基于联合策略提高FAD依赖的葡萄糖脱氢酶的酵母表达

doi:10.13560/j.cnki.biotech.bull.1985.2022-1255

摘要/Abstract

摘要：

为了提高了FAD依赖的葡萄糖脱氢酶在毕赤酵母中的表达量，本研究通过G418浓度梯度筛选，构建含有不同分子伴侣基因拷贝数共表达毕赤酵母重组菌株实现高效表达，优化摇瓶发酵条件。经G418浓度梯度筛选，得到1株高产重组菌，在此基础上分别构建了HAC1、Ero1、PDI三种分子伴侣1-4拷贝共表达重组菌株，RT-qPCR检测结果符合预期。试管水平酶活表明3拷贝PDI、3拷贝HAC1和2拷贝Ero1分别比1拷贝提高83%、77%和51%。总体比较而言，共表达3拷贝PDI的重组菌株酶活最高，比对照提高198%，优化摇瓶发酵条件表明培养基初始pH 7.0，摇瓶装液量50 mL，甲醇添加量1%，诱导温度28℃时，效果最佳。通过抗生素浓度筛选、增加分子伴侣的基因拷贝数策略可以提高FAD-GDH在毕赤酵母中的发酵产量。

关键词: FAD依赖的葡萄糖脱氢酶, 毕赤酵母, 抗生素梯度筛选, 分子伴侣多拷贝

Abstract:

In order to increase the expression of FAD-dependent glucose dehydrogenase（FAD-GDH）in Pichia pastoris, the recombinant strains of co-expressing different copy numbers of molecular chaperone genes were constructed to achieve high-efficiency expression through the G418 concentration gradient screening, and the shake flask fermentation conditions were optimized. One high-yielding recombinant strain was obtained by G418 concentration gradient screening. On this basis, the recombinant strains co-expressing 1-4 copies of three molecular chaperones, HAC1, Ero1 and PDI, were constructed respectively, and the results of RT-qPCR were in line with expectations. The enzyme activity at the test tube level showed that 3 copies of PDI, 3 copies of HAC1 and 2 copies of Ero1 increased by 83%, 77% and 51%, respectively, compared with 1 copy. The recombinant strain with 3 copies of PDI had the highest enzyme activity, which was 198% higher than the control. The optimal conditions of the recombinant strain were as follows: initial pH 7.0, liquid loading volume 50 mL, the methanol addition amount 1%, and induction temperature 28℃. The efficient expression of FAD-GDH in P. pastoris is achieved by screening of antibiotic concentration and increasing the gene copy number of molecular chaperones.

Key words: FAD-dependent glucose dehydrogenase, Pichia pastoris, antibiotic gradient screening, multiple copies of molecular chaperones

董聪, 高庆华, 王玥, 罗同阳, 王庆庆. 基于联合策略提高FAD依赖的葡萄糖脱氢酶的酵母表达[J]. 生物技术通报, 2023, 39(6): 316-324.

DONG Cong, GAO Qing-hua, WANG Yue, LUO Tong-yang, WANG Qing-qing. Increasing the Expression of FAD-dependent Glucose Dehydrogenase by Recombinant Pichia pastoris Using a Combined Strategy[J]. Biotechnology Bulletin, 2023, 39(6): 316-324.

图/表 10

表1 突变引物序列

Table 1 Mutation primer sequences

引物名称 Primer name	基因 Gene	引物序列 Primer sequence（5'-3'）
H- F	HAC1	CTCGCAGATCCTTGACTGAGGATCTGGACGAAG
H- R	HAC1	CCTCAGTCAAGGATCTGCGAGTGGATGTAGATG
E- F	Ero1	TGGAGAAACAAATCTTTTACCGATTGGTTTCTG
E- R	Ero1	GGTAAAAGATTTGTTTCTCCAAACAGAGGTCTTC
P1-F	PDI	CTGCTGCCGAAATCTTAAAGGACAATGAGCAGG
P1-R		CCTTTAAGATTTCGGCAGCAGAAACAAGTTCAG
P2-F		TTGACGGATCTACCTTCAAATCATATGCTGAAG
P2-F		ATTTGAAGGTAGATCCGTCAATGTCTCCAAATAG

图1 分子伴侣HAC1多拷贝表达盒载体的构建策略图

Fig. 1 Schematic representation of the recombinant vectors harboring multi-copy expression cassette for molecular chaperones HAC1

表2 RT-qPCR所用引物

Table 2 Primers used in RT-qPCR

引物名称Primer name	引物序列Primer sequence（5'-3'）
RT GAP up	CCAGCGGCAACAAGATCAAC
RT GAP down	CTCCTCGTTGACACCGACAA
RT GDH up	GGCAATACCAATCTGTCAACCAACCTTA
RT GDH down	AACCAGTGTTACCGATAGTTTCCCAAGC
RT HAC1 up	CTGAATATGACGACGAAGAA
RT HAC1 down	CTCCTGCTTGATAGATGTG
RT PDI up	ACCACATTTTACGGAGTTGCCGGT
RT PDI down	CCTCGCCAGGTCTGACAAGCA
RT Ero1 up	ATGAGGATAGTAAGGAGCGTAGC
RT Ero1 down	GTTGACCAGTTCCACCAGTTTG

表3 RT-qPCR检测基因拷贝数

Table 3 Copy numbers of genes detected by RT-qPCR

菌株 Strain	GDH基因拷贝数 Copy number of GDH	HAC1基因拷贝数 Copy number of HAC1	PDI基因拷贝数 Copy number of PDI	Ero1基因拷贝数 Copy number of Ero1
CK（X33/GDH）	3.20±0.20	Endogenous 1	Endogenous 1	Endogenous 1
CK-H	3.20±0.20	1.10±0.09	Endogenous 1	Endogenous 1
CK-2H	3.20±0.20	2.08±0.26	Endogenous 1	Endogenous 1
CK-3H	3.20±0.20	2.95±0.11	Endogenous 1	Endogenous 1
CK-4H	3.20±0.20	4.35±0.21	Endogenous 1	Endogenous 1
CK-P	3.20±0.20	Endogenous 1	1.02±0.11	Endogenous 1
CK-2P	3.20±0.20	Endogenous 1	2.32±0.19	Endogenous 1
CK-3P（X33/GDH-3PDI）	3.20±0.20	Endogenous 1	3.006±0.20	Endogenous 1
CK-4P	3.20±0.20	Endogenous 1	4.42±0.29	Endogenous 1
CK-E	3.20±0.20	Endogenous 1	Endogenous 1	1.21±0.13
CK-2E	3.20±0.20	Endogenous 1	Endogenous 1	1.98±0.34
CK-3E	3.20±0.20	Endogenous 1	Endogenous 1	3.22±0.17
CK-4E	3.20±0.20	Endogenous 1	Endogenous 1	4.19±0.20

图2 pPICZB-HAC1n（n=1,2,3或4）（A）、pPICZB-Ero1n（n=1,2,3或4）（B）、pPICZB-PDIn（n=1,2,3或4）（C）经双酶切后的琼脂糖凝胶电泳分析 M：DNA分子量标准；1-4：经Bgl II 和 BamH I双酶切后的pPICZB-HAC1/Ero1/PDIn（n=1,2,3或4）

Fig. 2 Agarose gel electrophoresis analysis for pPICZB-HAC1n（n=1,2,3或4）（A），pPICZB-Ero1n（n=1,2,3或4）（B），pPICZB-PDIn（n=1,2,3或4）（C）digested with double enzymes M: DNA ladder; lanes 1-4: pPICZB-HAC1/Ero1/PDIn（n=1,2,3或4）digested with BamH I and Bgl II

图3 试管水平不同分子伴侣共表达重组菌株相对酶活图中误差线表示标准偏差，小写字母表示不同菌株酶活差异达到（P<0.05）显著水平，下同

Fig. 3 Relative enzyme activities of recombinant strains co-expressed with different molecular chaperones at test tube level The error line in the figure refers to the standard deviation. The lowercase letters indicate that the difference among different strains reached a significant level（P<0.05）. The same below

图4 装液量对重组菌株酶活的影响

Fig. 4 Effects of liquid loading amount on the enzyme activity of recombinant strains

图5 培养基初始pH对重组菌株酶活的影响

Fig. 5 Effects of initial pH of the medium on the enzyme activity of recombinant strains

图6 诱导温度对重组菌株酶活的影响

Fig. 6 Effects of inducing temperature on the enzyme activity of recombinant strains

图7 甲醇添加量对重组菌株酶活的影响

Fig. 7 Effects of methanol addition amount on the enzyme activity of recombinant strains

参考文献 29

[1]	Fapyane D, Lee SJ, Kang SH, et al. High performance enzyme fuel cells using a genetically expressed FAD-dependent glucose dehydrogenase α-subunit of Burkholderia cepacia immobilized in a carbon nanotube electrode for low glucose conditions[J]. Phys Chem Chem Phys, 2013, 15(24): 9508-9512. doi: 10.1039/c3cp51864g pmid: 23695009
[2]	Southcott M, MacVittie K, Halámek J, et al. A pacemaker powered by an implantable biofuel cell operating under conditions mimicking the human blood circulatory system—battery not included[J]. Phys Chem Chem Phys, 2013, 15(17): 6278-6283. doi: 10.1039/c3cp50929j pmid: 23519144
[3]	Tang Z, Louie RF, Lee JH, et al. Oxygen effects on glucose meter measurements with glucose dehydrogenase- and oxidase-based test strips for point-of-care testing[J]. Crit Care Med, 2001, 29(5): 1062-1070. pmid: 11378622
[4]	Iwasa H, Ozawa K, Sasaki N, et al. Fungal FAD-dependent glucose dehydrogenases concerning high activity, affinity, and thermostability for maltose-insensitive blood glucose sensor[J]. Biochem Eng J, 2018, 140: 115-122. doi: 10.1016/j.bej.2018.09.014 URL
[5]	Yang YF, Huang L, Wang JF, et al. Efficient expression, purification, and characterization of a novel FAD-dependent glucose dehydrogenase from Aspergillus terreus in Pichia pastoris[J]. J Microbiol Biotechnol, 2014, 24(11): 1516-1524. doi: 10.4014/jmb.1401.01061 URL
[6]	Yang YF, Huang L, Wang JF, et al. Expression, characterization and mutagenesis of an FAD-dependent glucose dehydrogenase from Aspergillus terreus[J]. Enzyme Microb Technol, 2015, 68: 43-49. doi: 10.1016/j.enzmictec.2014.10.002 URL
[7]	Hohenblum H, Gasser B, Maurer M, et al. Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris[J]. Biotechnol Bioeng, 2004, 85(4): 367-375. doi: 10.1002/bit.10904 pmid: 14755554
[8]	Aw R, Polizzi KM. Can too many copies spoil the broth?[J]. Microb Cell Fact, 2013, 12: 128. doi: 10.1186/1475-2859-12-128 pmid: 24354594
[9]	黄猛猛. 利用组合策略促进毕赤酵母异源表达α-淀粉酶[D]. 上海: 华东理工大学, 2016.
	Huang MM. Enhancing expression of α-amylase in Pichia pastoris by combined strategies[D]. Shanghai: East China University of Science and Technology, 2016.
[10]	Sun J, Jiang J, Zhai XY, et al. Coexpression of Kex2 endoproteinase and Hac1 transcription factor to improve the secretory expression of bovine lactoferrin in Pichia pastoris[J]. Biotechnol Bioprocess Eng, 2019, 24(6): 934-941. doi: 10.1007/s12257-019-0176-5
[11]	Yu P, Zhu Q, Chen KF, et al. Improving the secretory production of the heterologous protein in Pichia pastoris by focusing on protein folding[J]. Appl Biochem Biotechnol, 2015, 175(1): 535-548. doi: 10.1007/s12010-014-1292-5 URL
[12]	Qiang L, Wang H, Farmer SR. Adiponectin secretion is regulated by SIRT1 and the endoplasmic Reticulum oxidoreductase Ero1-L alpha[J]. Mol Cell Biol, 2007, 27(13): 4698-4707. pmid: 17452443
[13]	Shen Q, Wu M, Wang HB, et al. The effect of gene copy number and co-expression of chaperone on production of albumin fusion proteins in Pichia pastoris[J]. Appl Microbiol Biotechnol, 2012, 96(3): 763-772. doi: 10.1007/s00253-012-4337-0 pmid: 22885695
[14]	闵琪. 耐热甘露聚糖酶ManA在毕赤酵母中的高效表达[D]. 武汉: 中南民族大学, 2020.
	Min Q. High-lever expression of thermostable mannanase ManA in Pichia pastoris[D]. Wuhan: South-central University for Nationalities, 2020.
[15]	钱晓芬, 吴涛, 赵理想, 等. 基因拷贝数对重组毕赤酵母的牛乳铁蛋白功能片段表达及细胞存活率的影响[J]. 食品与发酵工业, 2021, 47(4): 1-6.
	Qian XF, Wu T, Zhao LX, et al. Effect of gene copy number on the expression of bovine lactoferrin functional fragment and cell survival in recombinant Pichia pastoris[J]. Food Ferment Ind, 2021, 47(4): 1-6.
[16]	Huang MM, Gao YY, Zhou XS, et al. Regulating unfolded protein response activator HAC1p for production of thermostable raw-starch hydrolyzing α-amylase in Pichia pastoris[J]. Bioprocess Biosyst Eng, 2017, 40(3): 341-350. doi: 10.1007/s00449-016-1701-y URL
[17]	Inan M, Aryasomayajula D, Sinha J, et al. Enhancement of protein secretion in Pichia pastoris by overexpression of protein disulfide isomerase[J]. Biotechnol Bioeng, 2006, 93(4): 771-778. doi: 10.1002/(ISSN)1097-0290 URL
[18]	祁丽, 姜宁, 张爱忠, 等. 抗菌肽多基因表达技术与策略[J]. 黑龙江畜牧兽医, 2016(15): 52-56.
	Qi L, Jiang N, Zhang AZ, et al. Multi-gene expression technology and strategy of antibacterial peptides[J]. Heilongjiang Animal Sci Vet Med, 2016(15): 52-56.
[19]	董聪, 高庆华, 王玥, 等. 基于密码子优化的FAD依赖葡萄糖脱氢酶在毕赤酵母中的高效表达及酶学性质[J]. 生物技术通报, 2019, 35(7): 114-120. doi: 10.13560/j.cnki.biotech.bull.1985.2018-0941
	Dong C, Gao QH, Wang Y, et al. Expression and enzymatic characterization of Codon-optimized FAD-dependent glucose dehydrogenase in Pichia pastoris[J]. Biotechnol Bull, 2019, 35(7): 114-120.
[20]	Abad S, Kitz K, Hörmann A, et al. Real-time PCR-based determination of gene copy numbers in Pichia pastoris[J]. Biotechnol J, 2010, 5(4): 413-420. doi: 10.1002/biot.v5:4 URL
[21]	Li D, Wu JC, Chen J, et al. Optimized expression of classical swine fever virus E2 protein via combined strategy in Pichia pastoris[J]. Protein Expr Purif, 2020, 167: 105527. doi: 10.1016/j.pep.2019.105527 URL
[22]	Sygmund C, Staudigl P, Klausberger M, et al. Heterologous overexpression of Glomerella cingulata FAD-dependent glucose dehydrogenase in Escherichia coli and Pichia pastoris[J]. Microb Cell Fact, 2011, 10: 106. doi: 10.1186/1475-2859-10-106
[23]	马银鹏, 王玉文, 党阿丽, 等. 毕赤酵母表达系统研究进展[J]. 黑龙江科学, 2013, 4(9): 27-31.
	Ma YP, Wang YW, Dang AL, et al. Advances of Pichia pastoris expression system[J]. Heilongjiang Sci, 2013, 4(9): 27-31.
[24]	Hwang J, Qi L. Quality control in the endoplasmic Reticulum: crosstalk between ERAD and UPR pathways[J]. Trends Biochem Sci, 2018, 43(8): 593-605. doi: 10.1016/j.tibs.2018.06.005 URL
[25]	Yang J, Lu ZP, Chen JW, et al. Effect of cooperation of chaperones and gene dosage on the expression of porcine PGLYRP-1 in Pichia pastoris[J]. Appl Microbiol Biotechnol, 2016, 100(12): 5453-5465. doi: 10.1007/s00253-016-7372-4 pmid: 26883349
[26]	Kim JM, Jang SA, Yu BJ, et al. High-level expression of an antimicrobial peptide histonin as a natural form by multimerization and furin-mediated cleavage[J]. Appl Microbiol Biotechnol, 2008, 78(1): 123-130. pmid: 18094965
[27]	Cos O, Serrano A, Montesinos JL, et al. Combined effect of the methanol utilization(Mut)phenotype and gene dosage on recombinant protein production in Pichia pastoris fed-batch cultures[J]. J Biotechnol, 2005, 116(4): 321-335. doi: 10.1016/j.jbiotec.2004.12.010 URL
[28]	Song XP, Shao CS, Guo YG, et al. Improved the expression level of active transglutaminase by directional increasing copy of mtg gene in Pichia pastoris[J]. BMC Biotechnol, 2019, 19(1): 54. doi: 10.1186/s12896-019-0542-6
[29]	焦梁成. 毕赤酵母分子操作方法改进及其在米根霉脂肪酶高效表达中的应用[D]. 武汉: 华中科技大学, 2019.
	Jiao LC. Improvement of molecular manipulation method of Pichia pastoris and its application in high-level expression of Rhizopus oryzae lipase[D]. Wuhan: Huazhong University of Science and Technology, 2019.