Curr. Issues Mol. Biol., Volume 33, Issue 1 (October 2019) – 15 articles

Experimental evolution has become an increasingly common approach for studying evolutionary phenomena, as well as uncovering physiological connections in a manner complementary to traditional genetics. Here I describe the development of Methylobacterium as a model system for using experimental evolution to study questions at the intersection of metabolism and evolution. Each experiment was initiated to address a particular question inspired by patterns in natural methylotrophs, such as tradeoffs between single-carbon and multi-carbon growth, or the challenges involved in incorporating novel metabolic pathways or genes with poor codon usage that are acquired via horizontal gene transfer. What I could not have appreciated initially, however, was just how many fortuitous surprise findings would emerge. These have ranged from the repeatability of evolution, complex dynamics within populations, epistasis between beneficial mutations, and even the ability to use simple mathematical models to generate testable, quantitative hypotheses about the fitness landscape. Full article

One-carbon (C1) feedstocks can provide a vital link between cheap and sustainable abiotic resources and microbial bioproduction. Soluble C1 substrates, methanol and formate, could prove more suitable than gaseous feedstocks as they avoid mass transfer barriers. However, microorganisms that naturally assimilate methanol and formate are limited by a narrow product spectrum and a restricted genetic toolbox. Engineering biotechnological organisms to assimilate these soluble C1 substrates has therefore become an attractive goal. Here, we discuss the use of a step-wise, modular engineering approach for the implementation of C1-pathways. In this strategy, pathways are divided into metabolic modules, the activities of which are selected for in dedicated gene-deletion strains whose growth directly depends on module activity. This provides an easy way to identify and resolve metabolic barriers hampering pathway performance. Optimization of gene expression levels and adaptive laboratory evolution can be used to establish the desired activity if direct selection fails. We exemplify this approach using several pathways, focusing especially on the ribulose monophosphate cycle for methanol assimilation and the reductive glycine pathway for formate assimilation. We argue that such modular engineering and selection strategies will prove essential for rewiring microbial metabolism towards new growth phenotypes and sustainable bioproduction. Full article

Methanol, commercially generated from methane, is a renewable chemical feedstock that is highly soluble, relatively inexpensive, and easy to handle. The concept of native methylotrophic bacteria serving as whole cell catalysts for production of chemicals and materials using methanol as a feedstock is highly attractive. In recent years, the available omics data for methylotrophic bacteria, especially for Methylobacterium extorquens, the most well-characterized model methylotroph, have provided a solid platform for rational engineering of methylotrophic bacteria for industrial production. In addition, there is a strong interest in converting the more traditional heterotrophic production platforms toward the use of single carbon substrates, including methanol, through metabolic engineering. In this chapter, we review the recent progress toward achieving the desired growth and production yields from methanol, by genetically engineered native methylotrophic strains and by the engineered synthetic methylotrophs. Full article

Biosynthesized small molecules known as specialized metabolites often have valuable applications in fields such as medicine and agriculture. Consequently, there is always a demand for novel specialized metabolites and an understanding of their bioactivity. Methylotrophs are an underexplored metabolic group of bacteria that have several growth features that make them enticing in terms of specialized metabolite discovery, characterization, and production from cheap feedstocks such as methanol and methane gas. This chapter will examine the predicted biosynthetic potential of these organisms and review some of the specialized metabolites they produce that have been characterized so far. Full article

Methylotrophic yeasts, which are able to utilize methanol as the sole carbon and energy source, have been intensively studied in terms of physiological function and practical applications. When these yeasts grow on methanol, the genes encoding enzymes and proteins involved in methanol metabolism are strongly induced. Simultaneously, peroxisomes, organelles that contain the key enzymes for methanol metabolism, massively proliferate. These characteristics have made methylotrophic yeasts efficient hosts for heterologous protein production using strong and methanol-inducible gene promoters and also model organisms for the study of peroxisome dynamics. Much attention has been paid to the interaction between methylotrophic microorganisms and plants. In this chapter, we describe how methylotrophic yeasts proliferate and survive on plant leaves, focusing on their physiological functions and lifestyle in the phyllosphere. Our current understanding of the molecular basis of methanol-inducible gene expression, including methanol-sensing and its applications, is also summarized. Full article

In this review article, we cover the recent developments in understanding the principles and the mechanisms by which microbial communities participating in methane consumption in natural environmental niches are assembled, and the physiological and biochemical mechanisms and regulators that allow efficient carbon transfer within the communities. We first give a brief overview of methanotrophy. We then describe the recent evidence on non-random assembly of bacterial communities that utilize carbon from methane, based on stable isotope probing experiments as well as on results from natural community manipulations followed by metagenomic analysis. We follow up by highlighting results from synthetic methanotophic community manipulations identifying the importance of a lanthanide switch that regulates alternative methanol dehydrogenase enzymes in these communities. We further expand on the recently uncovered significance of lanthanides in methylotrophy and review data on the biochemical properties of representatives of two different clades of lanthanide-dependent enzymes. We also provide an overview of the occurrence and the distribution of the lanthanide-dependent alcohol dehydrogenases in the bacterial domain, these data strongly suggesting significance of these metals beyond methylotrophy. Full article

Methanethiol (MT) is an organic sulfur compound with a strong and disagreeable odour. It has biogeochemical relevance as an important compound in the global sulfur cycle, where it is produced as a reactive intermediate in a number of different pathways for synthesis and degradation of other globally significant sulfur compounds such as dimethylsulfoniopropionate, dimethylsulfide and methionine. With its low odour threshold and unpleasant smell, MT can be a significant cause of malodour originating from animal husbandry, composting, landfill operations, and wastewater treatment and is also associated with faeces, flatus and oral malodour (halitosis). A diverse range of microorganisms drives the production and degradation of MT, including its aerobic and anaerobic metabolism. MT producing and degrading organisms are known to be present in terrestrial, freshwater and marine environments but may also be important in association with plant and animal (including human) hosts. This chapter considers the role of MT as an intermediate of the global sulfur cycle and discusses current knowledge of microbial pathways of MT production and degradation. Full article

Chloromethane is a halogenated volatile organic compound, produced in large quantities by terrestrial vegetation. After its release to the troposphere and transport to the stratosphere, its photolysis contributes to the degradation of stratospheric ozone. A better knowledge of chloromethane sources (production) and sinks (degradation) is a prerequisite to estimate its atmospheric budget in the context of global warming. The degradation of chloromethane by methylotrophic communities in terrestrial environments is a major underestimated chloromethane sink. Methylotrophs isolated from soils, marine environments and more recently from the phyllosphere have been grown under laboratory conditions using chloromethane as the sole carbon source. In addition to anaerobes that degrade chloromethane, the majority of cultivated strains were isolated in aerobiosis for their ability to use chloromethane as sole carbon and energy source. Among those, the Proteobacterium Methylobacterium (recently reclassified as Methylorubrum) harbours the only characterisized 'chloromethane utilization' (cmu) pathway, so far. This pathway is not representative of chloromethane-utilizing populations in the environment as cmu genes are rare in metagenomes. Recently, combined 'omics' biological approaches with chloromethane carbon and hydrogen stable isotope fractionation measurements in microcosms, indicated that microorganisms in soils and the phyllosphere (plant aerial parts) represent major sinks of chloromethane in contrast to more recently recognized microbe-inhabited environments, such as clouds. Cultivated chloromethane-degraders lacking the cmu genes display a singular isotope fractionation signature of chloromethane. Moreover, ¹³CH₃Cl labelling of active methylotrophic communities by stable isotope probing in soils identify taxa that differ from the taxa known for chloromethane degradation. These observations suggest that new biomarkers for detecting active microbial chloromethane-utilizers in the environment are needed to assess the contribution of microorganisms to the global chloromethane cycle. Full article

Methylated amines (MAs) are ubiquitous in marine ecosystems, found from surface seawaters to sediment pore waters. These volatile ammonium analogs play important roles in biogeochemical cycles of carbon and nitrogen in the marine water column. They also contribute to the release of climate-active gases, being precursors of the potent greenhouse gas methane through methanogenesis in coastal sediments. Very recently, it also became acknowledged that MAs are important precursors for new particle growth, hence forming cloud-condensation nuclei in the marine atmosphere. Microbial metabolism of MAs has been demonstrated in the marine ecosystems for both Archaea and Bacteria. In this chapter, we summarize the latest developments in analytical methods for quantifying MA concentrations in marine surface water and sediments. We discuss the metabolic pathways leading to the formation and degradation of MAs by marine microbes and the novel biochemistry and structural biology of the enzymes for MA transformation. Lastly, we highlight the need for future research toward a better understanding of the microbiology and ecology of oceanic MA cycles. Full article

Paracoccus denitrificans Pd 1222 is a model methylotrophic bacterium. Its methylotrophy is based on autotrophic growth (enabled by the Calvin cycle) supported by energy from the oxidation of methanol or methylamine. The growing availability of genome sequence data has made it possible to investigate methylotrophy in other Paracoccus species. The examination of a large number of Paracoccus spp. genomes reveals great variability in C₁ metabolism, which have been shaped by different evolutionary mechanisms. Surprisingly, the methylotrophy schemes of many Paracoccus strains appear to have quite different genetic and biochemical bases. Besides the expected 'autotrophic methylotrophs', many strains of this genus possess another C₁ assimilatory pathway, the serine cycle, which seems to have at least three independent origins. Analysis of the co-occurrence of different methylotrophic pathways indicates, on the one hand, evolutionary linkage between the Calvin cycle and the serine cycle, and, on the other hand, that genes encoding some C₁ substrate-oxidizing enzymes occur more frequently in association with one or the other. This suggests that some genetic module combinations form more harmonious enzymatic sets, which act with greater efficiency in the methylotrophic process and thus undergo positive selection. Full article

Lanthanides were previously thought to be biologically inert owing to their low solubility; however, they have recently been shown to strongly impact the metabolism of methylotrophic bacteria. Leading efforts in this emergent field have demonstrated far-reaching impacts of lanthanide metabolism in biology; from the identification of novel roles of enzymes and pathways dependent on lanthanide-chemistry to the control of transcriptional regulatory networks to the modification of microbial community interactions. Even further, the recent discovery of lanthanide-dependent enzymes associated with multi-carbon metabolism in both methylotrophs and non-methylotrophs alike suggests that lanthanide biochemistry may be more widespread than initially thought. Current efforts aim to understand how lanthanide chemistry and lanthanide-dependent enzymes affect numerous ecosystems and metabolic functions. These efforts will likely have a profound impact on biotechnological processes involving methylotrophic communities and the biologically mediated recovery of these critical metals from a variety of waste streams while redefining our understanding of a fundamental set of metals in biology. Full article

This review is focused on recent studies of carbon metabolism in aerobic methanotrophs that specifically addressed the properties, distribution and phylogeny of some of the key enzymes involved in assimilation of carbon from methane. These include enzymes involved in sugar sythesis and cleavage, conversion of intermediates of the tricarboxylic acid cycle, as well as in osmoadaptation in halotolerant methanotrophs. Full article

Methanotrophic microorganisms utilize methane as an electron donor and a carbon source. To date, the capacity to oxidize methane is restricted to microorganisms from three bacterial and one archaeal phyla. Most of our knowledge of methanotrophic metabolism has been obtained using highly enriched or pure cultures grown in the laboratory. However, many methanotrophs currently evade cultivation, thus metagenomics provides a complementary approach for gaining insight into currently unisolated microorganisms. Here we synthesize the studies using metagenomics to glean information about methanotrophs. We complement this summary with an analysis of methanotroph marker genes from 235 publically available metagenomic datasets. We analyze the phylogenetic and environmental distribution of methanotrophs sampled by metagenomics. We also highlight metabolic insights that methanotroph genomes assembled from metagenomes are illuminating. In summary, metagenomics has increased methanotrophic foliage within the tree of life, as well as provided new insights into methanotroph metabolism, which collectively can guide new cultivation efforts. Lastly, given the importance of methanotrophs for biotechnological applications and their capacity to filter greenhouse gases from a variety of ecosystems, metagenomics will continue to be an important component in the arsenal of tools needed for understanding methanotroph diversity and metabolism in both engineered and natural systems. Full article

Microorganisms are important players in the global methane cycle. Anaerobic methanogenic archaea are largely responsible for methane production, while aerobic methanotrophic bacteria, as well as anaerobic methanotrophic bacteria and archaea, are involved in methane oxidation. In anoxic wetland soils, methanogens produce methane, while methanotrophs act as a filter and reduce methane emissions. In the predominantly oxic upland soils, aerobic methanotrophs oxidize atmospheric methane. This review gives an overview of the diversity of methanogenic and methanotrophic microorganisms, highlights recent discoveries and provides information concerning their occurrence in soils. Recent findings indicate that the methanogenic and methanotrophic lifestyles are more widespread in microorganisms than previously thought, and that the metabolic versatility of some methane-cycling organisms is broader than known from well characterized cultivated organisms. It also turned out that the control of methanogenic and methanotrophic bacteria by oxygen is more complex than previously thought. The implications this finding may have for the life of these microorganisms in soils and on soil methane fluxes is discussed. Full article

Aerobic methanotrophs are an intriguing group of microbes with the singular ability to consume methane as their sole source of carbon and energy. As such, methanotrophs are receiving increased attention to control methane emissions to limit future climate change. Methanotrophs have a wide range of other applications, including pollutant remediation and methane valorization (e.g. conversion of methane to protein, bioplastics, and biodiesel amongst other products). Methanotrophs also produce a novel copper-binding compound, methanobactin, that has significant potential for the treatment of copper-related human pathologies. Here we provide an overview of aerobic methanotrophy, describe current and future applications of these unique microbes, as well as discuss various strategies one can consider to better realize the opportunities these microbes present. Full article