Stabilized gene duplication enables long-term selection-free heterologous pathway expression

Abstract

Engineering robust microbes for the biotech industry typically requires high-level, genetically stable expression of heterologous genes and pathways. Although plasmids have been used for this task, fundamental issues concerning their genetic stability have not been adequately addressed. Here we describe chemically inducible chromosomal evolution (CIChE), a plasmid-free, high gene copy expression system for engineering Escherichia coli. CIChE uses E. coli recA homologous recombination to evolve a chromosome with approximately 40 consecutive copies of a recombinant pathway. Pathway copy number is stabilized by recA knockout, and the resulting engineered strain requires no selection markers and is unaffected by plasmid instabilities. Comparison of CIChE-engineered strains with equivalent plasmids revealed that CIChE improved genetic stability approximately tenfold and growth phase-specific productivity approximately fourfold for a strain producing the high metabolic burden-biopolymer poly-3-hydroxybutyrate. We also increased the yield of the nutraceutical lycopene by 60%. CIChE should be applicable in many organisms, as it only requires having targeted genomic integration methods and a recA homolog.

Overproduction of Magnetosomes by Genomic Amplification of Biosynthesis-Related Gene Clusters in a Magnetotactic Bacterium

“…For instance, Tang et al recently increased the production of the secondary metabolite spinosyn in the native host by partial gene cluster duplication (35). Using a chemically inducible chromosomal evolution approach, 40 copies of a poly-3-hydroxybutyrate gene cluster were consecutively inserted into the chromosome of E. coli, thereby causing a significant increase in the production of this biopolymer (36).…”

Implementation of stable and complex biological systems through recombinase-assisted genome engineering

“…Although RAGE was only applied towards integration of a single module in this example, it is a straightforward exercise to pursue the integration of multiple modules into a variety of backgrounds through the use of alternate mutually exclusive lox site pairings [34][35][36] . Finally, coupling this technique with adaptive evolution or direct ARTICLE genome editing 4,8,20 can further optimize the performance of the designed biological functions of interest. Indeed, the recovery of better ethanol producers after 50 generations of culturing clearly demonstrates the potential of generating additional improvements in phenotype through adaptive evolution and screening.…”

“…Due to the number of strain variants that may need to be generated, efficiency and simplicity are key assets of RAGE, particularly when the performance of the targeted biological system is dependent on other enzymes or genetic modules requiring parallel optimization 32,33 . As an added benefit, a stably integrated strain is also much more amenable to additional iterations of strain engineering, including directed or adaptive evolutionary processes aimed at improving phenotype 4,8,20 . We demonstrate the practical application of this method through the installation and optimization of a 34 kb heterologous gene cluster enabling alginate degradation and utilization in E. coli.…”

“…However, while the ease of manipulating and transferring extrachromosomal DNA makes plasmids an attractive tool for cursory proof-of-concept studies, their use introduces severe inaccuracies when carefully evaluating the performance of engineered biological systems. Indeed, the combined metabolic burden of both plasmid propagation/ maintenance 18,19 and pathway overexpression often results in segregational or genetic population events promoting plasmid loss or allele inactivation 20 . In addition, several studies have shown that cell-to-cell variability in copy number can be significant under different cellular growth states and conditions 21,22 , a phenomenon that can lead to erratic strain performance.…”

Synthetic Biology & Bioinformatics Prospects in the Cancer Arena

“…Plasmid-based expression and chromosomal integration are the two common vehicles for implementing synthetic metabolic pathways. Recently, the chemically inducible chromosomal evolution (CIChE) was proposed as a longwww.intechopen.com term expression alternative method (Tyo et al, 2009). This new method avoids complications associated with plasmid replication and segregation, and can be used to integrate multiple copies of genes into the genome.…”