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

  • Ting Zhang
  • Shuai Zhao
  • Lu-Sheng Liao
  • Cheng-Xi Li
  • Gui-Yan Liao
  • Jia-Xun FengEmail author
Original Paper


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.


Talaromyces 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


Relative unit


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).

Supplementary material

11274_2017_2331_MOESM1_ESM.pdf (820 kb)
Supplementary material 1 (PDF 819 KB)


  1. Buommino E, De Filippis A, Nicoletti R, Menegozzo M, Menegozzo S, Ciavatta ML, Rizzo A, Brancato V, Tufano MA, Donnarumma G (2012) Cell-growth and migration inhibition of human mesothelioma cells induced by 3-O-Methylfunicone from Penicillium pinophilum and cisplatin. Invest New Drug 30:1343–1351CrossRefGoogle Scholar
  2. Carvalho NDSP, Arentshorst M, Kwon MJ, Meyer V, Ram AFJ (2010) Expanding the ku70 toolbox for filamentous fungi: establishment of complementation vectors and recipient strains for advanced gene analyses. Appl Microbiol Biotechnol 87:1463–1473CrossRefGoogle Scholar
  3. Feng J, Li W, Hwang SF, Gossen BD, Strelkov SE (2012) Enhanced gene replacement frequency in KU70 disruption strain of Stagonospora nodorum. Microbiol Res 167:173–178CrossRefGoogle Scholar
  4. Gandia M, Xu S, Font C, Marcos JF (2016) Disruption of ku70 involved in non-homologous end-joining facilitates homologous recombination but increases temperature sensitivity in the phytopathogenic fungus Penicillium digitatum. Fungal Biol 120:317–323CrossRefGoogle Scholar
  5. Goins CL, Gerik KJ, Lodge JK (2006) Improvements to gene deletion in the fungal pathogen Cryptococcus neoformans: absence of Ku proteins increases homologous recombination, and co-transformation of independent DNA molecules allows rapid complementation of deletion phenotypes. Fungal Genet Biol 43:531–544CrossRefGoogle Scholar
  6. Guangtao Z, Hartl L, Schuster A, Polak S, Schmoll M, Wang TH, Seidl V, Seiboth B (2009) Gene targeting in a nonhomologous end joining deficient Hypocrea jecorina. J Biotechnol 139:146–151CrossRefGoogle Scholar
  7. Haarmann T, Lorenz N, Tudzynski P (2008) Use of a nonhomologous end joining deficient strain (Deltaku70) of the ergot fungus Claviceps purpurea for identification of a nonribosomal peptide synthetase gene involved in ergotamine biosynthesis. Fungal Genet Biol 45:35–44CrossRefGoogle Scholar
  8. Haki GD, Rakshit SK (2003) Developments in industrially important thermostable enzymes: a review. Bioresour Technol 89:17–34CrossRefGoogle Scholar
  9. Hasunuma T, Okazaki F, Okai N, Hara KY, Ishii J, Kondo A (2013) A review of enzymes and microbes for lignocellulosic biorefinery and the possibility of their application to consolidated bioprocessing technology. Bioresour Technol 135:513–522CrossRefGoogle Scholar
  10. He Y, Liu Q, Shao Y, Chen F (2013) Ku70 and ku80 null mutants improve the gene targeting frequency in Monascus ruber M7. Appl Microbiol Biotechnol 97:4965–4976CrossRefGoogle Scholar
  11. Hoff B, Kamerewerd J, Sigl C, Zadra I, Kück U (2010) Homologous recombination in the antibiotic producer Penicillium chrysogenum: strain DeltaPcku70 shows up-regulation of genes from the HOG pathway. Appl Microbiol Biotechnol 85:1081–1094CrossRefGoogle Scholar
  12. Hu YB, Xue HZ, Liu GD, Song X, Qu YB (2015) Efficient production and evaluation of lignocellulolytic enzymes using a constitutive protein expression system in Penicillium oxalicum. J Ind Microbiol Biotechnol 42:877–887CrossRefGoogle Scholar
  13. Ishibashi K, Suzuki K, Ando Y, Takakura C, Inoue H (2006) Nonhomologous chromosomal integration of foreign DNA is completely dependent on MUS-53 (human Lig4 homolog) in Neurospora. Proc Natl Acad Sci USA 103:14871–14876CrossRefGoogle Scholar
  14. Krappmann S, Sasse C, Braus GH (2006) Gene targeting in Aspergillus fumigatus by homologous recombination is facilitated in a nonhomologous end-joining-deficient genetic background. Eukaryot Cell 5:212–215CrossRefGoogle Scholar
  15. Kück U, Hoff B (2010) New tools for the genetic manipulation of filamentous fungi. Appl Microbiol Biotechnol 86:51–62CrossRefGoogle Scholar
  16. Kumar S (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874CrossRefGoogle Scholar
  17. Li ZH, Du CM, Zhong YH, Wang TH (2010) Development of a highly efficient gene targeting system allowing rapid genetic manipulations in Penicillium decumbens. Appl Microbiol Biotechnol 87:1065–1076CrossRefGoogle Scholar
  18. Li ZH, Yao GS, Wu RM, Gao LW, Kan QB, Liu M, Yang P, Liu GD, Qin YQ, Song X, Zhong YH, Fang X, Qu YB (2015) Synergistic and dose-controlled regulation of cellulase gene expression in Penicillium oxalicum. PLoS Genet 11:e1005509CrossRefGoogle Scholar
  19. Li CX, Zhao S, Zhang T, Xian L, Liao LS, Liu JL, Feng JX (2017) Genome sequencing and analysis of Talaromyces pinophilus provide insights into biotechnological applications. Sci Rep 7:490CrossRefGoogle Scholar
  20. Marín-Navarro J, Polaina J (2011) Glucoamylases: structural and biotechnological aspects. Appl Microbiol Biotechnol 89:1267–1273CrossRefGoogle Scholar
  21. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428CrossRefGoogle Scholar
  22. Møller MS, Svensson B (2016) Structural biology of starch-degrading enzymes and their regulation. Curr Opin Struct Biol 40:33–42CrossRefGoogle Scholar
  23. Nakazawa T, Ando Y, Kitaaki K, Nakahori K, Kamada T (2011) Efficient gene targeting in ΔCc.ku70 or △Cc.lig4 mutants of the agaricomycete Coprinopsis cinerea. Fungal Genet Biol48:939–946CrossRefGoogle Scholar
  24. Nayak T, Szewczyk E, Oakley E, Osmani A, Ukil L, Murray SL, Hyner MJ, Osmani SA, Oakley BR (2006) A versatile and efficient gene-targeting system for Aspergillus nidulans. Genetics 172:1557–1566CrossRefGoogle Scholar
  25. Ninomiya Y, Suzuki K, Ishii C, Inoue H (2004) Highly efficient gene replacements in Neurospora strains deficient for nonhomologous end-joining. Proc Natl Acad Sci USA 101:12248–12253CrossRefGoogle Scholar
  26. Oguro Y, Yamazaki H, Ara S, Shifa Y, Ogasawara W, Takagi M, Takaku H (2017) Efficient gene targeting in non-homologous end-joining-deficient Lipomyces starkeyi strains. Curr Genet. doi:  10.1007/s00294-017-0679-6 Google Scholar
  27. Petersen KL, Lehmbeck J, Christensen T (1999) A new transcriptional activator for amylase genes in Aspergillus. Mol Gen Genet 262:668–676CrossRefGoogle Scholar
  28. Poggeler S, Kuck U (2006) Highly efficient generation of signal transduction knockout mutants using a fungal strain deficient in the mammalian ku70 ortholog. Gene 378:1–10CrossRefGoogle Scholar
  29. Samson RA, Yilmaz N, Houbraken J, Spierenburg H, Seifert KA, Peterson SW, Varga J, Frisvad JC (2011) Phylogeny and nomenclature of the genus Talaromyces and taxa accommodated in Penicillium subgenus Biverticillium. Stud Mycol 70:159–183CrossRefGoogle Scholar
  30. Suzuki K, Tanaka M, Konno Y, Ichikawa T, Ichinose S, Hasegawa-Shiro S, Shintani T, Gomi K (2015) Distinct mechanism of activation of two transcription factors, AmyR and MalR, involved in amylolytic enzyme production in Aspergillus oryzae. Appl Microbiol Biotechnol 99:1805–1815CrossRefGoogle Scholar
  31. Uday USP, Choudhury P, Bandyopadhyay TK, Bhunia B (2016) Classification, mode of action and production strategy of xylanase and its application for biofuel production from water hyacinth. Int J Biol Macromol 82:1041–1054CrossRefGoogle Scholar
  32. VanKuyk PA, Benen JAE, Wösten HAB, Visser J, de Vries RP (2012) A broader role for AmyR in Aspergillus niger: regulation of the utilisation of D-glucose or D-galactose containing oligo- and polysaccharides. Appl Microbiol Biotechnol 93:285–293CrossRefGoogle Scholar
  33. Villalba F, Collemare J, Landraud P, Lambou K, Brozek V, Cirer B, Morin D, Bruel C, Beffa R, Lebrun MH (2008) Improved gene targeting in Magnaporthe grisea by inactivation of MgKU80 required for non-homologous end joining. Fungal Genet Biol 45:68–75CrossRefGoogle Scholar
  34. Visser EM, Falkoski DL, de Almeida MN, Maitan-Alfenas GP, Guimarães VM (2013) Production and application of an enzyme blend from Chrysoporthe cubensis and Penicillium pinophilum with potential for hydrolysis of sugarcane bagasse. Bioresour Technol 144:587–594CrossRefGoogle Scholar
  35. Xian L, Wang F, Luo X, Feng YL, Feng JX (2015) Purification and characterization of a highly efficient calcium-independent alpha-amylase from Talaromyces pinophilus 1–95. PLoS ONE 10:e0121531CrossRefGoogle Scholar
  36. Xu Q, Zhu CY, Wang MS, Sun XP, Li HY (2014) Improvement of a gene targeting system for genetic manipulation in Penicillium digitatum. J Zhejiang Univ Sci B 15:116–124CrossRefGoogle Scholar
  37. Zhai MM, Niu HT, Li J, Xiao H, Shi YP, Di DL, Crews P, Wu XY (2015) Talaromycolides A–C, novel phenyl-substituted phthalides isolated from the green Chinese onion-derived fungus Talaromyces pinophilus AF-02. J Agric Food Chem 63:9558–9564CrossRefGoogle Scholar
  38. Zhang JX, Mao ZH, Xue W, Li Y, Tang GM, Wang AQ, Zhang YJ, Wang HM (2011) Ku80 gene is related to non-homologous end-joining and genome stability in Aspergillus niger. Curr Microbiol 62:1342–1346CrossRefGoogle Scholar
  39. Zhang H, Wang S, Zhang XX, Ji W, Song FP, Zhao Y, Li J (2016) The amyR-deletion strain of Aspergillus niger CICC2462 is a suitable host strain to express secreted protein with a low background. Microb Cell Fact 15:68CrossRefGoogle Scholar
  40. Zhao S, Yan YS, He QP, Yang L, Yin X, Li CX, Mao LC, Liao LS, Huang JQ, Xie SB, Nong QD, Zhang Z, Jing L, Xiong YR, Duan CJ, Liu JL, Feng JX (2016) Comparative genomic, transcriptomic and secretomic profiling of Penicillium oxalicum HP7-1 and its cellulase and xylanase hyper-producing mutant EU2106, and identification of two novel regulatory genes of cellulase and xylanase gene expression. Biotechnol Biofuel 9:203CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2017

Authors and Affiliations

  1. 1.State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and TechnologyGuangxi UniversityNanningPeople’s Republic of China

Personalised recommendations