Systems analysis of methylerythritol-phosphate pathway flux in E. coli: insights into the role of oxidative stress and the validity of lycopene as an isoprenoid reporter metabolite

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.

A squalene synthase protein degradation method for improved sesquiterpene production in Saccharomyces cerevisiae

“…In order to increase availability of FPP in the presence of a heterologous sesquiterpene synthase, the entire MVA pathway as well as FPP synthase are typically overexpressed. This strategy is used both in yeast and in E. coli, the latter of which uses the alternative methylerythritol phosphate pathway for native isoprenoid production (Bongers et al, 2015;Ignea et al, 2011;Tsuruta et al, 2009;Yoon et al, 2009). In addition to the low capacity for prenyl phosphate (IPP/DMAPP/FPP) synthesis, non-productive consumption of FPP for sterol synthesis is a limitation for heterogeneous sesquiterpene production.…”

High density cultivation for efficient sesquiterpenoid biosynthesis inSynechocystissp. PCC 6803

“…While the culture dilutions themselves largely contribute to this effect, concomitantly stable volumetric productivities indicate a sustained carbon flux towards product formation when biomass accumulation rates slightly cease in phase 3. Sustained MEP pathway flux under stationary conditions was described for E. coli [54], and phototrophic patchoulol production in the green algae C. reinhardtii was shown to increase to some extent in the stationary phase [37]. As for cyanobacterial systems, the highest so far reported terpenoid yields using the example of isoprene were achieved in a 21-days cultivation experiment under a semi-batch regime with optimized carbon supply [13].…”

Chasing bacterial chassis for metabolic engineering: a perspective review from classical to non‐traditional microorganisms

“…Although IPP and DMAPP can be synthesized endogenously by E. coli through the methyleythritol-4-phosphate (MEP) pathway without generating MVA; this route has been shown to have a very limited flux (Ajikumar et al, 2010). Even though the endogenous MEP pathway is less efficient than expressing the heterologous MVA pathway (Morrone et al, 2010), quite some work has been devoted to optimize its performance and further understand its wiring (Kim and Keasling, 2001;Zhou et al, 2012;Bongers et al, 2015). The emerging picture indicates that further optimization is needed when the MVA pathway is used to release metabolic bottlenecks and reduce toxicity of some metabolic intermediates therein (Martin et al, 2003;Ajikumar et al, 2010;Ma et al, 2011;Wang et al, 2016a).…”