Spanning high-dimensional expression space using ribosome-binding site combinatorics

Abstract

Protein levels are a dominant factor shaping natural and synthetic biological systems. Although proper functioning of metabolic pathways relies on precise control of enzyme levels, the experimental ability to balance the levels of many genes in parallel is a major outstanding challenge. Here, we introduce a rapid and modular method to span the expression space of several proteins in parallel. By combinatorially pairing genes with a compact set of ribosome-binding sites, we modulate protein abundance by several orders of magnitude. We demonstrate our strategy by using a synthetic operon containing fluorescent proteins to span a 3D color space. Using the same approach, we modulate a recombinant carotenoid biosynthesis pathway in Escherichia coli to reveal a diversity of phenotypes, each characterized by a distinct carotenoid accumulation profile. In a single combinatorial assembly, we achieve a yield of the industrially valuable compound astaxanthin 4-fold higher than previously reported. The methodology presented here provides an efficient tool for exploring a high-dimensional expression space to locate desirable phenotypes.

Enhanced killing of antibiotic-resistant bacteria enabled by massively parallel combinatorial genetics

“…In future work, CombiGEM could also be extended to generate complex interactome datasets (49), screen for combinatorial regulators of desired phenotypes such as differentiation (36), identify potentiators of anticancer drugs (50), enhance pathway engineering (15), and characterize synthetic circuits at greater scales (51)(52)(53)(54)(55). The CombiGEM assembly strategy can be adapted to isothermal Gibson assembly (56) or recombinases (57) in situations where the library is incompatible with restriction sites.…”

“…Therefore, combinatorial libraries can be constructed in a pooled format without needing to barcode each combination within an individual reaction. A similar DNA assembly method was described for optimizing translational rates in metabolic pathways, in which hits were individually sequenced to identify the hit components (15). In contrast, we use high-throughput sequencing to census the distribution of library members within pooled populations in a highly multiplexed manner.…”

Toward metabolic engineering in the context of system biology and synthetic biology: advances and prospects

“…This strategy has yielded high-level production of fatty acids, (2S)-pinocembrin, and resveratrol in E. coli (Xu et al 2013c;Wu et al 2013a, b). In addition to promoter and expression copy number optimization, modular control of the ribosome binding site has been exploited to modularly control enzyme expression levels (Zelcbuch et al 2013). A series of ribosome binding sites with several orders of multitude in expression strength were applied in a modular cloning strategy for carotenoid biosynthesis pathway construction.…”

Fine-tuning of ecaA and pepc gene expression increases succinic acid production in Escherichia coli

“…However, the reliable and predicable gene regulation presents unique challenges as genetic parts such as promoter (Alper et al 2005;Cox et al 2007;Lescot et al 2002), ribosome binding site (RBS) (Barrick et al 1994;Chen et al 1994;Zelcbuch et al 2013;Levin-Karp et al 2013;Oliver et al 2014;Mutalik et al 2012;Salis et al 2009), and RNA (Sakai et al 2014;Laetitia and Martin 2006) often behave unexpectedly when they are used in novel designs. A gene regulation system that has been predicted analytically to perform well may perform poorly in practice.…”