Approaches to genetic tool development for rapid domestication of non-model microorganisms

Lauren A Riley,Adam M Guss

doi:10.1186/s13068-020-01872-z

Lauren A Riley, Adam M Guss

Open Access

https://doi.org/10.1186/s13068-020-01872-z

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Abstract

Non-model microorganisms often possess complex phenotypes that could be important for the future of biofuel and chemical production. They have received significant interest the last several years, but advancement is still slow due to the lack of a robust genetic toolbox in most organisms. Typically, “domestication” of a new non-model microorganism has been done on an ad hoc basis, and historically, it can take years to develop transformation and basic genetic tools. Here, we review the barriers and solutions to rapid development of genetic transformation tools in new hosts, with a major focus on Restriction-Modification systems, which are a well-known and significant barrier to efficient transformation. We further explore the tools and approaches used for efficient gene deletion, DNA insertion, and heterologous gene expression. Finally, more advanced and high-throughput tools are now being developed in diverse non-model microbes, paving the way for rapid and multiplexed genome engineering for biotechnology.

Highlights

The world’s energy and chemical demand is ever-increasing, and currently, the demand for fuels and chemicals is primarily met with fossil fuels
Pioneering work demonstrated transformation of Clostridium acetobutylicum by first methylating plasmid DNA in E. coli with the methyltransferase from B. subtilis phage phi3T [49]. This approach was expanded with a method called plasmid artificial modification (PAM), which introduced all the methyltransferases from a given strain into E. coli using plasmid-based expression, followed by isolation of the DNA of interest out of the PAM host to properly methylate it prior to transformation of the target organism [68]
Homologous recombination based genetic tools have been used in a variety of non-model organisms to increase biochemical production and enable nonnative carbon catabolism though the deletion of competing pathways and insertion of heterologous genes [119,120,121,122]

Summary

Introduction

The world’s energy and chemical demand is ever-increasing, and currently, the demand for fuels and chemicals is primarily met with fossil fuels. Two major barriers to the use of non-model organisms for metabolic engineering include a lack of genetic tools and limited knowledge of the organism’s physiology. The development of genetic tools requires the ability to efficiently transform DNA into a target organism.

Results

Conclusion