Soon Ho Hong

The genome sequence of the capnophilic rumen bacterium Mannheimia succiniciproducens

The rumen represents the first section of a ruminant animals stomach, where feed is collected and mixed with microorganisms for initial digestion. The major gas produced in the rumen is CO2 (65.5 mol%), yet the metabolic characteristics of capnophilic (CO2-loving) microorganisms are not well understood. Here we report the 2,314,078 base pair genome sequence of Mannheimia succiniciproducens MBEL55E, a recently isolated capnophilic Gram-negative bacterium from bovine rumen, and analyze its genome contents and metabolic characteristics. The metabolism of M. succiniciproducens was found to be well adapted to the oxygen-free rumen by using fumarate as a major electron acceptor. Genome-scale metabolic flux analysis indicated that CO2 is important for the carboxylation of phosphoenolpyruvate to oxaloacetate, which is converted to succinic acid by the reductive tricarboxylic acid cycle and menaquinone systems. This characteristic metabolism allows highly efficient production of succinic acid, an important four-carbon industrial chemical.

Recent advances in development of biomass pretreatment technologies used in biorefinery for the production of bio-based fuels, chemicals and polymers

Biochemical conversion of biomass into biofuels, biochemicals, and biopolymers has attracted much interest throughout the world in terms of biorefineries. Lignocellulosic biomass is one of the most plentifully available biomass resources on the earth. It is composed of three main biopolymers - cellulose, hemicelluloses, and lignin, all of which are cross-linked to each other to resist degradation by enzymes and microorganisms resulting in so-called biomass recalcitrance. The biorefinery process typically consists of three steps: pretreatment, hydrolysis, and fermentation. Energy and cost efficiency of biorefinery is predominantly dependent on how to produce inexpensive sugars from complex cell wall component of lignocellulosic biomass by overcoming biomass recalcitrance. There have been tremendous efforts to develop effective biomass pretreatment technologies for obtaining the highest yield of fermentable sugars from biomass feedstocks at the lowest cost. The present review discusses various pretreatment technologies to understand how to effectively break down biomass into fermentable sugars that are eventually used for microbial fermentation to produce biomass-based fuels, chemicals, and polymers.

Enhanced production of succinic acid by metabolically engineeredEscherichia coli with amplified activities of malic enzyme and fumarase

Apfl ldhA double mutantEscherichia coli strain NZN111 was used to produce succinic acid by overexpressing theE. coli malic enzyme gene (sfcA). This strain, however, produced a large amount of malic acid as well as succinic acid. After the analyses of the metabolic pathways, thefumB gene encoding the anaerobic fumarase ofE. coli was co-amplified to solve the problem of malic acid accumulation. A plasmid, pTrcMLFu, was constructed, which contains an artificial operon (sfcA-fumB) under the control of the inducibletrc promoter. From the batch culture of recombinantE. coli NZN111 harboring pTrcMLFu, 7 g/L of succinic acid was produced from 20 g/L of glucose, with no accumulation of malic acid. From the metabolic flux analysis the strain was found under reducing power limiting conditions by severe reorientation of metabolic fluxes.

Engineered fumarate sensing Escherichia coli based on novel chimeric two-component system

DcuS/DcuR two component system (TCS) was firstly employed for the expression of the gfp gene under the dcuB gene promoter in aerobic condition to develop high throughput screening system able to screen microorganisms producing high amount of fumarate. However, the DcuS/DcuR TCS could not produce a signal strong enough to mediate the expression of the gfp gene responding fumarate concentration. Thus, DcuS/DucR TCS was engineered by recruiting the EnvZ/OmpR system, the most-studied TCS in E. coli. A chimeric DcuS/EnvZ (DcuSZ) TCS was constructed by fusing the sensor histidine kinase of DcuS with the cytoplasmic catalytic domain of EnvZ, in which the expression of the gfp gene or the ompC gene was mediated by the ompC gene promoter through the cognate response regulator, OmpR. The output signals produced by the chimeric DcuSZ TCS were enough to detect fumarate concentration quantatively, in which the expressions of the gfp gene and the ompC gene were proportional to the fumarate concentration in the medium. Moreover, principal component analysis of C4-dicarboxylates showed that DcuSZ chimera was highly specific to fumarate but could also respond to other C4-dicarboxylates, which strongly suggests that TCS-based high throughput screening system able to screen microorganisms producing target chemicals can be developed.

Systems approaches to succinic acid-producing microorganisms

Succinic acid is a cellular metabolite belonging to the C4-dicarboxylic acid family, and the fermentative production of succinic acid via the use of recombinant microorganisms has recently become the focus of an increasing amount of attention. Considering the difficulty inherent to the direct application of natural succinic acid producers to the industrial process, a variety of systems biology studies have been conducted regarding the development of enhanced succinic acid production systems. This review shows how the metabolic processes of microorganisms, includingEscherichia coli andMannheimia succiniciproducens, have been optimized in order to achieve enhanced succinic acid production. First, their metabolic networks were constructed on the basis of complete genome sequences, after which their metabolic characteristics were estimated viain silico computer modeling. Metabolic engineering strategies were designed in accordance with the results ofin silico modeling and metabolically engineered versions of bothE. coli andM. succiniciproducens have been constructed. The succinic acid productivity and yield obtained using metabolically engineered bacteria was significantly higher than that obtained using wild-type bacteria.

Metabolically engineered Escherichia coli as a tool for the production of bioenergy and biochemicals from glycerol

Currently, a variety of feedstock is utilized by metabolically engineered bacteria for the production of bioenergy and biochemicals. Recent studies have shown that glycerol can be used as an alternative feedstock for glucose, considering its higher availability, lower price, and high degree of reduction. Hence, this review focuses on recent developments in the bioconversion of glycerol to bioenergy (ethanol and hydrogen) and biochemicals (1,3-propanediol, 1,2-propanediol, 3-hydroxypropionic acid, succinic acid, lactic acid, polyhydroxyalkanoates and Lphenyl alanine) using metabolically engineered Escherichia coli.

Facile synthesis of cysteine–functionalized graphene quantum dots for a fluorescence probe for mercury ions

Cystenine–functionalized graphene quantum dots (cys–GQDs) were synthesized by a simple and facile method, which involves chemical bond formation between L-cysteine and the graphene quantum dots (GQDs) prepared by the carbonization of citric acid. The obtained cys–GQDs show a strong green fluorescence and a uniform lateral size distribution with high crystallinity. The cys–GQDs are further demonstrated as highly sensitive and selective fluorescence probes for Hg2+. In the Hg2+ detection process, the cys–GQDs shows significant fluorescence quenching through a charge transfer process within the cys–GQDs and Hg2+ complex.

Bacteriorhodopsin production by cell recycle culture of Halobacterium halobium

When Halobacterium halobium R1 was cultured with cell recycle in a bioreactor equipped with an external hollow fiber membrane unit, the cell and bacteriorhodopsin concentrations reached in 10 days were 30.3 g cell dry weight/l and 282 mg/l, respectively. The productivity of bacteriorhodopsin (1.15 mg/l·h) was much higher than that (0.16 mg/l·h) obtained by typical batch fermentation.

Production of gamma-aminobutyric acid from glucose by introduction of synthetic scaffolds between isocitrate dehydrogenase, glutamate synthase and glutamate decarboxylase in recombinant Escherichia coli.

Escherichia coli were engineered for the direct production of gamma-aminobutyric acid from glucose by introduction of synthetic protein scaffold. In this study, three enzymes consisting GABA pathway (isocitrate dehydrogenase, glutamate synthase and glutamate decarboxylase) were connected via synthetic protein scaffold. By introduction of scaffold, 0.92g/L of GABA was produced from 10g/L of glucose while no GABA was produced in wild type E. coli. The optimum pH and temperature for GABA production were 4.5 and 30°C, respectively. When competing metabolic network was inactivated by knockout mutation, maximum GABA concentration of 1.3g/L was obtained from 10g/L glucose. The recombinant E. coli strain which produces GABA directly from glucose was successfully constructed by introduction of protein scaffold.

Construction of malate-sensing Escherichia coli by introduction of a novel chimeric two-component system

In an attempt to develop a high-throughput screening system for screening microorganisms which produce high amounts of malate, a MalKZ chimeric HK-based biosensor was constructed. Considering the sequence similarity among Escherichia coli (E. coli) MalK with Bacillus subtilis MalK and E. coli DcuS, the putative sensor domain of MalK was fused with the catalytic domain of EnvZ. The chimeric MalK/EnvZ TCS induced the ompC promoter through the cognate response regulator, OmpR, in response to extracellular malate. Real-time quantitative PCR and GFP fluorescence studies showed increased ompC gene expression and GFP fluorescence as malate concentration increased. By using this strategy, various chimeric TCS-based bacteria biosensors can be constructed, which may be used for the development of biochemical-producing recombinant microorganisms.