PHA production, with a particular focus on those exhibiting the highest PHA content in dry cell mass. The strains are organized according to their carbon source utilization, encompassing autotrophy (utilizing CO2, CO) and methylotrophy (utilizing reduced single-carbon substrates) to heterotrophy (utilizing more traditional and alternative substrates).

Scanning the active center of formolase to identify key residues for enhanced C1 to C3 bioconversion.

BACKGROUND: Formolase (FLS) is a computationally designed enzyme that catalyzes the carboligation of two or three C1 formaldehyde molecules into C2 glycolaldehyde or C3 dihydroxyacetone (DHA). FLS lays the foundation for several artificial carbon fixation and valorization pathways, such as the artificial starch anabolic pathway. However, the application of FLS is limited by its low catalytic activity and product promiscuity. FINDINGS: FLS, designed and engineered based on benzoylformate decarboxylase from Pseudomonas putida, was selected as a candidate for modification. To evaluate its catalytic activity, 25 residues located within an 8 Å distance from the active center were screened using single-point saturation mutagenesis. A screening approach based on the color reaction of the DHA product was applied to identify the desired FLS variants. After screening approximately 5,000 variants (approximately 200 transformants per site), several amino acid sites that were not identified by directed evolution were found to improve DHA formation. The serine-to-phenylalanine substitution at position 236 improved the activity towards DHA formation by 7.6-fold. Molecular dynamics simulations suggested that the mutation increased local hydrophobicity at the active site, predisposing the cofactor-C2 intermediate to nucleophilic attack by the third formaldehyde molecule for subsequent DHA generation. CONCLUSIONS: This study provides improved FLS variants and valuable information into the influence of residues adjacent to the active center affecting catalytic efficiency, which can guide the rational engineering or directed evolution of FLS to optimize its performance in artificial carbon fixation and valorization.

Glycine betaine metabolism is enabled in Methylorubrum extorquens PA1 by alterations to dimethylglycine dehydrogenase.

Low nutrient availability is a key characteristic of the phyllosphere (the aerial surface of plants). Phyllospheric bacteria utilize a wide array of carbon sources generated by plant hosts. Glycine betaine (GB) is a plant-derived compound that can be metabolized by certain members of the phyllosphere microbiota. Metabolism of glycine betaine generates formaldehyde, an intermediate of methylotrophic metabolism, leading us to investigate how the ubiquitous plant colonizing bacterium Methylorubrum extorquens PA1 might metabolize GB encountered in its native environment. M. extorquens PA1 cannot utilize GB as a sole carbon source. Through suppressor mutation analysis, we show that M. extorquens PA1 encodes a conserved GB utilization pathway that can be activated by single point mutations conferring GB utilization as a carbon source. We identified the gene cluster encoding the GB catabolic enzymes and found that gene expression was induced in the presence of GB. We show that utilization of GB is conserved among representative Methylobacterium species and generates the one-carbon metabolism intermediate formaldehyde, which M. extorquens utilizes as a source of energy. Our results support a model where suppressor mutations in Mext_3745 or ftsH (Mext_4840) prevent the degradation of the dimethylglycine dehydrogenase subunit DgcB by the membrane integral protease FtsH, conferring the ability to utilize GB by either (i) restoring stable membrane topology of DgcB or (ii) decreasing FtsH protease activity, respectively. Both mutations alleviate the bottleneck at the second step of GB degradation catalyzed by DgcAB.IMPORTANCEOvercoming low nutrient availability is a challenge many bacteria encounter in the environment. Facultative methylotrophs are able to utilize one-carbon and multi-carbon compounds as carbon and energy sources. The utilization of plant-derived glycine betaine (GB) represents a possible source of multi-carbon and one-carbon substrates. The metabolism of glycine betaine produces formaldehyde and glycine, which may be used simultaneously by facultative methylotrophs. However, the genes required for the utilization of GB in the ubiquitous plant-associated bacterium Methylorubrum extorquens have yet to be identified or described. Our work identifies and validates the genes required for glycine betaine metabolism in M. extorquens and shows that it directly intersects with methylotrophic metabolism through the production of formaldehyde.

Assessing Lanthanide-Dependent Methanol Dehydrogenase Activity: The Assay Matters.

Artificial dye-coupled assays have been widely adopted as a rapid and convenient method to assess the activity of methanol dehydrogenases (MDH). Lanthanide(Ln)-dependent XoxF-MDHs are able to incorporate different lanthanides (Lns) in their active site. Dye-coupled assays showed that the earlier Lns exhibit a higher enzyme activity than the late Lns. Despite widespread use, there are limitations: oftentimes a pH of 9 and activators are required for the assay. Moreover, Ln-MDH variants are not obtained by isolation from the cells grown with the respective Ln, but by incubation of an apo-MDH with the Ln. Herein, we report the cultivation of Ln-dependent methanotroph Methylacidiphilum fumariolicum SolV with nine different Lns, the isolation of the respective MDHs and the assessment of the enzyme activity using the dye-coupled assay. We compare these results with a protein-coupled assay using its physiological electron acceptor cytochrome cGJ (cyt cGJ ). Depending on the assay, two distinct trends are observed among the Ln series. The specific enzyme activity of La-, Ce- and Pr-MDH, as measured by the protein-coupled assay, exceeds that measured by the dye-coupled assay. This suggests that early Lns also have a positive effect on the interaction between XoxF-MDH and its cyt cGJ thereby increasing functional efficiency.

Engineering photo-methylotrophic Methylobacterium for enhanced 3-hydroxypropionic acid production during non-growth stage fermentation.

This study explored the potential of methanol as a sustainable feedstock for biomanufacturing, focusing on Methylobacterium extorquens, a well-established representative of methylotrophic cell factories. Despite this bacterium's long history, its untapped photosynthetic capabilities for production enhancement have remained unreported. Using genome-scale flux balance analysis, it was hypothesized that introducing photon fluxes could boost the yield of 3-hydroxypropionic acid (3-HP), an energy- and reducing equivalent-consuming chemicals. To realize this, M. extorquens was genetically modified by eliminating the negative regulator of photosynthesis, leading to improved ATP levels and metabolic activity in non-growth cells during a two-stage fermentation process. This modification resulted in a remarkable 3.0-fold increase in 3-HP titer and a 2.1-fold increase in its yield during stage (II). Transcriptomics revealed that enhanced light-driven methanol oxidation, NADH transhydrogenation, ATP generation, and fatty acid degradation were key factors. This development of photo-methylotrophy as a platform technology introduced novel opportunities for future production enhancements.

Methylotrophy in the Mire: direct and indirect routes for methane production in thawing permafrost.

While wetlands are major sources of biogenic methane (CH4), our understanding of resident microbial metabolism is incomplete, which compromises the prediction of CH4 emissions under ongoing climate change. Here, we employed genome-resolved multi-omics to expand our understanding of methanogenesis in the thawing permafrost peatland of Stordalen Mire in Arctic Sweden. In quadrupling the genomic representation of the site's methanogens and examining their encoded metabolism, we revealed that nearly 20% of the metagenome-assembled genomes (MAGs) encoded the potential for methylotrophic methanogenesis. Further, 27% of the transcriptionally active methanogens expressed methylotrophic genes; for Methanosarcinales and Methanobacteriales MAGs, these data indicated the use of methylated oxygen compounds (e.g., methanol), while for Methanomassiliicoccales, they primarily implicated methyl sulfides and methylamines. In addition to methanogenic methylotrophy, >1,700 bacterial MAGs across 19 phyla encoded anaerobic methylotrophic potential, with expression across 12 phyla. Metabolomic analyses revealed the presence of diverse methylated compounds in the Mire, including some known methylotrophic substrates. Active methylotrophy was observed across all stages of a permafrost thaw gradient in Stordalen, with the most frozen non-methanogenic palsa found to host bacterial methylotrophy and the partially thawed bog and fully thawed fen seen to house both methanogenic and bacterial methylotrophic activities. Methanogenesis across increasing permafrost thaw is thus revised from the sole dominance of hydrogenotrophic production and the appearance of acetoclastic at full thaw to consider the co-occurrence of methylotrophy throughout. Collectively, these findings indicate that methanogenic and bacterial methylotrophy may be an important and previously underappreciated component of carbon cycling and emissions in these rapidly changing wetland habitats.IMPORTANCEWetlands are the biggest natural source of atmospheric methane (CH4) emissions, yet we have an incomplete understanding of the suite of microbial metabolism that results in CH4 formation. Specifically, methanogenesis from methylated compounds is excluded from all ecosystem models used to predict wetland contributions to the global CH4 budget. Though recent studies have shown methylotrophic methanogenesis to be active across wetlands, the broad climatic importance of the metabolism remains critically understudied. Further, some methylotrophic bacteria are known to produce methanogenic by-products like acetate, increasing the complexity of the microbial methylotrophic metabolic network. Prior studies of Stordalen Mire have suggested that methylotrophic methanogenesis is irrelevant in situ and have not emphasized the bacterial capacity for metabolism, both of which we countered in this study. The importance of our findings lies in the significant advancement toward unraveling the broader impact of methylotrophs in wetland methanogenesis and, consequently, their contribution to the terrestrial global carbon cycle.

Different lanthanide elements induce strong gene expression changes in a lanthanide-accumulating methylotroph.

IMPORTANCE: Since its discovery, Ln-dependent metabolism in bacteria attracted a lot of attention due to its bio-metallurgical application potential regarding Ln recycling and circular economy. The physiological role of Ln is mostly studied dependent on presence and absence. Comparisons of how different (utilizable) Ln affect metabolism have rarely been done. We noticed unexpectedly pronounced changes in gene expression caused by different Ln supplementation. Our research suggests that strain RH AL1 distinguishes different Ln elements and that the effect of Ln reaches into many aspects of metabolism, for instance, chemotaxis, motility, and polyhydroxyalkanoate metabolism. Our findings regarding Ln accumulation suggest a distinction between individual Ln elements and provide insights relating to intracellular Ln homeostasis. Understanding comprehensively how microbes distinguish and handle different Ln elements is key for turning knowledge into application regarding Ln-centered biometallurgy.

Aerobic methanotrophy increases the net iron reduction in methanogenic lake sediments.

In methane (CH4) generating sediments, methane oxidation coupled with iron reduction was suggested to be catalyzed by archaea and bacterial methanotrophs of the order Methylococcales. However, the co-existence of these aerobic and anaerobic microbes, the link between the processes, and the oxygen requirement for the bacterial methanotrophs have remained unclear. Here, we show how stimulation of aerobic methane oxidation at an energetically low experimental environment influences net iron reduction, accompanied by distinct microbial community changes and lipid biomarker patterns. We performed incubation experiments (between 30 and 120 days long) with methane generating lake sediments amended with 13C-labeled methane, following the additions of hematite and different oxygen levels in nitrogen headspace, and monitored methane turnover by 13C-DIC measurements. Increasing oxygen exposure (up to 1%) promoted aerobic methanotrophy, considerable net iron reduction, and the increase of microbes, such as Methylomonas, Geobacter, and Desulfuromonas, with the latter two being likely candidates for iron recycling. Amendments of 13C-labeled methanol as a potential substrate for the methanotrophs under hypoxia instead of methane indicate that this substrate primarily fuels methylotrophic methanogenesis, identified by high methane concentrations, strongly positive δ13CDIC values, and archaeal lipid stable isotope data. In contrast, the inhibition of methanogenesis by 2-bromoethanesulfonate (BES) led to increased methanol turnover, as suggested by similar 13C enrichment in DIC and high amounts of newly produced bacterial fatty acids, probably derived from heterotrophic bacteria. Our experiments show a complex link between aerobic methanotrophy and iron reduction, which indicates iron recycling as a survival mechanism for microbes under hypoxia.

Directed evolution of a neutrophilic and mesophilic methanol dehydrogenase based on high-throughput and accurate measurement of formaldehyde.

Methanol is a promising one-carbon feedstock for biomanufacturing, which can be sustainably produced from carbon dioxide and natural gas. However, the efficiency of methanol bioconversion is limited by the poor catalytic properties of nicotinamide adenine dinucleotide (NAD+)-dependent methanol dehydrogenase (Mdh) that oxidizes methanol to formaldehyde. Herein, the neutrophilic and mesophilic NAD+-dependent Mdh from Bacillus stearothermophilus DSM 2334 (MdhBs) was subjected to directed evolution for enhancing the catalytic activity. The combination of formaldehyde biosensor and Nash assay allowed high-throughput and accurate measurement of formaldehyde and facilitated efficient selection of desired variants. MdhBs variants with up to 6.5-fold higher Kcat/KM value for methanol were screened from random mutation libraries. The T153 residue that is spatially proximal to the substrate binding pocket has significant influence on enzyme activity. The beneficial T153P mutation changes the interaction network of this residue and breaks the α-helix important for substrate binding into two short α-helices. Reconstructing the interaction network of T153 with surrounding residues may represent a promising strategy to further improve MdhBs, and this study provides an efficient strategy for directed evolution of Mdh.

Harnessing biological nitrogen fixation in plant leaves.

The importance of biological nitrogen fixation (BNF) in securing food production for the growing world population with minimal environmental cost has been increasingly acknowledged. Leaf surfaces are one of the biggest microbial habitats on Earth, harboring diverse free-living N2-fixers. These microbes inhabit the epiphytic and endophytic phyllosphere and contribute significantly to plant N supply and growth. Here, we summarize the contribution of phyllosphere-BNF to global N cycling, evaluate the diversity of leaf-associated N2-fixers across plant hosts and ecosystems, illustrate the ecological adaptation of N2-fixers to the phyllosphere, and identify the environmental factors driving BNF. Finally, we discuss potential BNF engineering strategies to improve the nitrogen uptake in plant leaves and thus sustainable food production.

Minor Actinides Can Replace Essential Lanthanides in Bacterial Life.

Certain f-block elements-the lanthanides-have biological relevance in the context of methylotrophic bacteria. The respective strains incorporate these 4 f elements into the active site of one of their key metabolic enzymes, a lanthanide-dependent methanol dehydrogenase. In this study, we investigated whether actinides, the radioactive 5 f elements, can replace the essential 4 f elements in lanthanide-dependent bacterial metabolism. Growth studies with Methylacidiphilum fumariolicum SolV and the Methylobacterium extorquens AM1 ΔmxaF mutant demonstrate that americium and curium support growth in the absence of lanthanides. Moreover, strain SolV favors these actinides over late lanthanides when presented with a mixture of equal amounts of lanthanides together with americium and curium. Our combined in vivo and in vitro results establish that methylotrophic bacteria can utilize actinides instead of lanthanides to sustain their one-carbon metabolism if they possess the correct size and a +III oxidation state.

Increasing lysine level improved methanol assimilation toward butyric acid production in Butyribacterium methylotrophicum.

BACKGROUND: Methanol, a promising non-food fermentation substrate, has gained increasing interest as an alternative feedstock to sugars for the bio-based production of value-added chemicals. Butyribacterium methylotrophicum, one of methylotrophic-acetogenic bacterium, is a promising host to assimilate methanol coupled with CO2 fixation for the production of organic acids, such as butyric acid. Although the methanol utilization pathway has been identified in B. methylotrophicum, little knowledge was currently known about its regulatory targets, limiting the rational engineering to improve methanol utilization. RESULTS: In this study, we found that methanol assimilation of B. methylotrophicum could be significantly improved when using corn steep liquor (CSL) as the co-substrate. The further investigation revealed that high level of lysine was responsible for enhanced methanol utilization. Through the transcriptome analysis, we proposed a potential mechanism by which lysine confers improved methylotrophy via modulating NikABCDE and FhuBCD transporters, both of which are involved in the uptake of cofactors essential for enzymes of methanol assimilation. The improved methylotrophy was also confirmed by overexpressing NikABCDE or FhuBCD operon. Finally, the de novo synthetic pathway of lysine was further engineered and the methanol utilization and butyric acid production of B. methylotrophicum were improved by 63.2% and 79.7%, respectively. After an optimization of cultivation medium, 3.69 g/L of butyric acid was finally achieved from methanol with a yield of 76.3%, the highest level reported to date. CONCLUSION: This study revealed a novel mechanism to regulate methanol assimilation by lysine in B. methylotrophicum and engineered it to improve methanol bioconversion to butyric acid, culminating in the synthesis of the highest butyric acid titer reported so far in B. methylotrophicum. What's more, our work represents a further advancement in the engineering of methylotrophic-acetogenic bacterium to improve C1-compound utilization.

Lanpepsy is a novel lanthanide-binding protein involved in the lanthanide response of the obligate methylotroph Methylobacillus flagellatus.

Lanthanides were recently discovered as metals required in the active site of certain methanol dehydrogenases. Since then, the characterization of the lanthanome, that is, proteins involved in sensing, uptake, and utilization of lanthanides, has become an active field of research. Initial exploration of the response to lanthanides in methylotrophs has revealed that the lanthanome is not conserved and that multiple mechanisms for lanthanide utilization must exist. Here, we investigated the lanthanome in the obligate model methylotroph Methylobacillus flagellatus. We used a proteomic approach to analyze differentially regulated proteins in the presence of lanthanum. While multiple known proteins showed induction upon growth in the presence of lanthanum (Xox proteins, TonB-dependent receptor), we also identified several novel proteins not previously associated with lanthanide utilization. Among these was Mfla_0908, a periplasmic 19 kDa protein without functional annotation. The protein comprises two characteristic PepSY domains, which is why we termed the protein lanpepsy (LanP). Based on bioinformatic analysis, we speculated that LanP could be involved in lanthanide binding. Using dye competition assays, quantification of protein-bound lanthanides by inductively coupled plasma mass spectrometry, as well as isothermal titration calorimetry, we demonstrated the presence of multiple lanthanide binding sites that showed selectivity over the chemically similar calcium ion. LanP thus represents the first member of the PepSY family that binds lanthanides. Although the physiological role of LanP is still unclear, its identification is of interest for applications toward the sustainable purification and separation of rare-earth elements.

A Methylotrophic Bacterium Growing with the Antidiabetic Drug Metformin as Its Sole Carbon, Nitrogen and Energy Source.

Metformin is one of the most prescribed antidiabetic agents worldwide and is also considered for other therapeutic applications including cancer and endocrine disorders. It is largely unmetabolized by human enzymes and its presence in the environment has raised concern, with reported toxic effects on aquatic life and potentially also on humans. We report on the isolation and characterisation of strain MD1, an aerobic methylotrophic bacterium growing with metformin as its sole carbon, nitrogen and energy source. Strain MD1 degrades metformin into dimethylamine used for growth, and guanylurea as a side-product. Sequence analysis of its fully assembled genome showed its affiliation to Aminobacter niigataensis. Differential proteomics and transcriptomics, as well as mini-transposon mutagenesis of the strain, point to genes and proteins essential for growth with metformin and potentially associated with hydrolytic C-N cleavage of metformin or with cellular transport of metformin and guanylurea. The obtained results suggest the recent evolution of the growth-supporting capacity of strain MD1 to degrade metformin. Our results identify candidate proteins of the enzymatic system for metformin transformation in strain MD1 and will inform future research on the fate of metformin and its degradation products in the environment and in humans.

Synthetic methylotrophic yeasts for the sustainable fuel and chemical production.

Global energy-related emissions, in particular carbon dioxide, are rapidly increasing. Without immediate and strong reductions across all sectors, limiting global warming to 1.5 °C and thus mitigating climate change is beyond reach. In addition to the expansion of renewable energies and the increase in energy efficiency, the so-called Carbon Capture and Utilization technologies represent an innovative approach for closing the carbon cycle and establishing a circular economy. One option is to combine CO2 capture with microbial C1 fermentation. C1-molecules, such as methanol or formate are considered as attractive alternative feedstock for biotechnological processes due to their sustainable production using only CO2, water and renewable energy. Native methylotrophic microorganisms can utilize these feedstock for the production of value-added compounds. Currently, constraints exist regarding the understanding of methylotrophic metabolism and the available genetic engineering tools are limited. For this reason, the development of synthetic methylotrophic cell factories based on the integration of natural or artificial methanol assimilation pathways in biotechnologically relevant microorganisms is receiving special attention. Yeasts like Saccharomyces cerevisiae and Yarrowia lipolytica are capable of producing important products from sugar-based feedstock and the switch to produce these in the future from methanol is important in order to realize a CO2-based economy that is independent from land use. Here, we review historical biotechnological applications, the metabolism and the characteristics of methylotrophic yeasts. Various studies demonstrated the production of a broad set of promising products from fine chemicals to bulk chemicals by applying methylotrophic yeasts. Regarding synthetic methylotrophy, the deep understanding of the methylotrophic metabolism serves as the basis for microbial strain engineering and paves the way towards a CO2-based circular bioeconomy. We highlight design aspects of synthetic methylotrophy and discuss the resulting chances and challenges using non-conventional yeasts as host organisms. We conclude that the road towards synthetic methylotrophic yeasts can only be achieved through a combination of methods (e.g., metabolic engineering and adaptive laboratory evolution). Furthermore, we presume that the installation of metabolic regeneration cycles such as supporting carbon re-entry towards the pentose phosphate pathway from C1-metabolism is a pivotal target for synthetic methylotrophy.

Methylothon: a Versatile Course-Based High School Research Experience in Microbiology and Bioinformatics with Pink Bacteria.

Methylothon 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 at http://methylothon.com), and relate lessons learned from adapting Methylothon for remote learning during the pandemic in spring of 2021. This curriculum description is intended not only for instructors but also for microbial ecology researchers with an interest in conducting K-12 outreach. 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.

Microbial functional diversity across biogeochemical provinces in the central Pacific Ocean.

Enzymes catalyze key reactions within Earth's life-sustaining biogeochemical cycles. Here, we use metaproteomics to examine the enzymatic capabilities of the microbial community (0.2 to 3 µm) along a 5,000-km-long, 1-km-deep transect in the central Pacific Ocean. Eighty-five percent of total protein abundance was of bacterial origin, with Archaea contributing 1.6%. Over 2,000 functional KEGG Ontology (KO) groups were identified, yet only 25 KO groups contributed over half of the protein abundance, simultaneously indicating abundant key functions and a long tail of diverse functions. Vertical attenuation of individual proteins displayed stratification of nutrient transport, carbon utilization, and environmental stress. The microbial community also varied along horizontal scales, shaped by environmental features specific to the oligotrophic North Pacific Subtropical Gyre, the oxygen-depleted Eastern Tropical North Pacific, and nutrient-rich equatorial upwelling. Some of the most abundant proteins were associated with nitrification and C1 metabolisms, with observed interactions between these pathways. The oxidoreductases nitrite oxidoreductase (NxrAB), nitrite reductase (NirK), ammonia monooxygenase (AmoABC), manganese oxidase (MnxG), formate dehydrogenase (FdoGH and FDH), and carbon monoxide dehydrogenase (CoxLM) displayed distributions indicative of biogeochemical status such as oxidative or nutritional stress, with the potential to be more sensitive than chemical sensors. Enzymes that mediate transformations of atmospheric gases like CO, CO2, NO, methanethiol, and methylamines were most abundant in the upwelling region. We identified hot spots of biochemical transformation in the central Pacific Ocean, highlighted previously understudied metabolic pathways in the environment, and provided rich empirical data for biogeochemical models critical for forecasting ecosystem response to climate change.

Toward Methanol-Based Biomanufacturing: Emerging Strategies for Engineering Synthetic Methylotrophy in Saccharomyces cerevisiae.

The global expansion of biomanufacturing is currently limited by the availability of sugar-based microbial feedstocks, which require farmland for cultivation and therefore cannot support large increases in production without impacting the human food supply. One-carbon feedstocks, such as methanol, present an enticing alternative to sugar because they can be produced independently of arable farmland from organic waste, atmospheric carbon dioxide, and hydrocarbons such as biomethane, natural gas, and coal. The development of efficient industrial microorganisms that can convert one-carbon feedstocks into valuable products is an ongoing challenge. This review discusses progress in the field of synthetic methylotrophy with a focus on how it pertains to the important industrial yeast, Saccharomyces cerevisiae. Recent insights generated from engineering synthetic methylotrophic xylulose- and ribulose-monophosphate cycles, reductive glycine pathways, and adaptive laboratory evolution studies are critically assessed to generate novel strategies for the future engineering of methylotrophy in S. cerevisiae.

Genome-Wide Transcription Start Sites Mapping in Methylorubrum Grown with Dichloromethane and Methanol.

Dichloromethane (DCM, methylene chloride) is a toxic halogenated volatile organic compound massively used for industrial applications, and consequently often detected in the environment as a major pollutant. DCM biotransformation suggests a sustainable decontamination strategy of polluted sites. Among methylotrophic bacteria able to use DCM as a sole source of carbon and energy for growth, Methylorubrum extorquens DM4 is a longstanding reference strain. Here, the primary 5'-ends of transcripts were obtained using a differential RNA-seq (dRNA-seq) approach to provide the first transcription start site (TSS) genome-wide landscape of a methylotroph using DCM or methanol. In total, 7231 putative TSSs were annotated and classified with respect to their localization to coding sequences (CDSs). TSSs on the opposite strand of CDS (antisense TSS) account for 31% of all identified TSSs. One-third of the detected TSSs were located at a distance to the start codon inferior to 250 nt (average of 84 nt) with 7% of leaderless mRNA. Taken together, the global TSS map for bacterial growth using DCM or methanol will facilitate future studies in which transcriptional regulation is crucial, and efficient DCM removal at polluted sites is limited by regulatory processes.

A putatively new family of alphaproteobacterial chloromethane degraders from a deciduous forest soil revealed by stable isotope probing and metagenomics.

BACKGROUND: Chloromethane (CH3Cl) is the most abundant halogenated organic compound in the atmosphere and substantially responsible for the destruction of the stratospheric ozone layer. Since anthropogenic CH3Cl sources have become negligible with the application of the Montreal Protocol (1987), natural sources, such as vegetation and soils, have increased proportionally in the global budget. CH3Cl-degrading methylotrophs occurring in soils might be an important and overlooked sink. RESULTS AND CONCLUSIONS: The objective of our study was to link the biotic CH3Cl sink with the identity of active microorganisms and their biochemical pathways for CH3Cl degradation in a deciduous forest soil. When tested in laboratory microcosms, biological CH3Cl consumption occurred in leaf litter, senescent leaves, and organic and mineral soil horizons. Highest consumption rates, around 2 mmol CH3Cl g-1 dry weight h-1, were measured in organic soil and senescent leaves, suggesting that top soil layers are active (micro-)biological CH3Cl degradation compartments of forest ecosystems. The DNA of these [13C]-CH3Cl-degrading microbial communities was labelled using stable isotope probing (SIP), and the corresponding taxa and their metabolic pathways studied using high-throughput metagenomics sequencing analysis. [13C]-labelled Metagenome-Assembled Genome closely related to the family Beijerinckiaceae may represent a new methylotroph family of Alphaproteobacteria, which is found in metagenome databases of forest soils samples worldwide. Gene markers of the only known pathway for aerobic CH3Cl degradation, via the methyltransferase system encoded by the CH3Cl utilisation genes (cmu), were undetected in the DNA-SIP metagenome data, suggesting that biological CH3Cl sink in this deciduous forest soil operates by a cmu-independent metabolism.