Abstract
BackgroundFilamentous fungi are 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.ResultsOver 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 36.5°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).ConclusionWe 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.
Highlights
Filamentous fungi are the most widely used eukaryotic biocatalysts in industrial and chemical applications
The only practical way to alter such complex phenotypes is using continuous culture to select for genetic variants that can adapt to gradual changes in environmental conditions – in essence, harnessing the power of evolution by natural selection [5]
Selection of thermostable M. anisopliae isolates An actively growing culture of M. anisopliae was inoculated inside the EvolugatorTM growth chamber at 28°C as described in the Methods section
Summary
Filamentous fungi are the most widely used eukaryotic biocatalysts in industrial and chemical applications. Metarhizium anisopliae is a broad host range entomopathogenic fungus currently under intensive investigation as a biologically based alternative to chemical pesticides 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. Single, or even multiple directed mutations often fail to produce the desired results Newer methods, such as gene prospecting [3], have been developed that are higher throughput, such methods are still limited by the fact that many favorable phenotypes, like sustained thermotolerance, often require genome-wide changes [4] that are difficult to select for by inserting individual genes or even groups of genes. The only practical way to alter such complex phenotypes is using continuous culture to select for genetic variants that can adapt to gradual changes in environmental conditions – in essence, harnessing the power of evolution by natural selection [5]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
