title: Development of genetic tools for Myceliophthora thermophila.
abstract: BACKGROUND
  The thermophilic filamentous fungus Myceliophthora thermophila has many suitable characteristics for industrial biotechnology and could be a promising new chassis system for synthetic biology, particularly the ATCC 42464 strain, whose genome was sequenced in 2011. However, metabolic engineering of this strain using genetic approaches has not been reported owing to a lack of genetic tools for this organism.
  
  
  RESULTS
  In the present study, we developed a high efficiency Agrobacterium tumefaciens mediated transformation system for M. thermophila, including an approach for targeted gene deletion using green fluorescence protein (GFP) as a marker for selection. Up to 145 transformants per 10(5) conidia were obtained in one transformation plate. Moreover, a ku70 deletion mutant was constructed in the ATCC 42464 background using the tools developed in present study and subsequently characterized. The ku70 deletion construct was designed using resistance to phosphinothricin as the selection marker. Additionally, a GFP-encoding cassette was incorporated that allowed for the selection of site-specific (no fluorescence) or ectopic (fluorescence) integration of the ku70 construct. Transformants with ectopically integrated ku70 deletion constructs were therefore identified using the fluorescent signal of GFP. PCR and Southern blotting analyses of non-fluorescent putative ku70 deletion transformants revealed all 11 tested transformants to be correct deletions. The deletion frequency in a pool of 116 transformants analyzed was 58 %. Moreover, the homologous rate improved about 3 folds under ku70 mutant using the pyrG as a test gene to disrupt in M. thermophila.
  
  
  CONCLUSIONS
  We successfully developed an efficient transformation and target gene disruption approach for M. thermophila ATCC 42464 mediated by A. tumefaciens. The tools and the ku70 deletion strain developed here should advance the development of M. thermophila as an industrial host through metabolic engineering and accelerate the elucidation of the mechanism of rapid cellulose degradation in this thermophilic fungus.
authors.affiliation: College of Life Sciences, Hubei University, Wuhan, 430062, China. xu_jing@tib.cas.cn.|Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China. xu_jing@tib.cas.cn.
authors.forename: Jing
authors.initials: J
authors.lastname: Xu
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China. li_jg@tib.cas.cn.
authors.forename: Jingen
authors.initials: J
authors.lastname: Li
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China. lin_lc@tib.cas.cn.
authors.forename: Liangcai
authors.initials: L
authors.lastname: Lin
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China. liu_q1@tib.cas.cn.
authors.forename: Qian
authors.initials: Q
authors.lastname: Liu
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China. sun_wl@tib.cas.cn.
authors.forename: Wenliang
authors.initials: W
authors.lastname: Sun
authors.affiliation: College of Life Sciences, Hubei University, Wuhan, 430062, China. huangbangquan@163.com.
authors.forename: Bangquan
authors.initials: B
authors.lastname: Huang
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China. tian_cg@tib.cas.cn.
authors.forename: Chaoguang
authors.initials: C
authors.lastname: Tian
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics*; D034961:Antigens, Nuclear / Q000235:genetics*; D002482:Cellulose / Q000378:metabolism; D004271:DNA, Fungal / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D017353:Gene Deletion; D049452:Green Fluorescent Proteins / Q000235:genetics; D000072200:Ku Autoantigen; D020037:Sordariales / Q000235:genetics* / Q000254:growth & development; D014170:Transformation, Genetic
chemical_list: D034961:Antigens, Nuclear; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D049452:Green Fluorescent Proteins; D002482:Cellulose; D000072200:Ku Autoantigen