Quantitative transcript analysis of the inducible expression system pSIP: comparison of the overexpression of <it>Lactobacillus </it>spp. β-galactosidases in <it>Lactobacillus plantarum</it>

• Main Authors: Eijsink Vincent GH, Mathiesen Geir, Schmelzer Philipp, Nguyen Thu-Ha, Maischberger Thomas, Nguyen Tien-Thanh, Haltrich Dietmar, Peterbauer Clemens K
• Format: Article
• Language: English
• Published: BMC 2011-06-01
• Series: Microbial Cell Factories
• Online Access: http://www.microbialcellfactories.com/content/10/1/46

<p>Abstract</p> <p>Background</p> <p>Two sets of overlapping genes, <it>lacLMReu </it>and <it>lacLMAci</it>, encoding heterodimeric β-galactosidases from <it>Lactobacillus reuteri </it>and <it>Lactobacillus acidophilus</it>, respectively, have previously been cloned and expressed using the pSIP vector system and <it>Lactobacillus plantarum </it>WCSF1 as host. Despite the high similarity between these <it>lacLM </it>genes and the use of identical cloning and expression strategies, strains harboring <it>lacLMReu </it>produced about twenty-fold more β-galactosidase than strains containing <it>lacLMAci</it>.</p> <p>Results</p> <p>In this study, the plasmid copy numbers (PCN) of expression vectors pEH9R (<it>lacLMReu</it>) and pEH9A (<it>lacLMAci</it>) as well as the transcription levels of both <it>lacLM </it>genes were compared using quantitative PCR methods. Analyses of parallel fermentations of <it>L. plantarum </it>harboring either pEH9R or pEH9A showed that the expression plasmids were present in similar copy numbers. However, transcript levels of <it>lacLM </it>from <it>L. reuteri </it>(pEH9R) were up to 18 times higher than those of <it>lacLM </it>from <it>L. acidophilus </it>(pEH9A). As a control, it was shown that the expression levels of regulatory genes involved in pheromone-induced promoter activation were similar in both strains.</p> <p>Conclusion</p> <p>The use of identical expression strategies for highly similar genes led to very different mRNA levels. The data indicate that this difference is primarily caused by translational effects that are likely to affect both mRNA synthesis rates and mRNA stability. These translational effects thus seem to be a dominant determinant for the success of gene expression efforts in lactobacilli.</p>