Mevalonate is a useful metabolite synthesized from three molecules of acetyl‐CoA, consuming two molecules of NADPH. Escherichia coli (
E. coli) catabolizes glucose to acetyl‐CoA via several routes, such as the Embden–Meyerhof–Parnas (EMP) and the oxidative pentose phosphate (oxPP) pathways. Although the oxPP pathway supplies NADPH, it is disadvantageous in terms of acetyl‐CoA supply, compared with the EMP pathway. In this study, the optimal flux ratio between the EMP and oxPP pathways on the mevalonate yield was investigated. Expression level of
pgi was controlled by isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) inducible promoter in an engineered mevalonate‐producing
E. coli strain. The relationship between the flux ratio and mevalonate yield was evaluated by changing the flux ratio by varying IPTG concentration. At the stationary phase, the mevalonate yield was maximum at an EMP flux of 39.7%, and was increased by 25% compared with that with no flux control (EMP flux of 70.4%). The optimal flux ratio was consistent with the theoretical value based on the mass balance of NADPH. The flux ratio between EMP and oxPP pathways affects the synthesis fluxes of mevalonate and acetate from acetyl‐CoA. Fine tuning of the flux ratio would be necessary to achieve an optimized production of metabolites that require NADPH.
An artificial metabolic route to an unnatural trichothecene was designed by taking advantage of the broad substrate specificities of the T-2 toxin biosynthetic enzymes of Fusarium sporotrichioides. By feeding 7-hydroxyisotrichodermin, a shunt pathway metabolite of F. graminearum, to a trichodiene synthase-deficient mutant of F. sporotrichioides, 7-hydroxy T-2 toxin (1) was obtained as the final metabolite. Such an approach may have future applications in the metabolic engineering of a variety of fungal secondary metabolites. The toxicity of 7-hydroxy T-2 toxin was 10 times lower than that of T-2 toxin in HL-60 cells.
Fusarium graminearum causes a disease of wheat and barley known as Fusarium head blight. It contaminates the grains with trichothecene mycotoxins such as deoxynivalenol (DON). As shunt intermediates in the DON biosynthetic pathway, 7-hydroxyisotrichodermin (7-HIT) and 8-hydroxyisotrichodermin (8-HIT) are known. However, their activities have not been previously evaluated. In this study, we performed toxicity assays of these trichothecenes by using a sensitive yeast bioassay that we have recently established. The IC 50 of 7-HIT and 8-HIT were in the range of 20-40 µg/ml, while the IC 50 of DON was approximately 1.5 µg/ml. Although the toxicity of these shunt metabolites remains to be investigated in animal systems, our present data indicate that 7-HIT and 8-HIT may not be major issues that require regulation in agricultural products.
Bioconversion of key intermediate metabolites such as
mevalonate
into various useful chemicals is a promising strategy for microbial
production. However, the conversion of mevalonate into isoprenoids
requires a supply of adenosine triphosphate (ATP). Light-driven ATP
regeneration using microbial rhodopsin is an attractive module for
improving the intracellular ATP supply. In the present study, we demonstrated
the ATP-consuming conversion of mevalonate to isoprenol using rhodopsin-expressing Escherichia coli cells as a whole-cell catalyst in
a medium that does not contain energy cosubstrate, such as glucose.
Heterologous genes for the synthesis of isoprenol from mevalonate,
which requires three ATP molecules for the series of reactions, and
a delta-rhodopsin gene derived from Haloterrigena turkmenica were cointroduced into E. coli. To
evaluate the conversion efficiency of mevalonate to isoprenol, the
cells were suspended in a synthetic medium containing mevalonate as
the sole carbon source and incubated under dark or light illumination
(100 μmol m–2 s–1). The
specific isoprenol production rates were 10.0 ± 0.9 and 20.4
± 0.7 μmol gDCW–1 h–1 for dark and light conditions, respectively. The conversion was
successfully enhanced under the light condition. Furthermore, the
conversion efficiency increased with increasing illumination intensity,
suggesting that ATP regenerated by the proton motive force generated
by rhodopsin using light energy can drive ATP-consuming reactions
in the whole-cell catalyst.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.