October 21, 2011 an article was published on line ahead of print, January 7, 2012, in Applied Environmental Microbiology, prior to be highlighted in the February issue. Click here

Experimental Evolution of a Facultative Thermophile from a Mesophilic Ancestor

Ian K. Blaby, Benjamin J. Lyons, Ewa Wroclawska-Hughes, Grier C. F. Phillips, Tyler P. Pyle, Stephen G. Chamberlin, Steven A. Benner, Thomas J. Lyons, Valérie de Crécy-Lagard and Eudes de Crécy

Abstract : Experimental evolution via continuous culture is a powerful approach to the alteration of complex phenotypes, such as optimal/maximal growth temperatures. The benefit of this approach is that phenotypic selection is tied to growth rate, allowing the production of optimized strains. Herein, we demonstrate the use of a recently described long-term culture apparatus called the Evolugator for the generation of a thermophilic descendant from a mesophilic ancestor (Escherichia coli MG1655). In addition, we used whole-genome sequencing of sequentially isolated strains throughout the thermal adaptation process to characterize the evolutionary history of the resultant genotype, identifying 31 genetic alterations that may contribute to thermotolerance, although some of these mutations may be adaptive for off-target environmental parameters, such as rich medium. We undertook preliminary phenotypic analysis of mutations identified in the glpF and fabA genes. Deletion of glpF in a mesophilic wild-type background conferred significantly improved growth rates in the 43-to-48°C temperature range and altered optimal growth temperature from 37°C to 43°C. In addition, transforming our evolved thermotolerant strain (EVG1064) with a wild-type allele of glpF reduced fitness at high temperatures. On the other hand, the mutation in fabA predictably increased the degree of saturation in membrane lipids, which is a known adaptation to elevated temperature. However, transforming EVG1064 with a wild-type fabA allele had only modest effects on fitness at intermediate temperatures. The Evolugator is fully automated and demonstrates the potential to accelerate the selection for complex traits by experimental evolution and significantly decrease development time for new industrial strains.

Appl. Environ. Microbiol. 2012, 78(1):144-55. DOI : Published Ahead of Print 21 October 2011. 10.1128/AEM.05773-11.


August 26, 2009 an article has been published on line for publication in BMC Biotechnology prior to the printed publication Click here

Directed evolution of a filamentous fungus for thermotolerance

Eudes de Crecy, Stefan Jaronski, Benjamin Lyons, Thomas J Lyons and Nemat O Keyhani

Background : Filamentous fungi represent the most widely used eukaryotic biocatalysts in industrial and chemical applications. Consequently, there is tremendous interest in methodology that can use the power of genetics to develop strains with improved performance. For example, Metarhizium anisopliae is a broad-host-range entomopathogenic fungus currently under intensive investigation as a biologically-based alternative to chemical pesticides. However, it use is limited by the relatively low tolerance of this species to abiotic stresses such as heat, with most strains displaying little to no growth between 35-37°C. In this study, we used a newly-developed automated continuous culture method called the Evolugator™, which takes advantage of a natural selection-adaptation strategy, to select for thermotolerant variants of M. anisopliae strain 2575 displaying robust growth at 37°C.

Results : Over a 4 month time course, 22 cycles of growth and dilution were used to select 2 thermotolerant variants of M. anisopliae. Both variants displayed robust growth at 35.5°C, whereas only one was able to grow at 37°C. Insect bioassays using Melanoplus sanguinipes (grasshoppers) were also performed to determine if thermotolerant variants of M. anisopliae retained entomopathogenicity. Assays confirmed that thermotolerant variants were, indeed, entomopathogenic, albeit with complex alterations in virulence parameters such as lethal dose responses (LD50) and median survival times (ST50).

Conclusion : We report the experimental evolution of a filamentous fungus via the novel application of a powerful new continuous culture device. This is the first example of using continuous culture to select for complex phenotypes such as thermotolerance. Temperature adapted variants of the insect-pathogenic, filamentous fungus M. anisopliae were isolated and demonstrated to show vigorous growth at a temperature that is inhibitory for the parent strain. Insect virulence assays confirmed that pathogenicity can be retained during the selection process. In principle, this technology can be used to adapt filamentous fungi to virtually any environmental condition including abiotic stress and growth substrate utilization.

BMC Biotechnology 2009, 9:74 doi:10.1186/1472-6750-9-74 Published : 26 August 2009


September 26, 2007 an article has been published on line for publication in Applied Microbiology & Biotechnology prior to the printed publication Click here.

Abstract Appl Microbiol Biotechnol. 2007 Nov ;77(2):489-96. Epub 2007 Sep 26.

Development of a novel continuous culture device for experimental evolution of bacterial populations.

de Crécy E, Metzgar D, Allen C, Pénicaud M, Lyons B, Hansen CJ, de Crécy-Lagard V

The availability of a robust and reliable continuous culture apparatus that eliminates wall growth problems would lead to many applications in the microbial field, including allowing genetically engineered strains to recover high fitness, improving biodegradation strains, and predicting likely antibiotic resistance mechanisms. We describe the design and implementation of a novel automated continuous culture machine that can be used both in time-dependent mode (similar to a chemostat) and turbidostat modes, in which wall growth is circumvented through the use of a long, variably divisible tube of growth medium. This tube can be restricted with clamps to create a mobile growth chamber region in which static portions of the tube and the associated medium are replaced together at equal rates. To functionally test the device as a tool for re-adaptation of engineered strains, we evolved a strain carrying a highly deleterious deletion of Elongation Factor P, a gene involved in translation. In 200 generations over 2 weeks of dilution cycles, the evolved strain improved in generation time by a factor of three, with no contaminations and easy manipulation.