AUTHOR=Xu Na , Li Li , Chen Fusheng 

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

JOURNAL=Frontiers in Microbiology

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2022.952323

DOI=10.3389/fmicb.2022.952323

ISSN=1664-302X

ABSTRACT=<p><italic>Monascus</italic> spp. are traditional medicinal and edible filamentous fungi in China, and can produce various secondary metabolites, such as <italic>Monascus</italic> 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 <italic>Monascus</italic> spp.. However, the resistance selection genes that can have been used for genetic modification in <italic>Monascus</italic> spp. are limited, and the gene replacement frequency (GRF) is usually &lt;5%. Therefore, we are committed to construct a highly efficient gene editing system without resistance selection marker gene. In this study, using <italic>M. ruber</italic> M7 as the starting strain, we successfully constructed a so-called markerlessly and highly genetic modification system including the mutants Δ<italic>mrpyrG</italic>Δ<italic>mrlig4</italic> and Δ<italic>mrpyrG</italic>Δ<italic>mrlig4</italic>::<italic>mrpyrG</italic>, in which we used the endogenous gene <italic>mrpyrG</italic> from <italic>M. ruber</italic> M7 instead of the resistance marker gene as the screening marker, and simultaneously deleted <italic>mrlig4</italic> related to non-homologous end joining in <italic>M. ruber</italic> 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 <italic>M. ruber</italic> M7. However, the U addition also has a certain effect on the orange and red pigments yield of <italic>M. ruber</italic> M7, which needs our further study. Finally, we applied the system to delete multiple genes from <italic>M. ruber</italic> M7 separately or continuously without any resistance marker gene, and found that the average GRF of Δ<italic>mrpyrG</italic>Δ<italic>mrlig4</italic> was about 18 times of that of <italic>M. ruber</italic> M7. The markerlessly and highly genetic modification system constructed in current study not only will be used for multi-gene simultaneous modification in <italic>Monascus</italic> spp., and also lays a foundation for investigating the effects of multi-genes modification on <italic>Monascus</italic> spp..</p>