Genome-Based Metabolic Engineering of <i>Mannheimia succiniciproducens</i> for Succinic Acid Production

TLDR

Succinic acid is a four‑carbon dicarboxylic acid produced as a fermentation product of anaerobic metabolism. The study aimed to use the genome sequence of *Mannheimia succiniciproducens* to perform gene knockouts that would elucidate its anaerobic metabolism and enable a strain that produces succinic acid without by‑products. The authors identified phosphoenolpyruvate carboxykinase as the key CO₂‑fixing enzyme for growth and succinic acid production, and showed that oxaloacetate derived from PEP is converted to succinic acid via malate dehydrogenase, fumarase, and fumarate reductase. Disruption of *ldhA*, *pflB*, *pta*, and *ackA* eliminated major by‑product pathways, and the engineered LPK7 strain produced 13.4 g L⁻¹ succinic acid from 20 g L⁻¹ glucose (0.97 mol mol⁻¹ yield) and 52.4 g L⁻¹ in fed‑batch (1.16 mol mol⁻¹ yield, 1.8 g L⁻¹ h⁻¹ productivity).

Abstract

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.