Methane is an essential component of the global carbon cycle and one of the most powerful greenhouse gases, yet it is also a promising alternative source of carbon for the biological production of ...value-added chemicals. Aerobic methane-consuming bacteria (methanotrophs) represent a potential biological platform for methane-based biocatalysis. Here we use a multi-pronged systems-level approach to reassess the metabolic functions for methane utilization in a promising bacterial biocatalyst. We demonstrate that methane assimilation is coupled with a highly efficient pyrophosphate-mediated glycolytic pathway, which under oxygen limitation participates in a novel form of fermentation-based methanotrophy. This surprising discovery suggests a novel mode of methane utilization in oxygen-limited environments, and opens new opportunities for a modular approach towards producing a variety of excreted chemical products using methane as a feedstock.
Methane, as natural gas or biogas, is the least expensive source of carbon for (bio)chemical synthesis. Scalable biological upgrading of this simple alkane to chemicals and fuels can bring new ...sustainable solutions to a number of industries with large environmental footprints, such as natural gas/petroleum production, landfills, wastewater treatment, and livestock. Microbial biocatalysis with methane as a feedstock has been pursued off and on for almost a half century, with little enduring success. Today, biological engineering and systems biology provide new opportunities for metabolic system modulation and give new optimism to the concept of a methane-based bio-industry. Here we present an overview of the most recent advances pertaining to metabolic engineering of microbial methane utilization. Some ideas concerning metabolic improvements for production of acetyl-CoA and pyruvate, two main precursors for bioconversion, are presented. We also discuss main gaps in the current knowledge of aerobic methane utilization, which must be solved in order to release the full potential of methane-based biosystems.
•Methanotrophs have the potential to play a role in our sustainable energy future.•A system-level understanding of methanotrophic metabolism will lay the groundwork for metabolic engineering to generate value-added products from methane.•A set of gaps in our fundamental knowledge of methanotrophy make predictive metabolic engineering more difficult, but approaches are available to accommodate the uncertainty.•A suite of available cultures and tools exist that will facilitate metabolic engineering efforts.•A set of approaches are suggested that have the potential to increase flux to key precursors for many value-added products.
We describe a computationally designed enzyme, formolase (FLS), which catalyzes the carboligation of three one-carbon formaldehyde molecules into one three-carbon dihydroxyacetone molecule. The ...existence of FLS enables the design of a new carbon fixation pathway, the formolase pathway, consisting of a small number of thermodynamically favorable chemical transformations that convert formate into a three-carbon sugar in central metabolism. The formolase pathway is predicted to use carbon more efficiently and with less backward flux than any naturally occurring one-carbon assimilation pathway. When supplemented with enzymes carrying out the other steps in the pathway, FLS converts formate into dihydroxyacetone phosphate and other central metabolites in vitro. These results demonstrate how modern protein engineering and design tools can facilitate the construction of a completely new biosynthetic pathway.
Significance This paper describes the development of a computationally designed enzyme that is the cornerstone of a novel metabolic pathway. This enzyme, formolase, performs a carboligation reaction, directly fixing one-carbon units into three-carbon units that feed into central metabolism. By combining formolase with several naturally occurring enzymes, we created a new carbon fixation pathway, the formolase pathway, which assimilates one-carbon units via formate. Unlike native carbon fixation pathways, this pathway is linear, not oxygen sensitive, and consists of a small number of thermodynamically favorable steps. We demonstrate in vitro pathway function as a proof of principle of how protein design in a pathway context can lead to new efficient metabolic pathways.
In the past few years, the field of methylotrophy has undergone a significant transformation in terms of discovery of novel types of methylotrophs, novel modes of methylotrophy, and novel metabolic ...pathways. This time has also been marked by the resolution of long-standing questions regarding methylotrophy and the challenge of long-standing dogmas. This chapter is not intended to provide a comprehensive review of metabolism of methylotrophic bacteria. Instead we focus on significant recent discoveries that are both refining and transforming the current understanding of methylotrophy as a metabolic phenomenon. We also review new directions in methylotroph ecology that improve our understanding of the role of methylotrophy in global biogeochemical processes, along with an outlook for the future challenges in the field.
Aerobic methanotrophs oxidize methane at ambient temperatures and pressures and are therefore attractive systems for methane-based bioconversions. In this work, we developed and validated genetic ...tools for Methylomicrobium buryatense, a haloalkaliphilic gammaproteobacterial (type I) methanotroph. M. buryatense was isolated directly on natural gas and grows robustly in pure culture with a 3-h doubling time, enabling rapid genetic manipulation compared to many other methanotrophic species. As a proof of concept, we used a sucrose counterselection system to eliminate glycogen production in M. buryatense by constructing unmarked deletions in two redundant glycogen synthase genes. We also selected for a more genetically tractable variant strain that can be conjugated with small incompatibility group P (IncP)-based broad-host-range vectors and determined that this capability is due to loss of the native plasmid. These tools make M. buryatense a promising model system for studying aerobic methanotroph physiology and enable metabolic engineering in this bacterium for industrial biocatalysis of methane.
Full exploitation of the information available in bacterial genome sequences requires the availability of facile tools for rapid genetic manipulation. One bacterium for which new genetic tools are ...needed is the methylotroph Methylobacterium extorquens AM1. IncQ and small IncP vectors were shown to be unsuitable for use in this bacterium, but a spontaneous mutant of a small IncP plasmid was isolated that functioned efficiently in M. extorquens AM1. This plasmid was sequenced and used as a base for developing improved broad-host-range cloning vectors. These vectors were found to replicate in a wide variety of bacterial species and have the following advantages: (1) high copy number in Escherichia coli; (2) small size (7.2 and 8.0 kb); (3) complete sequences; (4) variety of unique restriction sites; (5) blue-white screening via lacZalpha; (6) conjugative mobilization between bacterial species; and (7) readily adaptable into species-specific promoter-probe and expression vectors. Two low-background promoter-probe vectors were constructed based on these cloning vectors with either lacZ or xylE as reporter genes; these were shown to report gene expression effectively in M. extorquens AM1. Specific expression vectors were developed for use in M. extorquens AM1, which were shown to express foreign genes at significant levels, and a simple strategy is outlined to develop specific expression vectors for other bacteria. The strong mxaF promoter was used for expression, since E. coli lac-derived promoters were expressed at very low levels. This suite of genetic tools will enable a more sophisticated analysis of the physiology of M. extorquens AM1, and these vectors should also be valuable tools in the study of a variety of bacterial species.
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