Abstract
This work describes novel genetic tools for use in Clostridium thermocellum that allow creation of unmarked mutations while using a replicating plasmid. The strategy employed counter-selections developed from the native C. thermocellum hpt gene and the Thermoanaerobacterium saccharolyticum tdk gene and was used to delete the genes for both lactate dehydrogenase (Ldh) and phosphotransacetylase (Pta). The Δldh Δpta mutant was evolved for 2,000 h, resulting in a stable strain with 40:1 ethanol selectivity and a 4.2-fold increase in ethanol yield over the wild-type strain. Ethanol production from cellulose was investigated with an engineered coculture of organic acid-deficient engineered strains of both C. thermocellum and T. saccharolyticum. Fermentation of 92 g/liter Avicel by this coculture resulted in 38 g/liter ethanol, with acetic and lactic acids below detection limits, in 146 h. These results demonstrate that ethanol production by thermophilic, cellulolytic microbes is amenable to substantial improvement by metabolic engineering.
Highlights
This work describes novel genetic tools for use in Clostridium thermocellum that allow creation of unmarked mutations while using a replicating plasmid
Fermentation of 92 g/liter Avicel by this coculture resulted in 38 g/liter ethanol, with acetic and lactic acids below detection limits, in 146 h. These results demonstrate that ethanol production by thermophilic, cellulolytic microbes is amenable to substantial improvement by metabolic engineering
Plating experiments revealed that C. thermocellum is sensitive to AZH, and subsequent genetic analysis confirmed that open reading frame (ORF) Cthe2254 is an hpt homolog
Summary
This work describes novel genetic tools for use in Clostridium thermocellum that allow creation of unmarked mutations while using a replicating plasmid. Recent reports have described development of basic genetic tools for C. thermocellum, including transformation of plasmid DNA and the ability to make marked gene deletions [19, 28, 30] These tools do not provide the versatility associated with creating unmarked genetic mutations, and while they could be used to eliminate organic acid production, they would not be ideal. These approaches rely on replacement of gene targets with antibiotic resistance cassettes, limiting the number of gene deletions to the available number of antibiotic resistance markers. The cellular toxicity of fluoro-deoxyuracil (FUDR) is dependent on Downloaded from https://journals.asm.org/journal/aem on 08 November 2021 by 52.90.136.113
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