Genetic manipulation of the bifunctional gene, carRA, to enhance lycopene content in Blakeslea trispora

Abstract

Blakeslea trispora is an ideal natural source of lycopene. Here, we genetically manipulated the bifunctional protein gene, carRA, encoding a phytoene synthase and lycopene cyclase, to enhance lycopene production. carR- and carRA-knockout and carA overexpressing strains were obtained after several rounds of selection. Following combinational fermentation for 6 d, the lycopene content in carR-knockout strains increased from 0.47 to 1.40mg/gDW, while the total carotenoid content decreased from 17.05 to 3.52mg/gDW compared to the wild type. No carotenoid was produced in carRA-knockout strains. In carA overexpressing strains, lycopene content, β-carotene, and total carotenoid content increased 2.26-, 1.98-, and 2.02-fold, resepectively, with proportions similar to wild type. Dry weight increased 1.89–1.98-fold in carR- and carRA-knockout strains compared to the wild type, but not in carA overexpressing strains. qRT-PCR analysis revealed that disruption of CarR and CarRA down-regulated carotenoid synthesis genes, especially the isopentenyl pyrophosphate isomerase gene, whereas overexpression of carA up-regulated all examined genes, except the ergosterol synthase and the 4-hydroxybenzoate polyprenyl transferase genes. Tripropylamine was shown to target the R domain, rather than the A domain, of CarRA. Furthermore, the possibility of using a carR-knockout to obtain high-lycopene B. trispora strains for industrial production was explored.