Challenges in the Heterologous Production of Furanocoumarins in Escherichia coli

Abstract

Coumarins and furanocoumarins are plant secondary metabolites with known biological activities. As they are present in low amounts in plants, their heterologous production emerged as a more sustainable and efficient approach to plant extraction. Although coumarins biosynthesis has been positively established, furanocoumarin biosynthesis has been far more challenging. This study aims to evaluate if Escherichia coli could be a suitable host for furanocoumarin biosynthesis. The biosynthetic pathway for coumarins biosynthesis in E. coli was effectively constructed, leading to the production of umbelliferone, esculetin and scopoletin (128.7, 17.6, and 15.7 µM, respectively, from tyrosine). However, it was not possible to complete the pathway with the enzymes that ultimately lead to furanocoumarins production. Prenyltransferase, psoralen synthase, and marmesin synthase did not show any activity when expressed in E. coli. Several strategies were tested to improve the enzymes solubility and activity with no success, including removing potential N-terminal transit peptides and expression of cytochrome P450 reductases, chaperones and/or enzymes to increase dimethylallylpyrophosphate availability. Considering the results herein obtained, E. coli does not seem to be an appropriate host to express these enzymes. However, new alternative microbial enzymes may be a suitable option for reconstituting the furanocoumarins pathway in E. coli. Nevertheless, until further microbial enzymes are identified, Saccharomyces cerevisiae may be considered a preferred host as it has already been proven to successfully express some of these plant enzymes.

Reconstitution and Optimization of the Marmesin Biosynthetic Pathway in Yeast

“…coli, making the reconstitution of the furanocoumarin biosynthetic pathways in this host challenging. 7 The difficulty in expressing plant CYP450s inE. coli is partially attributed to the lack of endomembrane systems such as the endoplasmic reticulum in the bacteria.…”

“…Recently, the biosynthetic pathways for a few simple coumarins have been successfully constructed in microorganisms. ,− Yang et al constructed an engineered Escherichia coli harboring TAL , 4CL , and F6′H that can produce esculetin (52.3 mg/L), scopoletin (79.5 mg/L), and umbelliferone (82.9 mg/L) from their respective hydroxycinnamic acids precursors . Furthermore, they obtained strains for de novo biosynthesis of umbelliferone (66.1 mg/L) and esculetin (61.4 mg/L) by overexpressing tyrosine (TYR) biosynthetic genes and deleting a thioesterase to prevent degradation of the thioester intermediates for umbelliferone biosynthesis.…”

“…6 The biosynthetic pathways for furanocoumarins have been studied for decades, in which umbelliferone is produced as a key intermediate. 7 Umbelliferone is derived from paracoumaric acid (p-CA) in the phenylpropanoid pathway. In the linear furanocoumarin biosynthetic pathways, umbelliferone is first converted to 7-demethylsuberosin, which is sequentially converted to marmesin and psoralen by the marmesin synthase (MS) and psoralen synthase (PS), respectively (Figure 1A).…”

“…The biosynthetic pathways for furanocoumarins have been studied for decades, in which umbelliferone is produced as a key intermediate . Umbelliferone is derived from para -coumaric acid ( p -CA) in the phenylpropanoid pathway.…”

“…However, the PTs, MSs, and PSs involved in furanocoumarin biosynthesis could not be successfully expressed inE. coli, making the reconstitution of the furanocoumarin biosynthetic pathways in this host challenging . The difficulty in expressing plant CYP450s inE.…”