Cyanamide-inducible expression of homing nuclease ^(I−)SceI for selectable marker removal and promoter characterisation in Saccharomyces cerevisiae  

作　　者：Liam McDonnell Samuel Evans Zeyu Lu Mitch Suchoronczak Jonah Leighton Eugene Ordeniza Blake Ritchie Nik Valado Niamh Walsh James Antoney Chengqiang Wang Carlos Horacio Luna-Flores Colin Scott Robert Speight Claudia E.Vickers Bingyin Peng 

机构地区：[1]Centre of Agriculture and the Bioeconomy,School of Biology and Environmental Science,Faculty of Science,Queensland University of Technology,Brisbane,QLD,4000,Australia [2]ARC Centre of Excellence in Synthetic Biology,Australia [3]Australian Institute for Bioengineering and Nanotechnology(AIBN),The University of Queensland,Brisbane,QLD,4072,Australia [4]School of Biology and Environmental Science,Faculty of Science,Queensland University of Technology,Brisbane,QLD,4000,Australia [5]College of Life Sciences,Shandong Agricultural University,Taian,Shandong Province,271018,People's Republic of China [6]CSIRO Environment,Black Mountain Science and Innovation Park,Canberra,ACT,2601,Australia [7]Advanced Engineering Biology Future Science Platform,Commonwealth Scientific and Industrial Research Organisation(CSIRO),Black Mountain,ACT,2601,Australia

出　　处：《Synthetic and Systems Biotechnology》2024年第4期820-827,共8页合成和系统生物技术(英文)

基　　金：supported by the Australian Government through the Australian Research Council Centres of Excellence funding scheme(project CE200100029).

摘　　要：In synthetic biology,microbial chassis including yeast Saccharomyces cerevisiae are iteratively engineered with increasing complexity and scale.Wet-lab genetic engineering tools are developed and optimised to facilitate strain construction but are often incompatible with each other due to shared regulatory elements,such as the galactose-inducible(GAL)promoter in S.cerevisiae.Here,we prototyped the cyanamide-induced ^(I−)SceI expression,which triggered double-strand DNA breaks(DSBs)for selectable marker removal.We further combined cyanamide-induced ^(I−)SceI-mediated DSB and maltose-induced MazF-mediated negative selection for plasmid-free in situ promoter substitution,which simplified the molecular cloning procedure for promoter characterisation.We then characterised three tetracycline-inducible promoters showing differential strength,a non-leakyβ-estradiol-inducible promoter,cyanamide-inducible DDI2 promoter,bidirectional MAL32/MAL31 promoters,and five pairs of bidirectional GAL1/GAL10 promoters.Overall,alternative regulatory controls for genome engineering tools can be developed to facilitate genomic engineering for synthetic biology and metabolic engineering applications.

关 键 词：Metabolic engineering Genetic circuit Genome engineering Synthetic biology Yeast engineering Promoter engineering 

分 类 号：Q78[生物学—分子生物学]