Precise control of lycopene production to enable a fast-responding, minimal-equipment biosensor

Abstract

Pigmented metabolites have great potential for use in biosensors that target low-resource areas, since sensor output can be interpreted without any equipment. However, full repression of pigment production when undesired is challenging, as even small amounts of enzyme can catalyze the production of large, visible amounts of pigment. The red pigment lycopene could be particularly useful because of its position in the multi-pigment carotenoid pathway, but commonly used inducible promoter systems cannot repress lycopene production. In this paper, we designed a system that could fully repress lycopene production in the absence of an inducer and produce visible lycopene within two hours of induction. We engineered Lac, Ara, and T7 systems to be up to 10 times more repressible, but these improved systems could still not fully repress lycopene. Translational modifications proved much more effective in controlling lycopene. By decreasing the strength of the ribosomal binding sites on the crtEBI genes, we enabled full repression of lycopene and production of visible lycopene in 3–4 hours of induction. Finally, we added the mevalonate pathway enzymes to increase the rate of lycopene production upon induction and demonstrated that supplementation of metabolic precursors could decrease the time to coloration to about 1.5 hours. In total, this represents over an order of magnitude reduction in response time compared to the previously reported strategy. The approaches used here demonstrate the disconnect between fluorescent and metabolite reporters, help enable the use of lycopene as a reporter, and are likely generalizable to other systems that require precise control of metabolite production.

Metabolomics Analysis of the Toxic Effects of the Production of Lycopene and Its Precursors

“…Overcoming the toxicity effects of heterologous expression of the lycopene biosynthesis pathway from Pantoea ananatis is a challenge that has been encountered repeatedly in the field of metabolic engineering. In our previous work, we developed E. coli strains that could express different levels of lycopene with minimal effects on cell growth by replacing the native ribosomal binding sites (RBSs) on the crtEBI genes with RBSs of varying, but weaker, strength ( McNerney and Styczynski, 2017a ).…”

“…Escherichia coli K-12 DH10B (New England Biolabs, Ipswich, MA, United States) was used as the host strain for all metabolite production experiments, and all heterologous proteins were expressed from standard expression plasmids. A detailed description of plasmid assembly can be found in McNerney and Styczynski (2017a) . In short, the lycopene pathway genes crtE , crtB , and crtI were amplified from Part bba_k274100 of the Registry of Standard Biological Parts and placed under the control of an IPTG-inducible promoter.…”

“…Lycopene was extracted from cultures and analyzed as described previously ( McNerney and Styczynski, 2017a ). Briefly, 500 μL of bacterial culture was pelleted and resuspended in 50 μL of ultrapure water.…”

“…Specifically, we have used E. coli as the chassis organism for a whole-cell biosensor to measure zinc status in blood samples, with biosynthesis of differently colored pigments used as the readout for the sensor. In this application, the key challenges to date have included the precise control of production of only one pigment at a time, and the complete repression of any pigment production to enable a dense, colorless initial inoculum that allows a rapid output response ( McNerney and Styczynski, 2017a ). In this most recent work, we pursued multiple strategies to limit unwanted pigment production and speed up visible pigment production once desired, including precursor supplementation.…”

Self-Redirection of Metabolic Flux toward Squalene and Ethanol Pathways by Engineered Yeast

“…Expression of target genes can be regulated by using repressible promoters and therefore often employed to control the expression of genes competing for precursors with engineered pathways [23]. In this study, two novel metal ion repressible promoters AFT1p (iron) and CTR1p (copper) were also characterized and used to downregulate the expression of ERG1 for squalene overproduction in yeast.…”