Abstract
BackgroundIndustrial production of biofuels and other products by cellulolytic microorganisms is of interest but hindered by the nascent state of genetic tools. Although a genetic system for Clostridium thermocellum DSM1313 has recently been developed, available methods achieve relatively low efficiency and similar plasmids can transform C. thermocellum at dramatically different efficiencies.ResultsWe report an increase in transformation efficiency of C. thermocellum for a variety of plasmids by using DNA that has been methylated by Escherichia coli Dam but not Dcm methylases. When isolated from a dam+dcm+E. coli strain, pAMG206 transforms C. thermocellum 100-fold better than the similar plasmid pAMG205, which contains an additional Dcm methylation site in the pyrF gene. Upon removal of Dcm methylation, transformation with pAMG206 showed a four- to seven-fold increase in efficiency; however, transformation efficiency of pAMG205 increased 500-fold. Removal of the Dcm methylation site from the pAMG205 pyrF gene via silent mutation resulted in increased transformation efficiencies equivalent to that of pAMG206. Upon proper methylation, transformation efficiency of plasmids bearing the pMK3 and pB6A origins of replication increased ca. three orders of magnitude.ConclusionsE. coli Dcm methylation decreases transformation efficiency in C. thermocellum DSM1313. The use of properly methylated plasmid DNA should facilitate genetic manipulation of this industrially relevant bacterium.
Highlights
The transition to a sustainable resource base is one of the largest challenges facing humanity [1], with transportation being a among the largest and fastest-growing energy demands [2]
Due to the difficulties still involved in genetic modification of C. thermocellum, we aimed to understand the cause of this plasmid-to-plasmid variation in transformation efficiency
Plasmid pAMG205 transforms wild type C. thermocellum at very low efficiency when isolated from standard cloning strain E. coli Top10, whereas plasmid pAMG206 transforms at ca. 100-fold higher efficiency under identical conditions (Table 1)
Summary
The transition to a sustainable resource base is one of the largest challenges facing humanity [1], with transportation being a among the largest and fastest-growing energy demands [2]. While cellulosic biomass is a promising feedstock for the generation of renewable transport fuels, the cost of enzymatic hydrolysis of cellulose to soluble sugars is currently too high to be economically viable [3]. Combining the steps of enzyme production and sugar fermentation in a one-step process called consolidated bioprocessing (CBP) has the potential to address this limitation but requires the development of an organism that both degrades cellulose efficiently and produces fuel at high yield and titer [4]. The nascent state of genetic tools has hindered both fundamental and applied studies of C. thermocellum. Industrial production of biofuels and other products by cellulolytic microorganisms is of interest but hindered by the nascent state of genetic tools. A genetic system for Clostridium thermocellum DSM1313 has recently been developed, available methods achieve relatively low efficiency and similar plasmids can transform C. thermocellum at dramatically different efficiencies
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