Building a genome engineering toolbox in nonmodel prokaryotic microbes

Abstract

The realization of a sustainable bioeconomy requires our ability to understand and engineer complex design principles for the development of platform organisms capable of efficient conversion of cheap and sustainable feedstocks (e.g., sunlight, CO , and nonfood biomass) into biofuels and bioproducts at sufficient titers and costs. For model microbes, such as Escherichia coli, advances in DNA reading and writing technologies are driving the adoption of new paradigms for engineering biological systems. Unfortunately, microbes with properties of interest for the utilization of cheap and renewable feedstocks, such as photosynthesis, autotrophic growth, and cellulose degradation, have very few, if any, genetic tools for metabolic engineering. Therefore, it is important to develop "design rules" for building a genetic toolbox for novel microbes. Here, we present an overview of our current understanding of these rules for the genetic manipulation of prokaryotic microbes and the available genetic tools to expand our ability to genetically engineer nonmodel systems.

Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining

“…This result was expected for one of these strains (MGD15) because it should be incapable of pyrimidine biosynthesis due to deletion of the pyrF gene. Supplementation with uracil was able to restore growth of this deletion strain in minimal succinate medium, showing that pyrF could be used as an auxotrophic selection marker in ADP1 as it is in many other bacterial species (65).…”

Toward a genetic system in the marine cyanobacteriumProchlorococcus

“…Development of an efficient electroporation procedure . While electroporation is one of the most powerful techniques for delivering DNA to the inside of living cells [14], [63], and protocols have been developed for marine bacteria [64]- [67], it is a harsh treatment, typically resulting in significant cell loss [68]. Further, the required removal of salts results in osmotic stress -particularly challenging for a notoriously temperamental marine bacterium like Prochlorococcus [47].…”

“…An alternative strategy is to use integrative plasmids to serve as delivery vectors, relying on the target cell's homologous recombination machinery to recombine the exogenous DNA into the host genome [14]. This process has been successful in generating gene knockouts in marine Synechococcus strains [6] but required a lengthy procedure for Prochlorococcus [54], which was not reproducible in our hands (see section above on conjugation attempts).…”

“…Countless attempts have demonstrated that the creation of successful genetic transformation protocols requires tedious trial and error, and methods developed for one strain are often unsuccessful in close relatives (for a few examples see: [6]- [13]). There is no single solution for all bacteria, and no way to predict which techniques will succeed [14]. Thus, there is a pressing need for broad-spectrum, automatized, and standardized genetic transformation procedures [15] that could enable downstream genetic screening methods such as transposon insertion sequencing [5], [16]- [18].…”

Evolutionary Approaches for Engineering Industrially Relevant Phenotypes in Bacterial Cell Factories

“…Low product yields are often tied to a decrease in cell viability and genetic instability during scale‐up and under industrial production conditions, even after comprehensive optimization of biosynthetic pathways. In addition, establishing a genetic toolbox for the engineering of nonmodel microbes is still a daunting task . All of these aspects slow down the development of economically viable MCFs and bioprocesses, and, indeed, a relatively small number of these rationally engineered strains can be scaled up in an industrial context .…”