Recent advances in plasmid-based tools for establishing novel microbial chassis
Luísa Czamanski Noraa,1, Cauã Antunes Westmanna,1, María-Eugenia Guazzaronib, Chandranayaka Siddaiahc, Vijai Kumar Guptad, Rafael Silva-Rochaa
Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo 14049-900, Brazil.
A key challenge for domesticating alternative cultivable microorganisms with biotechnological potential lies in the development of innovative technologies. Within this framework, a myriad of genetic tools has flourished, allowing the design and manipulation of complex synthetic circuits and genomes to become the general rule in many laboratories rather than the exception. More recently, with the development of novel technologies such as DNA automated synthesis/sequencing and powerful computational tools, molecular biology has entered the synthetic biology era. In the beginning, most of these technologies were established in traditional microbial models (known as chassis in the synthetic biology framework) such as Escherichia coli and Saccharomyces cerevisiae, enabling fast advances in the field and the validation of fundamental proofs of concept. However, it soon became clear that these organisms, although extremely useful for prototyping many genetic tools, were not ideal for a wide range of biotechnological tasks due to intrinsic limitations in their molecular/physiological properties. Over the last decade, researchers have been facing the great challenge of shifting from these model systems to non-conventional chassis with endogenous capacities for dealing with specific tasks. The key to address these issues includes the generation of narrow and broad host plasmid-based molecular tools and the development of novel methods for engineering genomes through homologous recombination systems, CRISPR/Cas9 and other alternative methods. Here, we address the most recent advances in plasmid-based tools for the construction of novel cell factories, including a guide for helping with “build-your-own” microbial host.
Keywords: Genetic tools, Synthetic biology, Microbial chassis, Industrial microbiology, CRISPR/Cas9, mini-Transposons.