CRISPR-Cas12a system as a tool to construct boosted DMAPP-Escherichia coli strains towards de novo production of prenylflavonoids

• Author(s): Gomes, Daniela Filipa Correia ; Rodrigues, Joana Lúcia Lima Correia ; Scrutton, Nigel S. ; Rodrigues, L. R.
• Date: 2024
• Persistent ID: https://hdl.handle.net/1822/94706
• Origin: RepositóriUM - Universidade do Minho
• Document Type: Conference poster
• Language: English
• Contributor(s): Universidade do Minho

Description

Prenylflavonoids are flavonoid derived compounds with interesting bioactivities, namely estrogenic and anticancer. This study aimed to design and validate a biosynthetic pathway to de novo produce prenylnaringenins (PNs) in Escherichia coli. Firstly, tyrosine ammonia-lyase, 4-coumarate-CoA ligase, chalcone synthase, and chalcone isomerase were expressed to produce the intermediate naringenin in high amounts (689.5 mg/L). Eleven prenyltransferase (PT) were then evaluated to produce PNs. Due to a limitation in the availability of dimethylallyl pyrophosphate (DMAPP), PT extended substrate, CRISPR-Cas12a was used to construct boosted DMAPP-E. coli strains by improving the flux of 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and isopentenyl diphosphate isomerase (IDI) steps that are considered the rate limiting steps of the native methylerythritol phosphate pathway. Single and double integration of DXS and IDI genes with improved activities (DXS from Bacillus subtilis, IDI from Saccharomyces cerevisiae, and IDI from Bacillus licheniformis) 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 boosted DMAPP-E. coli strains were constructed, and deep well block experiments identified twelve strains capable of producing PNs, which were further evaluated at a flask scale (50 mL). E. coli M-PAR-121:EcDXS expressing the naringenin pathway and the soluble aromatic PT from Streptomyces roseochromogene (CloQ) was selected as the best producer strain and 71.7 µM of 3-PN and 1.7 µM of 6-PN were produced. As far as we know, this study marks the first successful attempt to de novo produce PNs in E. coli.