Laboratory Evolution and Multi-platform Genome Re-sequencing of the Cellulolytic Actinobacterium Thermobifida fusca

Abstract

Biological utilization of cellulose is a complex process involving the coordinated expression of different cellulases, often in a synergistic manner. One possible means of inducing an organism-level change in cellulase activity is to use laboratory adaptive evolution. In this study, evolved strains of the cellulolytic actinobacterium, Thermobifida fusca, were generated for two different scenarios: continuous exposure to cellobiose (strain muC) or alternating exposure to cellobiose and glucose (strain muS). These environmental conditions produced a phenotype specialized for growth on cellobiose (muC) and an adaptable, generalist phenotype (muS). Characterization of cellular phenotypes and whole genome re-sequencing were conducted for both the muC and muS strains. Phenotypically, the muC strain showed decreased cell yield over the course of evolution concurrent with decreased cellulase activity, increased intracellular ATP concentrations, and higher end-product secretions. The muS strain increased its cell yield for growth on glucose and exhibited a more generalist phenotype with higher cellulase activity and growth capabilities on different substrates. Whole genome re-sequencing identified 48 errors in the reference genome and 18 and 14 point mutations in the muC and muS strains, respectively. Among these mutations, the site mutation of Tfu_1867 was found to contribute the specialist phenotype and the site mutation of Tfu_0423 was found to contribute the generalist phenotype. By conducting and characterizing evolution experiments on Thermobifida fusca, we were able to show that evolutionary changes balance ATP energetic considerations with cellulase activity. Increased cellulase activity is achieved in stress environments (switching carbon sources), otherwise cellulase activity is minimized to conserve ATP.

Production of cellulolytic enzymes from ascomycetes: Comparison of solid state and submerged fermentation

“…reesei Rut-C30 [36], or by more targeted metabolic engineering. Furthermore, approaches using adaptive and directed evolution have provided novel strains with increased cellulase activities or improved functionality of cellulases for both fungi and bacteria [42,43]. With more and more genomes becoming available for ascomycetes it is advantageous to use bioinformatic tools in combination with metabolic engineering to express interesting enzymes.…”

Rumen microbial (meta)genomics and its application to ruminant production

“…These initial rumen microbial genomes were produced from type strains that were maintained as single cultures for years. It has to be noted that these type strain might have some differences with strains evolving in their natural habitat, as their genetic makeup can change over generations (Papadopoulos et al, 1999) and mutations can arise induced by the culture media (Deng and Fong, 2011). In addition, variability within single species also exists .…”

Visualizing evolution in real time to determine the molecular mechanisms of n-butanol tolerance in Escherichia coli

“…Advances in genomic tools have led to the development of reverse engineering tools for the determination of the underlying genetic determinants (points on the adaptive landscape) and molecular mechanisms associated with complex phenotypes; such as transcriptome analysis (Oh et al, 2002;Rutherford et al, 2010), genomic enrichment (Borden and Papoutsakis, 2007;Reyes et al, 2011), SCALEs (Lynch et al, 2007), TRMR (Warner et al, 2010), and coupling high throughput genomic technology with the use of adaptive laboratory evolution (Conrad et al, 2011;Deng and Fong, 2011;Minty et al, 2011). In adaptive laboratory evolution, adaptive mutants (strains with increased fitness) arise spontaneously and expand in a population under a specific selective pressure (Agrawal et al, 2011;Cooper and Lenski, 2010;Fong et al, 2005;Lee and Palsson, 2010;Minty et al, 2011;Paquin and Adams, 1983a;Paquin and Adams, 1983b).…”