Quantifying Methane and Methanol Metabolism of “<italic toggle="yes">Methylotuvimicrobium buryatense</italic>” 5GB1C under Substrate Limitation

ABSTRACT Methanotrophic bacteria are a group of prokaryotes capable of using methane as their sole carbon and energy source. Although efforts have been made to simulate and elucidate their metabolism via computational approaches or 13C tracer analysis, major gaps still exist in our understanding of...

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Autores principales:	Lian He, Yanfen Fu, Mary E. Lidstrom
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	13C metabolic flux analysis bioreactor chemostat isotopically nonstationary type I methanotroph Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/6bcb6fcdf5e346c1b024847a1ec1080f
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spelling	oai:doaj.org-article:6bcb6fcdf5e346c1b024847a1ec1080f2021-12-02T18:15:44ZQuantifying Methane and Methanol Metabolism of “<italic toggle="yes">Methylotuvimicrobium buryatense</italic>” 5GB1C under Substrate Limitation10.1128/mSystems.00748-192379-5077https://doaj.org/article/6bcb6fcdf5e346c1b024847a1ec1080f2019-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSystems.00748-19https://doaj.org/toc/2379-5077ABSTRACT Methanotrophic bacteria are a group of prokaryotes capable of using methane as their sole carbon and energy source. Although efforts have been made to simulate and elucidate their metabolism via computational approaches or 13C tracer analysis, major gaps still exist in our understanding of methanotrophic metabolism at the systems level. Particularly, direct measurements of system-wide fluxes are required to understand metabolic network function. Here, we quantified the central metabolic fluxes of a type I methanotroph, “Methylotuvimicrobium buryatense” 5GB1C, formerly Methylomicrobium buryatense 5GB1C, via 13C isotopically nonstationary metabolic flux analysis (INST-MFA). We performed labeling experiments on chemostat cultures by switching substrates from 12C to 13C input. Following the switch, we measured dynamic changes of labeling patterns and intracellular pool sizes of several intermediates, which were later used for data fitting and flux calculations. Through computational optimizations, we quantified methane and methanol metabolism at two growth rates (0.1 h−1 and 0.05 h−1). The resulting flux maps reveal a core consensus central metabolic flux phenotype across different growth conditions: a strong ribulose monophosphate cycle, a preference for the Embden-Meyerhof-Parnas pathway as the primary glycolytic pathway, and a tricarboxylic acid cycle showing small yet significant fluxes. This central metabolic consistency is further supported by a good linear correlation between fluxes at the two growth rates. Specific differences between methane and methanol growth observed previously are maintained under substrate limitation, albeit with smaller changes. The substrate oxidation and glycolysis pathways together contribute over 80% of total energy production, while other pathways play less important roles. IMPORTANCE Methanotrophic metabolism has been under investigation for decades using biochemical and genetic approaches. Recently, a further step has been taken toward understanding methanotrophic metabolism in a quantitative manner by means of flux balance analysis (FBA), a mathematical approach that predicts fluxes constrained by mass balance and a few experimental measurements. However, no study has previously been undertaken to experimentally quantitate the complete methanotrophic central metabolism. The significance of this study is to fill such a gap by performing 13C INST-MFA on a fast-growing methanotroph. Our quantitative insights into the methanotrophic carbon and energy metabolism will pave the way for future FBA studies and set the stage for rational design of methanotrophic strains for industrial applications. Further, the experimental strategies can be applied to other methane or methanol utilizers, and the results will offer a unique and quantitative perspective of diverse methylotrophic metabolism.Lian HeYanfen FuMary E. LidstromAmerican Society for Microbiologyarticle13C metabolic flux analysisbioreactorchemostatisotopically nonstationarytype I methanotrophMicrobiologyQR1-502ENmSystems, Vol 4, Iss 6 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	13C metabolic flux analysis bioreactor chemostat isotopically nonstationary type I methanotroph Microbiology QR1-502
spellingShingle	13C metabolic flux analysis bioreactor chemostat isotopically nonstationary type I methanotroph Microbiology QR1-502 Lian He Yanfen Fu Mary E. Lidstrom Quantifying Methane and Methanol Metabolism of “<italic toggle="yes">Methylotuvimicrobium buryatense</italic>” 5GB1C under Substrate Limitation
description	ABSTRACT Methanotrophic bacteria are a group of prokaryotes capable of using methane as their sole carbon and energy source. Although efforts have been made to simulate and elucidate their metabolism via computational approaches or 13C tracer analysis, major gaps still exist in our understanding of methanotrophic metabolism at the systems level. Particularly, direct measurements of system-wide fluxes are required to understand metabolic network function. Here, we quantified the central metabolic fluxes of a type I methanotroph, “Methylotuvimicrobium buryatense” 5GB1C, formerly Methylomicrobium buryatense 5GB1C, via 13C isotopically nonstationary metabolic flux analysis (INST-MFA). We performed labeling experiments on chemostat cultures by switching substrates from 12C to 13C input. Following the switch, we measured dynamic changes of labeling patterns and intracellular pool sizes of several intermediates, which were later used for data fitting and flux calculations. Through computational optimizations, we quantified methane and methanol metabolism at two growth rates (0.1 h−1 and 0.05 h−1). The resulting flux maps reveal a core consensus central metabolic flux phenotype across different growth conditions: a strong ribulose monophosphate cycle, a preference for the Embden-Meyerhof-Parnas pathway as the primary glycolytic pathway, and a tricarboxylic acid cycle showing small yet significant fluxes. This central metabolic consistency is further supported by a good linear correlation between fluxes at the two growth rates. Specific differences between methane and methanol growth observed previously are maintained under substrate limitation, albeit with smaller changes. The substrate oxidation and glycolysis pathways together contribute over 80% of total energy production, while other pathways play less important roles. IMPORTANCE Methanotrophic metabolism has been under investigation for decades using biochemical and genetic approaches. Recently, a further step has been taken toward understanding methanotrophic metabolism in a quantitative manner by means of flux balance analysis (FBA), a mathematical approach that predicts fluxes constrained by mass balance and a few experimental measurements. However, no study has previously been undertaken to experimentally quantitate the complete methanotrophic central metabolism. The significance of this study is to fill such a gap by performing 13C INST-MFA on a fast-growing methanotroph. Our quantitative insights into the methanotrophic carbon and energy metabolism will pave the way for future FBA studies and set the stage for rational design of methanotrophic strains for industrial applications. Further, the experimental strategies can be applied to other methane or methanol utilizers, and the results will offer a unique and quantitative perspective of diverse methylotrophic metabolism.
format	article
author	Lian He Yanfen Fu Mary E. Lidstrom
author_facet	Lian He Yanfen Fu Mary E. Lidstrom
author_sort	Lian He
title	Quantifying Methane and Methanol Metabolism of “<italic toggle="yes">Methylotuvimicrobium buryatense</italic>” 5GB1C under Substrate Limitation
title_short	Quantifying Methane and Methanol Metabolism of “<italic toggle="yes">Methylotuvimicrobium buryatense</italic>” 5GB1C under Substrate Limitation
title_full	Quantifying Methane and Methanol Metabolism of “<italic toggle="yes">Methylotuvimicrobium buryatense</italic>” 5GB1C under Substrate Limitation
title_fullStr	Quantifying Methane and Methanol Metabolism of “<italic toggle="yes">Methylotuvimicrobium buryatense</italic>” 5GB1C under Substrate Limitation
title_full_unstemmed	Quantifying Methane and Methanol Metabolism of “<italic toggle="yes">Methylotuvimicrobium buryatense</italic>” 5GB1C under Substrate Limitation
title_sort	quantifying methane and methanol metabolism of “<italic toggle="yes">methylotuvimicrobium buryatense</italic>” 5gb1c under substrate limitation
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/6bcb6fcdf5e346c1b024847a1ec1080f
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Quantifying Methane and Methanol Metabolism of “<italic toggle="yes">Methylotuvimicrobium buryatense</italic>” 5GB1C under Substrate Limitation

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