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.

Figure 1.

Modulation of enzyme expression levels is required for balanced pathway function. We used a simple quantitative model based on reversible Michaelis–Menten kinetics to depict the outcomes of an unbalanced enzyme expression (see Supplementary Results and Supplementary Figure S13 and S14 for full details). Considering two enzymes in a multi-step metabolic pathway, only a small region of the enzyme expression space (shown in white) sustains optimal production.

Figure 2.

(A) A small set of RBS sequences was designed to span several orders of magnitude of protein expression. The RBS set was composed of six pre-characterized RBS core sequences flanked by constant upstream and downstream insulators that were paired to the genes of interest, as detailed in Supplementary Figure S4 and Supplementary Methods. (B) Flow cytometry fluorescence measurements of cells expressing YFP, where in each clone a different RBS sequence (A–F) was located upstream to the coding sequence. (−) represents the autofluorescence of cells when no fluorescent protein is expressed.

Figure 3.

A modular cloning strategy for combinatorial assembly of multi-gene constructs. During the iterative assembly process, each gene of interest is joined with a chloramphenicol (Cm) resistance cassette and paired with the library of RBS sequences. To incorporate the next target gene, the marker is discarded, and the additional part is assembled into the vector. The newly formed construct contains the two RBS-modified genes and a resistance marker, enabling once again direct selection for positive constructs. This sequence of steps can be repeated to easily assemble a combinatorial library of RBS-modulated multi-gene operons.

Figure 4.

RBS modulation of three fluorescent proteins spans a color space. (A) We combinatorially joined CFP, YFP and mCherry with three representatives of our RBS set (sequences ‘A’, ‘C’ and ‘E’) and assembled the genes together into a synthetic operon. The resulting operon library differs only in the RBS sequences regulating gene expression. (B) Fluorescence microscopy imaging of E. coli colonies, transformed with the operon library. The observed colors represent additive combinations of the three primary colors, assigned to each of the fluorescent proteins. Irregular colony shapes are the result of touching boundaries of adjacent colonies. Some colonies harboring weak RBS for all three fluorescent reporters appear black. Inset: a bright-field microscopy image. (C) Fluorescence imaging of E. coli colonies containing the tri-color RBS-modulated operon. The images are arranged on a 3D grid where the position on each axis corresponds to the RBS strength of the fluorescent protein. (D) YFP and mCherry accumulation rates of clones sampled from a two-color operon library. RBS composition, as determined by barcode sequencing, is shown. Identical genotypes (each labeled by a distinct color) cluster together in the fluorescence space. The effect of translational coupling is also evident, where higher protein accumulation rate of YFP modulates the accumulation rate of mCherry.

Figure 5.

Carotenoid accumulation profile varies with the RBS sequences of biosynthetic genes. (A) We assembled a library of synthetic operons differing in the RBS sequences regulating each of the seven genes of the carotenoid biosynthesis pathway. (B) A binocular microscopy imaging of E. coli colonies transformed with the operon library. The color of the colony corresponds to the composition of the accumulated carotenoids, each having a characteristic color. Image was constructed by stitching multiple adjacent fields. (C) The carotenoid accumulation profile and RBS composition of clones isolated from the transformed library. The RBS composition of each clone was determined by sequencing (RBS encoding in barcode refers to the order of genes as illustrated in Figure 3A), and the carotenoid profile of each clone was analyzed using HPLC. Different genotypes result in distinct phenotypes, i.e. distinct carotenoid accumulation profiles. Circle area indicates the production yield of major carotenoid intermediates (>10% of total carotenoids), according to the metabolic pathway described on the right.