Enabling Unbalanced Fermentations by Using Engineered Electrode-Interfaced Bacteria

ABSTRACT Cellular metabolism is a series of tightly linked oxidations and reductions that must be balanced. Recycling of intracellular electron carriers during fermentation often requires substrate conversion to undesired products, while respiration demands constant addition of electron acceptors. T...

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Autores principales:	Jeffrey M. Flynn, Daniel E. Ross, Kristopher A. Hunt, Daniel R. Bond, Jeffrey A. Gralnick
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2010
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/6dd375a692e041fe99836c2b7b4e6eb3
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spelling	oai:doaj.org-article:6dd375a692e041fe99836c2b7b4e6eb32021-11-15T15:38:17ZEnabling Unbalanced Fermentations by Using Engineered Electrode-Interfaced Bacteria10.1128/mBio.00190-102150-7511https://doaj.org/article/6dd375a692e041fe99836c2b7b4e6eb32010-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00190-10https://doaj.org/toc/2150-7511ABSTRACT Cellular metabolism is a series of tightly linked oxidations and reductions that must be balanced. Recycling of intracellular electron carriers during fermentation often requires substrate conversion to undesired products, while respiration demands constant addition of electron acceptors. The use of electrode-based electron acceptors to balance biotransformations may overcome these constraints. To test this hypothesis, the metal-reducing bacterium Shewanella oneidensis was engineered to stoichiometrically convert glycerol into ethanol, a biotransformation that will not occur unless two electrons are removed via an external reaction, such as electrode reduction. Multiple modules were combined into a single plasmid to alter S. oneidensis metabolism: a glycerol module, consisting of glpF, glpK, glpD, and tpiA from Escherichia coli, and an ethanol module containing pdc and adh from Zymomonas mobilis. A further increase in product yields was accomplished through knockout of pta, encoding phosphate acetyltransferase, shifting flux toward ethanol and away from acetate production. In this first-generation demonstration, conversion of glycerol to ethanol required the presence of an electrode to balance the reaction, and electrode-linked rates were on par with volumetric conversion rates observed in engineered E. coli. Linking microbial biocatalysis to current production can eliminate redox constraints by shifting other unbalanced reactions to yield pure products and serve as a new platform for next-generation bioproduction strategies. IMPORTANCE All reactions catalyzed by whole cells or enzymes must achieve redox balance. In rare cases, conversion can be achieved via perfectly balanced fermentations, allowing all electron equivalents to be recovered in a single product. In most biotransformations, organisms must produce a mixture of acids, gasses, and/or alcohols, and no amount of enzyme or strain engineering can overcome this fundamental requirement. Stoichiometric conversion of glycerol, a waste product from biodiesel transesterification, into ethanol and CO2 with no side products represents such an impossible fermentation, due to the more reduced state of glycerol than of ethanol and CO2. The unbalanced conversion of glycerol to ethanol has been viewed as having only two solutions: fermenting glycerol to ethanol and potentially useful coproducts or “burning off” excess electrons via careful introduction of oxygen. Here, we use the glycerol-to-ethanol example to demonstrate a third strategy, using bacteria directly interfaced to electrodes.Jeffrey M. FlynnDaniel E. RossKristopher A. HuntDaniel R. BondJeffrey A. GralnickAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 1, Iss 5 (2010)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Jeffrey M. Flynn Daniel E. Ross Kristopher A. Hunt Daniel R. Bond Jeffrey A. Gralnick Enabling Unbalanced Fermentations by Using Engineered Electrode-Interfaced Bacteria
description	ABSTRACT Cellular metabolism is a series of tightly linked oxidations and reductions that must be balanced. Recycling of intracellular electron carriers during fermentation often requires substrate conversion to undesired products, while respiration demands constant addition of electron acceptors. The use of electrode-based electron acceptors to balance biotransformations may overcome these constraints. To test this hypothesis, the metal-reducing bacterium Shewanella oneidensis was engineered to stoichiometrically convert glycerol into ethanol, a biotransformation that will not occur unless two electrons are removed via an external reaction, such as electrode reduction. Multiple modules were combined into a single plasmid to alter S. oneidensis metabolism: a glycerol module, consisting of glpF, glpK, glpD, and tpiA from Escherichia coli, and an ethanol module containing pdc and adh from Zymomonas mobilis. A further increase in product yields was accomplished through knockout of pta, encoding phosphate acetyltransferase, shifting flux toward ethanol and away from acetate production. In this first-generation demonstration, conversion of glycerol to ethanol required the presence of an electrode to balance the reaction, and electrode-linked rates were on par with volumetric conversion rates observed in engineered E. coli. Linking microbial biocatalysis to current production can eliminate redox constraints by shifting other unbalanced reactions to yield pure products and serve as a new platform for next-generation bioproduction strategies. IMPORTANCE All reactions catalyzed by whole cells or enzymes must achieve redox balance. In rare cases, conversion can be achieved via perfectly balanced fermentations, allowing all electron equivalents to be recovered in a single product. In most biotransformations, organisms must produce a mixture of acids, gasses, and/or alcohols, and no amount of enzyme or strain engineering can overcome this fundamental requirement. Stoichiometric conversion of glycerol, a waste product from biodiesel transesterification, into ethanol and CO2 with no side products represents such an impossible fermentation, due to the more reduced state of glycerol than of ethanol and CO2. The unbalanced conversion of glycerol to ethanol has been viewed as having only two solutions: fermenting glycerol to ethanol and potentially useful coproducts or “burning off” excess electrons via careful introduction of oxygen. Here, we use the glycerol-to-ethanol example to demonstrate a third strategy, using bacteria directly interfaced to electrodes.
format	article
author	Jeffrey M. Flynn Daniel E. Ross Kristopher A. Hunt Daniel R. Bond Jeffrey A. Gralnick
author_facet	Jeffrey M. Flynn Daniel E. Ross Kristopher A. Hunt Daniel R. Bond Jeffrey A. Gralnick
author_sort	Jeffrey M. Flynn
title	Enabling Unbalanced Fermentations by Using Engineered Electrode-Interfaced Bacteria
title_short	Enabling Unbalanced Fermentations by Using Engineered Electrode-Interfaced Bacteria
title_full	Enabling Unbalanced Fermentations by Using Engineered Electrode-Interfaced Bacteria
title_fullStr	Enabling Unbalanced Fermentations by Using Engineered Electrode-Interfaced Bacteria
title_full_unstemmed	Enabling Unbalanced Fermentations by Using Engineered Electrode-Interfaced Bacteria
title_sort	enabling unbalanced fermentations by using engineered electrode-interfaced bacteria
publisher	American Society for Microbiology
publishDate	2010
url	https://doaj.org/article/6dd375a692e041fe99836c2b7b4e6eb3
work_keys_str_mv	AT jeffreymflynn enablingunbalancedfermentationsbyusingengineeredelectrodeinterfacedbacteria AT danieleross enablingunbalancedfermentationsbyusingengineeredelectrodeinterfacedbacteria AT kristopherahunt enablingunbalancedfermentationsbyusingengineeredelectrodeinterfacedbacteria AT danielrbond enablingunbalancedfermentationsbyusingengineeredelectrodeinterfacedbacteria AT jeffreyagralnick enablingunbalancedfermentationsbyusingengineeredelectrodeinterfacedbacteria
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Enabling Unbalanced Fermentations by Using Engineered Electrode-Interfaced Bacteria

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