Rapid prototyping of microbial cell factories via genome-scale engineering

Abstract

Advances in reading, writing and editing genetic materials have greatly expanded our ability to reprogram biological systems at the resolution of a single nucleotide and on the scale of a whole genome. Such capacity has greatly accelerated the cycles of design, build and test to engineer microbes for efficient synthesis of fuels, chemicals and drugs. In this review, we summarize the emerging technologies that have been applied, or are potentially useful for genome-scale engineering in microbial systems. We will focus on the development of high-throughput methodologies, which may accelerate the prototyping of microbial cell factories.

Keywords: Genome synthesis; Genome-scale engineering; High-throughput technology; Homologous recombination; Microbial cell factory; Transcriptome engineering.

Fig. 1. Overview of various genome-scale engineering tools

(A) A circular donor cassette can be integrated into the recognition site by an integrase. By flanking a target sequence with heterospecific sites, RMCE enables replacement of a target sequence using a donor cassette flanked by compatible sites. (B) The ss-oligos containing designed mutations are incorporated into the lagging strand of replicating DNA through recombineering. (C) HR or NHEJ is greatly promoted via DSBs using various endonucleases. (D) Site specific insertion can be achieved via group II introns. (E) Transcription factor (TF) libraries can be constructed by mutating the endogenous TFs (gTME) or introducing artificial TFs for large-scale perturbation on transcriptome. (F) Different regulatory non-coding RNAs (ncRNAs), including sRNAs, siRNAs and gRNAs, are used to modulate targeted gene expression in bacteria and yeast.

Fig. 2. Genome-wide strain libraries for high-throughput genotyping

Single mutations can be introduced through plasmid-borne libraries or directed genome editing. A double-mutation strain library can be created using the synthetic genetic array (SGA) method, whereby a query strain harboring the first mutation can be mated with a strain library to incorporate a genome-wide second mutation.

Fig. 3. Reporter based phenotyping

In the presence of a ligand, the specific activation of transcription factor (TF) is controlled by the expression of the reporter gene. A synthetic riboswitch acts as a biosensor for desired metabolites.

Fig. 4. Multiplex automated genome engineering (MAGE) and trackable multiplex recombineering (TRMR) accelerated E. coli genome evolution

MAGE enables rapid generation of sequence diversity via continuous delivery of ss-oligos into cells. With barcode incorporated oligos, TRMR enables simultaneous creation and tracking of multiple genetic modifications.

Fig. 5. RNAi-assisted genome evolution (RAGE) in S. cerevisiae

In the presence of a heterologous RNAi pathway, genome-wide knockdown screening can be performed with a double-stranded RNA library derived from genomic DNA. Iterative RNAi screen may help to accumulate beneficial genetic modifications in an evolving yeast genome for continuous improvement of a complex phenotype.