Journal of Biotechnology 157 (2012) 633–640 Contents lists available at ScienceDirect Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec Production of 3-hydroxypropionic acid via malonyl-CoA pathway using recombinant Escherichia coli strains Chelladurai Rathnasingh a , Subramanian Mohan Raj a,b,c , Youjin Lee a , Christy Catherine a , Somasundar Ashok a , Sunghoon Park a,∗ a Department of Chemical and Biomolecular Engineering, Pusan National University, Busan 609-735, Republic of Korea b Department of Biotechnology and Chemical Engineering, Kalasalingam University, TN 626-190, India c Center for Bioprocessing Research and Development, South Dakota School of Mines & Technology, Rapid City, SD 57701, USA article info Article history: Received 31 January 2011 Received in revised form 14 June 2011 Accepted 17 June 2011 Available online 23 June 2011 Keywords: 3-hydroxypropionic acid Malonyl-CoA Malonyl-CoA reductase Acetyl-CoA carboxylase Transhydrogenase Chloroflexus aurantiacus abstract Malonyl-CoA is an intermediary compound that is produced during fatty acid metabolism. Our study aimed to produce the commercially important platform chemical 3-hydroxypropionic acid (3-HP) from its immediate precursor malonyl-CoA by recombinant Escherichia coli strains heterologously expressing the mcr gene of Chloroflexus aurantiacus DSM 635, encoding an NADPH-dependent malonyl-CoA reduc- tase (MCR). The recombinant E. coli overexpressing mcr under the T5 promoter showed MCR activity of 0.015 U mg -1 protein in crude cell extract and produced 0.71 mmol/L of 3-HP in 24 h in shake flask cultivation under aerobic conditions with glucose as the sole source of carbon. When acetyl-CoA car- boxylase and biotinilase, encoded by the genes accADBCb (ACC) of E. coli K-12 were overexpressed along with MCR, the final 3-HP titer improved by 2-fold, which is 1.6 mM. Additional expression of the gene pntAB, encoding nicotinamide nucleotide transhydrogenase that converts NADH to NADPH, increased 3-HP production to 2.14 mM. The strain was further developed by deleting the sucAB gene, encoding -ketoglutarate dehydrogenase complex in tricarboxylic acid (TCA) cycle, or blocking lactate and acetate production pathways, and evaluated for the production of 3-HP. We report on the feasibility of producing 3-HP from glucose through the malonyl-CoA pathway. © 2011 Elsevier B.V. All rights reserved. 1. Introduction The platform chemical 3-hydroxypropionic acid (3-HP; C 3 H 6 O 3 , MW 90.08) has a wide range of industrial applications in the pro- duction of several specialty chemicals (Rathnasingh et al., 2009). Recently, we demonstrated the production of 3-HP from glycerol using a recombinant Escherichia coli expressing a B 12 -dependent glycerol dehydratase and an aldehyde dehydrogenase (Raj et al., 2008, 2009, 2010; Rathnasingh et al., 2009). Although the final titer was as high as 39 g/L, the use of expensive coenzyme B 12 was a major drawback for commercial application of this process. In this study, we investigated the feasibility of producing 3-HP from glucose via aB 12 -independent pathway in E. coli. To this end, we utilized the route to convert acetyl-CoA to malonyl-CoA and finally to 3-HP (Fig. 1). Conversion of acetyl-CoA to malonyl-CoA, catalyzed by acetyl- CoA carboxylase (ACC), is an essential reaction in fatty acid synthesis. In case of E. coli, ACC consists of four subunits, AccC, AccB, AccA and AccD. The AccC, biotin carboxylase, initiates trans- ∗ Corresponding author. Tel.: +1 82 51 510 2395; fax: +1 82 51 515 2716. E-mail address: parksh@pusan.ac.kr (S. Park). ferring CO 2 from bicarbonate to biotin carboxyl carrier protein (AccB) in an ATP-dependent manner. The active form of AccB requires covalent attachment of biotin, and the attachment is cat- alyzed by BirA (biotinilase). The CO 2 molecule in AccB-CO 2 is then incorporated to acetyl-CoA to form malonyl-CoA by carboxyltrans- ferases (AccA and AccD) (Davis et al., 2000). Malonyl-CoA reductase (MCR), which converts malonyl-CoA to 3-HP with the expense of two NADPH molecules, has been reported in several bacte- rial strains including Chloroflexus aurantiacus, C. aggregans, and Roseiflexus castenholzii (Hugler et al., 2002, 2003). They assimi- late CO 2 by the 3-HP cycle and MCR is an essential enzyme for the cycle. In 3-HP production processes, the route utilizing MCR is expected to have many advantages. First, it does not require coen- zyme B 12 as in the case of the glycerol dehydratase-dependent pathway. Second, various C 5 and C 6 sugars derived from lignocel- lulosic biomass can be used as raw material for 3-HP production since acetyl-CoA is a common intermediate of sugar metabolism. Third, a high conversion yield on glucose is expected since the pro- duction of 3-HP from glucose is energetically well balanced. The conversion of glucose to each mol of acetyl-CoA generates 2 mol of NADH, 1 mol of ATP and 1 mol of CO 2 ; while carboxylation of each mol of acetyl-CoA to malonyl-CoA utilizes 1 mol of CO 2 along with 0168-1656/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2011.06.008