Exploiting CRISPR-Cas12a to engineer DMAPP/SAM pathways in Escherichia coli towards de novo production of prenylflavonoids

• Autor(es): Gomes, Daniela Filipa Correia ; Rodrigues, Joana Lúcia Lima Correia ; Scrutton, Nigel S. ; Rodrigues, L. R.
• Data: 2025
• Identificador Persistente: https://hdl.handle.net/1822/98314
• Origem: RepositóriUM - Universidade do Minho
• Tipo de Documento: Outro
• Idioma: Inglês
• Contribuidor(es): Universidade do Minho

Descrição

Prenylflavonoids are flavonoid derived compounds with interesting bioactivities, namely estrogenic and anticancer. Among them, xanthohumol and prenylnaringenin (PN) isomers, including 3-prenylnaringenin (3-PN), 6-prenylnaringenin (6-PN), and 8-prenylnaringenin (8-PN) have attracted special interest. Due to their low production and accumulation in plants, the construction of microbial cell factories able to produce them appeared as a promising and sustainable solution. Herein, we aimed to engineer Escherichia coli strains towards de novo production of PN compounds and xanthohumol. Initially, efficient pathways for de novo production of the precursors naringenin chalcone and naringenin were constructed. E. coli M-PAR-121 expressing tyrosine ammonia lyase (TAL) from Flavobacterium johnsoniae (FjTAL), 4-coumarate:CoA ligase 1 (4CL-1) from Arabidopsis thaliana (At4CL), and chalcone synthase (CHS) from Curcubita maxima (CmCHS) was able to produce 560.2 mg/L of naringenin chalcone. Moreover, E. coli M-PAR-121 expressing FjTAL, At4CL, CmCHS, and chalcone isomerase (CHI) from Medicago sativa (MsCHI) was able to produce 769.5 mg/L of naringenin being the highest production reported so far in E. coli 1. Eleven prenyltransferase (PT) were evaluated to produce PNs. These elevens PTs were also tested in combination with the O-methyltransferase from Humulus lupulus (HlOMT1) to produce xanthohumol. However, no production of any PN compound or xanthohumol was detected in the wild-type strain. Since the final steps of the prenylflavonoids pathway depend on the availability of dimethylallyl pyrophosphate (DMAPP) and/or S-adenosylmethionine (SAM), clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a methodologies were exploited to engineer the endogenous E. coli pathways. Firstly, CRISPR-Cas12a was used to engineer the DMAPP pathway by improving the flux of the rate limiting steps 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and isopentenyl diphosphate isomerase (IDI). Single and double integration of DXS and IDI genes with improved activities (DXS from Bacillus subtilis (BsDXS), IDI from Saccharomyces cerevisiae (ScIDI), and IDI from Bacillus licheniformis (BlIDI)) were performed into E. coli M-PAR-121 genome. Alternatively, the single and double integration of the native DXS and IDI genes from E. coli were tested. Eight E. coli strains with engineered DMAPP pathway were constructed and the eleven PTs were individually expressed in combination with the naringenin biosynthetic pathway. E. coli M-PAR-121 strain with the integration of DXS from E. coli (EcDXS) (E. coli M-PAR-121:EcDXS) expressing the soluble aromatic PT from Streptomyces roseochromogenes (CloQ) (pCDFDuet_CloQ) and pRSFDuet_FjTAL_CmCHS_At4CL_MsCHI was selected as the best PN producer. This strain was able to produce 135.33 mg/L of 3-PN and 8.72 mg/L of 6-PN at a 2-L bioreactor scale 2. Moreover, native SAM synthase (metK), responsible for methionine convertion into SAM, was also integrated into the genome of the eight E. coli strains with engineered DMAPP pathway to improve SAM availability. The eleven PTs in combination with HlOMT1 and the naringenin chalcone biosynthetic pathway were expressed in the E. coli strains with engineered SAM/DMAPP pathways. E. coli M-PAR-121:BlIDI:metK expressing pRSFDuet_FjTAL_CmCHS_At4CL in combination with CdpC3PT from Neosartorya fischeri and HlOMT1 (pCDFDuet_CdpC3PT_HlOMT1) was selected as the best xanthohumol producing strain. Xanthohumol production reached 5.26 mg/L at a 2-L bioreactor scale. These productions represent the highest levels reported to date in any microbial host and the first reports of de novo production of these compounds in E. coli.