Elucidation of the co-metabolism of glycerol and glucose in Escherichia coli by genetic engineering, transcription profiling, and 13C metabolic flux analysis

Yao, Ruilian; Xiong, Dewang; Hu, Hongbo; Wakayama, Masataka; Yu, Wei; Zhang, Xuehong; Shimizu, Kazuyuki

doi:10.1186/s13068-016-0591-1

Cited by 31 publications

(28 citation statements)

References 69 publications

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“…5 c, d). In reality, inhibition of EMPP flux (e.g., removal of glucose transporter Δ ptsG or glucose-limiting chemostat cultures) allows E. coli to cometabolize glycerol with glucose [ 47 ].…”

Section: Discussionmentioning

confidence: 99%

Examining Escherichia coli glycolytic pathways, catabolite repression, and metabolite channeling using Δpfk mutants

Hollinshead

Rodrı́guez

Martín

et al. 2016

Biotechnol Biofuels

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BackgroundGlycolysis breakdowns glucose into essential building blocks and ATP/NAD(P)H for the cell, occupying a central role in its growth and bio-production. Among glycolytic pathways, the Entner Doudoroff pathway (EDP) is a more thermodynamically favorable pathway with fewer enzymatic steps than either the Embden–Meyerhof–Parnas pathway (EMPP) or the oxidative pentose phosphate pathway (OPPP). However, Escherichia coli do not use their native EDP for glucose metabolism.ResultsOverexpression of edd and eda in E. coli to enhance EDP activity resulted in only a small shift in the flux directed through the EDP (~20 % of glycolysis flux). Disrupting the EMPP by phosphofructokinase I (pfkA) knockout increased flux through OPPP (~60 % of glycolysis flux) and the native EDP (~14 % of glycolysis flux), while overexpressing edd and eda in this ΔpfkA mutant directed ~70 % of glycolytic flux through the EDP. The downregulation of EMPP via the pfkA deletion significantly decreased the growth rate, while EDP overexpression in the ΔpfkA mutant failed to improve its growth rates due to metabolic burden. However, the reorganization of E. coli glycolytic strategies did reduce glucose catabolite repression. The ΔpfkA mutant in glucose medium was able to cometabolize acetate via the citric acid cycle and gluconeogenesis, while EDP overexpression in the ΔpfkA mutant repressed acetate flux toward gluconeogenesis. Moreover, 13C-pulse experiments in the ΔpfkA mutants showed unsequential labeling dynamics in glycolysis intermediates, possibly suggesting metabolite channeling (metabolites in glycolysis are pass from enzyme to enzyme without fully equilibrating within the cytosol medium).ConclusionsWe engineered E. coli to redistribute its native glycolytic flux. The replacement of EMPP by EDP did not improve E. coli glucose utilization or biomass growth, but alleviated catabolite repression. More importantly, our results supported the hypothesis of channeling in the glycolytic pathways, a potentially overlooked mechanism for regulating glucose catabolism and coutilization of other substrates. The presence of channeling in native pathways, if proven true, would affect synthetic biology applications and metabolic modeling.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0630-y) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Examining Escherichia coli glycolytic pathways, catabolite repression, and metabolite channeling using Δpfk mutants

Hollinshead

Rodrı́guez

Martín

et al. 2016

Biotechnol Biofuels

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“…Strain SA‐B4 had the highest shikimate production in the culture medium with a 12:1 glycerol to glucose ratio portraying that changing the ratio of glycerol to glucose can influence the shikimic acid production. NADPH is an essential cofactor in the biosynthesis of shikimic acid . In this study, we demonstrated that NADPH availability had a positive correlation with shikimate production.…”

Section: Discussionmentioning

confidence: 99%

“…Carbon sources might influence the kinetics of desired product through a cellular response that redistributes the composition of the fermentation end‐products to maintain an oxidation–reduction (redox) equilibrium. The metabolic profiles resulting from varying carbon sources have been explicated by metabolic stoichiometric analysis and revealed the existence of different regulatory networks . Optimizing the carbon source is important to achieve elevated shikimate production with effective substrate conversion accompanied by diminished by‐product generation.…”

Section: Introductionmentioning

confidence: 99%

Systematically engineering Escherichia coli for enhanced shikimate biosynthesis co‐utilizing glycerol and glucose

Bilal

Yue

et al. 2018

Biofuels Bioprod Bioref

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In this study, an Escherichia coli strain with the capability to utilize glycerol/glucose simultaneously was genetically engineered to produce high shikimate. To achieve this, the ptsG gene encoding EIICBglc protein was inactivated in the BW25113 strain to alleviate carbon catabolite repression. The shikimate kinase genes aroK and aroL were also eliminated to construct the SA-B2 strain, which led to a 42.3% increase in shikimate production. Thereafter, three critical genes, namely mutant aroG fbr , ppsA, and tktA, were overexpressed in strain SA-B2, and the resulting engineered SA-B7 strain produced 0.92 g/L shikimate in shake flask cultures, which were 2.5 times higher than strain SA-B2. The influence of varying carbon mole numbers ratios of glycerol to glucose on the production of shikimate was also investigated, where the highest shikimate production was attained at a glycerolto-glucose ratio of 12:1 by SA-B7. Moreover, the amount of NADPH was 3.5-times higher in the mixed culture medium as compared to sole glycerol-based medium. RT-PCR data indicated that the addition of glucose upregulated the transcriptional levels of genes pertaining to shikimate biosynthesis. In conclusion, the findings demonstrated that E. coli could be modified as a potential cell factory to produce shikimate and other high-value derived aromatic compounds by genetic and metabolic engineering strategies.Spotlight: Engineering E. coli for enhanced shikimate biosynthesis M Bilal et al..

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“…We therefore co-expressed glycerol kinase (glpK) and sn-glycerol-3phosphate dehydrogenase (glpD) genes using the pPROLar.A122 vector backbone, since it was compatible with the plasmid used to express L-asparaginase in the DKO strain. Many previous studies have shown that these two genes play a critical role in enhancing glycerol uptake rates [20,[55][56][57][58].…”

Section: Growth and Substrate Utilization Pro Les Of The Dko Strain Pmentioning

confidence: 99%

Designing next generation recombinant protein expression platforms by modulating the cellular stress response in Escherichia coli

Guleria

Jain

Verma

et al. 2020

Preprint

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Background A cellular stress response (CSR) is triggered upon recombinant protein synthesis which acts as a global feedback regulator of protein expression. To remove this key regulatory bottleneck, we had previously proposed that genes that are up-regulated post induction could be part of the signaling pathways which activate the CSR. Knocking out some of these genes which were non-essential and belonged to the bottom of the E. coli regulatory network had provided higher expression of GFP and L-asparaginase . Results We chose the best performing double knockout E. coli BW25113 ΔelaAΔcysW and demonstrated its ability to enhance the expression of the toxic Rubella E1 glycoprotein by 2.5-fold by tagging it with sf GFP at the C-terminal end to better quantify expression levels. Transcriptomic analysis of this hyper-expressing mutant showed that a significantly lower proportion of genes got down-regulated post induction, which included genes for transcription, translation, protein folding and sorting, ribosome biogenesis, carbon metabolism, amino acid and ATP synthesis. This down-regulation which is a typical feature of the CSR was clearly blocked in the double knockout strain leading to its enhanced expression capability. Finally, we supplemented the expression of substrate uptake genes glpK and glpD whose down-regulation was not prevented in the double knockout, thus ameliorating almost all the negative effects of the CSR and obtained a further doubling in recombinant protein yields. Conclusion The study validated the hypothesis that these up-regulated genes act as signaling messengers which activate the CSR and thus, despite having no casual connection with recombinant protein synthesis, can improve cellular health and protein expression capabilities. Combining gene knockouts with supplementing the expression of key down-regulated genes can counter the harmful effects of CSR and help in the design of a truly superior host platform for recombinant protein expression.

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Elucidation of the co-metabolism of glycerol and glucose in Escherichia coli by genetic engineering, transcription profiling, and 13C metabolic flux analysis

Cited by 31 publications

References 69 publications

Examining Escherichia coli glycolytic pathways, catabolite repression, and metabolite channeling using Δpfk mutants

Examining Escherichia coli glycolytic pathways, catabolite repression, and metabolite channeling using Δpfk mutants

Systematically engineering Escherichia coli for enhanced shikimate biosynthesis co‐utilizing glycerol and glucose

Designing next generation recombinant protein expression platforms by modulating the cellular stress response in Escherichia coli

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