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1 生物工程学报 Chinese Journal of Biotechnology DOI: /j.cjb 肖梦榕等 / 大肠杆菌 PTS 系统改造及重组菌生长性能测定 October 25, 2014, 30(10): Chin J Biotech, All rights reserved 工业生物技术 1561 PTS 肖梦榕, 张梁, 刘双平, 石贵阳 ,,. PTS., 2014, 30(10): Xiao MR, Zhang L, Liu SP, et al. Transformation of phosphotransferase system in Escherichia coli. Chin J Biotech, 2014, 30(10): 摘要 : 利用 Red 重组系统对野生大肠杆菌 Escherichia coli 磷酸烯醇式丙酮酸糖磷酸转移酶系统 (Phosphoenolpyruvate: carbohydrate phosphotransferase system, PTS) 进行修饰改造, 敲除 PTS 系统中关键组分 EⅡCB Glc 的编码基因 (ptsg), 磷酸组氨酸搬运蛋白 HPr 的编码基因 (ptsi), 同时敲入来源于运动发酵单胞菌 Zymomonas mobilis 的葡萄糖易化体 (Glucose facilitator) 编码基因 (glf), 构建重组大肠杆菌, 比较测定并系统评价了基因敲除和敲入对细胞的生长葡萄糖代谢和乙酸积累的影响敲除基因 ptsg 和 ptsi 造成大肠杆菌 PTS 系统部分功能缺失, 细胞生长受到一定限制, 敲入 glf 基因后, 重组大肠杆菌能够利用 Glf-Glk ( 葡萄糖易化体 - 葡萄糖激酶 ) 途径, 消耗 ATP 将葡萄糖进行磷酸化并转运进入细胞通过该途径转运葡萄糖能够提高葡萄糖利用效率, 降低副产物乙酸生成, 同时能够使更多的碳代谢流进入后续相关合成途径, 预期能够提高相关产物产量 : 大肠杆菌, 运动发酵单胞菌,PTS 系统,glf,Red 同源重组, 代谢工程, 发酵 Received: December 23, 2013; Accepted: February 10, 2014 Supported by: National High Technology Research and Development Program of China (863 Program) (No. 2012AA021201), the Program for New Century Excellent Talents in University (No. NCET ), Innovative Research Team of Jiangsu Province, the Priority Academic Program Development of Jiangsu Higher Education Institutions (No. PADP), the Innovative Program for Graduate Student of Jiangsu Province (No. CXLX12_0733), the Fundamental Research Funds for the Central Universities (No. JUDCF12016). Corresponding author: Guiyang Shi. Tel: ; gyshi@jiangnan.edu.cn (863 ) (No. 2012AA021201) (No. NCET ) (No. PADP) 2012 (No. CXLX12_0733) (No. JUDCF12016)

2 1562 ISSN CN /Q Chin J Biotech October 25, 2014 Vol.30 No.10 Transformation of phosphotransferase system in Escherichia coli Mengrong Xiao, Liang Zhang, Shuangping Liu, and Guiyang Shi National Engineering Laboratory for Cereal Fermentation Technology, Key Laboratory of Industrial Biotechnology of Ministry of Education, Jiangnan University, Wuxi , Jiangsu, China Abstract: We constructed several recombinant Escherichia coli strains to transform phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS system) and compared the characteristics of growth and metabolism of the mutants. We knocked-out the key genes ptsi and ptsg in PTS system by using Red homologous recombination in E. coli and meanwhile we also knocked-in the glucose facilitator gene glf from Zymomonas mobilis in the E. coli chromosome. Recombinant E. coli strains were constructed and the effects of cell growth, glucose consumption and acetic acid accumulation were also evaluated in all recombinant strains. The deletion of gene ptsg and ptsi inactivated some PTS system functions and inhibited the growth ability of the cell. Expressing the gene glf can help recombinant E. coli strains re-absorb the glucose through Glf-Glk (glucose facilitator-glucokinase) pathway as it can use ATP to phosphorylate glucose and transport into cell. This pathway can improve the availability of glucose and also reduce the accumulation of acetic acid; it can also broaden the carbon flux in the metabolism pathway. Keywords: Escherichia coli, Zymomonas mobilis, PTS system, glf, Red homologous recombination, metabolic engineering, fermentation - (Phosphoenolpyruvate carbohydrate phosphotransferase system PTS ) [1] (PEP) TCA [2] PTS EⅠ (ptsh ) HPr (ptsi ) Ⅱ (EⅡ s) [3-4] EⅠ PEP HPr EⅡs EⅡs 20 EⅡs 20 EⅡ Glc EⅡ Man EⅡ Glc EⅡA Glc (crr ) EⅡCB Glc (ptsg ) [5] [5-8] PTS PTS Zymomonas mobilis Glf [9-12] K m = μmol/l Ren [13] Glf (CCR) Yi [14] PTS Glf Glk (glk )

3 肖梦榕等 / 大肠杆菌 PTS 系统改造及重组菌生长性能测定 1563 GalP-Glk Glf Glf Red [15] ptsg ptsi ptsi glf 1 材料与方法 1.1 材料 E. coli W3110 Zymomonas mobilis (ATCC 31821) pkd46 ( λ Gam Bet Exo) PKD13 pcp20 (CGSC E. coli Genetic Stock Center New Haven USA) [15] E. coli G1 I1 I DNA EcoRⅠ Hind Ⅲ Taq DNA dntp Mixture Rnase Fermentas (Amp) (Cm) (Kan) L- Sigma-Aldrich ( ) pmd18-t Vector Q5 NEB ( ) OXOID DNA LB (/L) 10 g 5 g 10 g NaCl SOB (/L) 20 g 5 g 0.5 g NaCl g KCl 0.95 g MgCl 2 1.5% 表 1 菌株与质粒 Table 1 Strains and plasmids Strains and plasmids Relevant characteristics Sources or reference Strains E. coli W3110 Wild type This Lab Zymomonas mobilis ZM4 ATCC This Lab E. coli G1 W3110 ( ptsg) This research E. coli I1 W3110 ( ptsi) This research E. coli I2 W3110 ( ptsi::glf) This research Plasmid PKD46 γ β exo (Red recombinase), temperature-conditional replicon This Lab PKD13 Amp and Kan markers [15] pcp20 Amp and Cm markers, helper plasmid [15] cjb@im.ac.cn

4 1564 ISSN CN /Q Chin J Biotech October 25, 2014 Vol.30 No.10 T (/L) 20 g 10 g 2 g KH 2 PO 4 1 g (NH 4 ) 2 SO 4 1 g MgSO 4 7H 2 O 28 FM (/L) 3 g MgSO 4 7H 2 O 3 g KH 2 PO 4 1 g NaCl 5 g (NH 4 ) 2 SO 4 [(μmol/l) FeCl 3 6H 2 O 8.88 CoCl 2 6H 2 O 1.26 CuCl 2 2H 2 O 0.88 ZnCl Na 2 MoO 4 2H 2 O 1.24 H 3 BO MnCl 2 4H 2 O 2 2.5] 50 g/l 10 g/l ( ) ph [16] 100 μg/ml 25 μg/ml 50 μg/ml 方法 DNA [17] glf Zymomonas mobilis glf (GenBank Accession No. ATCC 31281) IF1 IF2 ( 2) ptsi 表 2 本研究中所用引物 Table 2 Primers used in this research Primers Sequence (5ʹ 3ʹ) Size (bp) G1 TTTAAGAATGCATTTGCTAACCTGCAAAAGGTCGGTAAATCGCTGATGCTATTC CGGGGATCCGTCG 67 G2 TTAGTGGTTACGGATGTACTCATCCATCTCGGTTTTCAGGTTATCGGATTTGTAG GCTGGAGCTGCTTCG 70 YG1 ATTCCGGGGATCCGTCG 17 YG2 TGTAGGCTGGAGCTGCTTCG 20 I1 ATGATTTCAGGCATTTTAGCATCCCCGGGTATCGCTTTCGGTAAAGCTCTATTCC GGGGATCCGTCG 69 I2 TTAGCAGATTGTTTTTTCTTCAATGAACTTGTTAACCAGCGTCATTAACTTGTAG GCTGGAGCTGCTTCG 70 YI1 GTTAAACTGATGGCGGAACT 20 YI2 CAGTTTATCGAACAAACCCA 20 IF1 GAGTAATTTCCCGGGTTCTTTTAAAAATCAGTCACAAGTAAGGTAGGGTTATGA GTTCTGAAAGTAGTC 69 IF2 CGACGGATCCCCGGAATGTCCGCCCGCTTTATAC 34 KanI1 GTATAAAGCGGGCGGACATTCCGGGGATCCGTCG 34 TTAGCAGATTGTTTTTTCTTCAATGAACTTGTTAACCAGCGTCATTAACTTGTAG KanI2 GCTGGAGCTGCTTCG The underlined part is the homologous sequence of underknock-out gene. 70

5 肖梦榕等 / 大肠杆菌 PTS 系统改造及重组菌生长性能测定 1565 IF1 IF2 PCR PCR ddh 2 O 38.5 μl 10 Ex Taq 5 μl dntps 4 μl 1 μl 1 μl Ex Taq 0.5 μl PCR 95 5 min s s s min PCR pmd18 T-Vector JM109 LB Hind Ⅲ EcoRⅠ PCR PKD13 Q5 PCR PCR ddh 2 O 33.5 μl 5 Q5 10 μl dntps 4 μl 1 μl 1 μl Q5 0.5 μl PCR s s s s min PCR PKD46 10 μl 100 μl 1 mm V 1 ml SOB h Kan pcp20 Cm PCR E. coli G1 I1 I2 5 ml LB r/min 70 ml FM r/min 600 nm SOB r/min 8 h LB r/min 10 h 6 L 5 % 50 g/l 1 L/min 200 r/min (1 7 L/min) ( r/min) 30% 10% (V/V) ph h SBA-40C ( ) HPLC SHIMADAZU LC-10AT Dionex UltiMate Autosampler 3000 Shodex SH1011 Shodex RI mol/l H 2 SO ml/min cjb@im.ac.cn

1566 ISSN 1000-3061 CN 11-1998/Q Chin J Biotech October 25, 2014 Vol.30 No.10 2 结果与分析 2.1 目的基因的敲除 PCR E. coli G1 YG1/YG2 E.

6 1566 ISSN CN /Q Chin J Biotech October 25, 2014 Vol.30 No.10 2 结果与分析 2.1 目的基因的敲除 PCR E. coli G1 YG1/YG2 E. coliⅠ1 YI1/YI2 PCR ( 1) YG1/YG bp G1 ( ptsg::kan) bp G1 650 bp G1 ptsg ptsi YI1/YI bp ( 2) 图 2 ptsi 基因缺失菌株的 PCR 鉴定电泳图谱 Fig. 2 PCR identification of ptsi knockout mutant. M: 1 kb ladder marker; 1: W3110 PCR product; 2: I1 ( ptsi::kan) PCR product; 3: I1 ( ptsi) PCR product. Ⅰ1 ( ptsi::kan) PCR bp FRT Ⅰ1 200 bp Ⅰ1 ptsi ptsg ptsi E. coli G1 Ⅰ1 图 1 ptsg 基因缺失菌株的 PCR 鉴定电泳图谱 Fig. 1 PCR identification of ptsg knockout mutant. M: 1 kb ladder marker; 1: W3110 PCR product; 2: G1 ( ptsg::kan) PCR product; 3: G1 ( ptsg) PCR product. 2.2 外源基因在染色体上的整合表达 3 ptsi ::kan-glf glf ORF ptsi ATG glf glf ptsi ORF PCR

7 肖梦榕等 / 大肠杆菌 PTS 系统改造及重组菌生长性能测定 1567 E. coli I2 YI1/YI2 PCR ( 4) YI1/YI bp I2 ( ptsi::glf::kan) bp I2 PCR bp I2 ptsi glf ptsi glf E. coli I2 图 3 同源重组打靶片段 ptsi ::kan-glf 的制备 Fig. 3 Construction of ptsi ::kan-glf used for homologous recombination. cjb@im.ac.cn

1568 ISSN 1000-3061 CN 11-1998/Q Chin J Biotech October 25, 2014 Vol.30 No.

40 g/l (OD 600 =45.8) (OD 600 =25.1) 1.82 图 4 缺失 ptsi 基因同时插入 glf 基因菌株的 PCR 鉴定电泳图谱 Fig.

M: 1 kb ladder marker; 1: W3110 PCR product; 2: I2 ( ptsi::glf::kan) PCR product; 3: I2 (

37 g/(h L) W3110 (0.42 g/(h L)) (2.74 g/l) W3110 (2.34 g/l) I1 0.34 g/(h L) ( 3) W3110 3.

8 1568 ISSN CN /Q Chin J Biotech October 25, 2014 Vol.30 No.10 glf FM I2 I1 I g/(h L) I g/l I1 1.3 glf ( 6) E. coli I g/l (OD 600 =45.8) (OD 600 =25.1) 1.82 图 4 缺失 ptsi 基因同时插入 glf 基因菌株的 PCR 鉴定电泳图谱 Fig. 4 PCR identification of ptsi knock-out and glf knock-in mutant. M: 1 kb ladder marker; 1: W3110 PCR product; 2: I2 ( ptsi::glf::kan) PCR product; 3: I2 ( ptsi::glf) PCR product FM FM 2.3 E. coli W3110 G1 I1 及 I2 生长代谢特征比较 FM 5 FM G g/(h L) W3110 (0.42 g/(h L)) (2.74 g/l) W3110 (2.34 g/l) I g/(h L) ( 3) W g/l W I1 I1 图 5 重组大肠杆菌 E. coli W3110 G1 I1 I2 在摇瓶中的生长情况 Fig. 5 The growth curve of recombinant strains E. coli W3110 and its mutants G1, I1, I2.

肖梦榕等 / 大肠杆菌 PTS 系统改造及重组菌生长性能测定 1569 表 3 摇瓶发酵参数比较 Table 3 Comparison of parameters in shake-flask experiments Strains Biomass (g/l) Maximum specific growth rate (g/(h L)) Consumption of

74±0.51 5.64 I1 3.48±0.22 0.34±0.01 14.07±1.52 3.61±0.26 4.04 I2 4.68±0.19 0.63±0.00 21.99±1.14 1.74±0.18 4.69 W3110 G1 I1 I2 I2 687 g W3110 1 156 g 1.7 PTS Glf 图 6 重组大肠杆菌 E.

50 g/l 7 [18] 3 I2 W3110 G1 I1 W3110 G1 I2 I1 ptsi glf I2 (103.2 g) W3110 (75.4 g) W3110 (7.03 g/(h L)) I2 (4.75 g/(h L)) 图 7 重组大肠杆菌 E. coli W3110 G1 I1 I2 在摇瓶中的生长代谢情况 Fig.

9 肖梦榕等 / 大肠杆菌 PTS 系统改造及重组菌生长性能测定 1569 表 3 摇瓶发酵参数比较 Table 3 Comparison of parameters in shake-flask experiments Strains Biomass (g/l) Maximum specific growth rate (g/(h L)) Consumption of glucose (g/l) Concentration of acetic acid (g/l) Specific consumption rate of glucose (g/(h L)) W ± ± ± ± G1 2.74± ± ± ± I1 3.48± ± ± ± I2 4.68± ± ± ± W3110 G1 I1 I2 I2 687 g W g 1.7 PTS Glf 图 6 重组大肠杆菌 E. coli W3110 I2 在 6 L 发酵罐中的生长情况 Fig. 6 The growth curve of recombinant strains E. coli W3110 and its mutants I2 in a 6 L bioreactor. 50 g/l 7 [18] 3 I2 W3110 G1 I1 W3110 G1 I2 I1 ptsi glf I2 (103.2 g) W3110 (75.4 g) W3110 (7.03 g/(h L)) I2 (4.75 g/(h L)) 图 7 重组大肠杆菌 E. coli W3110 G1 I1 I2 在摇瓶中的生长代谢情况 Fig. 7 Acetic acid production and glucose consumption of strains E. coli W3110 and its mutants G1, I1, I2., : E. coli W3110;, : E. coli G1;, : E. coli I1;, : E. coli I2. Solid shape: consumption curve of glucose; Hollow shape: accumulation curve of acetic acid. cjb@im.ac.cn

10 1570 ISSN CN /Q Chin J Biotech October 25, 2014 Vol.30 No.10 3 讨论 Red PTS EⅡCB Glc (ptsg) HPr (ptsi) 6 L ptsg ptsi PTS ptsi PTS ptsg G1 ptsi I1 (GalP) [19-21] PEP GalP H + [19-21] ptsi I1 G1 PTS EⅡ Man [22] PEP PEP E. coli W3110 ptsi Zymononas Mobilis Glf I2 I1 glf Glf GalP GalP glf I2 Glf-glk ( - ) ATP Glf PTS Glf PTS (PEP) TCA [9-12] Glf [14] Glf GalP Glf Chandran [23] Glf Glk PTS Glf PTS Glf Glk Glf Glk PTS (ptsh-i-crr) (27%) 15 g/l 10 L Glf PTS

11 肖梦榕等 / 大肠杆菌 PTS 系统改造及重组菌生长性能测定 1571 Glf Tang [24] ATCC 8739 PTS Glf Glk GalP Glf 0.7 g/(h L) GalP 30% Glf Glk 2.13 g/(h L) Glf PEP PTS GalP [8,23,25-26] Glf PTS Glf Glf REFERENCES [1] Matsuoka Y, Shimizu K. Importance of understanding the main metabolic regulation in response to the specific pathway mutation for metabolic engineering of Escherichia coli. Comp Struct Biotechnol J, 2013, 3(4): e [2] Xiao MR, Zhang L, Shi GY. Improvements of shikimic acid production in Escherichia coli with ideal metabolic modification in biosynthetic pathway. Acta Microbiol Sin, 2014, 1: 002 (in Chinese).,,.., 2014, 1: 002. [3] Gosset G. Improvement of Escherichia coli production strains by modification of the phosphoenolpyruvate: sugar phosphotransferase system. Microb Cell Fact, 2005, 4(1): 14. [4] Escalante A, Cervantes AS, Gosset G, et al. Current knowledge of the Escherichia coli phosphoenolpyruvate carbohydrate phosphotransferase system: peculiarities of regulation and impact on growth and product formation. Appl Microbiol Biotechnol, 2012, 94(6): [5] Gabor E, Göhler AK, Kosfeld A, et al. The phosphoenol pyruvate-dependent glucose-phosphotransferase system from Escherichia coli K-12 as the center of a network regulating carbohydrate flux in the cell. Eur J Cell Biol, 2011, 90(9): [6] Flores S, Gosset G, Flores N, et al. Analysis of carbon metabolism in Escherichia coli strains with an inactive phosphotransferase system by (13) C labeling and NMR spectroscopy. Metab Eng, 2002, 4(2): 124. [7] De Anda R, Lara AR, Hernández V, et al. Replacement of the glucose phosphotransferase transport system by galactose permease reduces acetate accumulation and improves process performance of Escherichia coli for recombinant protein production without impairment of growth rate. Metab Eng, 2006, 8(3): [8] Wang Q, Wu C, Chen T, et al. Expression of galactose permease and pyruvate carboxylase in Escherichia coli ptsg mutant increases the growth rate and succinate yield under anaerobic conditions. Biotechnol Lett, 2006, 28(2): [9] Snoep JL, Arfman N, Yomano LP, et al. Reconstruction of glucose uptake and phosphorylation in a glucose-negative mutant of Escherichia coli by using Zymomonas mobilis cjb@im.ac.cn

12 1572 ISSN CN /Q Chin J Biotech October 25, 2014 Vol.30 No.10 genes encoding the glucose facilitator protein and glucokinase. J Bacteriol, 1994, 176(7): [10] Weisser P, Krämer R, Sahm H, et al. Functional expression of the glucose transporter of Zymomonas mobilis leads to restoration of glucose and fructose uptake in Escherichia coli mutants and provides evidence for its facilitator action. J Bacteriol, 1995, 177(11): [11] Parker C, Barnell WO, Snoep JL, et al. Characterization of the Zymomonas mobilis glucose facilitator gene product (glf) in recombinant Escherichia coli: examination of transport mechanism, kinetics and the role of glucokinase in glucose transport. Mol Microbiol, 1995, 15(5): [12] Siedler S, Bringer S, Blank LM, et al. Engineering yield and rate of reductive biotransformation in Escherichia coli by partial cyclization of the pentose phosphate pathway and PTS-independent glucose transport. Appl Microbiol Biotechnol, 2012, 93(4): [13] Ren C, Chen T, Zhang J, et al. An evolved xylose transporter from Zymomonas mobilis enhances sugar transport in Escherichia coli. Microb Cell Fact, 2009, 8(66): 1 9. [14] Yi J, Draths KM, Li K, et al. Altered glucose transport and shikimate pathway product yields in E. coli. Biotechnol Prog, 2003, 19(5): [15] Baba T, Ara T, Hasegawa M, et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol, 2006, 2(1): [16] Liu SP, Xiao MR, Zhang L, et al. Production of L phenylalanine from glucose by metabolic engineering of wild type Escherichia coli W3110. Process Biochem, 2013, 48(3): [17] Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual. 3rd ed. Beijing: Science Press, 2002: Sambrook J, Russell DW..,. 3. :, 2002: [18] Qi YZ, Wang SX. Biochemical Reaction Kinetics and Bioreactor. 3rd Ed. Beijing: Chemical Industry Press, 2007: 8 40 (in Chinese) :, 2007: [19] Martínez K, de Anda R, Hernández G, et al. Coutilization of glucose and glycerol enhances the production of aromatic compounds in an Escherichia coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system. Microb Cell Fact, 2008, 7(1): [20] Lu J, Tang J, Liu Y, et al. Combinatorial modulation of galp and glk gene expression for improved alternative glucose utilization. Appl Microbiol Biotechnol, 2012, 93(6): [21] Hernández Montalvo V, Martínez A, Hernández Chavez G, et al. Expression of galp and glk in a Escherichia coli PTS mutant restores glucose transport and increases glycolytic flux to fermentation products. Biotechnol Bioeng, 2003, 83(6): [22] Zou YK, Zhou JZ, Sun X, et al. Construction of shikimic acid producing engineered Escherichia coli strains based on ptshicrr mutants. Microbiol China, 2011, 38(8): (in Chinese).,,,. PTS., 2011, 38(8): [23] Chandran SS, Yi J, Draths KM, et al. Phosphoenolpyruvate availability and the biosynthesis of shikimic acid. Biotechnol Prog, 2003, 19(3): [24] Tang J, Zhu X, Lu J, et al. Recruiting alternative glucose utilization pathways for improving succinate production. Appl Microbiol Biotechnol, 2013: 1 8. [25] Escalante A, Calderón R, Valdivia A, et al. Metabolic engineering for the production of shikimic acid in an evolved Escherichia coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system. Microb Cell Fact, 2010, 9(1): 21. [26] Li MM, Chen XZ, Zhou L, et al. Rational design and construction of an overproducing shikimic acid Escherichia coli by metabolic engineering. Chin J Biotech, 2013, 29(1): (in Chinese).,,,.., 2013, 29(1): ( 本文责编郝丽芳 )

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc 微生物学通报 Microbiology China tongbao@im.ac.cn Apr. 20, 2016, 43(4): 793 797 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.150504 高校教改纵横生物工程专业发酵课程群建设探索 * 赵宏宇