Construction of gene modification system with highly efficient and markerless for Monascus ruber M7

Abstract

Monascus spp. are traditional medicinal and edible filamentous fungi in China, and can produce various secondary metabolites, such as Monascus pigments (MPs) and citrinin (CIT). Genetic modification methods, such as gene knock-out, complementation, and overexpression, have been used extensively to investigate the function of related genes in Monascus spp.. However, the resistance selection genes that can have been used for genetic modification in Monascus spp. are limited, and the gene replacement frequency (GRF) is usually <5%. Therefore, we are committed to construct a highly efficient gene editing system without resistance selection marker gene. In this study, using M. ruber M7 as the starting strain, we successfully constructed a so-called markerlessly and highly genetic modification system including the mutants ΔmrpyrGΔmrlig4 and ΔmrpyrGΔmrlig4::mrpyrG, in which we used the endogenous gene mrpyrG from M. ruber M7 instead of the resistance marker gene as the screening marker, and simultaneously deleted mrlig4 related to non-homologous end joining in M. ruber M7. Then, the morphology, the growth rate, the production of MPs and CIT of the mutants were analyzed. And the results show that the mutant strains have normal mycelia, cleistothecia and conidia on PDA+Uridine(U) plate, the biomass of each mutant is also no different from M. ruber M7. However, the U addition also has a certain effect on the orange and red pigments yield of M. ruber M7, which needs our further study. Finally, we applied the system to delete multiple genes from M. ruber M7 separately or continuously without any resistance marker gene, and found that the average GRF of ΔmrpyrGΔmrlig4 was about 18 times of that of M. ruber M7. The markerlessly and highly genetic modification system constructed in current study not only will be used for multi-gene simultaneous modification in Monascus spp., and also lays a foundation for investigating the effects of multi-genes modification on Monascus spp..

Keywords: Monascus ruber M7; genetic modification system; mrlig4; mrpyrG; resistance selection marker.

Figure 1

Deletion of mrpyrG and mrlig4 in M. ruber M7. (A) Strategy to construct mrpyrG markerless deletion strain. (B) Strategy to construct mrlig4 markerless deletion strain.

Figure 2

PCR analysis of ΔmrpyrG, ΔpyrG+lig4::pyrG, and ΔpyrG+lig4. (A) Confirmation of mrpyrG deletion. Lane 1: ΔmrpyrG; Lane 2: M7; M: Trans 2K plus II marker; (B) Confirmation of mrlig4 markerless deletion in ΔmrpyrG strain. Lane 1: ΔpyrG+lig4::pyrG; Lane 2: ΔmrpyrG; (C) Confirmation of mrpyrG homologous recombination events in ΔpyrG+lig4::pyrG strain. Lane 1: ΔpyrG+lig4; Lane 2: M7; M: Trans 2K plus II marker.

Figure 3

Morphologies and biomasses of M. ruber M7 and derivative markerless deletion strains. (I) Colonial morphologies on PDA, PDA+U and PDA+U+ 5-FOA plates. (II) Cleistothecia and conidia formation on PDA and PDA+U plates. (III) Biomass (dry cell weight).

Figure 4

Production of intracellular MPs and extracellular CIT by M. ruber M7 and derivative markerless deletion strains on the PDA supplied with/without uridine. (I) The yield of yellow pigments; (II) The yield of orange pigments; (III) The yield of red pigments; and (IV) The yield of CIT.