Journal of Biotechnology 129 (2007) 510–515 Genome-shuffling improved acid tolerance and l-lactic acid volumetric productivity in Lactobacillus rhamnosus Yuhua Wang a,b , Yan Li a , Xiaolin Pei a , Lei Yu a,b , Yan Feng a,∗ a Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, 2519 Jiefang Road, Changchun 130023, PR China b College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, PR China Received 15 September 2006; received in revised form 6 January 2007; accepted 16 January 2007 Abstract Genome shuffling is an efficient approach for the rapid improvement of industrially important microbial phenotypes. Here we improved the acid tolerance and volumetric productivity of an industrial strain Lactobacillus rhamnosus ATCC 11443 by genome shuffling. Five strains with subtle improvements in pH tolerance and volumetric productivity were obtained from the populations generated by ultraviolet irradiation and nitrosoguanidine mutagenesis, and then they were subjected for recursive protoplast fusion. A library that was more likely to yield positive colonies was created by fusing the lethal protoplasts obtained from both ultraviolet irradiation and heat treatments. After three rounds of genome shuffling, four strains that could grow at pH 3.6 were obtained. We observed 3.1- and 2.6-fold increases in lactic acid production and cell growth of the best performing at pH 3.8, respectively. The maximum volumetric productivity was 5.77 ± 0.05 g/l h when fermented with 10% glucose under neutralizing condition with CaCO 3 , which was 26.5 ± 1.5% higher than the wild type. © 2007 Elsevier B.V. All rights reserved. Keywords: Genome shuffling; Lactobacillus rhamnosus; l-Lactic acid; Acid tolerance; Productivity 1. Introduction Lactic acid is extensively used in food and pharmaceutical industries. It is increasingly used as feedstocks for potential biodegradable plastics from low-cost and renewable carbohy- drates (Boswell, 2001; Datta et al., 1995). Lactic acid has a chiral center and exists as d and l enantiomers. Enantiomeric purity is important for industrial uses because the physical properties of polylactide and the rate of biodegradation depend on the stereo- chemistry of the lactic acid molecule. The greater demand is for the l isomer (Tsuji, 2002). Microbial fermentation technology can select lactic acid isomers. The most important industrial microorganisms that produce lactic acid belong to the genera Lactobacillus (Benninga, 1990) and Rhizopus (Benninga, 1990). Rhizopus is initially used for l-lactic acid production because it directly consumes starch and gives high optical purify of the product. Recently, Lactobacillus strains have been particularly ∗ Corresponding author. Tel.: +86 431 88987975; fax: +86 431 88987975. E-mail address: yfeng@mail.jlu.edu.cn (Y. Feng). useful due to their high yield, high growth rates and can be genetically engineered for selective production of d or l optical isomers (Benthin and Villadsen, 1995; Kari et al., 2000; Lapierre et al., 1999; Gottschalk, 1985). L. rhamnosus is a homofermenter that produces solely l- lactic acid (Gottschalk, 1985). Further improvement of L. rhamnosus includes increases in volumetric productivity and acid tolerance. Lactic acid fermentation is a typical example of a product-inhibited bioconversion. Accumulation of lactic acid in an undissociated form inhibits both cell growth and production. The addition of Ca(OH) 2 , CaCO 3 , NaOH or NH 4 OH to neu- tralize the lactic acid is a conventional operation to minimize the negative effects of undissociated lactic acid accumulation in industrial processes (Porro et al., 1999; Madzingaido et al., 2002). However, the neutralization of lactic acid during fermen- tation has disadvantages that additional operations are required to regenerate undissociated lactic acid from its salt and to dis- pose of or recycle the neutralizing cation (Porro et al., 1999; Madzingaido et al., 2002). This complicates downstream pro- cess and yields large amounts of gypsum as a by-product which is normally dumped as waste (Madzingaido et al., 2002; U.S. 0168-1656/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2007.01.011