Improving growth of Cupriavidus necator H16 on formate using adaptive laboratory evolution-informed engineering

Christopher H. Calvey; Violeta Sànchez i Nogué; Aleena M. White; Colin M. Kneucker; Sean P. Woodworth; Hannah M. Alt; Carrie A. Eckert; Christopher W. Johnson

doi:10.1016/j.ymben.2022.10.016

Improving growth of Cupriavidus necator H16 on formate using adaptive laboratory evolution-informed engineering

Christopher H. Calvey, Violeta Sànchez i Nogué, Aleena M. White, Colin M. Kneucker, Sean P. Woodworth, Hannah M. Alt, Carrie A. Eckert, Christopher W. Johnson

Synthetic Biology

Research output: Contribution to journal › Article › peer-review

22 Scopus citations

Abstract

Conversion of CO₂ to value-added products presents an opportunity to reduce GHG emissions while generating revenue. Formate, which can be generated by the electrochemical reduction of CO₂, has been proposed as a promising intermediate compound for microbial upgrading. Here we present progress towards improving the soil bacterium Cupriavidus necator H16, which is capable of growing on formate as its sole source of carbon and energy using the Calvin–Benson–Bassham (CBB) cycle, as a host for formate utilization. Using adaptive laboratory evolution, we generated several isolates that exhibited faster growth rates on formate. The genomes of these isolates were sequenced, and resulting mutations were systematically reintroduced by metabolic engineering, to identify those that improved growth. The metabolic impact of several mutations was investigated further using RNA-seq transcriptomics. We found that deletion of a transcriptional regulator implicated in quorum sensing, PhcA, reduced expression of several operons and led to improved growth on formate. Growth was also improved by deleting large genomic regions present on the extrachromosomal megaplasmid pHG1, particularly two hydrogenase operons and the megaplasmid CBB operon, one of two copies present in the genome. Based on these findings, we generated a rationally engineered ΔphcA and megaplasmid-deficient strain that exhibited a 24% faster maximum growth rate on formate. Moreover, this strain achieved a 7% growth rate improvement on succinate and a 19% increase on fructose, demonstrating the broad utility of microbial genome reduction. This strain has the potential to serve as an improved microbial chassis for biological conversion of formate to value-added products.

Original language	English
Pages (from-to)	78-90
Number of pages	13
Journal	Metabolic Engineering
Volume	75
DOIs	https://doi.org/10.1016/j.ymben.2022.10.016
State	Published - Jan 2023

Funding

This work was authored by the National Renewable Energy Laboratory, operated by Alliance for Sustainable Energy, LLC, for the U.S. Department of Energy (DOE) under Contract No. DE-AC36-08GO28308. Funding was provided by the U.S. Department of Energy Office of Energy Efficiency and Renewable Energy Bioenergy Technologies Office. The views expressed in the article do not necessarily represent the views of the DOE or the U.S. Government. The U.S. Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this work, or allow others to do so, for U.S. Government purposes. We thank George Peabody and Adam Guss at Oak Ridge National Laboratory for the gift of pQP307, Ian Rowe at DOE BETO for helpful discussions, and Allison Werner at NREL for critical reading of the manuscript. This work was authored by the National Renewable Energy Laboratory, operated by Alliance for Sustainable Energy, LLC, for the U.S. Department of Energy (DOE) under Contract No. DE-AC36-08GO28308. Funding was provided by the U.S. Department of Energy Office of Energy Efficiency and Renewable Energy Bioenergy Technologies Office. The views expressed in the article do not necessarily represent the views of the DOE or the U.S. Government. The U.S. Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this work, or allow others to do so, for U.S. Government purposes. We thank George Peabody and Adam Guss at Oak Ridge National Laboratory for the gift of pQP307, Ian Rowe at DOE BETO for helpful discussions, and Allison Werner at NREL for critical reading of the manuscript.

Funders	Funder number
U.S. Department of Energy Office of Energy Efficiency and Renewable Energy BioEnergy Technologies Office
U.S. Government
U.S. Department of Energy	DE-AC36-08GO28308
Oak Ridge National Laboratory
National Renewable Energy Laboratory

Keywords

Adaptive laboratory evolution
Cupriavidus necator H16
Formate
Genome minimization
Metabolic engineering
Quorum sensing
phcA

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1016/j.ymben.2022.10.016

Cite this

@article{9813463ee2d84af58ec09c4113633371,

title = "Improving growth of Cupriavidus necator H16 on formate using adaptive laboratory evolution-informed engineering",

abstract = "Conversion of CO2 to value-added products presents an opportunity to reduce GHG emissions while generating revenue. Formate, which can be generated by the electrochemical reduction of CO2, has been proposed as a promising intermediate compound for microbial upgrading. Here we present progress towards improving the soil bacterium Cupriavidus necator H16, which is capable of growing on formate as its sole source of carbon and energy using the Calvin–Benson–Bassham (CBB) cycle, as a host for formate utilization. Using adaptive laboratory evolution, we generated several isolates that exhibited faster growth rates on formate. The genomes of these isolates were sequenced, and resulting mutations were systematically reintroduced by metabolic engineering, to identify those that improved growth. The metabolic impact of several mutations was investigated further using RNA-seq transcriptomics. We found that deletion of a transcriptional regulator implicated in quorum sensing, PhcA, reduced expression of several operons and led to improved growth on formate. Growth was also improved by deleting large genomic regions present on the extrachromosomal megaplasmid pHG1, particularly two hydrogenase operons and the megaplasmid CBB operon, one of two copies present in the genome. Based on these findings, we generated a rationally engineered ΔphcA and megaplasmid-deficient strain that exhibited a 24% faster maximum growth rate on formate. Moreover, this strain achieved a 7% growth rate improvement on succinate and a 19% increase on fructose, demonstrating the broad utility of microbial genome reduction. This strain has the potential to serve as an improved microbial chassis for biological conversion of formate to value-added products.",

keywords = "Adaptive laboratory evolution, Cupriavidus necator H16, Formate, Genome minimization, Metabolic engineering, Quorum sensing, phcA",

author = "Calvey, {Christopher H.} and {S{\`a}nchez i Nogu{\'e}}, Violeta and White, {Aleena M.} and Kneucker, {Colin M.} and Woodworth, {Sean P.} and Alt, {Hannah M.} and Eckert, {Carrie A.} and Johnson, {Christopher W.}",

note = "Publisher Copyright: {\textcopyright} 2022 International Metabolic Engineering Society",

year = "2023",

month = jan,

doi = "10.1016/j.ymben.2022.10.016",

language = "English",

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AU - Calvey, Christopher H.

AU - Sànchez i Nogué, Violeta

AU - White, Aleena M.

AU - Kneucker, Colin M.

AU - Woodworth, Sean P.

AU - Alt, Hannah M.

AU - Eckert, Carrie A.

AU - Johnson, Christopher W.

PY - 2023/1

Y1 - 2023/1

N2 - Conversion of CO2 to value-added products presents an opportunity to reduce GHG emissions while generating revenue. Formate, which can be generated by the electrochemical reduction of CO2, has been proposed as a promising intermediate compound for microbial upgrading. Here we present progress towards improving the soil bacterium Cupriavidus necator H16, which is capable of growing on formate as its sole source of carbon and energy using the Calvin–Benson–Bassham (CBB) cycle, as a host for formate utilization. Using adaptive laboratory evolution, we generated several isolates that exhibited faster growth rates on formate. The genomes of these isolates were sequenced, and resulting mutations were systematically reintroduced by metabolic engineering, to identify those that improved growth. The metabolic impact of several mutations was investigated further using RNA-seq transcriptomics. We found that deletion of a transcriptional regulator implicated in quorum sensing, PhcA, reduced expression of several operons and led to improved growth on formate. Growth was also improved by deleting large genomic regions present on the extrachromosomal megaplasmid pHG1, particularly two hydrogenase operons and the megaplasmid CBB operon, one of two copies present in the genome. Based on these findings, we generated a rationally engineered ΔphcA and megaplasmid-deficient strain that exhibited a 24% faster maximum growth rate on formate. Moreover, this strain achieved a 7% growth rate improvement on succinate and a 19% increase on fructose, demonstrating the broad utility of microbial genome reduction. This strain has the potential to serve as an improved microbial chassis for biological conversion of formate to value-added products.

AB - Conversion of CO2 to value-added products presents an opportunity to reduce GHG emissions while generating revenue. Formate, which can be generated by the electrochemical reduction of CO2, has been proposed as a promising intermediate compound for microbial upgrading. Here we present progress towards improving the soil bacterium Cupriavidus necator H16, which is capable of growing on formate as its sole source of carbon and energy using the Calvin–Benson–Bassham (CBB) cycle, as a host for formate utilization. Using adaptive laboratory evolution, we generated several isolates that exhibited faster growth rates on formate. The genomes of these isolates were sequenced, and resulting mutations were systematically reintroduced by metabolic engineering, to identify those that improved growth. The metabolic impact of several mutations was investigated further using RNA-seq transcriptomics. We found that deletion of a transcriptional regulator implicated in quorum sensing, PhcA, reduced expression of several operons and led to improved growth on formate. Growth was also improved by deleting large genomic regions present on the extrachromosomal megaplasmid pHG1, particularly two hydrogenase operons and the megaplasmid CBB operon, one of two copies present in the genome. Based on these findings, we generated a rationally engineered ΔphcA and megaplasmid-deficient strain that exhibited a 24% faster maximum growth rate on formate. Moreover, this strain achieved a 7% growth rate improvement on succinate and a 19% increase on fructose, demonstrating the broad utility of microbial genome reduction. This strain has the potential to serve as an improved microbial chassis for biological conversion of formate to value-added products.

KW - Adaptive laboratory evolution

KW - Cupriavidus necator H16

KW - Formate

KW - Genome minimization

KW - Metabolic engineering

KW - Quorum sensing

KW - phcA

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SN - 1096-7176

VL - 75

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EP - 90

JO - Metabolic Engineering

JF - Metabolic Engineering

ER -

Improving growth of Cupriavidus necator H16 on formate using adaptive laboratory evolution-informed engineering

Abstract

Funding

Keywords

UN SDGs

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