Abstract
BackgroundHigh-throughput screening methods assume that the output measured is representative of changes in metabolic flux toward the desired product and is not affected by secondary phenotypes. However, metabolic engineering can result in unintended phenotypes that may go unnoticed in initial screening. The red pigment lycopene, a carotenoid with antioxidant properties, has been used as a reporter of isoprenoid pathway flux in metabolic engineering for over a decade. Lycopene production is known to vary between wild-type Escherichia coli hosts, but the reasons behind this variation have never been fully elucidated.ResultsIn an examination of six E. coli strains we observed that strains also differ in their capacity for increased lycopene production in response to metabolic engineering. A combination of genetic complementation, quantitative SWATH proteomics, and biochemical analysis in closely-related strains was used to examine the mechanistic reasons for variation in lycopene accumulation. This study revealed that rpoS, a gene previously identified in lycopene production association studies, exerts its effect on lycopene accumulation not through modulation of pathway flux, but through alteration of cellular oxidative status. Specifically, absence of rpoS results in increased accumulation of reactive oxygen species during late log and stationary phases. This change in cellular redox has no effect on isoprenoid pathway flux, despite the presence of oxygen-sensitive iron-sulphur cluster enzymes and the heavy redox requirements of the methylerythritol phosphate pathway. Instead, decreased cellular lycopene in the ΔrpoS strain is caused by degradation of lycopene in the presence of excess reactive oxygen species.ConclusionsOur results demonstrate that lycopene is not a reliable indicator of isoprenoid pathway flux in the presence of oxidative stress, and suggest that caution should be exercised when using lycopene as a screening tool in genome-wide metabolic engineering studies. More extensive use of systems biology for strain analysis will help elucidate such unpredictable side-effects in metabolic engineering projects.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0381-7) contains supplementary material, which is available to authorized users.
Highlights
High-throughput screening methods assume that the output measured is representative of changes in metabolic flux toward the desired product and is not affected by secondary phenotypes
We aimed to identify global regulators of flux through the MEP pathway in E. coli using lycopene as a read-out
It was found that lycopene production varies significantly in wild-type and engineered E. coli strains from different phylogenetic groups, and that strains respond differently to overexpression of the assumed primary rate-limiting enzyme Dxs
Summary
High-throughput screening methods assume that the output measured is representative of changes in metabolic flux toward the desired product and is not affected by secondary phenotypes. Due to its red color, differences in lycopene content can be estimated by visual inspection and quantified with a quick and inexpensive spectrophotometric method This has led to lycopene becoming the metabolite of choice in highthroughput metabolic engineering studies of isoprenoid pathway flux [11,12,13,14,15,16,17,18]. Several studies focused on identifying E. coli genes not directly involved in core MEP pathway reactions that affect lycopene production [12, 14, 19, 20] Differential expression of these non-MEP pathway genes was used to increase lycopene production, often without an in-depth analysis of the mechanism leading to this phenotype. These association studies inferred a link between the identified overexpression- or knockout targets and changes in carbon flux towards lycopene
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