The oxidative TCA cycle operates during methanotrophic growth of the Type I methanotroph Methylomicrobium buryatense 5GB1 |
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| Affiliation: | 1. Department of Chemical Engineering, University of Washington, 616 NE, Northlake Place, Seattle, WA 98195, USA;2. Department of Microbiology, University of Washington, 616 NE, Northlake Place, Seattle, WA 98195, USA;1. Department of Biotechnology, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan;2. Research Center for Solar Energy Chemistry, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan;3. Department of Chemistry, Graduate School of Engineering Science, Osaka University, Machikaneyama, Toyonaka, Osaka 560-8531, Japan;4. Education Academy of Computational Life Sciences, Tokyo Institute of Technology, 2-12-1 Ookayama, Meguro-ku, Tokyo 152-8550, Japan;5. Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo, Hokkaido 062-8517, Japan;6. Department of Life Science and Technology, Graduated School of Bioscience and Biotechnology, Tokyo Institute of Technology, 2-12-1 Ookayama, Meguro-ku, Tokyo 152-8550, Japan;1. Department of Civil and Environmental Engineering, Stanford University, CA, United States;2. Department of Mechanical Engineering, Stanford University, CA, United States;3. Department of Materials Science and Engineering, Stanford University, CA, United States;1. Department of Technology, Savitribai Phule Pune University, Pune, India;2. Praj Matrix - R&D Centre, division of Praj Industries Limited, Urawade, Pune, India;1. Biology Department, San Diego State University, San Diego, CA 92182-4614, United States;2. Department of Chemical Engineering, Seattle, WA 98195, United States;3. Department of Microbiology, University of Washington, Seattle, WA 98195, United States |
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| Abstract: | Methanotrophs are a group of bacteria that use methane as sole carbon and energy source. Type I methanotrophs are gamma-proteobacterial methanotrophs using the ribulose monophosphate cycle (RuMP) cycle for methane assimilation. In order to facilitate metabolic engineering in the industrially promising Type I methanotroph Methylomicrobium buryatense 5GB1, flux analysis of cellular metabolism is needed and 13C tracer analysis is a foundational tool for such work. This biological system has a single-carbon input and a special network topology that together pose challenges to the current well-established methodology for 13C tracer analysis using a multi-carbon input such as glucose, and to date, no 13C tracer analysis of flux in a Type I methanotroph has been reported. In this study, we showed that by monitoring labeling patterns of several key intermediate metabolites in core metabolism, it is possible to quantitate the relative flux ratios for important branch points, such as the malate node. In addition, it is possible to assess the operation of the TCA cycle, which has been thought to be incomplete in Type I methanotrophs. Surprisingly, our analysis provides direct evidence of a complete, oxidative TCA cycle operating in M. buryatense 5GB1 using methane as sole carbon and energy substrate, contributing about 45% of the total flux for de novo malate production. Combined with mutant analysis, this method was able to identify fumA (METBUDRAFT_1453/MBURv2__60244) as the primary fumarase involved in the oxidative TCA cycle, among 2 predicted fumarases, supported by 13C tracer analysis on both fumA and fumC single knockouts. Interrupting the oxidative TCA cycle leads to a severe growth defect, suggesting that the oxidative TCA cycle functions to not only provide precursors for de novo biomass synthesis, but also to provide reducing power to the system. This information provides new opportunities for metabolic engineering of M. buryatense for the production of industrially relevant products. |
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| Keywords: | Methanotroph Methane TCA cycle |
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