共表达透明颤菌血红蛋白对毕赤酵母产Β-甘露聚糖酶发酵过程的影响

doi:10.13560/j.cnki.biotech.bull.1985.2015.12.028

生物技术通报 ›› 2015, Vol. 31 ›› Issue (12): 193-199.doi: 10.13560/j.cnki.biotech.bull.1985.2015.12.028

共表达透明颤菌血红蛋白对毕赤酵母产Β-甘露聚糖酶发酵过程的影响

张晓龙¹,肖静¹,王瑞明¹,汪俊卿¹,王兴吉²,王正祥³

1. 齐鲁工业大学生物工程学院山东省微生物重点实验室，济南 250353；
2. 山东隆利特酶制剂有限公司, 临沂 276000；
3. 天津科技大学，天津 300222

收稿日期:2015-04-23 出版日期:2015-12-19 发布日期:2015-12-19
作者简介:张晓龙，男，硕士研究生，专业方向:微生物酶技术；E-mail:qingshuang0302@163.com
基金资助:
山东省轻工生物基产品清洁生产与炼制协同创新中心项目(03082001)

Effect of Co-expression of Vitreoscilla Hemoglobin on Expression of Β-mannanase in Pichia pastoris

Zhang Xiaolong¹, Xiao Jing¹, Wang Ruiming¹, Wang Junqing¹, Wang Xingji², Wang Zhengxiang³

1. Institute of Biological Engineering of Qilu University of Technology，Shandong Province Key Laboratory of Microbial Engineering，Jinan 250353
2. Linyi Longkete Enzyme Preparation Co. Ltd，Linyi 276000
3. Tianjin University of Science and Technology，Tianjin 300222

Received:2015-04-23 Published:2015-12-19 Online:2015-12-19

摘要/Abstract

摘要：

为了改善高密度发酵生产Β-甘露聚糖酶过程中的溶氧限制，提高Β-甘露聚糖酶产量，将VHb和Β--甘露聚糖酶基因置于AOX1启动子调控之下，进行VHb和Β--甘露聚糖酶基因在毕赤酵母中的共表达。经密码子优化合成VHb基因，插入表达载体pPICZαA，整合到Β--甘露聚糖酶工程菌中，通过G418和Zeocin抗性筛选共表达VHb基因的重组酵母工程菌。在30 L发酵罐水平上分析共表达VHb菌株(VHb⁺)与初始菌株(VHb^-)对Β--甘露聚糖酶表达的差异。结果显示，限氧条件下，VHb⁺菌株的Β--甘露聚糖酶的表达量比对照菌株VH b^-高90%，且透明颤菌血红蛋白提高了甲醇耐受性，缩短发酵周期约40 h。

关键词: 透明颤菌血红蛋白, &#x00392, -甘露聚糖酶, 毕赤酵母, 高密度发酵表达

Abstract:

In order to improve the limitation of dissolved oxygen and increase Β-mannanase production in the process of high-density fermentation, the Β-mannanase gene and Vitreoscilla hemoglobin gene(VHb)were co-expressed in Pichia pastoris under the control of AOX1 promoter. The VHb gene was synthesized by codon optimization and inserted into expression vector pPICZαA, then integrated into the engineering bacterium of Β-mannanase gene, P. pastoris GS115/Ppic9K-Β-MAN. The recombinant P. pastoris of co-expressing VHb was selected by G418 and Zeocin resistance screening. Finally, the discrepancies in Β-mannanase expression between the co-expressed strain VHb⁺ and initial strain VHb^-were analyzed in 30 L fermenter. The results showed as below, compared with the controlled strain, the expression of Β-mannanase by VHb⁺ strain was increased by 90% under oxygen-limited condition. Furthermore, Vitreoscilla hemoglobin was improved on the methanol tolerance, and fermentation period was shorted about 40 h, which could be an important industrial value.

Key words: Vitreoscilla hemoglobin, &#x00392, -mannanase, Pichia pastoris, high-density fermentation

张晓龙,肖静,王瑞明,汪俊卿,王兴吉,王正祥. 共表达透明颤菌血红蛋白对毕赤酵母产Β-甘露聚糖酶发酵过程的影响[J]. 生物技术通报, 2015, 31(12): 193-199.

Zhang Xiaolong, Xiao Jing, Wang Ruiming, Wang Junqing, Wang Xingji, Wang Zhengxiang. Effect of Co-expression of Vitreoscilla Hemoglobin on Expression of Β-mannanase in Pichia pastoris[J]. Biotechnology Bulletin, 2015, 31(12): 193-199.

参考文献

[1]Simon C, Peter B, Thomas K, et al. Human chymotrypsinogen B production with Pichia pastoris by integrated development of fermentation and downstream processing. Part 1[J]. Fermentation Biotechnology Progress, 2001, 17(8):495-502.
[2]Wei XX, Chen GQ. Applications of the VHb gene vgb for improved microbial fermentation processes[J]. Methodds in Enzymology, 2008, 436(5):273-287.
[3]Zhang L, Li YJ, Wang ZN, et al. Recent developments and future prospects of Vitreoscilla hemoglobin application in metabolic engineering[J]. Biotechnol Adv, 2007, 25(2):123-136.
[4]Chen HX, Chu J, Zhang SL, et, al. Intracellular expression of Vitreoscilla hemoglobin improves S-adenosylmethionine production in a recombinant Pichia pastoris[J]. Appl Microbiol Biotechnol, 2007, 74(6):1205-1212.
[5]Khosravi M, Webster DA, Stark BC. Presence of the bacterial hemoglobin gene improves α-amylase production of a recombinant Escherichia coli strain[J]. Plasmid, 1990, 24:190-194.
[6]王清路, 张锐, 倪万潮. 透明颤菌血红蛋白基因vgb和腈水解酶基因bxn在毕赤酵母中的表达研究[J]. 生物工程学报, 2004, 20(5):730-735.
[7]李兵, 李术娜, 周艳芬. 透明颤菌血红蛋白基因在产黄青霉中的克隆与表达[J]. 中国抗生素杂志, 2006, 31(7):400-402.
[8]辛总秀. Β-甘露聚糖酶在家禽饲料中应用的研究进展[J]. 畜牧与饲料料学, 2011, 32(5):28-30.
[9]余河斌, 余绍文, 等. 血红蛋白基因在产CBHⅡ毕赤酵母工程菌中的表达[J]. 生物技术, 2007, 17(4):25-29.
[10]林俊涵. 毕赤酵母高密度发酵工艺的研究[J]. 中国生物工程杂志, 2009, 29(5):120-125.
[11]Zhang L, Li YJ, Wang ZN, et al. Recent developments and future prospects of Vitreoscilla hemoglobin application in metabolic engineering[J]. Biotechnology Advances, 2006, 11(3):123-136.
[12]孟志刚, 张锐, 陈毓荃, 等. 利用透明颤菌血红蛋白基因vgb改造毕赤酵母分泌性表达载体pPIC9K的研究[J]. 西北农业学报, 2005, 14(4):166-170.
[13]唐辉桂, 黄火清, 罗会颖, 等. 利用透明颤菌血红蛋白在低氧条件下提高毕赤酵母中植酸酶的表达[J]. 中国农业科技导报, 2008, 10(3):84-89.
[14]Rinaldi AC, Bonamore A, Macone A, et al. Interaction of Vitreoscilla hemoglobin with membrane lipeds[J]. Biochemistry, 2006, 45(13):4069-4076.
[15] Wu JM, Hsu TA, Lee CK. Expression of the gene coding for bacterial hemoglobin improves Β-galactosidase production in a recombinant Pichia pastoris[J]. Biotechnol Lett, 2003, 25(17):1457-1462.
[16]Chien LJ, Lee CK. Expression of bacterial hemoglobin in the yeast, Pichia pastoris, with a low O₂-induced promoter[J]. Biotechnol Lett, 2005, 27(19):1591-1497.
[17]卢俊裕, 林影, 梁书利, 叶燕锐. 共表达透明颤菌血红蛋白提高脂肪酶在毕赤酵母中的表达[J]. 现代食品科技, 2014, 30(6):110-115.
[18]Hu XQ, Chu J, Zhang SL, et al. A nobel feeding strategy during the production phase for enhancing the enzymatic synthesis of S-adenosyl-L-methionine by methylotrophic Pichia pastoris[J]. Enzyme Microb Techinol, 2007, 40(4):669-674.
[19]Chen RZ, Bailey JE. Energetic Effect of Vitreoscilla hemoglobin expression in Escherichia coli:An online 31PNMR and a saturation transfer study[J]. Biotechnology Progress, 1994, 10(7):360-364.
[20]Rinaldi AC, Bonamore A, Macone A, et al. Interaction of Vitreoscilla hemoglobin with membrane lipids[J]. Biochemistry, 2006, 45(13):4069-4076.
[21]Gekil H, Stark BC, Webster DA. Cell growth and oxygen uptake of Escherichia coli and Pseudomonas aeruginosa aer differently effected by the genetically engineered Vitreoscilla hemoglobin gene[J]. J Biotechinol, 2001, 85(9):57-66.
[22]Hikmet G, Salih G, Huseyin K, et al. Genetic engineering of Enterobacter aerogines with the Vireoscilla hemoglobin:Cell growth, survival, and antioxidant enzyme status under oxidative stress[J]. Research in Mirobiol, 2003, 154(6):425-431.
[23]Mayson BE, Douglas G. Effects of methanol concentration on expression leves of recombinant protein in fed-batch cultures of Pichia methanolica[J]. Biotechnol Bioeng, 2003, 81(3):291-298.
[24]Hong F, Meinander NQ, Joensson LJ. Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris[J]. Biotechnol Bioeng, 2002, 79(4):438-449.

共表达透明颤菌血红蛋白对毕赤酵母产Β-甘露聚糖酶发酵过程的影响

Effect of Co-expression of Vitreoscilla Hemoglobin on Expression of Β-mannanase in Pichia pastoris

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