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1 微生物学通报 Microbiology China May 20, 2017, 44(5): DOI: /j.microbiol.china 研究报告黑曲霉阿魏酸酯酶基因密码子优化及在毕赤酵母中的高效表达 * 李兵蔡国林朱德伟陆健 ( ) 摘要 : 目的对黑曲霉(Aspergillus niger) 阿魏酸酯酶基因进行克隆和密码子优化, 使其在毕赤酵母 (Pichia pastoris X-33) 中高效表达方法以黑曲霉基因组为模板, 经重叠延伸 PCR 扩增得到阿魏酸酯酶基因 (AnfaeA), 并对 AnfaeA 基因进行毕赤酵母密码子偏好性随机优化和一对一优化, 全基因合成后分别与表达载体 ppiczαa 连接, 构建表达载体 ppiczαa-anfaea ppiczαa-opanfaea I 和 ppiczαa-opanfaea II 经 Sac I 线性化后电转化至 P. pastoris X-33 中, 筛选阳性转化子摇瓶发酵 4.5 d 后, 测定并比较重组阿魏酸酯酶 (reanfaea) 酶活结果密码子优化前阿魏酸酯酶酶活为 6.8±0.1 U/mL, 基因一对一优化和随机优化后的重组酶酶活分别为 5.2±0.1 U/mL 和 39.9±0.1 U/mL, 随机优化后酶活比优化前提高了近 6 倍, 而一对一优化后酶活仅为优化前酶活的 76.5% 重组阿魏酸酯酶的最适 ph 为 5.5, 且在 ph 稳定性较好 ; 最适反应温度 50 C, 在 C 较稳定结论阿魏酸酯酶基因经密码子随机优化后进行重组表达, 酶活显著提高, 对研究阿魏酸酯酶在毕赤酵母及其它宿主中的高效表达具有一定的借鉴意义, 也为大规模工业化应用奠定了基础关键词 : 黑曲霉, 毕赤酵母, 阿魏酸酯酶, 密码子优化 Codon optimization of feruloyl esterase gene of Aspergillus niger and its high expression in Pichia pastoris LI Bing CAI Guo-Lin ZHU De-Wei LU Jian * (Key Laboratory of Industrial Biotechnology, Ministry of Education, National Engineering Laboratory for Cereal Fermentation Technology, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu , China) Abstract: [Objective] Gene cloning and codon optimization of the feruloyl esterase from Aspergillus niger (AnfaeA) for its inducible expression in Pichia pastoris X-33. [Methods] The AnfaeA gene was Foundation item: National High Technology Research and Development Program of China (863 Program) (No. 2013AA102109); Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions *Corresponding author: Tel/Fax: ; jlu@jiangnan.edu.cn Received: August 02, 2016; Accepted: November 01, 2016; Published online ( December 05, 2016 基金项目国家高技术研究发展计划 (863 计划 ) 项目 (No. 2013AA102109); 江苏高校优势学科建设工程资助项目 * 通讯作者 :Tel/Fax: ; jlu@jiangnan.edu.cn 收稿日期 : ; 接受日期 : ; 优先数字出版日期 (

2 1066 微生物学通报 Microbiol. China 2017, Vol.44, No.5 amplified by overlap extension PCR using the genome of A. niger as template. At the same time, the AnfaeA gene was optimized by one amino acid one codon and codon randomization and then synthesized. The three kinds of ferulic acid esterase gene were cloned into the expression vector ppiczαa, respectively, obtaining the recombinant expression vectors ppiczαa-anfaea, ppiczαa-opanfaea I and ppiczαa-opanffaea II. The plasmids were then linearized by Sac I, and transformed into P. pastoris X-33. The positive transformants of each gene type were identified by PCR, and further screened by determination of feruloyl esterase activity in fermentations using high performance liquid chromatography (HPLC). [Results] The feruloyl esterase activity of FaeA-ori, FaeA-opt I and FaeA-opt II were 6.8±0.1 U/mL, 5.2±0.1 U/mL, and 39.9±0.1 U/mL, respectively. By codon randomization optimization, the activity of recombinant enzyme was 6 times higher than that coded by original AnfaeA gene. However, the feruloyl esterase coded by one amino acid one codon optimized gene was only 76.5% of the original enzyme. The optimal temperature and ph for recombinant AnfaeA (reanfaea) was 50 C and 5.5 respectively In addition, reanfaea showed stability when incubated in C and ph [Conclusions] By codon randomization optimization, the resultant recombinant feruloyl esterase expressed in P. pastoris X-33 was 6 times higher than that coded by original AnfaeA, reaching the highest activity among existed recombinant feruloyl esterase until now. The results provided large-scale application potential of feruloyl esterase in industrial, and laid the experimental foundation for the further improvement the enzyme activity. Keywords: Aspergillus niger, Pichia pastoris, Feruloyl esterase, Codon optimization (E.C Feruloyl esterase FAE) [1-2] 1991 Faulds [3] (Streptomyces olitrochromogenes) 30 [4] (Aspergillus sp.) (A. niger) (A. oryzae) (A. awamori) (A. nidulans) [5-8] U/mL [9] 2001 Juge 300 mg/l [10] GS U/mL [11] [12] [13] ppiczαa-anfaea 1 材料与方法 1.1 材料菌株和质粒 :(Aspergillus niger) CBS (Escherichia coli) JM109 X-33 ppiczαa pmd19-t Simple Vector TaKaRa

3 : 主要试剂仪器及培养基 :PCR Sangon PCR DNA Marker Marker (Zeocin) TaKaRa Sigma AKTA avant25 HisTrap TM excel (1 ml) GE Healthcare Agilent 1260 Mastercycler pro PCR Eppendorf YPD BMGY BMMY Invitrogen 2% 300 μl 10% ( ) 1.2 方法黑曲霉阿魏酸酯酶基因的克隆及基因优化合成 : (GenBank Y ) Signal P3.0 PCR ( 1) CTAB [14] DNA 1 (F1/R1 F2/R2) 1 2 PCR 95 C 4 min 95 C 15 s 52 C 15 s 72 C 30 s C 10 min PCR (1:1 ) F1 R2 PCR 60 s 1 PCR pmd19-t JM109 pmd19-anfaea 生物工程有限公司 G+C%CAI- ( 2 [15] 生物工程有限公司 pmd19-t pmd19-opanfaea I pmd19-opanfaea II 阿魏酸酯酶的活性测定 : Zhang [16] 900 µl (1 mmol/l ph 6.0 ) 1.5 ml EP 50 C 5 min 100 µl 10 min 400 µl ( 0.22 μm) 表 1 实验所用的引物序列及酶切位点 Table 1 Primers used in this study Primer Sequence (5 3 ) Restriction site F1 CGGAATTCGCCTCCACGCAAGGCATCTC EcoR I R1 CTATGGCCTGTCACGGTAAGC None F2 CGCTTACCGTGACAGGCCATAGTCTGGGAGCGTCGATGGC None R2 ATAAGAATGCGGCCGCTTACCAAGTACAAGCTCCGC Not I Opt I-F CGGAATTCGCCTCAACACAAGGTATCAG EcoR I Opt I-R ATAAGAATGCGGCCGCCCAAGTACAAGCGCCACTTG Not I Opt II-F CGGAATTCGCTTCTACTCAAGGTATTTCTG EcoR I Opt II-R ATAAGAATGCGGCCGCCCAAGTACAAGCACCACTAG Not I. Note: The underlined are restriction enzyme cutting sites.

4 1068 微生物学通报 Microbiol. China 2017, Vol.44, No.5 Amino acid Codon Optimal codon 表 2 毕赤酵母高表达偏好性密码子 Table 2 The optimal codon list in P. pastoris Amino Optimal Amino Optimal Codon Codon acid codon acid codon Amino acid Codon Phe TTT * Ser TCT * Tyr TAT Cys TGT * TTC TCC TAC * TGC Leu TTA TCA His CAT Arg CGT TTG * TCG CAC * CGC CTT AGT Gln CAA * CGA CTC AGC CAG CGG CTA Pro CCT Asn AAT AGA * CTG CCC AAC * AGG Ile ATT * CCA * Lys AAA Gly GGT * ATC CCG AAG * GGC ATA Thr ACT * Asp GAT GGA Val GTT * ACC GAC * GGG GTC ACA Glu GAA * Ala GCT * GTA ACG GAG GCC GTG *. Note: *: Optimal codon. GCA GCG Optimal codon [17] 400 µl 1 μmol 1 U 毕赤酵母重组表达载体的构建及阳性转化子的筛选 : pmd19-anfaea pmd19-opanfaea I pmd19-opanfaea II EcoR I Not I ppiczαa JM109 PCR ppiczαa-anfaea ppiczαa-opanfaea I ppiczαa-opanfaea II 3 Sac I X-33 Zeocin YPD 30 C 3 5 d PCR ( Opt I-F/R Opt II-F/R PCR ) 30 C 1 h 重组阿魏酸酯酶的纯化 : X-33 ppiczαa His (Ni) (0.22 μm) HisTrap TM excel 10 ml SDS-PAGE 重组阿魏酸酯酶特性研究 : ph ph 1.2.2

: 1069 ph (ph 3.0 7.0)- (50 mmol/l) ph ph (ph 3.0 7.5)- (50 mmol/l) 100 µl 35 C 1 h ph 1.2.2 (30 75 C) 5 min 40 50 60 C 10 60 min EDTA (Na + K + Ca 2+ Fe 2+ Mg 2+ Co 2+ Mn 2+ Zn 2+ ) EDTA ( 2.

jp/codon) ( 15%) CCG(Pro) GCG (Ala) CTC(Leu) TCG/AGC(Ser) ACG(Thr) CGG/C(Arg) 2 6 AnfaeA G+C% 54.4% (AnfaeA-opt I) (AnfaeA-opt II) G+C% 45.1% 40.9% CAI- 0.51 0.91 G+C% CAI- (CAI 0.8 1.

5 : 1069 ph (ph )- (50 mmol/l) ph ph (ph )- (50 mmol/l) 100 µl 35 C 1 h ph (30 75 C) 5 min C min EDTA (Na + K + Ca 2+ Fe 2+ Mg 2+ Co 2+ Mn 2+ Zn 2+ ) EDTA ( 2.0 mmol/l)35 C 1 h 50 C 5 min EDTA 35 C 1 h ( 100%) EDTA 2 结果与分析 2.1 黑曲霉阿魏酸酯酶基因的克隆和基因优化后合成 DNA PCR ( ) 750 bp ( 1) 780 bp NCBI (GenBank Y ) BLAST 99.64% ( or.jp/codon) ( 15%) CCG(Pro) GCG (Ala) CTC(Leu) TCG/AGC(Ser) ACG(Thr) CGG/C(Arg) 2 6 AnfaeA G+C% 54.4% (AnfaeA-opt I) (AnfaeA-opt II) G+C% 45.1% 40.9% CAI G+C% CAI- (CAI ) [15] 3 ( 2) pmd19-t JM 重组表达载体的构建及阳性转化子的筛选 3 EcoR I Not I ppiczαa AOX1 ppiczαa-anfaea ( 3A) ppiczαa-opanfaea I ppiczαa-opanfaea II Sac I X-33 PCR 3 d ( 3B) 5 FaeA-ori FaeA-opt I FaeA-opt II 图 1 AnfaeA 基因的琼脂糖电泳检测 Figure 1 Agarose gel electrophoresis of AnfaeA M DNA marker 1 2 PCR. Note: M: DNA marker; 1 2: PCR products of AnfaeA.

6 1070 微生物学通报 Microbiol. China 2017, Vol.44, No.5 图 2 基因优化前后 AnfaeA 基因的核苷酸序列 Figure 2 Alignment of nucleotide of the optimized and original AnfaeA genes. Note: Characters with shadow are the same nucleotides, and others are different ones. 2.3 重组阿魏酸酯酶在毕赤酵母中的表达 1% FaeA-ori FaeA-opt I FaeA-opt II 12 h d FaeA-ori 6.8 U/mL FaeA-opt II 5.2 U/mL 76.5% FaeA-opt I 39.9 U/mL 重组阿魏酸酯酶的纯化及 SDS-PAGE 分析 HisTrap TM excel SDS-PAGE 33 kd ( 5) (28 kd) ppiczαa/ X-33 ppiczαa ppiczαa His c-myc

Marker 1 2 ( ppiczαa/x-33) 3. Note: M: Standard protein marker; 1: Fermentation supernatant of recombinant strain; 2: ppiczαa/x-33; 3: Purification of reanfaea. 2.5 kd NetNGlyc 1.0 Server (http://www.

7 : 1071 Figure 3 图 3 重组载体 ppiczαa-anfaea 构建图谱 (A) 和阿魏酸乙酯平板法检测酶活 (B) ppiczαa-anfaea plasmid map (A) and the feruloyl esterase assay of P. pastoris transformants (B) 图 4 3 株重组菌诱导发酵生产阿魏酸酯酶 Figure 4 Fermentation and secretion curve of recombinant AnfaeA from yeast 图 5 reanfaea 分离纯化的 SDS-PAGE 图谱 Figure 5 SDS-PAGE analysis of reanfaea M Marker 1 2 ( ppiczαa/x-33) 3. Note: M: Standard protein marker; 1: Fermentation supernatant of recombinant strain; 2: ppiczαa/x-33; 3: Purification of reanfaea. 2.5 kd NetNGlyc 1.0 Server ( (Asn-Tyr-Thr-Leu) N- N- 2 kd 5 kd 2.5 重组阿魏酸酯酶的酶学特性研究 C 50 C (37 C) [18] (45 C) [19] 60 C C 50 C 2 h 50%55 C 1 h 20% 7 ph ph 5.5 [19] [20] ph ph (ph ) 35 C 1 h EDTA 8 EDTA 85% Na + K + Ca 2+ Mg 2+ Zn 2+

stability of recombinant feruloyl esterase Fe 2+ Co 2+ Mn 2+ EDTA 3 讨论图 7 ph 对重组阿魏酸酯酶活力及其稳定性的影响 Figure 7 Effects of ph on the

8 1072 微生物学通报 Microbiol. China 2017, Vol.44, No.5 Figure 6 图 6 温度对重组阿魏酸酯酶活力的影响及重组阿魏酸酯酶的温度稳定性 Effects of temperature on the activity of recombinant feruloyl esterase and the stability of recombinant feruloyl esterase Fe 2+ Co 2+ Mn 2+ EDTA 3 讨论图 7 ph 对重组阿魏酸酯酶活力及其稳定性的影响 Figure 7 Effects of ph on the activity and stability of recombinant feruloyl esterase 图 8 金属离子和 EDTA 对 reanfaea 酶活力的影响 Figure 8 Effects of various metal ions and EDTA on the activity of the reanfaea [21] 2.11 U/mL [22] U/mL [19] 39.9 U/mL

9 : % G+C% CAI- ph Fe 2+ 参考文献 [1] Kroon PA, Garcia-Conesa MT, Fillingham IJ, et al. Release of ferulic acid dehydrodimers from plant cell walls by feruloylesterases[j]. Journal of the Science of Food and Agriculture, 1999, 79(3): [2] Hu XS, Li XL. Review on cloning, expression, regulation and applications of feruloyl esterase genes[j]. Biotechnology Bulletin, 2009(12): (in Chinese),. [J]., 2009(12): [3] Faulds CB, Williamson GJ. The purification and characterization of 4-hydroxy-3-methoxycinnamic (ferulic) acid esterase from Streptomyces olivochromogenes[j]. Journal of General Microbiology, 1991, 137(10): [4] Topakas E, Vafiadi C, Christakopoulos P. Microbial production, characterization and applications of feruloylesterases[j]. Process Biochemistry, 2007, 42(4): [5] Zhang SB, Wu ZL. Identification of amino acid residues responsible for increased thermostability of feruloyl esterase a from Aspergillus niger using the PoPMuSiC algorithm[j]. Bioresource Technology, 2011, 102(2): [6] Koseki T, Hori A, Seki S, et al. Characterization of two distinct feruloylesterases, AoFaeB and AoFaeC, from Aspergillus oryzae[j]. Applied Microbiology and Biotechnology, 2009, 83(4): [7] Fazary AE, Hamad HA, Lee JC, et al. Expression of feruloyl esterase from Aspergillus awamori in Escherichia coli: characterization and crystal studies of the recombinant enzyme[j]. International Journal of Biological Macromolecules, 2010, 46(4): [8] Debeire P, Khoune P, Jeltsch JM, et al. Product patterns of a feruloyl esterase from Aspergillus nidulans on largeferuloylarabino-xylo-oligosaccharides from wheat bran[j]. Bioresource Technology, 2012, 119: [9] Fazary AE, Ju YH. Feruloyl esterases as biotechnological tools: current and future perspectives[j]. Acta Biochimica et Biophysica Sinica, 2007, 39(11): [10] Juge N, Williamson G, Puigserver A, et al. High-level production of recombinant Aspergillus niger cinnamoyl esterase (FaeA) in the methylotrophic yeast Pichia pastoris[j]. FEMS Yeast Research, 2001, 1(2): [11] Zhang SB, Pei XQ, Wu ZL. Cloning and expression of feruloyl esterase A from Aspergillus niger, and establishment of fast activity detection methods[j]. Chinese Journal of Applied & Environmental Biology, 2009, 15(2): (in Chinese),,. A [J]., 2009, 15(2): [12] Huang GR, Tian LC, Huang SP, et al. High expression of codon optimized phytase-encoding gene appa-p in Pichia pastoris[j]. Journal of Hubei University (Natural Science), 2007, 29(3): (in Chinese),,,. appa-p [J]. :, 2007, 29(3): [13] Menzella HG. Comparison of two codon optimization strategies to enhance recombinant protein production in Escherichia coli[j]. Microbial Cell Factories, 2011, 10(1): [14] Zhang XL, Lü XL, Shen YN, et al. Four methods of DNA extraction for Aspergillus niger[j]. Chinese Journal of Mycology, 2011, 6(3): (in Chinese),,,. 4 DNA [J]., 2011, 6(3): [15] Zhao X, Huo KK, Li YY. Synonymous codon usage in Pichia pastoris[j]. Chinese Journal of Biotechnology, 2000, 16(3): [16] Zhang SB, Pei XQ, Wu ZL. Multiple amino acid substitutions significantly improve the thermostability of feruloyl esterase A from Aspergillus niger[j]. Bioresource Technology, 2012, 117: [17] Chen YH, Yang JT, Chau KH. Determination of the helix and β form of proteins in aqueous solution by circular dichroism[j]. Biochemistry, 1974, 13(16): [18] Donaghy J, McKay AM. Purification and characterisation of a feruloyl esterase from the fungus Penicillium expansum[j]. Journal of Applied Microbiology, 1997, 83(6): [19] Gong YY, Yin X, Wu MC, et al. Cloning, expression and enzymatic characterization of feruloyl esterase A from Aspergillus usamii[j]. Journal of Food Science and Biotechnology, 2013, 32(7): (in Chinese),,,. A [J]., 2013, 32(7): [20] Koseki T, Takahashi K, Fushinobu S, et al. Mutational analysis of a feruloyl esterase from Aspergillus awamori involved in substrate discrimination and ph dependence[j]. Biochimica et Biophysica Acta (BBA)-General Subjects, 2005, 1722(2): [21] Huang XY, Zhang L, Li Y, et al. Constitutive expression of feruloyl esterase from Aspergillus niger in Pichia pastoris[j]. Microbiology China, 2017, 44(1): (in Chinese),,,. [J]., 2017, 44(1): [22] Zeng Y, Gong YY, Wu MC, et al. Gene cloning, expression of a feruloyl esterase and purification of its hydrolysis products[j]. Chinese Journal of Biotechnology, 2014, 30(3): (in Chinese),,,. A [J]., 2014, 30(3):

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