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

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

Zymomonas diversity and potential for biofuel production.

BackgroundZymomonas mobilis is an aerotolerant α-proteobacterium, which has been genetically engineered for industrial purposes for decades. However, a comprehensive comparison of existing strains on the genomic level in conjunction with phenotype analysis has yet to be carried out. We here performed whole-genome comparison of 17 strains including nine that were sequenced in this study. We then compared 15 available Zymomonas strains for their natural abilities to perform under conditions relevant to biofuel synthesis. We tested their growth in anaerobic rich media, as well as growth, ethanol production and xylose utilization in lignocellulosic hydrolysate. We additionally compared their tolerance to isobutanol, flocculation characteristics, and ability to uptake foreign DNA by electroporation and conjugation.ResultsUsing clustering based on 99% average nucleotide identity (ANI), we classified 12 strains into four clusters based on sequence similarity, while five strains did not cluster with any other strain. Strains belonging to the same 99% ANI cluster showed similar performance while significant variation was observed between the clusters. Overall, conjugation and electroporation efficiencies were poor across all strains, which was consistent with our finding of coding potential for several DNA defense mechanisms, such as CRISPR and restriction-modification systems, across all genomes. We found that strain ATCC31821 (ZM4) had a more diverse plasmid profile than other strains, possibly leading to the unique phenotypes observed for this strain. ZM4 also showed the highest growth of any strain in both laboratory media and lignocellulosic hydrolysate and was among the top 3 strains for isobutanol tolerance and electroporation and conjugation efficiency.ConclusionsOur findings suggest that strain ZM4 has a unique combination of genetic and phenotypic traits that are beneficial for biofuel production and propose investing future efforts in further engineering of ZM4 for industrial purposes rather than exploring new Zymomonas isolates

Zymomonas diversity and potential for biofuel production.

BackgroundZymomonas mobilis is an aerotolerant α-proteobacterium, which has been genetically engineered for industrial purposes for decades. However, a comprehensive comparison of existing strains on the genomic level in conjunction with phenotype analysis has yet to be carried out. We here performed whole-genome comparison of 17 strains including nine that were sequenced in this study. We then compared 15 available Zymomonas strains for their natural abilities to perform under conditions relevant to biofuel synthesis. We tested their growth in anaerobic rich media, as well as growth, ethanol production and xylose utilization in lignocellulosic hydrolysate. We additionally compared their tolerance to isobutanol, flocculation characteristics, and ability to uptake foreign DNA by electroporation and conjugation.ResultsUsing clustering based on 99% average nucleotide identity (ANI), we classified 12 strains into four clusters based on sequence similarity, while five strains did not cluster with any other strain. Strains belonging to the same 99% ANI cluster showed similar performance while significant variation was observed between the clusters. Overall, conjugation and electroporation efficiencies were poor across all strains, which was consistent with our finding of coding potential for several DNA defense mechanisms, such as CRISPR and restriction-modification systems, across all genomes. We found that strain ATCC31821 (ZM4) had a more diverse plasmid profile than other strains, possibly leading to the unique phenotypes observed for this strain. ZM4 also showed the highest growth of any strain in both laboratory media and lignocellulosic hydrolysate and was among the top 3 strains for isobutanol tolerance and electroporation and conjugation efficiency.ConclusionsOur findings suggest that strain ZM4 has a unique combination of genetic and phenotypic traits that are beneficial for biofuel production and propose investing future efforts in further engineering of ZM4 for industrial purposes rather than exploring new Zymomonas isolates

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

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

Towards patterned bioelectronics: facilitated immobilization of exoelectrogenic Escherichia coli with heterologous pili.

Biosensors detect signals using biological sensing components such as redox enzymes and biological cells. Although cellular versatility can be beneficial for different applications, limited stability and efficiency in signal transduction at electrode surfaces represent a challenge. Recent studies have shown that the Mtr electron conduit from Shewanella oneidensis MR-1 can be produced in Escherichia coli to generate an exoelectrogenic model system with well-characterized genetic tools. However, means to specifically immobilize this organism at solid substrates as electroactive biofilms have not been tested previously. Here, we show that mannose-binding Fim pili can be produced in exoelectrogenic E. coli and can be used to selectively attach cells to a mannose-coated material. Importantly, cells expressing fim genes retained current production by the heterologous Mtr electron conduit. Our results demonstrate the versatility of the exoelectrogenic E. coli system and motivate future work that aims to produce patterned biofilms for bioelectronic devices that can respond to various biochemical signals