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.

Constructing E. coli Co‐Cultures for De Novo Biosynthesis of Natural Product Acacetin

“…Modular co‐culture engineering has recently emerged as a robust method for biosynthesis of a variety of natural products. [ 12–17 ] In particular, a series of two‐strain co‐culture systems has been developed for producing flavonoid natural products, such as naringenin, [ 18,19 ] apigetrin, [ 20 ] resveratrol, [ 21 ] and pyranoanthocyanins, [ 22 ] all of which showed improved production performance. On the other hand, co‐culture systems composed of three or more strains have also been developed for producing natural products with complex biosynthesis pathways, such as rosmarinic acid and anthocyanins, [ 23,24 ] which demonstrates the potential of using polycultures to better balance the biosynthetic pathway.…”

Advances in the development and application of microbial consortia for metabolic engineering

“…(2018)Oxygenated taxanes E. coli- S. cerevisiae Xylose33 mg/L100%Zhou et al. (2015)Apigetrin E. coli - E. coli Glucose and p-coumaric acid16.6 mg/L2.5 foldThuan et al. (2018)…”

Metabolic Engineering of Microorganisms for the Production of Flavonoids

“…Second, the flavonoids biosynthetic pathway involves lengthy reaction steps and complicated regulations that significantly limit production yield. To overcome this obstacle, some researchers attempted to construct coculture or polyculture system to distribute metabolic burden by dividing the entire biosynthetic pathway into several independent host strains (Ganesan et al, 2017;Thuan et al, 2018;Wang et al, 2020). However, the metabolite transport limitation and the difficulty in keeping stable and reliable coculture system are the primary challenges for a wide-range application of this approach (Jones et al, 2016b(Jones et al, , 2017.…”

“…The first strain contains 4CL from Nicotiana tabacum, CHS from P. hybrida, CHI from M. sativa, and FNS from Parsley to generate apigenin from p-coumaric acid, whereas the second strain carries PaGT3 and an UDP-glucose overproduction system to convert apigenin into apigenin-7-O-β-D-glucoside. By optimization of initial inoculum ratio of the two strains, the titer of apigenin-7-O-β-D-glucoside reached 16.6 mg/L, which was 2.5 times higher than monoculture producing strain (Thuan et al, 2018). Breviscapine is a crude extract of flavonoids isolated from Erigeron breviscapus.…”