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

He, Lian; Xiu, Yu; Jones, J. Andrew; Baidoo, Edward E. K.; Keasling, Jay D.; Tang, Yinjie J.; Koffas, Mattheos A. G.

doi:10.1016/j.ymben.2016.12.008

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

Journal Article · Fri Dec 23 00:00:00 EST 2016 · Metabolic Engineering

DOI:https://doi.org/10.1016/j.ymben.2016.12.008· OSTI ID:1393105

He, Lian ^[1]; Xiu, Yu ^[2]; Jones, J. Andrew ^[3]; Baidoo, Edward E. K. ^[4]; Keasling, Jay D. ^[5]; Tang, Yinjie J. ^[1]; Koffas, Mattheos A. G. ^[6]

Washington Univ., St. Louis, MO (United States)
Rensselaer Polytechnic Inst., Troy, NY (United States); Beijing Univ. of Chemical Technology (China)
Rensselaer Polytechnic Inst., Troy, NY (United States); Hamilton College, Clinton, NY (United States)
Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States)
Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Technical Univ. of Denmark, Lyngby (Denmark)
Rensselaer Polytechnic Inst., Troy, NY (United States)

Microbial fermentation conditions are dynamic, due to transcriptional induction, nutrient consumption, or changes to incubation conditions. In this paper, 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 1.5 mmol/L. On the contrary, ~0.02 mmol/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. Finally, 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).

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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER); National Science Foundation (NSF)

Grant/Contract Number:: AC02-05CH11231; MCB 1616619; DBI1356669; MCB 1448657

OSTI ID:: 1393105

Alternate ID(s):: OSTI ID: 1414313

Journal Information:: Metabolic Engineering, Vol. 39; ISSN 1096-7176

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 28 works

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

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