title: Heterologous Expression of Mannanase and Developing a New Reporter Gene System in Lactobacillus casei and Escherichia coli.
abstract: Reporter gene systems are useful for studying bacterial molecular biology, including the regulation of gene expression and the histochemical analysis of protein products. Here, two genes, β-1,4-mannanase (manB) from Bacillus pumilus and β-glucuronidase (gusA) from Escherichia coli K12, were cloned into the expression vector pELX1. The expression patterns of these reporter genes in Lactobacillus casei were investigated by measuring their enzymatic activities and estimating their recombinant protein yields using western blot analysis. Whereas mannanase activity was positively correlated with the accumulation of ManB during growth, GusA activity was not; western blot analysis indicated that while the amount of GusA protein increased during later growth stages, GusA activity gradually decreased, indicating that the enzyme was inactive during cell growth. A similar trend was observed in E. coli JM109. We chose to use the more stable mannanase gene as the reporter to test secretion expression in L. casei. Two pELX1-based secretion vectors were constructed: one carried the signal peptide of the unknown secretion protein Usp45 from Lactococcus lactis (pELSH), and the other contained the full-length SlpA protein from the S-layer of L. acidophilus (pELWH). The secretion of ManB was detected in the supernatant of the pELSH-ManB transformants and in the S-layer of the cell surface of the pELWH-ManB transformants. This is the first report demonstrating that the B. pumilus manB gene is a useful reporter gene in L. casei and E.coli.
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.|State Key Laboratory of Dairy Biotechnology, Technology Center of Bright Dairy & Food Co., Ltd., 1518 Jiangchang Road (W), Shanghai, 200436, China.
authors.forename: Jinzhong
authors.initials: J
authors.lastname: Lin
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.
authors.forename: Yexia
authors.initials: Y
authors.lastname: Zou
authors.affiliation: State Key Laboratory of Dairy Biotechnology, Technology Center of Bright Dairy & Food Co., Ltd., 1518 Jiangchang Road (W), Shanghai, 200436, China.
authors.forename: Chengjie
authors.initials: C
authors.lastname: Ma
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.|Department of Biology, University of Copenhagen, Biocenter, Ole Maaloes Vej 5, DK-2200, Copenhagen N, Denmark.
authors.forename: Qunxin
authors.initials: Q
authors.lastname: She
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.
authors.forename: Yunxiang
authors.initials: Y
authors.lastname: Liang
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.
authors.forename: Zhengjun
authors.initials: Z
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.
authors.forename: Xiangyang
authors.initials: X
authors.lastname: Ge
mesh_terms: D001407:Bacillus / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D005966:Glucuronidase / Q000235:genetics* / Q000378:metabolism; D007780:Lacticaseibacillus casei / Q000235:genetics* / Q000378:metabolism; D008361:Mannosidases / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D008361:Mannosidases; D005966:Glucuronidase; C041867:endo-1,4-beta-D-mannanase