Please cite this article in press as: Wang Z, et al. Metabolic engineering of Propionibacterium freudenreichii subsp. shermanii for enhanced propionic acid fermentation: Effects of overexpressing three biotin-dependent carboxylases. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.11.012 ARTICLE IN PRESS G Model PRBI-10287; No. of Pages 11 Process Biochemistry xxx (2014) xxx–xxx Contents lists available at ScienceDirect Process Biochemistry jo ur nal home p age: www.elsevier.com/locate/procbio Metabolic engineering of Propionibacterium freudenreichii subsp. shermanii for enhanced propionic acid fermentation: Effects of overexpressing three biotin-dependent carboxylases Zhongqiang Wang a , Meng Lin a , Liqun Wang a,b , Ehab M. Ammar a , Shang-Tian Yang a,∗ a William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, 140 W 19th Ave., Columbus, OH43210, United States b School of Pharmaceutical Engineering and Life Sciences, Changzhou University, 1 Ge Hu Road, Jiangsu 213164, China a r t i c l e i n f o Article history: Received 19 May 2014 Received in revised form 17 November 2014 Accepted 22 November 2014 Available online xxx Keywords: Propionic acid fermentation Propionibacteria Metabolic engineering Methylmalonyl-CoA carboxyltransferase Methylmalonyl-CoA decarboxylase Pyruvate carboxylase a b s t r a c t Bio-based propionic acid production by propionibacteria is increasingly attractive because of its potential to replace petroleum-based chemical synthesis processes. Metabolic engineering of Propionibacterium freudenreichii subsp. shermanii DSM 4902 for enhanced propionic acid fermentation is made possible by the recently available whole genome sequence and genetic engineering tools. Pyruvate carboxylase (PYC), methylmalonyl-CoA decarboxylase (MMD), and methylmalonyl-CoA carboxyltransferase (MMC) are three biotin-dependent carboxylases in the dicarboxylic acid pathway controlling the carbon flux in the Wood–Werkman cycle. These carboxylases were overexpressed in P. shermanii to study their effects on propionic acid fermentation in serum bottles with glucose and glycerol as substrates. Com- pared to the wild-type, the mutants overexpressing MMC and MMD showed a significant increase in the metabolic fluxes toward the biosynthesis of propionic acid, against acetic and succinic acids, with significantly increased yield (up to 14% increase) and productivity (up to 17% increase), especially in the co-fermentation of glycerol and glucose. On the other hand, the mutant overexpressing PYC grew slower, produced more succinate, and had a lower production of propionate (up to 12% decrease in productivity). This is the first study demonstrating enhanced propionic acid production by overexpressing MMC and MMD in propionibacteria. © 2014 Elsevier Ltd. All rights reserved. 1. Introduction Propionibacterium freudenreichii subsp. shermanii, a Gram- positive, facultative anaerobic, rod-shaped bacterium, has long been used in Swiss-type cheese ripening and vitamin B 12 manu- facturing [1]. It has also been extensively studied for production of value-added products, including propionic acid [2,3], n-propanol [4], trehalose [5], bifidogenic growth stimulator [6], vitamin B 12 [7], and porphyrin [8]. Propionibacteria is able to convert various carbon sources, including glucose, xylose, lactose, sucrose, glycerol, and lactic acid, to propionic acid as the major fermentation product with acetic and succinic acids as by-products. Propionic acid and its salts are mainly used as preservatives in animal feed, grains, and human food. Currently, propionic acid is almost exclusively pro- duced through petrochemical processes. Although there are high interests to produce bio-based propionic acid by fermentation with ∗ Corresponding author. Tel.: +1 614 292 6611; fax: +1 614 292 3769. E-mail address: yang.15@osu.edu (S.-T. Yang). propionibacteria such as P. shermanii with the GRAS status and pro- biotic properties [3,9], propionic acid fermentation is limited by its relatively low productivity, yield, and product purity. Metabolic engineering is a powerful approach widely used to improve micro- bial strains for fermentation production of chemicals. Several shuttle vectors have been developed and used to express genes in propionibacteria [10–12], and the genomic sequences of several dairy propionibacteria strains have also been published recently [1,9,13]. However, to date very little has been done on metabolic engineering of propionibacteria [4,14,15] because of the limited genetic engineering tools and difficulty in transforming propioni- bacteria, especially P. acidipropionici [11]. Three biotin-dependent carboxylases, pyruvate carboxylase (PYC), methylmalonyl-CoA carboxyltransferase (MMC), and methylmalonyl-CoA decarboxylase (MMD), play important roles in the metabolism of propionibacteria. MMC and PYC are two enzymes around the pyruvate node in the dicarboxylic acid path- way of propionibacteria (Fig. 1). Both of them carboxylate pyruvate to form oxaloacetate, which is then converted successively into malic, fumaric, succinic, and eventually propionic acids. Thus, they http://dx.doi.org/10.1016/j.procbio.2014.11.012 1359-5113/© 2014 Elsevier Ltd. All rights reserved.