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Deletion of TpKu70 facilitates gene targeting in Talaromyces pinophilus and identification of TpAmyR involvement in amylase production

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Abstract

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.

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Abbreviations

benA :

Benomyl resistance gene

DIG:

Digoxigenin

hph :

Hygromycin resistant gene

LCM:

Liquid complete medium

NHEJ:

Non-homologous end-joining pathway

PDA:

Potato dextrose agar

qRT-PCR:

Quantitative reverse transcription-polymerase chain reaction

RU:

Relative unit

SLM:

Standard liquid medium

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Acknowledgements

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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  1. Ting Zhang and Shuai Zhao have contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University, 100 Daxue Road, Nanning, 530004, Guangxi, People’s Republic of China

    Ting Zhang, Shuai Zhao, Lu-Sheng Liao, Cheng-Xi Li, Gui-Yan Liao & Jia-Xun Feng

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  1. Ting Zhang
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  2. Shuai Zhao
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Correspondence to Jia-Xun Feng.

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Zhang, T., Zhao, S., Liao, LS. et al. Deletion of TpKu70 facilitates gene targeting in Talaromyces pinophilus and identification of TpAmyR involvement in amylase production. World J Microbiol Biotechnol 33, 171 (2017). https://doi.org/10.1007/s11274-017-2331-5

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