TARSyn: Tunable Antibiotic Resistance Devices Enabling Bacterial Synthetic Evolution and Protein Production

Abstract

Evolution can be harnessed to optimize synthetic biology designs. A prominent example is recombinant protein production-a dominating theme in biotechnology for more than three decades. Typically, a protein coding sequence (cds) is recombined with genetic elements, such as promoters, ribosome binding sites and terminators, which control expression in a cell factory. A major bottleneck during production is translational initiation. Previously we identified more effective translation initiation regions (TIRs) by creating sequence libraries and then selecting for a TIR that drives high-level expression-an example of synthetic evolution. However, manual screening limits the ability to assay expression levels of all putative sequences in the libraries. Here we have solved this bottleneck by designing a collection of translational coupling devices based on a RNA secondary structure. Exchange of different sequence elements in this device allows for different coupling efficiencies, therefore giving the devices a tunable nature. Sandwiching these devices between the cds and an antibiotic selection marker that functions over a broad dynamic range of antibiotic concentrations adds to the tunability and allows expression levels in large clone libraries to be probed using a simple cell survival assay on the respective antibiotic. The power of the approach is demonstrated by substantially increasing production of two commercially interesting proteins, a Nanobody and an Affibody. The method is a simple and inexpensive alternative to advanced screening techniques that can be carried out in any laboratory.

A synbio approach for selection of highly expressed gene variants in Gram-positive bacteria

“…We first set out to explore the applicability of a translational coupling design that was previously developed in E. coli [ 25 ]. To this end we constructed a plasmid for L. lactis and B. subtilis that contained the gene encoding green fluorescent protein ( gfp ), coupled by a sequence with hairpin-forming (hp) propensity to a chloramphenicol resistance gene ( gfp - hp - Cm R ) (Fig.…”

“…To address this issue, we developed a simple growth based selection system based on translational coupling of an antibiotic resistance gene with the upstream gene sequence that leaves the protein of interest unaltered. Different from previous attempts, our system focuses on Gram-positive bacteria [ 25 ], works on genomically integrated targets and does not require tagging of the protein of interest [ 32 ]. Nonetheless, tags can be purposefully added to the protein if desired.…”

“…For this purpose, we have established a phenotypic screening approach for TIR libraries in E. coli [ 25 ]. The approach utilises an antibiotic resistance gene that is translationally coupled to the gene of interest.…”

New tools for recombinant protein production in Escherichia coli: A 5‐year update

“…Briefly, the cloning scar sequence was changed in all possible combinations by PCR with degenerate primers. The library was transformed in E. coli , and protein production was screened via cell sorting as the coding sequence of interest was fused to green fluorescent protein (or more recently, by translationally coupling the coding sequence to an antibiotic resistance gene). The authors reported a 1000‐fold difference between low and high expression vectors, which highlights the importance of optimizing the translation initiation region.…”

Increased production of periplasmic proteins in Escherichia coli by directed evolution of the translation initiation region

“…We were able to screen these TIR LIBRARIES as we fused signal peptides to the β-lactamase protein, which confers resistance to β-lactam antibiotics. For proteins where no simple screening assay is available it is possible to directly couple [18], or translationally-couple β-lactamase to the recombinant protein [40][41][42]. It is also possible to use the pET28a-based TIRs EVOLVED , which we identified in this study and which improved production yields of a single chain antibody fragment, a hormone and another recombinant protein in Escherichia coli.…”