Isolation and characterization of a new mucoid-free Klebsiella pneumoniae strain for 2,3-butanediol production

The secretion of mucoid substances by Klebsiella pneumoniae, a natural 2,3-butanediol (2,3-BD) hyper-producer, hinders its application in large-scale fermentation because of pathogenicity, fermentation instability, and downstream difficulty. In this study, 14 K. pneumoniae strains were isolated from a waste water treatment plant and their 2,3-BD production efficiencies were assessed with the strain K. pneumoniae DSM2026. Among various strains isolated, K. pneumoniae GSC010 and GSC112 produced relatively large amounts of 2,3-BD compared to other isolates; and their 2,3-BD production was consistent with DSM2026. Meanwhile, mucoidic characteristics of GSC010 were more or less similar to DSM2026, which was observed by scanning electron microscope (SEM) as a characteristic intercalated thread anchored on the surface of the cells. However, no polysaccharide materials were found in a non-mucoid cell, GSC112. Fed-batch culture of GSC112 with continuous glucose feeding resulted in the production of 2,3-BD at 52.4 g/l with 2,3-BD yield and overall productivity of 0.27 g/g glucose and 0.52 g/l/h, respectively. These results strongly suggest that the newly isolated mucoid-free K. pneumoniae GSC112 has potential for industrial production of 2,3-BD.Keywords: 2,3-Butanediol, Klebsiella pneumoniae, isolation, capsular polysaccharides, scanning electron microscop

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

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

The proteome of Mannheimia succiniciproducens, a capnophilic rumen bacterium

Mannheimia succiniciproducens MBEL55E isolated from bovine rumen is an industrially important bacterium as an efficient succinic acid producer. Recently, its full genome sequence was determined. In the present study, we analyzed the M. succiniciproducens proteome based on the genome information using 2-DE and MS. We established proteome reference map of M. succiniciproducens by analyzing whole cellular proteins, membrane proteins, and secreted proteins. More than 200 proteins were identified and characterized by MS/MS supported by various bioinformatic tools. The presence of proteins previously annotated as hypothetical proteins or proteins having putative functions were also confirmed. Based on the proteome reference map, cells in the different growth phases were analyzed at the proteome level. Comparative proteome profiling revealed valuable information to understand physiological changes during growth, and subsequently suggested target genes to be manipulated for the strain improvement.11Nsciescopu

Effects of dissolved CO2 levels on the growth of Mannheimia succiniciproducens and succinic acid production

A capnophilic rumen bacterium Mannheimia succiniciproducens produces succinic acid as a major fermentation end product under CO2-rich anaerobic condition. Since succinic acid is produced by carboxylation of C3 compounds during the fermentation, intracellular CO2 availability is important for efficient succinic acid formation. Here, we investigated the metabolic responses of M. succiniciproducens to the different dissolved CO2 concentrations (0-260 mM). Cell growth was severely suppressed when the dissolved CO2 concentration was below 8.74 mM. On the other hand, cell growth and succinic acid production increased proportionally as the dissolved CO2 concentration increased from 8.74 to 141 mM. The yields of biomass and succinic acid on glucose obtained at the dissolved CO2 concentration of 141 mM were 1.49 and 1.52 times higher, respectively, than those obtained at the dissolved CO2 concentration of 8.74 mM. It was also found that the additional CO2 source provided in the form of NaHCO3, MgCO3, or CaCO3 had positive effects 1 on cell growth and succinic acid production. However, growth inhibition was observed when excessive bicarbonate salts were added. By the comparison of the activities of key enzymes, it was found that PEP carboxylation by PEP carboxykinase (PckA) is the most important for succinic acid production as well as the growth of M. succiniciproducens by providing additional ATP.11Nsciescopu

From genome sequence to integrated bioprocess for succinic acid production by Mannheimia succiniciproducens

Mannheimia succiniciproducens is a capnophilic gram-negative bacterium isolated from bovine rumen. Wild-type M. succiniciproducens can produce succinic acid as a major fermentation product with acetic, formic, and lactic acids as byproducts during the anaerobic cultivation using several different carbon sources. Succinic acid is an important C4 building block chemical for many applications. Here, we review the progress made with M. succiniciproducens for efficient succinic acid production; the approaches taken towards the development of an integrated process for succinic acid production are described, which include strain isolation and characterization, complete genome sequencing and annotation, development of genetic tools for metabolic engineering, strain development by systems approach of integrating omics and in silico metabolic analysis, and development of fermentation and recovery processes. We also describe our current effort on further improving the performance of M. succiniciproducens and optimizing the mid- and downstream processes. Finally, we finish this mini-review by discussing the issues that need to be addressed to make this process of fermentative succinic acid production employing M. succiniciproducens to reach the industrial-scale process.11Nsciescopu

Effects of mutation of 2,3-butanediol formation pathway on glycerol metabolism and 1,3-propanediol production by Klebsiella pneumoniae J2B

The current study investigates the impact of mutation of 2,3-butanediol (BDO) formation pathway on glycerol metabolism and 1,3-propanediol (PDO) production by lactate dehydrogenase deficient mutant of Klebsiella pneumoniae J2B. To this end, BDO pathway genes, budA, budB, budC and budO (whole-bud operon), were deleted from K. pneumoniae J2B Delta ldhA and the mutants were studied for glycerol metabolism and alcohols (PDO, BDO) production. Delta budO-mutant-only could completely abolish BDO production, but with reductions in cell growth and PDO production. By modifying the culture medium, the Delta budO mutant could recover its performance on the flask scale. However, in bioreactor experiments, the Delta budO mutant accumulated a significant amount of pyruvate (>73 mM) in the late phase and PDO production stopped concomitantly. Glycolytic intermediates of glycerol, especially glyceraldehyde-3-phosphate (G3P) was highly inhibitory to glycerol dehydratase (GDHt); its accumulation, followed by pyruvate accumulation, was assumed to be responsible for the Delta budO mutant's low PDO production.ope