Bacterial genome reductions: Tools, applications, and challenges

Abstract

Bacterial cells are widely used to produce value-added products due to their versatility, ease of manipulation, and the abundance of genome engineering tools. However, the efficiency of producing these desired biomolecules is often hindered by the cells' own metabolism, genetic instability, and the toxicity of the product. To overcome these challenges, genome reductions have been performed, making strains with the potential of serving as chassis for downstream applications. Here we review the current technologies that enable the design and construction of such reduced-genome bacteria as well as the challenges that limit their assembly and applicability. While genomic reductions have shown improvement of many cellular characteristics, a major challenge still exists in constructing these cells efficiently and rapidly. Computational tools have been created in attempts at minimizing the time needed to design these organisms, but gaps still exist in modelling these reductions in silico. Genomic reductions are a promising avenue for improving the production of value-added products, constructing chassis cells, and for uncovering cellular function but are currently limited by their time-consuming construction methods. With improvements to and the creation of novel genome editing tools and in silico models, these approaches could be combined to expedite this process and create more streamlined and efficient cell factories.

Keywords: bacteria; genome engineering; genome reduction; minimal genome; synthetic biology.

FIGURE 1

Schematic diagram of the construction of a reduced genome cell. Identification of essential genes using experimental and computational methods, genomic reduction by a top-down gene deletion or bottom-up synthesis approach, and evaluation of modifications of this strain.

FIGURE 2

Experimental methods of determining gene essentiality. TnSeq inactivates a random gene by randomly inserting a transposon from a vector. LoxTnSeq deletes a random genomic region by inserting two transposons containing LoxP sites and activating recombination by Cre. CRISPRi-Seq inactivates a random gene by expressing a random gRNA and dCas9 that will bind and repress expression. For all three, the mutants will be pooled and subjected to various conditions followed by sequencing to identify remaining transposon location or guide RNA.

FIGURE 3

Deletion methods used to make large genomic reductions. (A) Site-specific recombination deletion method using two recombination sites (RS) which are acted on by a recombinase to result in recombination and deletion of the target. (B) λ red recombineering with I-SceI uses a linear DNA fragment with I-SceI cut sites (S) to cause a double-stranded break with recombination between the homologous regions (HR) to repair, resulting in a deletion. (C) Homologous recombination mediated deletions using a counterselectable marker (CS) to select for the second recombination event resulting in the deletion or return of wild-type. (D) CRISPR-based deletions employing Cas9 to cause a double-stranded break, forcing the cell to repair using homologous recombination, resulting in the deletion.