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Deciphering flux adjustments of engineered E. coli cells during fermentation with changing growth conditions.

Abstract：

:Microbial fermentation conditions are dynamic, due to transcriptional induction, nutrient consumption, or changes to incubation conditions. In this study, 13C-metabolic flux analysis was used to characterize two violacein-producing E. coli strains with vastly different productivities, and to profile their metabolic adjustments resulting from external perturbations during fermentation. The two strains were first grown at 37°C in stage 1, and then the temperature was transitioned to 20°C in stage 2 for the optimal expression of the violacein synthesis pathway. After induction, violacein production was minimal in stage 3, but accelerated in stage 4 (early production phase) and 5 (late production phase) in the high producing strain, reaching a final concentration of 1.5mmol/L. On the contrary, ~0.02mmol/L of violacein was obtained from the low producing strain. To have a snapshot of the temporal metabolic changes in each stage, we performed 13C-MFA via isotopomer analysis of fast-turnover free metabolites. The results indicate strikingly stable flux ratios in the central metabolism throughout the early growth stages. In the late stages, however, the high producer rewired its flux distribution significantly, which featured an upregulated pentose phosphate pathway and TCA cycle, reflux from acetate utilization, negligible anabolic fluxes, and elevated maintenance loss, to compensate for nutrient depletion and drainage of some building blocks due to violacein overproduction. The low producer with stronger promoters shifted its relative fluxes in stage 5 by enhancing the flux through the TCA cycle and acetate overflow, while exhibiting a reduced biomass growth and a minimal flux towards violacein synthesis. Interestingly, the addition of the violacein precursor (tryptophan) in the medium inhibited high producer but enhanced low producer's productivity, leading to hypotheses of unknown pathway regulations (such as metabolite channeling).

journal_name

Metab Eng

journal_title

Metabolic engineering

authors

He L,Xiu Y,Jones JA,Baidoo EEK,Keasling JD,Tang YJ,Koffas MAG

doi

10.1016/j.ymben.2016.12.008

subject

Has Abstract

pub_date

2017-01-01 00:00:00

pages

247-256

eissn

1096-7176

issn

1096-7184

pii

S1096-7176(16)30226-9

journal_volume

pub_type

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Deciphering flux adjustments of engineered E. coli cells during fermentation with changing growth conditions.