Production of succinic acid from sucrose and sugarcane molasses by metabolically engineered Escherichia coli Sitha Chan, Sunthorn Kanchanatawee, Kaemwich Jantama ⇑ School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, 111 University Ave., Suranaree, Muang, Nakhon Ratchasima 30000, Thailand article info Article history: Received 6 August 2011 Received in revised form 22 September 2011 Accepted 23 September 2011 Available online 7 October 2011 Keywords: Succinate Escherichia coli Metabolic engineering Sucrose Molasses abstract Sucrose-utilizing genes (cscKB and cscA) from Escherichia coli KO11 were cloned and expressed in a metabolically engineered E. coli KJ122 to enhance succinate production from sucrose. KJ122 harboring a recombinant plasmid, pKJSUC, was screened for the efficient sucrose utilization by growth-based selec- tion and adaptation. KJ122-pKJSUC-24T efficiently utilized sucrose in a low-cost medium to produce high succinate concentration with less accumulation of by-products. Succinate concentrations of 51 g/L (productivity equal to 1.05 g/L/h) were produced from sucrose in anaerobic bottles, and concentrations of 47 g/L were produced in 10 L bioreactor within 48 h. Antibiotics had no effect on the succinate produc- tion by KJ122-pKJSUC-24T. In addition, succinate concentrations of 62 g/L were produced from sugarcane molasses in anaerobic bottles, and concentrations of 56 g/L in 10 L bioreactor within 72 h. These results demonstrated that KJ122-pKJSUC-24T would be a potential strain for bio-based succinate production from sucrose and sugarcane molasses. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Succinic acid has been identified by the US Department of En- ergy as one of ten organic acids as key chemical building blocks. Succinic acid acts as a precursor for many specialty chemicals in food, pharmaceuticals, green solvents, and biodegradable plastics (Song and Lee, 2006). Due to worldwide demand for oil for fuel and production of commodity and specialty chemicals, the use of maleic anhydride, which is derived from oil and petroleum, for succinate production should be replaced. This is due to the decline in oil reserves, the increasing price, and pollution. Alternatively, succinate can be produced from agricultural feedstock by microbial fermentation. However, a combination of technology in systems biology, synthetic biology, and metabolic engineering is required to construct new microorganisms for an efficient succinate produc- tion under simple fermentation conditions (Adsul et al., 2011). Inexpensive carbon sources and media for fermentation, as well as high succinate titer with good productivity are also important constraints in developing industrial succinate production by fermentation processes (Sauer et al., 2008). Previous studies were extensively investigated the succinate production from glucose by many microorganisms such as Actinobacillus succinogenes, Mannheimia succiniciproducens, and metabolically engineered Escherichia coli (Adsul et al., 2011). Recently, a metabolically engi- neered E. coli was developed for succinate production from a mix- ture of glycerol and fatty acid but the succinate yield and productivity were still low (Kang et al., 2010; Kang et al., 2011). For an economical production, succinate could be produced from lignocellulosic biomass such as corn stover hydrolysate and rice straw (Li et al., 2011; Zheng et al., 2009; Zheng et al., 2010). How- ever, a pretreatment process of these materials usually releases significant amounts of sugars and lignin degradation products that are inhibitory to succinate fermentation. Therefore, a pretreatment step is currently considered as the bottleneck in the production of cellulosic succinate (Adsul et al., 2011). Sucrose and sugarcane molasses are common and inexpensive carbon substrates. In 2008, the USDA reported that raw cane sugar and sugarcane molasses were sold at prices less than $0.50 per kg. The prices of both substrates are lower than that of glucose. There- fore sucrose and sugarcane molasses can be considered as target substrates to produce lower-price biosuccinate. Lui et al. (2008) re- ported that a succinate concentration of 40 g/L with a productivity of 0.66 g/L/h was produced from sucrose by A. succinogenes CGMCC1593. Wang et al. (2011) also showed that succinate con- centration and productivity of 32.65 g/L and 0.34 g/L/h, respec- tively, can be obtained from sucrose by E. coli SBS550MG harboring the plasmids, pHL413 (harboring pyc gene from Lacto- coccus lactis) and pUR400 (harboring scrKYABR genes from E. coli K12). However, the production of succinate by both strains re- quired tryptone, yeast extract, rich sources of nutrients, carbon dioxide gas, and even antibiotics and IPTG for maintenance and expression of plasmids; this requires an increase in production price and purification. 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.09.096 ⇑ Corresponding author. Tel.: +66 44 224562; fax: +66 44 224154. E-mail address: kaemwich@sut.ac.th (K. Jantama). Bioresource Technology 103 (2012) 329–336 Contents lists available at SciVerse ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech