Construction of a Food-Grade Expression Vector Based on pMG36e by Using an α-Galactosidase Gene as a Selectable Marker

doi:10.1016/S2095-3119(13)60625-5

Journal of Integrative Agriculture ›› 2014, Vol. 13 ›› Issue (8): 1802-1808.DOI: 10.1016/S2095-3119(13)60625-5

Construction of a Food-Grade Expression Vector Based on pMG36e by Using an α-Galactosidase Gene as a Selectable Marker

GU Xin-xi, TAN Jian-xin, TIAN Hong-tao, ZHANG Yu-lan, LUO Yun-bo , GUO Xing-hua

1、College of Food Science and Technology, Agricultural University of Hebei, Baoding 071001, P.R.China
2、College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, P.R.China
3、Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, P.R.China

收稿日期:2013-04-17 出版日期:2014-08-01 发布日期:2014-08-02
通讯作者: TIAN Hong-tao, Mobile: 13126883987, E-mail: tht631022@163.com
作者简介:GU Xin-xi, Tel: +86-312-7528423, E-mail: helloguxinxi@163.com; TAN Jian-xin, Tel: +86-312-7528180, E-mail: jianxintan@sina.com
基金资助:
This work was supported by the National High-Tech R&D Program of China (863 Program, 2006AA10Z317).

Construction of a Food-Grade Expression Vector Based on pMG36e by Using an α-Galactosidase Gene as a Selectable Marker

GU Xin-xi, TAN Jian-xin, TIAN Hong-tao, ZHANG Yu-lan, LUO Yun-bo , GUO Xing-hua

1、College of Food Science and Technology, Agricultural University of Hebei, Baoding 071001, P.R.China
2、College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, P.R.China
3、Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, P.R.China

Received:2013-04-17 Online:2014-08-01 Published:2014-08-02
Contact: TIAN Hong-tao, Mobile: 13126883987, E-mail: tht631022@163.com
About author:GU Xin-xi, Tel: +86-312-7528423, E-mail: helloguxinxi@163.com; TAN Jian-xin, Tel: +86-312-7528180, E-mail: jianxintan@sina.com
Supported by:
This work was supported by the National High-Tech R&D Program of China (863 Program, 2006AA10Z317).

摘要/Abstract

摘要： Construction of a food-grade expression vector for application to lactic acid bacteria (LAB) is of importance for dairy fermentation system. An α-galactosidase (aga) gene encoding an enzyme degrading melibiose was amplified by PCR from the plasmid pRAF800 of Lactococcus lactis NZ9000. The aga gene was introduced into pMG36e to substitute the primary antibiotic selectable marker of pMG36e, resulting in construction of a new food-grade expression vector pMG36-aga. To testify the expression efficiency of exogenous gene in pMG36-aga, a 1.5 kb long α-amylase (amy) gene from Bacillus licheniformis was cloned by PCR and introduced into the plasmid pMG36-aga. The resultant plasimd pMG36-aga-amy was transformed into L. lactis ML23 by electroporation. The positive clones were selected with the medium containing melibiose as the sole carbon source. The selection efficiency of aga was 8.71×103 CFU with a standard deviation of 9.1×102 CFU mg-1 DNA of pMG36-aga. Furthermore, the SDS-PAGE analysis showed that the pMG36-aga-amy expressed a 56.4 kDa protein which was the same as the putative molecular weight of α-amylase. The starch plate assay also indicated that L. lactis ML23 displayed high activity of α-amylase by expressing of amy gene of pMG36-aga-amy.

关键词: food-grade expression vector , Lactococcus lactis , &alpha, -galactosidase gene , amylase gene , pMG36e

Abstract: Construction of a food-grade expression vector for application to lactic acid bacteria (LAB) is of importance for dairy fermentation system. An α-galactosidase (aga) gene encoding an enzyme degrading melibiose was amplified by PCR from the plasmid pRAF800 of Lactococcus lactis NZ9000. The aga gene was introduced into pMG36e to substitute the primary antibiotic selectable marker of pMG36e, resulting in construction of a new food-grade expression vector pMG36-aga. To testify the expression efficiency of exogenous gene in pMG36-aga, a 1.5 kb long α-amylase (amy) gene from Bacillus licheniformis was cloned by PCR and introduced into the plasmid pMG36-aga. The resultant plasimd pMG36-aga-amy was transformed into L. lactis ML23 by electroporation. The positive clones were selected with the medium containing melibiose as the sole carbon source. The selection efficiency of aga was 8.71×103 CFU with a standard deviation of 9.1×102 CFU mg-1 DNA of pMG36-aga. Furthermore, the SDS-PAGE analysis showed that the pMG36-aga-amy expressed a 56.4 kDa protein which was the same as the putative molecular weight of α-amylase. The starch plate assay also indicated that L. lactis ML23 displayed high activity of α-amylase by expressing of amy gene of pMG36-aga-amy.

Key words: food-grade expression vector , Lactococcus lactis , α-galactosidase gene , amylase gene , pMG36e

GU Xin-xi, TAN Jian-xin, TIAN Hong-tao, ZHANG Yu-lan, LUO Yun-bo , GUO Xing-hua. Construction of a Food-Grade Expression Vector Based on pMG36e by Using an α-Galactosidase Gene as a Selectable Marker[J]. Journal of Integrative Agriculture, 2014, 13(8): 1802-1808.

参考文献

Abdullah A M, Sugimoto S, Higashi C, Matsumoto S, Sonomoto K. 2010. Improvement of multiple-stress tolerance and lactic acid production in Lactococcus lactisNZ9000 under conditions of thermal stress by heterologous expression of Escherichia coli DnaK. Applied andEnvironmental Microbiology, 76, 4277-4285

Boucher I, Parrot M, Gaudreau H, Champagne C P,Vadeboncoeur C, Moineau S. 2002. Novel food-gradeplasmid vector based on melibiose fermentation for thegenetic engineering of Lactococcus lactis. Applied andEnvironmental Microbiology, 68, 6152-6161

Bron P A, Benchimol M G, Lambert J, Palumbo E, DeghorainM, Delcour J, de Vos W M, Kleerebezem M, Hols P.2002. Use of the alr gene as a food-grade selectionmarker in lactic acid bacteria. Applied and EnvironmentalMicrobiology, 68, 5663-5670

Brurberg M B, Haandrikman A J, Leenhouts K J, Venema G,Nes I F. 1994. Expression of a chitinase gene from Serratiamarcescens in Lactococcus lactis and Lactobacillus plantarum. Applied and Environmental Microbiology,42, 108-115

Casalta E, Montel M C. 2008. Safety assessment of dairymicroorganisms: The Lactococcus genus. InternationalJournal of Food Microbiology, 126, 271-273

Durmaz E, Higgins D L, Klaenhammer T R. 1992. Molecularcharacterization of a second abortive phage resistance genepresent in Lactococcus lactis subsp. lactis ME2. Journalof Bacteriology, 174, 7463-7469

van de Guchte M, Kodde J, Vossen J M, Kok J, Venema G. 1990. Heterologous gene expression in Lactococcus lactis subsp. lactis: Synthesis, secretion, and processingof the Bacillus subtilis neutral protease. Applied andEnvironmental Microbiology, 56, 2606-2011

van de Guchte M, van der Vossen J M, Kok J, Venema G.(1989). Construction of a lactococcal expression vector:expression of hen egg white lysozyme in Lactococcus lactissubsp. lactis. Applied and Environmental Microbiology,55, 224-228

Guo Q, Zhang W, Ma L L, Chen Q H, Chen J C, Zhang H B,Ruan H, He G Q. 2010. A food-grade industrial armingyeast expressing β-1,3-1,4-glucanase with enhancedthermal stability Journal of Zhejiang University (ScienceB), 11, 41-51

Haaber J, Moineau S, Hammer K. 2009a. Activation andtransfer of the chromosomal phage resistance mechanismAbiV in Lactococcus lactis. Applied and EnvironmentalMicrobiology, 75, 3358-3361

Haaber J, Rousseau G M, Hammer K, Moineau S. 2009b.Identification and characterization of the phage gene sav, involved in sensitivity to the lactococcal abortiveinfection mechanism AbiV. Applied and EnvironmentalMicrobiology, 75, 2484-2494

Jeong D W, Choi Y C, Lee J M, Kim J H, Lee J H, Kim K H,Lee H J. 2006. Isolation and characterization of promotersfrom Lactococcus lactis ssp. cremoris LM0230. FoodMicrobiology, 23, 82-89

Labrie S, Bart C, Vadeboncoeur C, Moineau S. 2005. Use ofan α-galactosidase gene as a food-grade selection markerfor Streptococcus thermophilus. Journal of Dairy Science,88, 2341-2347

Laemmli U K. 1970. Cleavage of structural proteins duringthe assembly of the head of bacteriophage T4. Nature,227, 680-685

Machielsen R, Siezen R J, van Hijum S A, van HylckamaVlieg J E. 2011. Molecular description and industrial potential of Tn6098 conjugative transfer conferring alpha-galactoside metabolism in Lactococcus lactis. Applied andEnvironmental Microbiology, 77, 555-563

McAuliffe O, Hill C, Ross R P. 2000. Identification andoverexpression of ltnl, a novel gene which confers immunity to the two-component lantibiotic lacticin 3147.Microbiology, 146, 129-138

Michelsen O, Hansen F G, Albrechtsen B, Jensen P R. 2010 The MG1363 and IL1403 laboratory strains of Lactococcus lactis and several dairy strains are diploid. Journal of Bacteriology, 192, 1058-1065

Miller G L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 3, 426-428

Park J Y, Jung J H, Seo D H, Ha S J, Yoon J W, Kim Y C, Shim J H, Park C S. 2010. Microbial production of palatinose through extracellular expression of a sucrose isomerase from Enterobacter sp. FMB-1 in Lactococcus lactis MG1363 Bioresource Technology, 101, 8828-8833

Platteeuw C, Van A B I, Van S S, Maarten V W. 1996. Food- grade cloning and expression system for Lactococcus lactis. Applied and Environmental Microbiology, 62, 1008-1013

Raha A R, Chang L Y, Sipat A, Yusoff K, Haryanti T. 2006. Expression of a thermostable xylanase gene from Bacillus coagulans ST-6 in Lactococcus lactis Letters in Applied Microbiology, 42, 210-214

Sridhar V R, Smeianov V V, Steele J L. 2006. Construction and evaluation of food-grade vectors for Lactococcus lactis using aspartate aminotransferase and alpha-galactosidase as selectable markers. Journal of Applied Microbiology, 101, 161-171

Takala M, Saris P E. 2002. A food-grade cloning vector for lactic acid bacteria based on the nisin immunity gene nisI. Applied and Environmental Microbiology, 59, 467-471

Tian H T, Ma L H, Zhang L F, Gu X X, Guo X H, Luo Y B. 2011. Cloning and heterologous expression of the plasmid encoded shsp gene of Streptococcus thermophilus isolated from Chinese dairy. African Journal of Biotechnology, 10, 1233-1245

Wang C, Zhang C W, Liu H C, Yu Q, Pei X F. 2008. Non- fusion and fusion expression of beta-galactosidase from Lactobacillus bulgaricus in Lactococcus lactis. Biomedical and Environmental Sciences, 21, 389-397

Willemoës M, Kilstrup M, Roepstorff P, Hammer K. 2002. Proteome analysis of a Lactococcus lactis strain overexpressing gapA suggests that the gene product is an auxiliary glyceraldehyde 3-phosphate dehydrogenase. Proteomics, 2, 1041-1046

Zhang W, Wang C, Huang C, Yu Q, Liu H, Zhang C, Pei X. 2011. Construction and expression of food-grade β-galactosidase gene in Lactococcus Lactis. Current Microbiology, 62, 639-644.

[1]	ZENG Qiu-feng, YANG Xia, ZHENG Ping, ZHANG Ke-ying, LUO Yu-heng, DING Xue-mei, BAI Shi-ping, WANG Jian-ping, XUAN Yue, SU Zhuo-wei. Effects of low ambient temperatures and dietary vitamin C supplementation on pulmonary vascular remodeling and hypoxic gene expression of 21-d-old broilers[J]. Journal of Integrative Agriculture, 2016, 15(1): 183-190.
[2]	JIANG Dai-xun, ZHANG Mei-hua, ZHANG Qian, CHEN Yi-shan, MA Wen-jing, WU Wei-peng, MU Xiang. Influence of gallic acid on porcine neutrophils phosphodiesterase 4, IL-6, TNF-α and rat arthritis model[J]. Journal of Integrative Agriculture, 2015, 14(4): 758-764.

Construction of a Food-Grade Expression Vector Based on pMG36e by Using an α-Galactosidase Gene as a Selectable Marker

Construction of a Food-Grade Expression Vector Based on pMG36e by Using an α-Galactosidase Gene as a Selectable Marker

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 2

编辑推荐

Metrics