Abstract
High-level expression of β-mannanase has been reported in Pichia pastoris under control of the GAP promoter. Two factors that strongly influence protein production and fermentation process development in Pichia pastoris protein expression system are gene dosage and cultivation temperature. The aim of this research was to improve the expression level of β-mannanase in Pichia pastoris by proper increasing the gene dosage and decreasing the culture temperature. To this end, a panel of strains harboring different copy numbers of β-mannanase gene were obtained by multiple zeocin concentration gradients screening, the influence of gene copy number on the expression of β-mannanase in Pichia pastoris X33 was investigated. With the constitutive GAP promoter, the four copies strain exhibited a 4.04-fold higher β-mannanase yield and a 1.83-fold higher total secretion proteins than the one copy strain, but an increase of the copy number above four resulted in a decrease of expression. Furthermore, the effects of culture temperature were studied in flask. The decreased culture temperature of four copies strain resulted in a 1.8-fold (26 °C) and 3.5-fold (22 °C) higher β-mannanase activity compared to that at 30 °C. A fed-batch strategy was successfully used for high cell-density fermentation and β-mannanase activity reached 2124 U/mL after cultivation for 72 h in a 5 L fermenter.
摘要
使用GAP 启动子可以实现β-甘露聚糖酶基因在毕赤酵母中的组成型表达。为了提高β-甘露聚 糖酶在毕赤酵母中的产量,本研究通过梯度抗生素浓度筛选法构建了携带多拷贝β-甘露聚糖酶的毕赤 酵母菌株,并利用实时荧光定量PCR 技术确定了各菌株的拷贝数。考察了不同基因拷贝数对β-甘露 聚糖酶在毕赤酵母中表达的影响,同时研究了不同拷贝数下,培养温度对β-甘露聚糖酶分泌表达的影 响。研究获得了β-甘露聚糖酶基因拷贝数为1、3、4、5、7 的毕赤酵母重组菌株,结果显示β-甘露聚 糖酶的mRNA 水平随着拷贝数的增加而升高,β-甘露聚糖酶产量随着拷贝数的增加而提高,其中拷贝 数为4 时产酶水平最高,是单拷贝菌株的4.04 倍,当拷贝数大于4 时,酶的产量不再随着拷贝数的增 加而增加。培养温度实验结果显示,较低的温度对重组菌株生产β-甘露聚糖酶有促进作用,当温度降 低至22 °C 时,携带4 拷贝的重组菌株的酶活是30 °C 时的3.5 倍。为了探索上述实验结果在工业发 酵上的应用潜力,我们进行了5 L 反应器水平的发酵实验,4 拷贝菌株在发酵72 h 后,酶活达到2124 U/mL。
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
DHAWAN S, KAUR J. Microbial mannanases: An overview of production and applications [J]. Critical Reviews in Biotechnology, 2007, 27(4): 197–216.
TUNTRAKOOL P, KEAWSOMPONG S. Kinetic properties analysis of beta-mannanase from Klebsiella oxytoca KUB-CW2-3 expressed in Escherichia coli [J]. Protein Expression and Purification, 2018, 146: 23–26.
EOM G T, OH J Y, PARK J H, JEGAL J, SONG J K. Secretory production of enzymatically active endo-ß-1, 4-mannanase from Bacillus subtilis by ABC exporter in Escherichia coli [J]. Process Biochemistry, 2016, 51(8): 999–1005.
HATADA Y, TAKEDA N, HIRASAWA K, OHTA Y, USAMI R, YOSHIDA Y, GRANT W D, ITO S, HORIKOSHI K. Sequence of the gene for a high-alkaline mannanase from an alkaliphilic Bacillus sp. strain JAMB-750, its expression in Bacillus subtilis and characterization of the recombinant enzyme [J]. Extremophiles, 2005, 9(6): 497–500.
SONG Ya-feng, FU Gang, DONG Hui-na, LI Jian-jun, DU Yu-guang, ZHANG Da-wei. High-efficiency secretion of ß-mannanase in Bacillus subtilis through protein synthesis and secretion optimization [J]. Journal of Agricultural and Food Chemistry, 2017, 65(12): 2540–2548.
PRIMA A, HARA K Y, DJOHAN A C, KASHIWAGI N, KAHAR P, ISHII J, NAKAYAMA H, OKAZAKI F, PRASETYA B, KONDO A, YOPI, OGINO C. Glutathione production from mannan-based bioresource by mannanase/mannosidase expressing Saccharomyces cerevisiae [J]. Bioresource Technology, 2017, 245(12): 1400–1406.
LI Yan-xiao, YI Ping, LIU Jun, YAN Qiao-juan, JIANG Zheng-qiang. High-level expreßsion of an engineered ß-mannanase (mRmMan5A) in Pichia pastoris for manno-oligosaccharide production using steam explosion pretreated palm kernel cake [J]. Bioresource Technology, 2018, 256: 30–37.
LUO Zhang-cai, MIAO Jing, LI Guo-ying, DU Yao, YU Xiao-bin. A recombinant highly thermostable ß-mannanase (ReTMan26) from thermophilic bacillus subtilis (TBS2) expressed in Pichia pastoris and its pH and temperature stability [J]. Applied Biochemistry and Biotechnology, 2017, 182(4): 1259–1275.
YANG Hong, SHI Peng-jun, LU Hai-qiang, WANG Hui-min, LUO Hui-ying, HUANG Huo-qing, YANG Pei-long, YAO Bin. A thermophilic ß-mannanase from Neosartorya fischeri P1 with broad pH stability and significant hydrolysis ability of various mannan polymers [J]. Food Chemistry, 2015, 173: 283–289.
YU Shi, LI Zhe-zhe, WANG Ya-ping, CHEN Wan-ping, FU Lin, TANG Wei, CHEN Chen, LIU Yunyun, ZHANG Xue, MA Li-xin. High-level expreßsion and characterization of a thermophilic ß-mannanase from Aspergillus niger in Pichia pastoris [J]. Biotechnology Letters, 2015, 37(9): 1853–1859.
HSU Y, KOIZUMI H, OTAGIRI M, MORIYA S, ARIOKA M. Trp residue at subsite-5 plays a critical role in the substrate binding of two protistan GH26 ß-mannanases from a termite hindgut [J]. Applied microbiology and Biotechnology, 2018: 1–11.
JIAO Liang-cheng, ZHOU Qing-hua, SU Zhi-xin, LI Xu, YAN Yun-jun. High-level extracellular production of Rhizopus oryzae lipase in Pichia pastoris via a strategy combining optimization of gene-copy number with co-expression of ERAD-related proteins [J]. Protein Expression and Purification, 2018, 147: 1–12.
TENG Da, XI Di, ZHANG Jun, WANG Xiu-min, MAO Ruo-yu, ZHANG Yong, WANG Jian-hua. Multiple copies of the target gene enhances plectasin secretion in Pichia pastoris X-33 [J]. Process Biochemistry, 2015, 50(4): 553–560.
CÁMARA E, LANDES N, ALBIOL J, GASSER B, MATTANOVICH D, FERRER P. Increased dosage of AOX1 promoter-regulated expression cassettes leads to transcription attenuation of the methanol metabolism in Pichia pastoris [J]. Scientific Reports, 2017, 7: 44302.
YANG Hu, ZHAI Chao, YU Xian-hong, LI Zhe-zhe, TANG Wei, LIU Yun-yun, MA Xiao-jian, ZHONG Xing, LI Guo-long, WU Di, MA Li-xin. High-level expression of Proteinase K from Tritirachium album Limber in Pichia pastoris using multi-copy expression strains [J]. Protein Expression and Purification, 2016, 122: 38–44.
NORDÉN K, AGEMARK M, DANIELSON J Å H, ALEXANDERSSON E, KJELLBOM P, JOHANSON U. Increasing gene dosage greatly enhances recombinant expression of aquaporins in Pichia pastoris [J]. BMC Biotechnology, 2011, 11(1): 47.
COS O, SERRANO A, MONTESINOS J L, MONTESIONS J L, FERRER P, CREGG J M, VALERO F. Combined effect of the methanol utilization (Mut) phenotype and gene dosage on recombinant protein production in Pichia pastoris fed-batch cultures [J]. Journal of Biotechnology, 2005, 116(4): 321–335.
HOHENBLUM H, GASSER B, MAURER M, BORTH N, MATTANOVICH D. Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris [J]. Biotechnology and Bioengineering, 2004, 85(4): 367–375.
ZHU Tai-cheng, GUO Mei-jin, SUN Chen, QIAN Jiangchao, ZHUANG Ying-ping, CHU Ju, ZHANG Si-liang. A systematical investigation on the genetic stability of multi-copy Pichia pastoris strains [J]. Biotechnology Letters, 2009, 31(5): 679–684.
INAN M, ARYASOMAYAJULA D, SINHA J, MEAGHER M M. Enhancement of protein secretion in Pichia pastoris by overexpression of protein disulfide isomerase [J]. Biotechnology and Bioengineering, 2006, 93(4): 771–778.
INAN M, FANDERS S A, ZHANG Wen-hui, HOTEZ P J, ZHAN Bin, MEAGHER M M. Saturation of the secretory pathway by overexpression of a hookworm (Necator americanus) Protein (Na-ASP1) [C]// Pichia Protocols. Totowa: Humana Press, 2007: 65–75.
ZHAN Rong-rong, MU Wan-meng, JIANG Bo, ZHOU Liu-ming, ZHANG Tao. Efficient secretion of inulin fructotransferase in Pichia pastoris using the formaldehyde dehydrogenase 1 promoter [J]. Journal of Industrial Microbiology & Biotechnology, 2014, 41(12): 1783–1791.
SCORER C A, CLARE J J, MCCOMBIE W R, ROMANOS M A. SREEKRISHNA K. Rapid selection using G418 of high copy number transformants of Pichia pastoris for high–level foreign gene expression [J]. Nature Biotechnology, 1994, 12(2): 181–184.
VASSILEVA A, CHUGH D A, SWAMINATHAN S, KHANNA N. Expression of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris using the GAP promoter [J]. Journal of Biotechnology, 2001, 88(1): 21–35.
CREGG J M. Pichia protocols [M]. Totowa: Humana Press, 2007.
SINHA J, PLANTZ B A, INAN M, MEAGHER M M. Causes of proteolytic degradation of secreted recombinant proteins produced in methylotrophic yeast Pichia pastoris: Case study with recombinant ovine interferon-t [J]. Biotechnology and Bioengineering, 2005, 89(1): 102–112.
DAMASCENO L M, HUANG C J, BATT C A. Protein secretion in Pichia pastoris and advances in protein production [J]. Applied Microbiology and Biotechnology, 2012, 93(1): 31–39.
MATTANOVICH D, GASSER B, HOHENBLUM H, SAUER M. Stress in recombinant protein producing yeasts [J]. Journal of Biotechnology, 2004, 113(1): 121–135.
PEI Xiao-lin, WANG Qiu-yan, MENG Li-jun, LI Jing, YANG Zheng-fen, YIN Xiao-pu, YANG Li-rong, CHEN Shao-yun, WU Jian-ping. Chaperones-assisted soluble expression and maturation of recombinant Co-type nitrile hydratase in Escherichia coli to avoid the need for a low induction temperature [J]. Journal of Biotechnology, 2015, 203: 9–16.
OVERTON T W. Recombinant protein production in bacterial hosts [J]. Drug Discovery Today, 2014, 19(5): 590–601.
CHEN Wen-bo, NIE Yao, XU Yan, XIAO Rong. Enhancement of extracellular pullulanase production from recombinant Escherichia coli by combined strategy involving auto-induction and temperature control [J]. Bioprocess and Biosystems Engineering, 2014, 37(4): 601–608.
GAO Min-jie, ZHAN Xiao-bei, GAO Peng, ZHANG Xu, DONG Shi-juan, LI Zhen, SHI Zhong-ping, LIN Chi-chung. Improving performance and operational stability of porcine interferon-a production by Pichia pastoris with combinational induction strategy of low temperature and methanol/sorbitol co-feeding [J]. Applied Biochemistry and Biotechnology, 2015, 176(2): 493–504.
TAO Hui, GUO Dao-yi, ZHANG Yu-chen, DENG Zi-xin, LIU Tian-gang. Metabolic engineering of microbes for branched-chain biodiesel production with low-temperature property [J]. Biotechnology for Biofuels, 2015, 8(1): 92.
JAHIC M, WALLBERG F, BOLLOK M, GARCIA P, ENFORS S O. Temperature limited fed-batch technique for control of proteolysis in Pichia pastoris bioreactor cultures [J]. Microbial Cell Factories, 2003, 2(1): 6.
SCHEIN C H, NOTEBORN M H M. Formation of soluble recombinant proteins in Escherichia coli is favored by lower growth temperature [J]. Nature Biotechnology, 1988, 6(3): 291–294.
ZHAO Wei, ZHENG Jia, ZHOU Hong-bo. A thermotolerant and cold-active mannan endo-1, 4-ß-mannosidase from Aspergillus niger CBS 513.88: Constitutive overexpression and high-density fermentation in Pichia pastoris [J]. Bioresource Technology, 2011, 102(16): 7538–7547.
WATERHAM H R, DIGAN M E, KOUTZ P J, LAIR S V, GREGG J M. Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter [J]. Gene, 1997, 186(1): 37–44.
WANG Meng-fan, YOU Sheng-ping, ZHANG Shuai-shuai, QI Wei, LIU Zhao-hui, WU Wei-na, SU Rong-xin, HE Zhi-min. Purification, characterization, and production of ß-mannanase from Bacillus subtilis TJ-102 and its application in gluco-mannooligosaccharides preparation [J]. European Food Research and Technology, 2013, 237(3): 399–408.
CEREGHINO J L, CREGG J M. Heterologous protein expression in the methylotrophic yeast Pichia pastoris [J]. FEMS Microbiology Reviews, 2000, 24(1): 45–66.
ZHU Tai-cheng, GUO Mei-jin, ZHUANG Ying-ping, CHU Ju, ZHANG Si-liang. Understanding the effect of foreign gene dosage on the physiology of Pichia pastoris by transcriptional analysis of key genes [J]. Applied Microbiology and Biotechnology, 2011, 89(4): 1127–1135.
MALHOTRA J D, KAUFMAN R J. Endoplasmic reticulum stress and oxidative stress: a vicious cycle or a double-edged sword? [J]. Antioxidants & Redox Signaling, 2007, 9(12): 2277–2294.
INAN M, ARYASOMAYAJULA D, SINHA J, MEAGHER M M. Enhancement of protein secretion in Pichia pastoris by overexpression of protein disulfide isomerase [J]. Biotechnology and Bioengineering, 2006, 93(4): 771–778.
DAMASCENO L M, ANDERSON K A, RITTER G, GREGG J M, OLD L J, BATT C A. Cooverexpression of chaperones for enhanced secretion of a single-chain antibody fragment in Pichia pastoris [J]. Applied Microbiology and Biotechnology, 2007, 74(2): 381–389.
DALY R, HEARN M T W. Expression of heterologous proteins in Pichia pastoris: A useful experimental tool in protein engineering and production [J]. Journal of Molecular Recognition, 2005, 18(2): 119–138.
GASSER B, SALOHEIMO M, RINAS U, DRAGOSITS M, ESCARLATA R C, BAUMANN K, GIULIANI M, PARRILLI E, BRANDUARDI P, LANG C, PORRO D, FERRER P, TUTINO M L, MATTANOVICH D, VILLAVERDE A. Protein folding and conformational stress in microbial cells producing recombinant proteins: A host comparative overview [J]. Microbial Cell Factories, 2008, 7(1): 11.
WANG Ye, ZHENG Jia, LIN Fu-lai, ZHOU Hong-bo. Improved extracellular endo-1, 4-ß-mannosidase activity of recombinant Pichia pastoris by optimizing signal peptide [J]. Journal of Central South University, 2015, 22: 2088–2095
LUO Hui-ying, WANG Ya-ru, WANG Hui, YANG Jun, YANG Yu-hui, HUANG Huo-qing, YANG Pei-long, BAI Ying-guo, SHI Peng-jun, FAN Yun-liu, YAO Bin. A novel highly acidic ß-mannanase from the acidophilic fungus Bispora sp. MEY-1: Gene cloning and overexpression in Pichia pastoris [J]. Applied Microbiology and Biotechnology, 2009, 82(3): 453–461.
YU Shi, LI Zhe-zhe, WANG Ya-ping, CHEN Wan-ping, FU Lin, TANG Wei, CHEN Chen, LIU Yun-yun, ZHANG Xue, MA Li-xin. High-level expreßsion and characterization of a thermophilic ß-mannanase from Aspergillus niger in Pichia pastoris [J]. Biotechnology Letters, 2015, 37(9): 1853–1859.
Author information
Authors and Affiliations
Corresponding author
Additional information
Foundation item: Project(31870115) supported by the National Natural Science Foundation of China; Project(2015JJ5006) supported by the Natural Science of Hunan Province & Changde City Joint Foundation, China; Projects(2015zzts268, ZY2015823) supported by the Fundamental Research Funds for the Central Universities, China
Rights and permissions
About this article
Cite this article
Tang, Sz., Lin, Fl., Zheng, J. et al. Effect of gene dosage and incubation temperature on production of β-mannanase by recombinant Pichia pastoris. J. Cent. South Univ. 26, 184–195 (2019). https://doi.org/10.1007/s11771-019-3992-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11771-019-3992-z