Promiscuous plasmid replication in thermophiles: Use of a novel hyperthermophilic replicon for genetic manipulation of Clostridium thermocellum at its optimum growth temperature

Joseph Groom; Daehwan Chung; Daniel G. Olson; Lee R. Lynd; Adam M. Guss; Janet Westpheling

doi:10.1016/j.meteno.2016.01.004

Promiscuous plasmid replication in thermophiles: Use of a novel hyperthermophilic replicon for genetic manipulation of Clostridium thermocellum at its optimum growth temperature

Joseph Groom, Daehwan Chung, Daniel G. Olson, Lee R. Lynd, Adam M. Guss, Janet Westpheling

Synthetic Biology

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

14 Scopus citations

Abstract

Clostridium thermocellum is a leading candidate for the consolidated bioprocessing of lignocellulosic biomass for the production of fuels and chemicals. A limitation to the engineering of this strain is the availability of stable replicating plasmid vectors for homologous and heterologous expression of genes that provide improved and/or novel pathways for fuel production. Current vectors relay on replicons from mesophilic bacteria and are not stable at the optimum growth temperature of C. thermocellum. To develop more thermostable genetic tools for C. thermocellum, we constructed vectors based on the hyperthermophilic Caldicellulosiruptor bescii replicon pBAS2. Autonomously replicating shuttle vectors based on pBAS2 reproducibly transformed C. thermocellum at 60 °C and were maintained in multiple copy. Promoters, selectable markers and plasmid replication proteins from C. bescii were functional in C. thermocellum. Phylogenetic analyses of the proteins contained on pBAS2 revealed that the replication initiation protein RepL is unique among thermophiles. These results suggest that pBAS2 may be a broadly useful replicon for other thermophilic Firmicutes.

Original language	English
Pages (from-to)	30-38
Number of pages	9
Journal	Metabolic Engineering Communications
Volume	3
DOIs	https://doi.org/10.1016/j.meteno.2016.01.004
State	Published - Dec 1 2016

Funding

We thank Kelly Dyer and Caitlin Conn for assistance with the phylogenic analysis. JG was supported by an NIH 5T32GM007103 Predoctoral Training Grant to the Genetics Department of the University of Georgia. This work was supported by the BioEnergy Science Center, a U.S. DOE Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science . Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. DOE under contract DE-AC05-00OR22725 . The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Funders	Funder number
BioEnergy Science Center
DOE Office of Science
Genetics Department of the University of Georgia
Office of Biological and Environmental Research
National Institutes of Health
U.S. Department of Energy
Oak Ridge National Laboratory	DE-AC05-00OR22725

Keywords

Clostridia
Genetic tools
Plasmid
Thermophile
Transformation

UN SDGs

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

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10.1016/j.meteno.2016.01.004

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title = "Promiscuous plasmid replication in thermophiles: Use of a novel hyperthermophilic replicon for genetic manipulation of Clostridium thermocellum at its optimum growth temperature",

abstract = "Clostridium thermocellum is a leading candidate for the consolidated bioprocessing of lignocellulosic biomass for the production of fuels and chemicals. A limitation to the engineering of this strain is the availability of stable replicating plasmid vectors for homologous and heterologous expression of genes that provide improved and/or novel pathways for fuel production. Current vectors relay on replicons from mesophilic bacteria and are not stable at the optimum growth temperature of C. thermocellum. To develop more thermostable genetic tools for C. thermocellum, we constructed vectors based on the hyperthermophilic Caldicellulosiruptor bescii replicon pBAS2. Autonomously replicating shuttle vectors based on pBAS2 reproducibly transformed C. thermocellum at 60 °C and were maintained in multiple copy. Promoters, selectable markers and plasmid replication proteins from C. bescii were functional in C. thermocellum. Phylogenetic analyses of the proteins contained on pBAS2 revealed that the replication initiation protein RepL is unique among thermophiles. These results suggest that pBAS2 may be a broadly useful replicon for other thermophilic Firmicutes.",

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author = "Joseph Groom and Daehwan Chung and Olson, {Daniel G.} and Lynd, {Lee R.} and Guss, {Adam M.} and Janet Westpheling",

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Promiscuous plasmid replication in thermophiles: Use of a novel hyperthermophilic replicon for genetic manipulation of Clostridium thermocellum at its optimum growth temperature. / Groom, Joseph; Chung, Daehwan; Olson, Daniel G. et al.
In: Metabolic Engineering Communications, Vol. 3, 01.12.2016, p. 30-38.

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

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Promiscuous plasmid replication in thermophiles: Use of a novel hyperthermophilic replicon for genetic manipulation of Clostridium thermocellum at its optimum growth temperature

Abstract

Funding

Keywords

UN SDGs

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