RESEARCH High-Level Expression of Heme-Dependent Catalase Gene katA from Lactobacillus Sakei Protects Lactobacillus Rhamnosus from Oxidative Stress Haoran An • Hui Zhou • Ying Huang • Guohong Wang • Chunguang Luan • Jing Mou • Yunbo Luo • Yanling Hao Ó Springer Science+Business Media, LLC 2010 Abstract Lactic acid bacteria (LAB) are generally sen- sitive to hydrogen peroxide (H 2 O 2 ), Lactobacillus sakei YSI8 is one of the very few LAB strains able to degrade H 2 O 2 through the action of a heme-dependent catalase. Lactobacillus rhamnosus strains are very important probi- otic starter cultures in meat product fermentation, but they are deficient in catalase. In this study, the effect of heter- ologous expression of L. sakei catalase gene katA in L. rhamnosus on its oxidative stress resistance was tested. The recombinant L. rhamnosus AS 1.2466 was able to decompose H 2 O 2 and the catalase activity reached 2.85 lmol H 2 O 2 /min/10 8 c.f.u. Furthermore, the expres- sion of the katA gene in L. rhamnosus conferred enhanced oxidative resistance on the host. The survival ratios after short-term H 2 O 2 challenge were increased 600 and 10 4 -fold at exponential and stationary phase, respectively. Further, viable cells were 100-fold higher in long-term aerated cultures. Simulation experiment demonstrated that both growth and catalase activity of recombinant L. rhamnosus displayed high stability under environmental conditions similar to those encountered during sausage fermentation. Keywords Heme-dependent catalase Á Heterologous expression Á Lactobacillus rhamnosus Á Oxidative stress Introduction Lactic acid bacteria (LAB) are widely used in a large variety of food fermentations including dairy, meat and vegetable products. Their main functions are to produce acid which lowers the pH and prevents the growth of spoilage bacteria [1]. Meanwhile, their secondary metabolites, such as acetic acid, ethanol and exopolysaccharide, are beneficial to the development of a desirable flavor and texture of the fer- mented products [2]. It has been reported that L. rhamnosus strains, for example, L. rhamnosus GG and E-97800, are suitable for use as the probiotic starter cultures in fermented dry sausage [3]. Furthermore, L. rhamnosus is commercially used for enhancing human gastro-intestinal health and immune system [4]. For this reason, a new market for the fermented foods which contain L. rhamnosus has rapidly been emerging in recent years. In industrial processes, LAB often suffers from oxidative stress when reactive oxygen species (ROS) accumulate in the cell. ROS originate from partial reduction of molecular oxygen to superoxide (O 2 - ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radical (OH • ), which can cause damage to macromolecules, such as proteins, lipids and nucleotides, leading to the growth arrest and cell death [5, 6]. Catalase plays an important role in reducing oxidative stress by decomposing H 2 O 2 . Until now, two distinct families of catalase including the heme-dependent and the manganese- containing catalase have been discovered in some LAB, such as Lactobacillus, Pediococcus, and Leuconostoc [7–11]. In recent years, in order to improve the oxidative stress resistance of LAB deficient in catalase activity, the strategy used was to express the catalase gene from cata- lase-positive Lactobacillus and Bacillus subtilis in heter- ologous hosts, which greatly improved the survival of modified strains in oxidative stress conditions [7, 12–14]. H. An Á H. Zhou Á Y. Huang Á G. Wang Á C. Luan Á J. Mou Á Y. Luo Á Y. Hao (&) Key Laboratory of Functional Dairy of Ministry of Education of the People’s Republic of China & Municipal Government of Beijing, College of Food Science & Nutritional Engineering, China Agricultural University, 17 Qing Hua East Road, Hai Dian District, Beijing 100083, China e-mail: haoyl@cau.edu.cn Mol Biotechnol DOI 10.1007/s12033-010-9254-9