Skip to main content

Advertisement

Log in

Engineering co-culture system for production of apigetrin in Escherichia coli

  • Metabolic Engineering and Synthetic Biology - Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

Microbial cells have extensively been utilized to produce value-added bioactive compounds. Based on advancement in protein engineering, DNA recombinant technology, genome engineering, and metabolic remodeling, the microbes can be re-engineered to produce industrially and medicinally important platform chemicals. The emergence of co-culture system which reduces the metabolic burden and allows parallel optimization of the engineered pathway in a modular fashion restricting the formation of undesired byproducts has become an alternative way to synthesize and produce bioactive compounds. In this study, we present genetically engineered E. coli-based co-culture system to the de novo synthesis of apigetrin (APG), an apigenin-7-O-β-d-glucopyranoside of apigenin. The culture system consists of an upstream module including 4-coumarate: CoA ligase (4CL), chalcone synthase, chalcone flavanone isomerase (CHS, CHI), and flavone synthase I (FNSI) to synthesize apigenin (API) from p-coumaric acid (PCA). Whereas, the downstream system contains a metabolizing module to enhance the production of UDP-glucose and expression of glycosyltransferase (PaGT3) to convert API into APG. To accomplish this improvement in titer, the initial inoculum ratio of strains for making the co-culture system, temperature, and media component was optimized. Following large-scale production, a yield of 38.5 µM (16.6 mg/L) of APG was achieved. In overall, this study provided an efficient tool to synthesize bioactive compounds in microbial cells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

References

  1. Alwahsh MA, Khairuddean M, Chong WK (2015) Chemical constituents and antioxidant activity of Teucrium barbeyanum Aschers. Rec Nat Prod 9:159–163

    Google Scholar 

  2. Atsumi S, Cann AF, Connor MR et al (2008) Metabolic engineering of Escherichia coli for 1-butanol production. Metab Eng 10:305–311. https://doi.org/10.1016/j.ymben.2007.08.003

    Article  CAS  PubMed  Google Scholar 

  3. Beekwilder J, Wolswinkel R, Jonker H et al (2006) Production of resveratrol in recombinant microorganisms. Appl Environ Microbiol 72:5670–5672. https://doi.org/10.1128/AEM.00609-06

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Bhan N, Xu P, Koffas MAG (2013) Pathway and protein engineering approaches to produce novel and commodity small molecules. Curr Opin Biotechnol 24:1137–1143. https://doi.org/10.1016/j.copbio.2013.02.019

    Article  CAS  PubMed  Google Scholar 

  5. Brazier-Hicks M, Edwards R (2013) Metabolic engineering of the flavone-C-glycoside pathway using polyprotein technology. Metab Eng 16:11–20. https://doi.org/10.1016/j.ymben.2012.11.004

    Article  CAS  PubMed  Google Scholar 

  6. Brazier-Hicks M, Evans KM, Gershater MC et al (2009) The C-glycosylation of flavonoids in cereals. J Biol Chem 284:17926–17934. https://doi.org/10.1074/jbc.M109.009258

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Brenner K, You L, Arnold FH (2008) Engineering microbial consortia: a new frontier in synthetic biology. Trends Biotechnol 26:483–489. https://doi.org/10.1016/j.tibtech.2008.05.004

    Article  CAS  PubMed  Google Scholar 

  8. Buettner FFR, Ashikov A, Tiemann B et al (2013) C. elegans DPY-19 is a C-mannosyltransferase glycosylating thrombospondin repeats. Mol Cell 50:295–302. https://doi.org/10.1016/j.molcel.2013.03.003

    Article  CAS  PubMed  Google Scholar 

  9. Camacho-Zaragoza JM, Hernández-Chávez G, Moreno-Avitia F et al (2016) Engineering of a microbial coculture of Escherichia coli strains for the biosynthesis of resveratrol. Microb Cell Fact 15:163. https://doi.org/10.1186/s12934-016-0562-z

    Article  PubMed  PubMed Central  Google Scholar 

  10. Chanos P, Mygind T (2016) Co-culture-inducible bacteriocin production in lactic acid bacteria. Appl Microbiol Biotechnol 100:4297–4308. https://doi.org/10.1007/s00253-016-7486-8

    Article  CAS  PubMed  Google Scholar 

  11. Chaudhary AK, Dhakal D, Sohng JK (2013) An insight into the “-omics” based engineering of Streptomycetes for secondary metabolite overproduction. Biomed Res Int 2013:968518. https://doi.org/10.1155/2013/968518

    Article  PubMed  PubMed Central  Google Scholar 

  12. Giang PM, Son PT (2004) Apigenin 7-O-β-d-glucoside from the leaves of Acanthus integrifolius T. Anders., Acanthaceae. J Chem 42:496–498

    Google Scholar 

  13. Gurung RB, Kim EH, Oh TJ, Sohng JK (2013) Enzymatic synthesis of apigenin glucosides by glucosyltransferase (YjiC) from Bacillus licheniformis DSM 13. Mol Cells 36:355–361. https://doi.org/10.1007/s10059-013-0164-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Hamilton ML, Caulfield JC, Pickett JA, Hooper AM (2009) C-Glucosylflavonoid biosynthesis from 2-hydroxynaringenin by Desmodium uncinatum (Jacq.) (Fabaceae). Tetrahedron Lett 50:5656–5659. https://doi.org/10.1016/j.tetlet.2009.07.118

    Article  CAS  Google Scholar 

  15. Havsteen BH (2002) The biochemistry and medical significance of the flavonoids. Pharmacol Ther 96(2–3):67–202

    Article  CAS  PubMed  Google Scholar 

  16. Hwang EI, Kaneko M, Ohnishi Y, Horinouchi S (2003) Production of plant-specific flavanones by Escherichia coli containing an artificial gene cluster. Appl Environ Microbiol 69:2699–2706. https://doi.org/10.1128/aem.69.5.2699

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Jones JA, Koffas MAG (2016) Optimizing metabolic pathways for the improved production of natural products. In: O’Connor SE (ed) Synthetic biology and metabolic engineering in plants and microbes part a: metabolism in microbes, 1st edn. Elsevier Inc, pp 179–193. https://doi.org/10.1016/bs.mie.2016.02.010

  18. Jones JA, Vernacchio VR, Sinkoe AL et al (2016) Experimental and computational optimization of an Escherichia coli co-culture for the efficient production of flavonoids. Metab Eng 35:55–63. https://doi.org/10.1016/j.ymben.2016.01.006

    Article  CAS  PubMed  Google Scholar 

  19. Kaneko M, Il Hwang E, Ohnishi Y, Horinouchi S (2003) Heterologous production of flavanones in Escherichia coli: potential for combinatorial biosynthesis of flavonoids in bacteria. J Ind Microbiol Biotechnol 30:456–461. https://doi.org/10.1007/s10295-003-0061-1

    Article  CAS  PubMed  Google Scholar 

  20. Kim HL, Kim AH, Park MB et al (2013) Altered sugar donor specificity and catalytic activity of pteridine glycosyltransferases by domain swapping or site-directed mutagenesis. BMB reports 46:37–40

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kren V, Martínková L (2001) Glycosides in medicine: “The role of glycosidic residue in biological activity”. Curr Med Chem 8:1303–1328. https://doi.org/10.2174/0929867013372193

    Article  CAS  PubMed  Google Scholar 

  22. Langenhan JM, Peters NR, Guzei IA et al (2005) Enhancing the anticancer properties of cardiac glycosides by neoglycorandomization. Proc Natl Acad Sci USA 102:12305–12310. https://doi.org/10.1073/pnas.0503270102

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Lee H, Kim BG, Kim M, Ahn JH (2015) Biosynthesis of two flavones, apigenin and genkwanin, in Escherichia coli. J Microbiol Biotechnol 25:1442–1448. https://doi.org/10.4014/jmb.1503.03011

    Article  CAS  PubMed  Google Scholar 

  24. Leonard E, Lim KH, Saw PN, Koffas MAG (2007) Engineering central metabolic pathways for high-level flavonoid production in Escherichia coli. Appl Environ Microbiol 73:3877–3886. https://doi.org/10.1128/AEM.00200-07

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Leonard E, Yan Y, Fowler ZL et al (2008) Strain improvement of recombinant Escherichia coli for efficient production of plant flavonoids. Mol Pharm 5:257–265. https://doi.org/10.1021/mp7001472

    Article  CAS  PubMed  Google Scholar 

  26. Leonard E, Yan Y, Lim KH, Koffas MAG (2005) Investigation of two distinct flavone synthases for plant-specific flavone biosynthesis in Saccharomyces cerevisiae. Appl Environ Microbiol 71:8241–8248. https://doi.org/10.1128/AEM.71.12.8241-8248.2005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Lim HS, Kim OS, Kim BY, Jeong SJ (2016) Apigetrin from Scutellaria baicalensis Georgi inhibits neuroinflammation in BV-2 microglia and exerts neuroprotective effect in HT22 hippocampal cells. J Med Food 19:1032–1040. https://doi.org/10.1089/jmf.2016.0074

    Article  CAS  PubMed  Google Scholar 

  28. Lin XH, Pan JB, Zhang XJ (2017) Anti-inflammatory and anti-oxidant effects of apigetrin on LPS-induced acute lung injury by regulating Nrf2 and AMPK pathways. Biochem Biophys Res Commun. https://doi.org/10.1016/j.bbrc.2017.07.071

    Google Scholar 

  29. Liu T, Khosla C (2010) Genetic engineering of Escherichia coli for biofuel production. Annu Rev Genet 44:53–69. https://doi.org/10.1146/annurev-genet-102209-163440

    Article  CAS  PubMed  Google Scholar 

  30. Magdouli S, Brar SK, Blais JF (2016) Co-culture for lipid production: advances and challenges. Biomass Bioenerg 92:20–30. https://doi.org/10.1016/j.biombioe.2016.06.003

    Article  CAS  Google Scholar 

  31. Malla S, Koffas MAG, Kazlauskas RJ, Kim BG (2012) Production of 7-O-methyl aromadendrin, a medicinally valuable flavonoid, in Escherichia coli. Appl Environ Microbiol 78:684–694

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Marín L, Gutiérrez-Del-Río I, Yagüe P et al (2017) De novo biosynthesis of apigenin, luteolin, and eriodictyol in the actinomycete Streptomyces albus and production improvement by feeding and spore conditioning. Front Microbiol 8:921. https://doi.org/10.3389/fmicb.2017.00921

    Article  PubMed  PubMed Central  Google Scholar 

  33. Martens S, Forkmann G, Matern U, Lukačin R (2001) Cloning of parsley flavone synthase I. Phytochemistry 58:43–46. https://doi.org/10.1016/S0031-9422(01)00191-1

    Article  CAS  PubMed  Google Scholar 

  34. Miyahisa I, Funa N, Ohnishi Y et al (2006) Combinatorial biosynthesis of flavones and flavonols in Escherichia coli. Appl Microbiol Biotechnol 71:53–58. https://doi.org/10.1007/s00253-005-0116-5

    Article  CAS  PubMed  Google Scholar 

  35. Miyahisa I, Kaneko M, Funa N et al (2005) Efficient production of (2S)-flavanones by Escherichia coli containing an artificial biosynthetic gene cluster. Appl Microbiol Biotechnol 68:498–504. https://doi.org/10.1007/s00253-005-1916-3

    Article  CAS  PubMed  Google Scholar 

  36. Nasr Bouzaiene N, Chaabane F, Sassi A et al (2016) Effect of apigenin-7-glucoside, genkwanin and naringenin on tyrosinase activity and melanin synthesis in B16F10 melanoma cells. Life Sci 144:80–85. https://doi.org/10.1016/j.lfs.2015.11.030

    Article  CAS  PubMed  Google Scholar 

  37. Noguchi A, Kunikane S, Homma H et al (2009) Identification of an inducible glucosyltransferase from Phytolacca americana L. cells that are capable of glucosylating capsaicin. Plant Biotechnol 26:285–292.  https://doi.org/10.5511/plantbiotechnology.26.285

    Article  CAS  Google Scholar 

  38. Oyama K, Kondo T (2004) Total synthesis of apigenin 7,4′-di-O-β-glucopyranoside, a component of blue flower pigment of Salvia patens, and seven chiral analogues. Tetrahedron 60:2025–2034. https://doi.org/10.1016/j.tet.2004.01.001

    Article  CAS  Google Scholar 

  39. Ozaki S, Imai H, Iwakiri T et al (2012) Regioselective glucosidation of trans-resveratrol in Escherichia coli expressing glucosyltransferase from Phytolacca americana. Biotech Lett 34:475–481. https://doi.org/10.1007/s10529-011-0784-4

    Article  CAS  Google Scholar 

  40. Pandey RP, Malla S, Simkhada D et al (2012) Production of 3-O-xylosyl quercetin in Escherichia coli. Appl Microbiol Biotechnol 97(5):1889–1901. https://doi.org/10.1007/s00253-012-4438-9

    Article  PubMed  Google Scholar 

  41. Park JH, Oh JE, Lee KH et al (2012) Rational design of Escherichia coli for l-isoleucine production. ACS Synth Biol 1:532–540. https://doi.org/10.1021/sb300071a

    Article  CAS  PubMed  Google Scholar 

  42. Ramos A, Olano C, Braña AF et al (2009) Modulation of deoxysugar transfer by the elloramycin glycosyltransferase ElmGT through site-directed mutagenesis. J Bacteriol 191:2871–2875. https://doi.org/10.1128/JB.01747-08

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, third. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  44. Samet I, Villareal MO, Motojima H et al (2015) Olive leaf components apigenin 7-glucoside and luteolin 7-glucoside direct human hematopoietic stem cell differentiation towards erythroid lineage. Differentiation 89:146–155. https://doi.org/10.1016/j.diff.2015.07.001

    Article  CAS  PubMed  Google Scholar 

  45. Takamura Y, Nomura G (1988) Changes in the intracellular concentration of acetyl-CoA and Malonyl-CoA in relation to the carbon and energy metabolism of Escherichia coli K12. Microbiology 134:2249–2253. https://doi.org/10.1099/00221287-134-8-2249

    Article  CAS  Google Scholar 

  46. Thorson JS, Vogt T (2005) Glycosylated natural products. In: Wong CH (ed) Carbohydrate-based drug discovery. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, pp 685–711

    Chapter  Google Scholar 

  47. Thuan NH, Pandey RP, Thuy TTT et al (2013) Improvement of regio-specific production of myricetin-3-O-α-l-rhamnoside in engineered Escherichia coli. Appl Biochem Biotechnol 171:1956–1967. https://doi.org/10.1007/s12010-013-0459-9

    Article  CAS  PubMed  Google Scholar 

  48. Thuan NH, Park JW, Sohng JK (2013) Toward the production of flavone-7-O-β-d-glucopyranosides using Arabidopsis glycosyltransferase in Escherichia coli. Process Biochem 48:1744–1748. https://doi.org/10.1016/j.procbio.2013.07.005

    Article  CAS  Google Scholar 

  49. Thuan NH, Sohng JK (2013) Recent biotechnological progress in enzymatic synthesis of glycosides. J Ind Microbiol Biotechnol 40(12):1329–1356. https://doi.org/10.1007/s10295-013-1332-0

    Article  PubMed  Google Scholar 

  50. Wang J, Zhou R-G, Wu T et al (2012) Total synthesis of apigenin. J Chem Res 36:121–122. https://doi.org/10.3184/174751912X13285269293913

    Article  Google Scholar 

  51. Watts K, Lee P, Schmidt-Dannert C (2006) Biosynthesis of plant-specific stilbene polyketides in metabolically engineered Escherichia coli. BMC Biotechnol 6:22. https://doi.org/10.1186/1472-6750-6-22

    Article  PubMed  PubMed Central  Google Scholar 

  52. Xu S, Shang MY, Liu GX et al (2013) Chemical constituents from the Rhizomes of Smilax glabra and their antimicrobial activity. Molecules 18:5265–5287. https://doi.org/10.3390/molecules18055265

    Article  CAS  PubMed  Google Scholar 

  53. Yan Y, Li Z, Koffas MAG (2008) High-yield anthocyanin biosynthesis in engineered Escherichia coli. Biotechnol Bioeng 100:126–140. https://doi.org/10.1002/bit.21721

    Article  CAS  PubMed  Google Scholar 

  54. Yu O, Shi J, Hession AO et al (2003) Metabolic engineering to increase isoflavone biosynthesis in soybean seed. Phytochemistry 63:753–763. https://doi.org/10.1016/S0031-9422(03)00345-5

    Article  CAS  PubMed  Google Scholar 

  55. Zhang H, Li Z, Pereira B, Stephanopoulos G (2015) Engineering E. coli-E. coli cocultures for production of muconic acid from glycerol. Microb Cell Fact 14:134. https://doi.org/10.1186/s12934-015-0319-0

    Article  PubMed  PubMed Central  Google Scholar 

  56. Zhang H, Pereira B, Li Z, Stephanopoulos G (2015) Engineering Escherichia coli coculture systems for the production of biochemical products. Proc Natl Acad Sci USA 112:8266–8271. https://doi.org/10.1073/pnas.1506781112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Zhang H, Stephanopoulos G (2016) Co-culture engineering for microbial biosynthesis of 3-amino-benzoic acid in Escherichia coli. Biotechnol J 11:981–987. https://doi.org/10.1002/biot.201600013

    Article  CAS  PubMed  Google Scholar 

  58. Zhou K, Qiao K, Edgar S, Stephanopoulos G (2015) Distributing a metabolic pathway among a microbial consortium enhances production of natural products. Nat Biotechnol 33:377–383. https://doi.org/10.1038/nbt.3095

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Foundation for Science and Technology Development of Vietnam (NAFOSTED) (106-NN.02-2014.25). We are grateful to Dr. Jules Beekwilder (Plant Research International, The Netherlands) for kindly providing pAC-4CL-STS plasmid, Dr. Shin-ichi Ozaki (Yamaguchi University, Japan) for providing plasmid pQE3-PaGT3 plasmid, Dr. Stefan Marten (Biotecnologia dei Prodotti Naturali, San Michele all’Adige, Italy) for FNSI, Dr. Mattheos A. G. Koffas (Rensselaer Polytechnic Institute, Troy, New York 12180, United States) and Dr. Sailesh Malla (Technical University of Denmark) for CHS and CHI genes, respectively.

Author information

Authors and Affiliations

Authors

Contributions

NHT and ACK conceived the study, designed experiments, analyzed data, and wrote the manuscript. NHT, DVC, and NXC performed the experiments, and NXC performed NMR study and analyzed the NMR data. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Nguyen Huy Thuan.

Ethics declarations

Conflict of interest

The authors declare no competing financial interests.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 1241 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Thuan, N.H., Chaudhary, A.K., Van Cuong, D. et al. Engineering co-culture system for production of apigetrin in Escherichia coli. J Ind Microbiol Biotechnol 45, 175–185 (2018). https://doi.org/10.1007/s10295-018-2012-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10295-018-2012-x

Keywords

Navigation