摘要：Deinococcus radiodurans’high resistance to various stressors combined with its ability to utilize sustainable carbon sources makes it an attractive bacterial chassis for synthetic biology and industrial ***,to fully harness the capabilities of this microbe,further strain engineering and tool development are *** for creating seamless genome modifications are an essential part of the microbial genetic toolkit to enable strain ***,we report the development of the SLICER method,which can be used to create seamless gene deletions in *** process involves(a)integration of a seamless deletion cassette replacing a target gene,(b)introduction of the pSLICER plasmid to mediate cassette excision by I-SceI endonuclease cleavage and homologous recombination,and(c)curing of the helper *** demonstrate the utility of SLICER for creating multiple gene deletions in *** by sequentially targeting 5 putative restriction-modification system genes,recycling the same selective and screening markers for each subsequent *** we observed no significant increase in transformation efficiency for most of the knockout strains,we demonstrated SLICER as a promising method to create a fully restriction-minus strain to expand the synthetic biology applications of ***,including its potential as an in vivo DNA assembly platform.

摘要：Fungi are nature’s recyclers,allowing for ecological nutrient cycling and,in turn,the continuation of life on *** fungi inhabit the human microbiome where they can provide health benefits,while others are opportunistic pathogens that can cause ***,members of the fungal kingdom,have been domesticated by humans for the production of beer,bread,and,recently,medicine and ***,the great untapped potential exists within the diverse fungal ***,many yeasts are intractable,preventing their use in biotechnology or in the development of novel treatments for pathogenic ***,as a first step for the domestication of new fungi,an efficient DNA delivery method needs to be ***,we report the creation of superior conjugative plasmids and demonstrate their transfer via conjugation from bacteria to 7 diverse yeast species including the emerging pathogen Candida *** create our superior plasmids,derivatives of the 57 kb conjugative plasmid pTA-Mob 2.0 were built using designed gene deletions and insertions,as well as some unintentional ***,a cluster mutation in the promoter of the conjugative gene traJ had the most significant effect on improving conjugation to *** addition,we created Golden Gate assembly-compatible plasmid derivatives that allow for the generation of custom plasmids to enable the rapid insertion of designer genetic ***,we demonstrated that designer conjugative plasmids harboring engineered restriction endonucleases can be used as a novel antifungal agent,with important applications for the development of next-generation antifungal therapeutics.