Deletion of TpKu70 facilitates gene targeting in Talaromyces pinophilus and identification of TpAmyR involvement in amylase production
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Talaromyces pinophilus is a promising filamentous fungus for industrial production of biomass-degrading enzymes used in biorefining, and its genome was recently sequenced and reported. However, functional analysis of genes in T. pinophilus is rather limited owing to lack of genetic tools. In this study, a putative TpKu70 encoding the Ku70 homolog involved in the classic non-homologous end-joining pathway was deleted in T. pinophilus 1-95. ΔTpKu70 displayed no apparent defect in vegetative growth and enzyme production, and presented similar sensitivity to benomyl, bleomycin, and UV, when compared with the wild-type T. pinophilus strain 1-95. Seven genes that encode putative transcription factors, including TpAmyR, were successfully knocked out in ΔTpKu70 at 61.5–100% of homologous recombination frequency, which is significantly higher than that noted in the wild-type. Interestingly, ΔTpAmyR produced approximately 20% of amylase secreted by the parent strain ΔTpKu70 in medium containing soluble starch from corn as the sole carbon source. Real-time quantitative reverse transcription PCR showed that TpAmyR positively regulated the expression of genes encoding α-amylase and glucoamylase. Thus, this study provides a useful tool for genetic analysis of T. pinophilus, and identification of a key role for the transcription factor TpAmyR in amylase production in T. pinophilus.
KeywordsTalaromyces pinophilus TpKu70 Gene targeting system Transcriptional regulation TpAmyR
Benomyl resistance gene
Hygromycin resistant gene
Liquid complete medium
Non-homologous end-joining pathway
Potato dextrose agar
Quantitative reverse transcription-polymerase chain reaction
Standard liquid medium
We thank Baoshan Chen from State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University for providing us with the plasmid pLPMBn containing benA gene. This work was financially supported by the Guangxi BaGui Scholars Program Foundation (Grant No. 2011A001), the ‘One Hundred Person’ Project of Guangxi and 2017 Excellent Teaching Program of Guangxi High Education-Program of Advantage and Characteristic Specialty (High Quality of Undergraduate Program).
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