CymA and Exogenous Flavins Improve Extracellular Electron Transfer and Couple It to Cell Growth in Mtr-Expressing Escherichia coli

Introducing extracellular electron transfer pathways into heterologous organisms offers the opportunity to explore fundamental biogeochemical processes and to biologically alter redox states of exogenous metals for various applications. While expression of the MtrCAB electron nanoconduit from Shewanella oneidensis MR-1 permits extracellular electron transfer in Escherichia coli, the low electron flux and absence of growth in these cells limits their practicality for such applications. Here we investigate how the rate of electron transfer to extracellular Fe(III) and cell survival in engineered E. coli are affected by mimicking different features of the S. oneidensis pathway: the number of electron nanoconduits, the link between the quinol pool and MtrA, and the presence of flavin-dependent electron transfer. While increasing the number of pathways does not significantly improve the extracellular electron transfer rate or cell survival, using the native inner membrane component, CymA, significantly improves the reduction rate of extracellular acceptors and increases cell viability. Strikingly, introducing both CymA and riboflavin to Mtr-expressing E. coli also allowed these cells to couple metal reduction to growth, which is the first time an increase in biomass of an engineered E. coli has been observed under Fe2O3 (s) reducing conditions. Overall, this work provides engineered E. coli strains for modulating extracellular metal reduction and elucidates critical factors for engineering extracellular electron transfer in heterologous organisms

Photoreduction of Shewanella oneidensis Extracellular Cytochromes by Organic Chromophores and Dye-Sensitized TiO2.

The transfer of photoenergized electrons from extracellular photosensitizers across a bacterial cell envelope to drive intracellular chemical transformations represents an attractive way to harness nature's catalytic machinery for solar-assisted chemical synthesis. In Shewanella oneidensis MR-1 (MR-1), trans-outer-membrane electron transfer is performed by the extracellular cytochromes MtrC and OmcA acting together with the outer-membrane-spanning porin⋅cytochrome complex (MtrAB). Here we demonstrate photoreduction of solutions of MtrC, OmcA, and the MtrCAB complex by soluble photosensitizers: namely, eosin Y, fluorescein, proflavine, flavin, and adenine dinucleotide, as well as by riboflavin and flavin mononucleotide, two compounds secreted by MR-1. We show photoreduction of MtrC and OmcA adsorbed on RuII -dye-sensitized TiO2 nanoparticles and that these protein-coated particles perform photocatalytic reduction of solutions of MtrC, OmcA, and MtrCAB. These findings provide a framework for informed development of strategies for using the outer-membrane-associated cytochromes of MR-1 for solar-driven microbial synthesis in natural and engineered bacteria.This work was supported by the UK Biotechnology and Biological Sciences Research Council (grants BB/K009753/1, BB/K010220/1, BB/K009885/1, and BB/K00929X/1), the Engineering and Physical Sciences Research Council (EP/M001989/1, PhD studentship 1307196 to E.V.A.), a Royal Society Leverhulme Trust Senior Research Fellowship to J.N.B., the Christian Doppler Research Association, and OMV group

Electroactivity across the cell wall of Gram-positive bacteria

Funding Information: This work was supported by national funds through FCT? Funda??o para a Ci?ncia e a Tecnologia, I.P. Project UIDB/04612/2020, UIDP/04612/2020 and PTDC/BIA-BQM/30176/2017, and by the European Union's Horizon 2020 research and innovation programme under grant agreement No 810856. Funding Information: This work was supported by national funds through FCT– Fundação para a Ciência e a Tecnologia, I.P., Project UIDB/04612/2020, UIDP/04612/2020 and PTDC/BIA-BQM/30176/2017, and by the European Union’s Horizon 2020 research and innovation programme under grant agreement No 810856. Publisher Copyright: © 2020 The Author(s) Copyright: Copyright 2021 Elsevier B.V., All rights reserved.The growing interest on sustainable biotechnological processes for the production of energy and industrial relevant organic compounds have increased the discovery of electroactive organisms (i.e. organisms that are able to exchange electrons with an electrode) and the characterization of their extracellular electron transfer mechanisms. While most of the knowledge on extracellular electron transfer processes came from studies on Gram-negative bacteria, less is known about the processes performed by Gram-positive bacteria. In contrast to Gram-negative bacteria, Gram-positive bacteria lack an outer-membrane and contain a thick cell wall, which were thought to prevent extracellular electron transfer. However, in the last decade, an increased number of Gram-positive bacteria have been found to perform extracellular electron transfer, and exchange electrons with an electrode. In this mini-review the current knowledge on the extracellular electron transfer processes performed by Gram-positive bacteria is introduced, emphasising their electroactive role in bioelectrochemical systems. Also, the existent information of the molecular processes by which these bacteria exchange electrons with an electrode is highlighted. This understanding is fundamental to advance the implementation of these organisms in sustainable biotechnological processes, either through modification of the systems or through genetic engineering, where the organisms can be optimized to become better catalysts.publishersversionpublishe

Pathways to cellular supremacy in biocomputing

Synthetic biology uses living cells as the substrate for performing human-defined computations. Many current implementations of cellular computing are based on the “genetic circuit” metaphor, an approximation of the operation of silicon-based computers. Although this conceptual mapping has been relatively successful, we argue that it fundamentally limits the types of computation that may be engineered inside the cell, and fails to exploit the rich and diverse functionality available in natural living systems. We propose the notion of “cellular supremacy” to focus attention on domains in which biocomputing might offer superior performance over traditional computers. We consider potential pathways toward cellular supremacy, and suggest application areas in which it may be found.A.G.-M. was supported by the SynBio3D project of the UK Engineering and Physical Sciences Research Council (EP/R019002/1) and the European CSA on biological standardization BIOROBOOST (EU grant number 820699). T.E.G. was supported by a Royal Society University Research Fellowship (grant UF160357) and BrisSynBio, a BBSRC/ EPSRC Synthetic Biology Research Centre (grant BB/L01386X/1). P.Z. was supported by the EPSRC Portabolomics project (grant EP/N031962/1). P.C. was supported by SynBioChem, a BBSRC/EPSRC Centre for Synthetic Biology of Fine and Specialty Chemicals (grant BB/M017702/1) and the ShikiFactory100 project of the European Union’s Horizon 2020 research and innovation programme under grant agreement 814408

American Gut: an Open Platform for Citizen Science Microbiome Research

McDonald D, Hyde E, Debelius JW, et al. American Gut: an Open Platform for Citizen Science Microbiome Research. mSystems. 2018;3(3):e00031-18

SUSTAINABLE INTENSIFICATION OF FOOD PRODUCTION SYSTEMS IN MALAWI

Smallholder farmers in southeastern Africa are constrained by poor rainwater-use efficiency, soil degradation, and limited financial resources. Conservation agriculture (CA), based on the principles of minimal soil disturbance, year-round ground cover, and diverse crop rotations, is being promoted to sustainably improve crop production, food security, and smallholder farm income. Adoption of CA principles in the region has predominately been limited to eliminating tillage and retaining residues, with little adoption of crop rotations. In this study, three cropping systems--continuous no-till maize, CA rotation, and conventional tillage rotation--were established on smallholder farms in the Nkhotakota and Dowa districts, two distinct agro-ecological zones in Malawi. Three-year crop rotations of cassava, cowpea, and maize and cassava, soybean, maize were implemented in CA and conventional tillage, respectively, in Nkhotakota. In Dowa, a 3-year rotation of sweet potato, bean, and maize was implemented in both CA and conventional tillage. Cropping systems were analyzed for their impacts on crop production, soil-water relations, soil quality, and financial returns from 2011 to 2014. No-till maize and CA improved infiltration and the soil water balance compared to conventional tillage in Nkhotakota but did not affect soil-water relations in Dowa. No-till maize and CA increased exchangeable K, Ca, and Mg and reduced soil erosion compared to conventional tillage. In no-till maize, retention of low quality residue resulted in N immobilization. Conservation agriculture improved plant available N and nutrient cycling compared to no-till maize, but less residue cover increased bulk density compared to no-till maize and conventional tillage. Soils in conventional tillage had the most plant available N, which could lead to N leaching and reduced fertilizer-use efficiency. Tillage and residue management did not affect yields of cassava, sweet potato, cowpea, soybean, or bean. Crop rotation, regardless of tillage practice, increased maize yields compared to no-till maize. Net revenue was highest in no-till maize and labor productivity and gross margins were higher in no-till and conventional tillage than in CA rotation. Before widespread adoption of CA can occur, further research is needed to improve alternative crop production under CA management and identify the most appropriate agroecological zones for successful CA

Biological Limitations Of Shewanella Oneidensis Mr-1 In Bioelectrochemical Systems

Shewanella oneidensis MR-1 is a model microbe for use in bioelectrochemical systems (BESs) for several reasons, including its ability to produce electric current in the presence of oxygen and its use of endogenous electron shuttles for electron transfer. I performed in-depth studies on the growth and physiology of S. oneidensis to gain insight into BES performance with this microbe. In the first study, I analyzed changes in current production when oxygen was added to batch- and continuously-fed BESs with S. oneidensis. These experiments revealed that oxygen is more beneficial under continuously-fed conditions because it allows S. oneidensis to grow and produce flavins at a faster rate, and therefore decreases flavin washout. In the second study I optimized poised electrode potentials, because previous research suggested that electrodes poised at oxidizing potentials may cause a stress response in S. oneidensis. I grew S. oneidensis in continuously-fed BESs with potentiostatically poised electrodes at 5 different redox potentials and concluded that oxidizing electrode potentials do not cause a general stress response, but decrease current production by direct damage of biofilm cells at the electrode surface. In the third study, I compared the transcriptomes of S. oneidensis grown in a wide variety of conditions and used machine learning to discover genes important to anode- and iron-respiration in this organism. This meta-analysis revealed that some putative members of the electron transport chain, including an NADH dehydrogenase and a cytochrome oxidase, were important under anode- or Fe(III)-respiring conditions. Knockouts strains with these genes deleted confirmed their role in the anaerobic electron transport chain of S. oneidensis. Future work is needed to better characterize the efficiency of the anaerobic electron transport chain in S. oneidensis. The overall finding of this work is that S. oneidensis is not appropriate for use in BES applications that require strict anaerobic conditions or quick exchange of medium (e.g., wastewater treatment), because it performs better when mediators and planktonic cells are not washed out of the system