Production of Recombinant β-Galactosidase in Lactobacillus plantarum, using a pSIP-Based Food-Grade Expression System Numphon Thaiwong 1, a , Siwatt Thaiudom 1, b * , Dietmar Haltrich 2, c and Montarop Yamabhai 3, d 1 School of Food Technology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, Thailand 30000 2 Division of Food Biotechnology, Department of Food Sciences and Technology, BOKU University of Natural Resources and Applied Life Sciences Vienna, Austria 3 School of biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, Thailand 30000 a nthaiwong@hotmail.com, b thaiudomi@sut.ac.th, c dietmar.haltrich@buku.ac.at, d montarop@sut.ac.th Keywords: Food-grade expression systems, pSIP system, Lactic acid bacteria, β-galactosidases, Alanine racemase gene Abstract. Food-grade expression systems based on using food-grade microorganisms have been developed for the production of recombinant enzymes used in food applications. Lactic acid bacteria (LAB), especially Lactobacilli, have been widely used for various purposes in food and recognized as a promising host of food-grade enzyme production. In this study, the pSIP409 vectors, originally containing the erm gene, were used to replace this selection marker by the alr gene resulting in the production of the pSIP609 expression vector in L. planatarum. This vector could express high amounts of β-galactosidases, showing both high volumetric as well a specific enzymatic activity. Thus, the food-grade recombinant enzyme production in L. planatarum harboring pSIP609 was very fruitful and useful for food industries. Introduction Lactic acid bacteria (LAB) play important roles in foods, amongst others because they have been recognized as a food-grade additive. Several LAB, and especially Lactobacilli, have been developed as cell factories relating to an inducible gene expression for food applications in which they are used for the production of a range of interesting proteins. Such enzyme production can be obtained using the Nisin-Controlled Expression (NICE) or the pheromone-inducible (pSIP) systems, which are the well-known overexpression systems for LAB [1-5]. Furthermore, the vectors in the pSIP system permit all parts of the plasmid, to be easily exchanged and to be used amongst different LAB, especially Lactobacillus spp., while the plasmids in the NICE system are suitable mainly for Lactococcus spp. [1, 6]. Moreover, it was shown that the pSIP system resulted in higher levels of overexpressed enzyme than the NICE system. Thus, recently, the use of the pSIP system has been increasingly attractive in food applications. Yet, the pSIP system still has a major limitation when it is applied in foods because the erythromycin antibiotic resistance gene (erm) is used as a selection marker in the original system. Consequently, the erythromycin used for the cell selection stage of enzyme production might affect the microflora in human body, and the erm gene might be transferred to other organisms resulting in resistance to this antibiotic. To avoid the addition of undesirable antibiotics to the enzyme expression process, the alanine racemase gene (alr) can be used as a selection marker instead of the erm gene, which is safer in terms of human food consumption. Using pSIP vectors carrying the alr gene as selection marker have been successfully applied in complementation approaches both in Lactococci and Lactobacilli [7]. The alanine racemase enzyme is important for cell wall biosynthesis, especially in LAB. It converts L- alanine to D-alanine, which is an essential component for the growth and cell wall biosynthesis of prokaryotic cells [8]. Thus, using the alr gene as a selection marker together with the lacZ gene, Advanced Materials Research Vols. 931-932 (2014) pp 1518-1523 © (2014) Trans Tech Publications, Switzerland doi:10.4028/www.scientific.net/AMR.931-932.1518 All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of TTP, www.ttp.net. (ID: 202.28.41.16-30/04/14,09:34:33)