文献类型：期刊文献

英文题名：Light-inducible genetic engineering and control of non-homologous end-joining in industrial eukaryotic microorganisms: LML 3.0 and OFN 1.0

作者：Zhang, Lei[1];Zhao, Xihua[2];Zhang, Guoxiu[1];Zhang, Jiajia[1];Wang, Xuedong[1];Zhang, Suping[3];Wang, Wei[1];Wei, Dongzhi[1]

机构：[1]E China Univ Sci & Technol, New World Inst Biotechnol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Jiangxi Normal Univ, Coll Life Sci, Nanchang 330022, Peoples R China;[3]E China Univ Sci & Technol, Res Ctr Biomass Energy, Shanghai 200237, Peoples R China

年份：2016

卷号：6

外文期刊名：SCIENTIFIC REPORTS

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000369656500002)】；

基金：We wish to thank Astrid R. Mach-Aigner (Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, TU Wien, Austria) for providing the cre-containing plasmid pKBxyn1-cre. This research was supported by the National Natural Science Foundation of China (No. C010302-31500066), the National Basic Research Program of China (973, Program No. 2012CB721103), and the National High Technology Research and Development Program of China (863, Program No. 2012AA101806 and 2012AA022206).

语种：英文

摘要：Filamentous fungi play important roles in the production of plant cell-wall degrading enzymes. In recent years, homologous recombinant technologies have contributed significantly to improved enzymes production and system design of genetically manipulated strains. When introducing multiple gene deletions, we need a robust and convenient way to control selectable marker genes, especially when only a limited number of markers are available in filamentous fungi. Integration after transformation is predominantly nonhomologous in most fungi other than yeast. Fungal strains deficient in the nonhomologous end-joining (NHEJ) pathway have limitations associated with gene function analyses despite they are excellent recipient strains for gene targets. We describe strategies and methods to address these challenges above and leverage the power of resilient NHEJ deficiency strains. We have established a foolproof light-inducible platform for one-step unmarked genetic modification in industrial eukaryotic microorganisms designated as 'LML 3.0', and an on-off control protocol of NHEJ pathway called 'OFN 1.0', using a synthetic light-switchable transactivation to control Cre recombinase-based excision and inversion. The methods provide a one-step strategy to sequentially modify genes without introducing selectable markers and NHEJ-deficiency. The strategies can be used to manipulate many biological processes in a wide range of eukaryotic cells.