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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

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. Lastly, these results suggest that pBAS2 may be a broadly useful replicon for other thermophilic Firmicutes.
Authors:
 [1] ;  [2] ;  [3] ;  [3] ;  [4] ;  [1]
  1. Univ. of Georgia, Athens, GA (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
  2. Univ. of Georgia, Athens, GA (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); National Renewable Energy Lab. (NREL), Golden, CO (United States)
  3. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Dartmouth College, Hanover, NH (United States)
  4. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Publication Date:
OSTI Identifier:
1240243
Report Number(s):
NREL/JA--2700-65970
Journal ID: ISSN 2214-0301
Grant/Contract Number:
AC36-08GO28308
Type:
Accepted Manuscript
Journal Name:
Metabolic Engineering Communications
Additional Journal Information:
Journal Volume: 3; Related Information: Metabolic Engineering Communications; Journal ID: ISSN 2214-0301
Publisher:
Elsevier
Research Org:
NREL (National Renewable Energy Laboratory (NREL), Golden, CO (United States))
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANAYLYTICAL CHEMISTRY plasmid; genetic tools; transformation; thermophile; clostridia