CRISPR-Cas12a-driven engineering of DMAPP and SAM pathways in E. coli for de novo Prenylflavonoid Biosynthesis

• Author(s): Gomes, Daniela Filipa Correia ; Rodrigues, Joana Lúcia Lima Correia ; Scrutton, Nigel S. ; Rodrigues, L. R.
• Date: 2025
• Persistent ID: https://hdl.handle.net/1822/98315
• Origin: RepositóriUM - Universidade do Minho
• Document Type: Other
• Language: English
• Contributor(s): Universidade do Minho

Description

Prenylflavonoids, including xanthohumol and prenylnaringenin (PN) (3-PN, 6-PN, and 8-PN), have been reported as bioactive compounds. However, their low accumulation in plants limits its broader utilization. We engineered Escherichia coli strains for the de novo biosynthesis of PNs and xanthohumol. Stepwise optimization was performed to construct naringenin chalcone and naringenin pathways. E. coli M-PAR-121 expressing tyrosine ammonia lyase from Flavobacterium johnsoniae (FjTAL), 4-coumarate:CoA ligase from Arabidopsis thaliana (At4CL), and chalcone synthase from Curcubita maxima (CmCHS) was able to produce 560.2 mg/L of naringenin chalcone. Adding chalcone isomerase from Medicago sativa (MsCHI), 769.5 mg/L of naringenin were produced. As the final prenylation and methylation steps rely on dimethylallyl pyrophosphate (DMAPP) and S-adenosylmethionine (SAM) availability, CRISPR-Cas12a was used to improve these pathways. The best PN-producing strain, E. coli M-PAR-121 strain with the integration of native 1-deoxy- D-xylulose-5-phosphate synthase (EcDXS) (E. coli M-PAR-121:EcDXS) expressing CloQ from Streptomyces roseochromogenes and pRSFDuet_FjTAL_CmCHS_At4CL_MsCHI, was able to produce 135.33 mg/L of 3- PN and 8.72 mg/L of 6-PN. Moreover, E. coli M-PAR-121:BlIDI:metK, with integration of SAM synthase (metK) and isopentenyl diphosphate isomerase from Bacillus licheniformis (BlIDI) expressing pRSFDuet_FjTAL_CmCHS_At4CL and CdpC3PT from Neosartorya fischeri and O-methyltransferase from Humulus lupulus (HlOMT1) (pCDFDuet_CdpC3PT_HlOMT) was selected as the best xanthohumol producing strain. This strain produced 5.26 mg/L of xanthohumol. These represent the highest production levels reported in any microorganism and the first de novo production of 3-PN, 6-PN, and xanthohumol in E. coli.