Metabolic engineering of Mannheimia succiniciproducens for malic acid production using dimethylsulfoxide as an electron acceptor.

Microbial production of various TCA intermediates and related chemicals through the reductive TCA cycle has been of great interest. However, rumen bacteria that naturally possess strong reductive TCA cycle have been rarely studied to produce these chemicals, except for succinic acid, due to their dependence on fumarate reduction to transport electrons for ATP synthesis. In this study, malic acid (MA), a dicarboxylic acid of industrial importance, was selected as a target chemical for mass production using Mannheimia succiniciproducens, a rumen bacterium possessing a strong reductive branch of the TCA cycle. The metabolic pathway was reconstructed by eliminating fumarase to prevent MA conversion to fumarate. The respiration system of M. succiniciproducens was reconstructed by introducing the Actinobacillus succinogenes dimethylsulfoxide (DMSO) reductase to improve cell growth using DMSO as an electron acceptor. Also, the cell membrane was engineered by employing Pseudomonas aeruginosa cis-trans isomerase to enhance MA tolerance. High inoculum fed-batch fermentation of the final engineered strain produced 61 g/L of MA with an overall productivity of 2.27 g/L/h, which is the highest MA productivity reported to date. The systems metabolic engineering strategies reported in this study will be useful for developing anaerobic bioprocesses for the production of various industrially important chemicals.

Membrane engineering via trans-unsaturated fatty acids production improves succinic acid production in Mannheimia succiniciproducens.

Engineering of microorganisms to produce desired bio-products with high titer, yield, and productivity is often limited by product toxicity. This is also true for succinic acid (SA), a four carbon dicarboxylic acid of industrial importance. Acid products often cause product toxicity to cells through several different factors, membrane damage being one of the primary factors. In this study, cis-trans isomerase from Pseudomonas aeruginosa was expressed in Mannheimia succiniciproducens to produce trans-unsaturated fatty acid (TUFA) and to reinforce the cell membrane of M. succiniciproducens. The engineered strain showed significant decrease in membrane fluidity as production of TUFA enabled tight packing of fatty acids, which made cells to possess more rigid cell membrane. As a result, the membrane-engineered M. succiniciproducens strain showed higher tolerance toward SA and increased production of SA compared with the control strain without membrane engineering. The membrane engineering approach employed in this study will be useful for increasing tolerance to, and consequently enhancing production of acid products.

Metabolic engineering of Mannheimia succiniciproducens for succinic acid production based on elementary mode analysis with clustering.

Mannheimia succiniciproducens, a capnophilic gram-negative rumen bacterium, has been employed for the efficient production of succinic acid. Although M. succiniciproducens metabolism was previously studied using a genome-scale metabolic model, more metabolic characteristics are to be understood. To this end, elementary mode analysis accompanied with clustering ('EMC' analysis) is used to gain further insights on metabolic characteristics of M. succiniciproducens allowing efficient succinic acid production. Elementary modes (EMs) generated from the central carbon metabolic network of M. succiniciproducens are clustered to systematically analyze succinic acid production routes. Based on the results of EMC analysis, zwf gene is identified as a novel overexpression target for the improved succinic acid production. This gene is overexpressed in a previously constructed succinic acid-overproducing M. succiniciproducens LPK7 strain. Heterologous NADPH-dependent mdh is later intuitively selected for overexpression to synergistically improve succinic acid production by utilizing abundant NADPH pool mediated by the overexpressed zwf. The LPK7 strains co-expressing mdh alone and both zwf and mdh genes are subjected to fed-batch fermentation to better examine their succinic acid production performances. Strategies of EMC analysis will be useful for further metabolic engineering of M. succiniciproducens and other microorganisms to improve production of succinic acid and other chemicals of interest.

Highly selective production of succinic acid by metabolically engineered Mannheimia succiniciproducens and its efficient purification.

Succinic acid (SA) is one of the fermentative products of anaerobic metabolism, and an important industrial chemical that has been much studied for its bio-based production. The key to the economically viable bio-based SA production is to develop an SA producer capable of producing SA with high yield and productivity without byproducts. Mannheimia succiniciproducens is a capnophilic rumen bacterium capable of efficiently producing SA. In this study, in silico genome-scale metabolic simulations were performed to identify gene targets to be engineered, and the PALK strain (ΔldhA and Δpta-ackA) was constructed. Fed-batch culture of PALK on glucose and glycerol as carbon sources resulted in the production of 66.14 g/L of SA with the yield and overall productivity of 1.34 mol/mol glucose equivalent and 3.39 g/L/h, respectively. SA production could be further increased to 90.68 g/L with the yield and overall productivity of 1.15 mol/mol glucose equivalent and 3.49 g/L/h, respectively, by utilizing a mixture of magnesium hydroxide and ammonia solution as a pH controlling solution. Furthermore, formation of byproducts was drastically reduced, resulting in almost homo-fermentative SA production. This allowed the recovery and purification of SA to a high purity (99.997%) with a high recovery yield (74.65%) through simple downstream processes composed of decolorization, vacuum distillation, and crystallization. The SA producer and processes developed in this study will allow economical production of SA in an industrial-scale. Biotechnol. Bioeng. 2016;113: 2168-2177. © 2016 Wiley Periodicals, Inc.

Exploration of the arrest peptide sequence space reveals arrest-enhanced variants.

Translational arrest peptides (APs) are short stretches of polypeptides that induce translational stalling when synthesized on a ribosome. Mechanical pulling forces acting on the nascent chain can weaken or even abolish stalling. APs can therefore be used as in vivo force sensors, making it possible to measure the forces that act on a nascent chain during translation with single-residue resolution. It is also possible to score the relative strengths of APs by subjecting them to a given pulling force and ranking them according to stalling efficiency. Using the latter approach, we now report an extensive mutagenesis scan of a strong mutant variant of the Mannheimia succiniciproducens SecM AP and identify mutations that further increase the stalling efficiency. Combining three such mutations, we designed an AP that withstands the strongest pulling force we are able to generate at present. We further show that diproline stretches in a nascent protein act as very strong APs when translation is carried out in the absence of elongation factor P. Our findings highlight critical residues in APs, show that certain amino acid sequences induce very strong translational arrest and provide a toolbox of APs of varying strengths that can be used for in vivo force measurements.

The CpxRA two-component system is involved in the maintenance of the integrity of the cell envelope in the rumen bacterium Mannheimia succiniciproducens.

In this study, we characterized the CpxRA two-component signal transduction system of the rumen bacterium Mannheimia succiniciproducens. The truncated form of the CpxA sensor kinase protein without its transmembrane domain was able to autophosphorylate and transphosphorylate the CpxR response regulator protein in vitro. We identified 152 putative target genes for the Cpx system in M. succiniciproducens, which were differentially expressed by more than twofold upon overexpression of the CpxR protein. Genes of a putative 16-gene operon related to the cell wall and lipopolysaccharide biosynthesis were induced strongly upon CpxR overexpression. The promoter region of the first gene of this operon, wecC encoding UDP-N-acetyl-D-mannosaminuronate dehydrogenase, was analyzed and found to contain a sequence homologous to the CpxR box of Escherichia coli. An electrophoretic mobility shift assay showed that the phosphorylated CpxR proteins were able to bind specifically to PCR-amplified DNA fragments containing the promoter sequence of wecC. Furthermore, a cpxR-disrupted mutant strain exhibited increased envelope permeability compared with a wild-type strain. These results suggest that the Cpx system of M. succiniciproducens is involved in the maintenance of the integrity of the cell envelope.

Mannheimia succiniciproducens phosphotransferase system for sucrose utilization.

The succinic acid producer Mannheimia succiniciproducens can efficiently utilize sucrose as a carbon source, but its metabolism has not been understood. This study revealed that M. succiniciproducens uses a sucrose phosphotransferase system (PTS), sucrose 6-phosphate hydrolase, and a fructose PTS for the transport and utilization of sucrose.

The effect of protectants and pH changes on the cellular growth and succinic acid yield of Mannheimia succiniciproducens LPK7.

The harmful effects of succinic acid and oxidative stress on cell growth were determined during batch fermentation with Mannheimia succiniciproducens LPK7, a powerful succinic acid-producing strain, and conditions were optimized to minimize these effects. In terms of toxicity, the cell concentration decreased as the concentration of succinic acid increased. By changing the pH from 6.5 to 7 during fermentation, the cell concentration increased by about 10%, and the level of succinic acid production was 6% higher than that of the control. In addition, by introducing protectants, the cell concentration increased by about 10%, and the level of succinic acid produced was increased by 3%.

Continuous production of succinic acid using an external membrane cell recycle system.

Succinic acid was produced by continuous fermentation of Actinobacillus succinogenes sp. 130Z in an external membrane cell recycle reactor to improve viable cell concentration and productivity. Using this system, cell concentration increased to 16.4 g/l at the dilution rate 0.2 h-1, up to 3 times higher than that of batch culture, and the volumetric productivity of succinic acid increased up to 6.63 g/l/h at the dilution rate 0.5 h-1, 5 times higher than that of batch fermentation. However, in the continuous culture using a high dilution rate, operational problems including severe membrane fouling and contamination by lactic acid producer were observed. Another succinic acid producer, Mannheimia succiniciproducens MBEL55E, was also utilized in this system, and the cell concentration and productivity of succinic acid at the dilution rate of 0.3 h-1 were found to be above 3 and 2.3 times higher, respectively, compared with those obtained at the dilution rate of 0.1 h-1. These observations give a deep insight into the process design for a continuous succinic acid production by microorganisms.

In silico analysis of the effects of H2 and CO2 on the metabolism of a capnophilic bacterium Mannheimia succiniciproducens.

This study presents an in-depth study on the physiological behavior of Mannheimia succiniciproducens, a capnophilic bacterium and an efficient succinic acid producer, under varying gas conditions as H(2) and CO(2) play important roles in the production of succinic acid. Constraints-based flux analysis of the genome-scale metabolic model of M. succiniciproducens was performed to estimate the production patterns of several organic acids in response to varying H(2), CO(2), and glucose uptake rates. Results from controlled cultivations performed previously and constraints-based flux analyses of M. succiniciproducens in this study revealed that there is an optimal range of CO(2) level in the medium for enhancing cell growth and succinic acid production at a given glucose uptake rate. Furthermore, the uptake rates of H(2) and CO(2) from the medium have a direct relationship with each other, significantly influencing the rates of cell growth and succinic acid production. Predictions made in this study quantitatively describe the physiological changes of the cell in response to varying H(2), CO(2), and glucose uptake rates, which consequently allow us to identify the feasible physiological states of the cell with respect to cell growth rate and succinic acid production rate.

Optimization and scale-up of succinic acid production by Mannheimia succiniciproducens LPK7.

The effects of culture conditions on succinic acid production and its possible scale-up have been studied. Mannheimia succiniciproducens LPK7, engineered for enhanced production of succinic acid and reduced by-product secretion, was used for the experiments. Mannheimia succiniciproducens LPK7 is a knock-out strain of wild type deficient in the ldhA, pflB, and pta-ackA genes, and is derived from Mannheimia succiniciproducens MBEL55E. Process optimization of factors including optimal temperature, pH, carbon source, and nitrogen source was performed to enhance the production of succinic acid in flasks. To observe scale-up effects, batch fermentation was carried out at various working volumes. At a working volume of 7.0 l, the final succinic acid concentration and yield were 15.4 g/l and 0.86 g/g. This result shows similar amount of succinic acid obtained in lab-scale fermentation, and it is possible to scale up to larger fermentors without major problems.

Succinic acid production from continuous fermentation process using Mannheimia succiniciproducens LPK7.

To achieve a higher succinic acid productivity and evaluate the industrial applicability, this study used Mannheimia succiniciproducens LPK7 (knock-out: lahA, pflB, pta-ackA), which was recently designed to enhance the productivity of succinic acid and reduce by-product secretion. Anaerobic continuous fermentation of Mannheimia succiniciproducens LPK7 was carried out at different glucose feed concentrations and dilution rates. After extensive fermentation experiments, a succinic acid yield and productivity of 0.38 mol/mol and 1.77 g/l/h, respectively, were achieved with a glucose feed concentration of 18.0 g/l and 0.2 h-1 dilution rate. A similar amount of succinic acid production was also produced in batch culture experiments. Therefore, these optimal conditions can be industrially applied for the continuous production of succinic acid. To examine the quantitative balance of the metabolism, a flux distribution analysis was also performed using the metabolic network model of glycolysis and the pentose phosphate pathway.

Development of chemically defined medium for Mannheimia succiniciproducens based on its genome sequence.

This study presents a novel methodology for the development of a chemically defined medium (CDM) using genome-scale metabolic network and flux balance analysis. The genome-based in silico analysis identified two amino acids and four vitamins as non-substitutable essential compounds to be supplemented to a minimal medium for the sustainable growth of Mannheimia succiniciproducens, while no substitutable essential compounds were identified. The in silico predictions were verified by cultivating the cells on a CDM containing the six non-substitutable essential compounds, and it was further demonstrated by observing no cell growth on the CDM lacking any one of the non-substitutable essentials. An optimal CDM for the enhancement of cell growth and succinic acid production, as a target product, was formulated with a single-addition technique. The fermentation on the optimal CDM increased the succinic acid productivity by 36%, the final succinic acid concentration by 17%, and the succinic acid yield on glucose by 15% compared to the cultivation using a complex medium. The optimal CDM also lowered the sum of the amounts of by-products (acetic, formic, and lactic acids) by 30%. The strategy reported in this paper should be generally applicable to the development of CDMs for other organisms, whose genome sequences are available.

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.

Recovery of succinic acid produced by fermentation of a metabolically engineered Mannheimia succiniciproducens strain.

There have recently been much advances in the production of succinic acid, an important four-carbon dicarboxylic acid for many industrial applications, by fermentation of several natural and engineered bacterial strains. Mannheimia succiniciproducens MBEL55E isolated from bovine rumen is able to produce succinic acid with high efficiency, but also produces acetic, formic and lactic acids just like other anaerobic succinic acid producers. We recently reported the development of an engineered M. succiniciproducens LPK7 strain which produces succinic acid as a major fermentation product while producing much reduced by-products. Having an improved succinic acid producer developed, it is equally important to develop a cost-effective downstream process for the recovery of succinic acid. In this paper, we report the development of a simpler and more efficient method for the recovery of succinic acid. For the recovery of succinic acid from the fermentation broth of LPK7 strain, a simple process composed of a single reactive extraction, vacuum distillation, and crystallization yielded highly purified succinic acid (greater than 99.5% purity, wt%) with a high yield of 67.05wt%. When the same recovery process or even multiple reactive extraction steps were applied to the fermentation broth of MBEL55E, lower purity and yield of succinic acid were obtained. These results suggest that succinic acid can be purified in a cost-effective manner by using the fermentation broth of engineered LPK7 strain, showing the importance of integrating the strain development, fermentation and downstream process for optimizing the whole processes for succinic acid production.

Construction and characterization of shuttle vectors for succinic acid-producing rumen bacteria.

Shuttle vectors carrying the origins of replication that function in Escherichia coli and two capnophilic rumen bacteria, Mannheimia succiniciproducens and Actinobacillus succinogenes, were constructed. These vectors were found to be present at ca. 10 copies per cell. They were found to be stably maintained in rumen bacteria during the serial subcultures in the absence of antibiotic pressure for 216 generations. By optimizing the electroporation condition, the transformation efficiencies of 3.0 x 10(6) and 7.1 x 10(6) transformants/mug DNA were obtained with M. succiniciproducens and A. succinogenes, respectively. A 1.7-kb minimal replicon was identified that consists of the rep gene, four iterons, A+T-rich regions, and a dnaA box. It was found that the shuttle vector replicates via the theta mode, which was confirmed by sequence analysis and Southern hybridization. These shuttle vectors were found to be suitable as expression vectors as the homologous fumC gene encoding fumarase and the heterologous genes encoding green fluorescence protein and red fluorescence protein could be expressed successfully. Thus, the shuttle vectors developed in this study should be useful for genetic and metabolic engineering of succinic acid-producing rumen bacteria.

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 CO(2)-rich anaerobic condition. Since succinic acid is produced by carboxylation of C3 compounds during the fermentation, intracellular CO(2) availability is important for efficient succinic acid formation. Here, we investigated the metabolic responses of M. succiniciproducens to the different dissolved CO(2) concentrations (0-260 mM). Cell growth was severely suppressed when the dissolved CO(2) concentration was below 8.74 mM. On the other hand, cell growth and succinic acid production increased proportionally as the dissolved CO(2) concentration increased from 8.74 to 141 mM. The yields of biomass and succinic acid on glucose obtained at the dissolved CO(2) concentration of 141 mM were 1.49 and 1.52 times higher, respectively, than those obtained at the dissolved CO(2) concentration of 8.74 mM. It was also found that the additional CO(2) source provided in the form of NaHCO(3), MgCO(3), or CaCO(3) had positive effects 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.

Genome-scale analysis of Mannheimia succiniciproducens metabolism.

Mannheimia succiniciproducens MBEL55E isolated from bovine rumen is a capnophilic gram-negative bacterium that efficiently produces succinic acid, an industrially important four carbon dicarboxylic acid. In order to design a metabolically engineered strain which is capable of producing succinic acid with high yield and productivity, it is essential to optimize the whole metabolism at the systems level. Consequently, in silico modeling and simulation of the genome-scale metabolic network was employed for genome-scale analysis and efficient design of metabolic engineering experiments. The genome-scale metabolic network of M. succiniciproducens consisting of 686 reactions and 519 metabolites was constructed based on reannotation and validation experiments. With the reconstructed model, the network structure and key metabolic characteristics allowing highly efficient production of succinic acid were deciphered; these include strong PEP carboxylation, branched TCA cycle, relative weak pyruvate formation, the lack of glyoxylate shunt, and non-PTS for glucose uptake. Constraints-based flux analyses were then carried out under various environmental and genetic conditions to validate the genome-scale metabolic model and to decipher the altered metabolic characteristics. Predictions based on constraints-based flux analysis were mostly in excellent agreement with the experimental data. In silico knockout studies allowed prediction of new metabolic engineering strategies for the enhanced production of succinic acid. This genome-scale in silico model can serve as a platform for the systematic prediction of physiological responses of M. succiniciproducens to various environmental and genetic perturbations and consequently for designing rational strategies for strain improvement.

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.

Enhanced proteome profiling by inhibiting proteolysis with small heat shock proteins.

Proteolytic degradation is one of the critical problems in two-dimensional electrophoresis (2-DE). Here we report that small heat shock proteins (sHsps), including IbpA(Ec) and IbpB(Ec) from Escherichia coli and Hsp26(Sc) from Saccharomyces cerevisiae, are able to protect proteins in vitro from proteolytic degradation. Addition of sHsps during 2-DE of human serum or whole cell extracts of E. coli, Mannheimia succinciproducens, Arabidopsis thaliana, and human kidney cells allowed detection of up to 50% more protein spots than those obtainable with currently available protease inhibitors. Therefore, the use of sHsps during 2-DE significantly improves proteome profiling by generally enabling the detection of many more protein spots that could not be seen previously.