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

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.

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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.

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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.

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Correspondence to Nguyen Huy Thuan.

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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

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Keywords

  • Apigetrin
  • Co-culture
  • E. coli
  • Fermentation
  • Recombinant