Genome-Based Metabolic Engineering of Mannheimia succiniciproducens for Succinic Acid Production

Abstract

Succinic acid is a four-carbon dicarboxylic acid produced as one of the fermentation products of anaerobic metabolism. Based on the complete genome sequence of a capnophilic succinic acid-producing rumen bacterium, Mannheimia succiniciproducens, gene knockout studies were carried out to understand its anaerobic fermentative metabolism and consequently to develop a metabolically engineered strain capable of producing succinic acid without by-product formation. Among three different CO 2 -fixing metabolic reactions catalyzed by phosphoenolpyruvate (PEP) carboxykinase, PEP carboxylase, and malic enzyme, PEP carboxykinase was the most important for the anaerobic growth of M. succiniciproducens and succinic acid production. Oxaloacetate formed by carboxylation of PEP was found to be converted to succinic acid by three sequential reactions catalyzed by malate dehydrogenase, fumarase, and fumarate reductase. Major metabolic pathways leading to by-product formation were successfully removed by disrupting the ldhA, pflB, pta, and ackA genes. This metabolically engineered LPK7 strain was able to produce 13.4 g/liter of succinic acid from 20 g/liter glucose with little or no formation of acetic, formic, and lactic acids, resulting in a succinic acid yield of 0.97 mol succinic acid per mol glucose. Fed-batch culture of M. succiniciproducens LPK7 with intermittent glucose feeding allowed the production of 52.4 g/liter of succinic acid, with a succinic acid yield of 1.16 mol succinic acid per mol glucose and a succinic acid productivity of 1.8 g/liter/h, which should be useful for industrial production of succinic acid.Succinic acid, also known as butanedioic or amber acid, is a four-carbon dicarboxylic acid synthesized as an intermediate of the tricarboxylic acid cycle and also as one of the mixed-acid fermentation products. There is growing interest in the production of succinic acid from renewable resources by microbial fermentation because succinic acid can be used in numerous applications (36,37). Some anaerobic bacteria, such as Anaerobiospirillum succiniciproducens (6) and Actinobacillus succinogenes (10), produce succinic acid as a major fermentation product of their metabolism. During anaerobic fermentation, the greenhouse gas CO 2 is fixed by carboxylation reactions, converting C 3 to C 4 metabolites. Although A. succiniciproducens and A. succinogenes are able to produce relatively more succinic acid than other microorganisms, concomitant production of metabolic by-products, such as acetic, formic, and lactic acids, is problematic because it reduces the succinic acid yield and makes the purification process difficult and costly.With an aim to reduce by-product formation, several different approaches have been taken. In one example, Guettler et al. (9) developed a monofluoroacetate-resistant mutant strain of A. succinogenes that produced less acetic and formic acids than the parent strain. In other studies, Lee et al. used glycerol as a carbon source (22) and supplied hydrogen gas (21) during fermentation to reduce b...

Industrial systems biology

“…Their results confirmed that a mutant capable of virtually no lactate, fumarate, or acetate formation was feasible, and that PEP carboxykinase was most critical for anaerobic growth and maximizing succinic acid production (Lee et al, 2006a). The resulting metabolically engineered strain, M. succiniciproducens LPK7 under batch fermentation conditions produced 0.97 mol succinic acid per mol glucose, and under fed-batch fermentation conditions reached a maximum titer, productivity, and yield of 52.4 g/L, 1.8 g/L/h, and 1.16 mol succinic acid per mol glucose, respectively (Lee et al, 2006a). The theoretical carbon yield of succinate under excess reducing power and CO 2 carboxylation, is 2 mol succinic acid per mol glucose ðDG o 0 ¼ À317 kJ=molÞ (McKinlay et al, 2007).…”

“…As a consequence of the simulations, they note, ''Based on these findings, we now design metabolic engineering strategies for the enhanced production of succinic acid; one such strategy will be increasing the PEP carboxylation flux while decreasing the fluxes to acetic, formic, and lactic acid'' (Hong et al, 2004). In 2006, the authors constructed a series of knock-out mutants of M. succiniciproducens MBEL55E that included disruption of three CO 2 catalyzing reactions (PEP carboxykinase, PEP carboxylase, malic enzyme) and disruption of four genes responsible for by-product formation of lactate, formate, and acetate (ldhA, pflB, pta, and ackA genes) (Lee et al, 2006a). Their results confirmed that a mutant capable of virtually no lactate, fumarate, or acetate formation was feasible, and that PEP carboxykinase was most critical for anaerobic growth and maximizing succinic acid production (Lee et al, 2006a).…”

“…In 2006, the authors constructed a series of knock-out mutants of M. succiniciproducens MBEL55E that included disruption of three CO 2 catalyzing reactions (PEP carboxykinase, PEP carboxylase, malic enzyme) and disruption of four genes responsible for by-product formation of lactate, formate, and acetate (ldhA, pflB, pta, and ackA genes) (Lee et al, 2006a). Their results confirmed that a mutant capable of virtually no lactate, fumarate, or acetate formation was feasible, and that PEP carboxykinase was most critical for anaerobic growth and maximizing succinic acid production (Lee et al, 2006a). The resulting metabolically engineered strain, M. succiniciproducens LPK7 under batch fermentation conditions produced 0.97 mol succinic acid per mol glucose, and under fed-batch fermentation conditions reached a maximum titer, productivity, and yield of 52.4 g/L, 1.8 g/L/h, and 1.16 mol succinic acid per mol glucose, respectively (Lee et al, 2006a).…”

Biorefinery Concept: An Overview of Producing Energy, Fuels and Materials from Biomass Feedstocks

“…Succinic acid, a derivative of glucose is very important and reactive molecule currently produced commercially by chemical processes. Interest in production of industrial chemicals from renewable resources has led to the development of several microorganisms that could produce succinic acid at concentrations sufficiently high to make the development of commercial fermentation processes economically feasible and attractive (Lee, Song, & Lee, 2006). It has been demonstrated that carbon dioxide produced in an ethanol fermentor can be used directly for succinic acid production in an adjacent fermentor without any impurities removal treatment (Nghiem, Hicks, & Johnston, 2010).…”

The elucidation of metabolic pathways and their improvements using stable optimization of large-scale kinetic models of cellular systems

“…Microbial population studies can now be complemented by nuclear magnetic resonance (NMR) experiments to measure intracellular fluxes and metabolite levels, which can be economically designed using multi-level computational optimization-based frameworks employing stoichiomertic reaction networks (Ghosh et al, 2006). Credible overproduction strategies have recently been suggested based on cellular stoichiometry alone (Alper et al 2005;Burgard et al 2003Burgard et al , 2004Ibarra et al 2002;Lee et al 2005Lee et al , 2006Pharkya et al 2004;Pharkya and Maranas 2006). Because stoichiometry does correctly define overall barriers and limits for steady state reaction fluxes under fixed 'defined medium' constraints, genome-scale stoichiometric models have been very successful in many instances in fundamental and applied research (Palsson 2006).…”