Assessing Glycolytic Flux Alterations Resulting from Genetic Perturbations in E. Coli Using a Biosensor

Overview

Journal Metab Eng

Specialties Biomedical Engineering
Endocrinology

Date 2017 Jul 16

PMID 28709932

Citations 10

Authors

Christina E Lehning

Solvej Siedler

Mostafa M H Ellabaan

Morten O A Sommer

Affiliations

Soon will be listed here.

Abstract

We describe the development of an optimized glycolytic flux biosensor and its application in detecting altered flux in a production strain and in a mutant library. The glycolytic flux biosensor is based on the Cra-regulated ppsA promoter of E. coli controlling fluorescent protein synthesis. We validated the glycolytic flux dependency of the biosensor in a range of different carbon sources in six different E. coli strains and during mevalonate production. Furthermore, we studied the flux-altering effects of genome-wide single gene knock-outs in E. coli in a multiplex FlowSeq experiment. From a library consisting of 2126 knock-out mutants, we identified 3 mutants with high-flux and 95 mutants with low-flux phenotypes that did not have severe growth defects. This approach can improve our understanding of glycolytic flux regulation improving metabolic models and engineering efforts.

Citing Articles

Insights into the regulatory mechanisms and application prospects of the transcription factor Cra.

Huang Y, Jia K, Zhao W, Zhu L Appl Environ Microbiol. 2024; 90(11):e0122824.

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Flow-Seq Method: Features and Application in Bacterial Translation Studies.

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References

Siedler S, Stahlhut S, Malla S, Maury J, Neves A . Novel biosensors based on flavonoid-responsive transcriptional regulators introduced into Escherichia coli. Metab Eng. 2013; 21:2-8. DOI: 10.1016/j.ymben.2013.10.011. View

Monk J, Koza A, Campodonico M, Machado D, Seoane J, Palsson B . Multi-omics Quantification of Species Variation of Escherichia coli Links Molecular Features with Strain Phenotypes. Cell Syst. 2016; 3(3):238-251.e12. PMC: 5058344. DOI: 10.1016/j.cels.2016.08.013. View

Tang S, Qian S, Akinterinwa O, Frei C, Gredell J, Cirino P . Screening for enhanced triacetic acid lactone production by recombinant Escherichia coli expressing a designed triacetic acid lactone reporter. J Am Chem Soc. 2013; 135(27):10099-103. DOI: 10.1021/ja402654z. View

Weickert M, Adhya S . Isorepressor of the gal regulon in Escherichia coli. J Mol Biol. 1992; 226(1):69-83. DOI: 10.1016/0022-2836(92)90125-4. View

Skinner A, Cooper R . The regulation of ribose-5-phosphate isomerisation in Escherichia coli K12. FEBS Lett. 1971; 12(5):293-296. DOI: 10.1016/0014-5793(71)80202-8. View

Mahr R, Gatgens C, Gatgens J, Polen T, Kalinowski J, Frunzke J . Biosensor-driven adaptive laboratory evolution of l-valine production in Corynebacterium glutamicum. Metab Eng. 2015; 32:184-194. DOI: 10.1016/j.ymben.2015.09.017. View

Cavaleiro A, Kim S, Seppala S, Nielsen M, Norholm M . Accurate DNA Assembly and Genome Engineering with Optimized Uracil Excision Cloning. ACS Synth Biol. 2015; 4(9):1042-6. DOI: 10.1021/acssynbio.5b00113. View

Lutz R, Bujard H . Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res. 1997; 25(6):1203-10. PMC: 146584. DOI: 10.1093/nar/25.6.1203. View

Binder S, Schendzielorz G, Stabler N, Krumbach K, Hoffmann K, Bott M . A high-throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell level. Genome Biol. 2012; 13(5):R40. PMC: 3446293. DOI: 10.1186/gb-2012-13-5-r40. View

10.

Hebisch E, Knebel J, Landsberg J, Frey E, Leisner M . High variation of fluorescence protein maturation times in closely related Escherichia coli strains. PLoS One. 2013; 8(10):e75991. PMC: 3796512. DOI: 10.1371/journal.pone.0075991. View

11.

Kosuri S, Goodman D, Cambray G, Mutalik V, Gao Y, Arkin A . Composability of regulatory sequences controlling transcription and translation in Escherichia coli. Proc Natl Acad Sci U S A. 2013; 110(34):14024-9. PMC: 3752251. DOI: 10.1073/pnas.1301301110. View

12.

Siedler S, Bringer S, Blank L, Bott M . Engineering yield and rate of reductive biotransformation in Escherichia coli by partial cyclization of the pentose phosphate pathway and PTS-independent glucose transport. Appl Microbiol Biotechnol. 2011; 93(4):1459-67. PMC: 3275745. DOI: 10.1007/s00253-011-3626-3. View

13.

Kosuri S, Church G . Large-scale de novo DNA synthesis: technologies and applications. Nat Methods. 2014; 11(5):499-507. PMC: 7098426. DOI: 10.1038/nmeth.2918. View

14.

Bonde M, Kosuri S, Genee H, Sarup-Lytzen K, Church G, Sommer M . Direct mutagenesis of thousands of genomic targets using microarray-derived oligonucleotides. ACS Synth Biol. 2014; 4(1):17-22. PMC: 4304438. DOI: 10.1021/sb5001565. View

15.

Wetmore K, Price M, Waters R, Lamson J, He J, Hoover C . Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons. mBio. 2015; 6(3):e00306-15. PMC: 4436071. DOI: 10.1128/mBio.00306-15. View

16.

Lennen R, Herrgard M . Combinatorial strategies for improving multiple-stress resistance in industrially relevant Escherichia coli strains. Appl Environ Microbiol. 2014; 80(19):6223-42. PMC: 4178669. DOI: 10.1128/AEM.01542-14. View

17.

Cao H, Butler K, Hossain M, Lewis J . Variation in the fitness effects of mutations with population density and size in Escherichia coli. PLoS One. 2014; 9(8):e105369. PMC: 4133409. DOI: 10.1371/journal.pone.0105369. View

18.

Shimada T, Yamamoto K, Ishihama A . Novel members of the Cra regulon involved in carbon metabolism in Escherichia coli. J Bacteriol. 2010; 193(3):649-59. PMC: 3021228. DOI: 10.1128/JB.01214-10. View

19.

Heller K, Wilson T . Selectivity of the Escherichia coli outer membrane porins ompC and ompF. FEBS Lett. 1981; 129(2):253-5. DOI: 10.1016/0014-5793(81)80177-9. View

20.

Jiang W, Bikard D, Cox D, Zhang F, Marraffini L . RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol. 2013; 31(3):233-9. PMC: 3748948. DOI: 10.1038/nbt.2508. View