Gomes, D.; Rodrigues, J. L.; Scrutton, N. S.; Rodrigues, L. R.

Prenylnaringenin (PN) compounds, namely 8-prenylnaringenin (8-PN), 3\'-prenylnaringenin (3\'-PN), and 6-prenylnaringenin (6-PN), are reported to have several interesting bioactivities. This study aimed to validate a biosynthetic pathway for de novo production of PN in Escherichia coli. A previously optimized E. coli chassis capable of efficiently de novo producing naringenin was used to evaluate eleven prenyltransferases (PTs) for the production of PN compounds. As PT reaction requires dimethylallyl pyrophosphate (DMAPP) as extended substrate that has limited availability inside the cells, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 12a (Cas12a) (CRISPR-Cas12a) was used to construct ten boosted DMAPP-E. coli strains. All the PTs, in combination with the naringenin biosynthetic pathway, were tested in these strains. Experiments in 96-well deep well plates identified twelve strains capable of producing PN. E. coli M-PAR-121 with the integration of the 1-deoxy-D-xylulose-5-phosphate synthase (DXS) gene from E. coli (EcDXS) into the lacZ locus of the genome (E. coli M-PAR-121:EcDXS) expressing the soluble aromatic PT from Streptomyces roseochromogenes (CloQ) and the naringenin biosynthetic pathway was selected as the best producer strain. After optimizing the production media in shake flasks, 160.57 M of 3\'-PN, 4.4 M of 6-PN, and 2.66 M of 8-PN were obtained. The production was then evaluated at the bioreactor scale and 397.57 M of 3\'-PN (135.33 mg/L) and 25.61 M of 6-PN (8.72 mg/L) were obtained. To the best of our knowledge, this work represents the first report of de novo production of PN compounds using E. coli as a chassis.