Jeong Eun Hyeon

Production of minicellulosomes for the enhanced hydrolysis of cellulosic substrates by recombinant Corynebacterium glutamicum

Although cellulosic materials of plant origin are the most abundant utilizable biomass resource, the amino acid-producing organism Corynebacterium glutamicum can not utilize these materials. Here we report the engineering of a C. glutamicum strain expressing functional minicellulosomes containing chimeric endoglucanase E bound to miniCbpA from Clostridium cellulovorans that can hydrolyze cellulosic materials. The chimeric endoglucanase E consists of the endoglucanase E catalytic backbone of Clostridium thermocellum fused with the endoglucanase B dockerin domain of C. cellulovorans. The resulting strain degraded cellulose efficiently by substrate targeting via the carbohydrate binding module. The assembly of minicellulosomes increased the activity against carboxymethyl cellulose approximately 2.8-fold compared with that for the corresponding enzymes alone. This is the first report of the formation of Clostridium minicellulosomes by C. glutamicum. The development of C. glutamicum strain that is capable of more effective cellulose hydrolysis brings about a realization of consolidated bioprocessing for the utilization of cellulosic biomass.

Cellulosome-based, Clostridium-derived multi-functional enzyme complexes for advanced biotechnology tool development: Advances and applications

The cellulosome is one of natures most elegant and elaborate nanomachines and a key biological and biotechnological macromolecule that can be used as a multi-functional protein complex tool. Each protein module in the cellulosome system is potentially useful in an advanced biotechnology application. The high-affinity interactions between the cohesin and dockerin domains can be used in protein-based biosensors to improve both sensitivity and selectivity. The scaffolding protein includes a carbohydrate-binding module (CBM) that attaches strongly to cellulose substrates and facilitates the purification of proteins fused with the dockerin module through a one-step CBM purification method. Although the surface layer homology (SLH) domain of CbpA is not present in other strains, replacement of the cell surface anchoring domain allows a foreign protein to be displayed on the surface of other strains. The development of a hydrolysis enzyme complex is a useful strategy for consolidated bioprocessing (CBP), enabling microorganisms with biomass hydrolysis activity. Thus, the development of various configurations of multi-functional protein complexes for use as tools in whole-cell biocatalyst systems has drawn considerable attention as an attractive strategy for bioprocess applications. This review provides a detailed summary of the current achievements in Clostridium-derived multi-functional complex development and the impact of these complexes in various areas of biotechnology.

Bi-functional cellulases complexes displayed on the cell surface of Corynebacterium glutamicum increase hydrolysis of lignocelluloses at elevated temperature.

Introducing cellulases into Corynebacterium glutamicum leads to the direct degradation of lignocellulosic materials for energy sources. In this study, a cellulase complex containing two cellulolytic enzymes, endoglucanase E (CelE) and β-glucosidase A (BglA), was established to completely degrade cellulose to glucose. The cellulases complexes were displayed on the cell surface of C. glutamicum by using the mechanosensitive channel (Msc) to anchor enzymes in the cytoplasmic membrane. As confirmed by comparison enzyme activities in the cell pellet fraction and supernatant and dual color based immunofluorescence microscopy, the cellulolytic enzymes was successfully associated with the cell surface of C. glutamicum. The displayed cellulases complexes had a synergic effect on the direct conversion of biomass to reducing sugars leading to 3.1- to 6.0-fold increase compared to the conversion by the secreted cellulases complexes. In addition, the displayed cellulases complexes increased the residual activities of cCelE and cBglA at 70°C from 28.3% and 24.3% in the secreted form to 65.1% and 82.8%, respectively. The display of cellulases complexes on the cell surface of C. glutamicum enhances the polysaccharide equivalent and the direct saccharification of low cost biomass via the action of multi-thermostable enzyme complexes.

GntR-Type Transcriptional Regulator PckR Negatively Regulates the Expression of Phosphoenolpyruvate Carboxykinase in Corynebacterium glutamicum

The pck (cg3169) gene of Corynebacterium glutamicum encodes a phosphoenolpyruvate carboxykinase (PEPCK). Here, a candidate transcriptional regulator that binds to the promoter region of pck was detected using a DNA affinity purification approach. An isolated protein was identified to be PckR (Cg0196), a GntR family transcriptional regulator which consists of 253 amino acids with a mass of 27 kDa as measured by peptide mass fingerprinting. The results of electrophoretic mobility shift assays verified that PckR specifically binds to the pck promoter. The putative regulator binding region extended from position -44 to -27 (an 18-bp sequence) relative to the transcriptional start point of the pck gene. We measured the expression of pck in a pckR deletion mutant by using quantitative real-time reverse transcription-PCR. The expression level of pck in the pckR mutant was 7.6 times higher than that in wild-type cells grown in glucose. Comparative DNA microarray hybridizations and bioinformatic searches revealed the gene composition of the transcriptional regulon of C. glutamicum. Based on these results, PckR seemed to play an important role in the regulation of PEPCK in C. glutamicum grown in glucose. In particular, these assays revealed that PckR acts as a repressor of pck expression during glucose metabolism.

Identification and characterization of a transcriptional regulator, SucR, that influences sucCD transcription in Corynebacterium glutamicum.

We have identified and characterized a novel transcriptional regulator that binds to the promoter region of succinyl-CoA synthetase (sucCD) in Corynebacterium glutamicum. Using biotin-labeled DNA affinity beads, we identified a DeoR-type transcriptional regulator, SucR (Cg0146), which is a protein consisting of 282 amino acids with a mass of 31 kDa and RamB (Cg0444). The results of electrophoretic mobility shift assays verified that these regulators specifically bind to the sucCD promoter region. The putative SucR binding region extends from position -155 to -146 (a 10 bp sequence, ACTCTAGGGG) relative to the transcriptional start point of the sucCD operon. The expression level of sucCD in a sucR deletion mutant was seven times higher than that in wild-type cells grown on acetate. The increase in succinyl-CoA synthetase levels caused by inactivation of sucR. These assays revealed that SucR acts as a repressor of sucCD expression during acetate metabolism.

5-Aminolevulinic acid production in engineered Corynebacterium glutamicum via C5 biosynthesis pathway.

ALA (5-aminolevulinic acid) is an important intermediate in the synthesis of tetrapyrroles and the use of ALA has been gradually increasing in many fields, including medicine and agriculture. In this study, improved biological production of ALA in Corynebacterium glutamicum was achieved by overexpressing glutamate-initiated C5 pathway. For this purpose, copies of the glutamyl t-RNA reductase HemA from several bacteria were mutated by site-directed mutagenesis of which a HemA version from Salmonella typhimurium exhibited the highest ALA production. Cultivation of the HemA-expressing strain produced approximately 204 mg/L of ALA, while co-expression with HemL (glutamate-1-semialdehyde aminotransferase) increased ALA concentration to 457 mg/L, representing 11.6- and 25.9-fold increases over the control strain (17 mg/L of ALA). Further effects of metabolic perturbation were investigated, leading to penicillin addition that further improves ALA production to 584 mg/L. In an optimized flask fermentation, engineered C. glutamicum strains expressing the HemA and hemAL operon produced up to 1.1 and 2.2g/L ALA, respectively, under glutamate-producing conditions. The final yields represent 10.7- and 22.0-fold increases over the control strain (0.1g/L of ALA). From these findings, ALA biosynthesis from glucose was successfully demonstrated and this study is the first to report ALA overproduction in C. glutamicum via metabolic engineering.

Enhanced hydrolysis of lignocellulosic biomass: Bi‐functional enzyme complexes expressed in Pichia pastoris improve bioethanol production from Miscanthus sinensis

Lignocellulosic biomass is the most abundant utilizable natural resource. In the process of bioethanol production from lignocellulosic biomass, an efficient hydrolysis of cellulose and hemicellulose to release hexose and pentose is essential. We have developed a strain of Pichia pastoris that can produce ethanol via pentose and hexose using an assembly of enzyme complexes. The use of enzyme complexes is one of the strategies for effective lignocellulosic biomass hydrolysis. Xylanase XynB from Clostridium cellulovorans and a chimeric endoglucanase cCelE from Clostridium thermocellum were selected as enzyme subunits, and were bound to a recombinant scaffolding protein mini‐CbpA from C. cellulovorans to assemble the enzyme complexes. These complexes efficiently degraded xylan and carboxymethylcellulose (CMC), producing approximately 1.18 and 1.07 g/L ethanol from each substrate, respectively, which is 2.3‐fold and 2.7‐fold higher than that of the free‐enzyme expressing strain. Miscanthus sinensis was investigated as the lignocellulosic biomass for producing bioethanol, and 1.08 g/L ethanol was produced using our recombinant P. pastoris strain, which is approximately 1.9‐fold higher than that of the wild‐type strain. In future research, construction of enzyme complexes containing various hydrolysis enzymes could be used to develop biocatalysts that can completely degrade lignocellulosic biomass into valuable products such as biofuels.

Design of nanoscale enzyme complexes based on various scaffolding materials for biomass conversion and immobilization

The utilization of scaffolds for enzyme immobilization involves advanced bionanotechnology applications in biorefinery fields, which can be achieved by optimizing the function of various enzymes. This review presents various current scaffolding techniques based on proteins, microbes and nanomaterials for enzyme immobilization, as well as the impact of these techniques on the biorefinery of lignocellulosic materials. Among them, architectural scaffolds have applied to useful strategies for protein engineering to improve the performance of immobilized enzymes in several industrial and research fields. In complexed enzyme systems that have critical roles in carbon metabolism, scaffolding proteins assemble different proteins in relatively durable configurations and facilitate collaborative protein interactions and functions. Additionally, a microbial strain, combined with designer enzyme complexes, can be applied to the immobilizing scaffold because the in vivo immobilizing technique has several benefits in enzymatic reaction systems related to both synthetic biology and metabolic engineering. Furthermore, with the advent of nanotechnology, nanomaterials possessing ideal physicochemical characteristics, such as mass transfer resistance, specific surface area and efficient enzyme loading, can be applied as novel and interesting scaffolds for enzyme immobilization. Intelligent application of various scaffolds to couple with nanoscale engineering tools and metabolic engineering technology may offer particular benefits in research.

Efficient enzymatic degradation process for hydrolysis activity of the Carrageenan from red algae in marine biomass.

Carrageenan is a generic name for a family of polysaccharides obtained from certain species of red algae. New methods to produce useful cost-efficiently materials from red algae are needed to convert enzymatic processes into fermentable sugars. In this study, we constructed chimeric genes cCgkA and cCglA containing the catalytic domain of κ-carrageenase CgkA and λ-carrageenase CglA from Pseudoalteromonas carrageenovora fused with a dockerin domain. Recombinant strains expressing the chimeric carrageenase resulted in a halo formation on the carrageenan plate by alcian blue staining. The recombinant cCgkA and cCglA were assembled with scaffoldin miniCbpA via cohesin and dockerin interaction. Carbohydrate binding module (CBM) in scaffoldin was used as a tag for cellulose affinity purification using cellulose as a support. The hydrolysis process was monitored by the amount of reducing sugar released from carrageenan. Interestingly, these results indicated that miniCbpA, cCgkA and cCglA assembled into a complex and that the dockerin-fused enzymes on the scaffoldin had synergistic activity in the degradation of carrageenan. The observed enhancement of activity by carrageenolytic complex was 3.1-fold-higher compared with the corresponding enzymes alone. Thus, the assemblies of advancement of active enzyme complexes will facilitate the commercial production of useful products from red algae biomass which represents inexpensive and sustainable feed-stocks.

Enhanced thermostability of mesophilic endoglucanase Z with a high catalytic activity at active temperatures

This is the first study for therrmostable mutants of mesophilic endoglucanase EngZ from Clostridium cellulovorans using by site-directed mutagenesis. K94R, S365P and their double mutant K94R/S365P had a wide range of active temperatures (30-60 °C). In addition, the optimal temperature of K94R/S365P was increased by 7.5 °C. K94R/S365P retained 78.3% relative activity at 70 °C, while the wild type retained only 5.8%. Especially, K94R/S365P remained 45.1-fold higher activity than the wild type at 70 °C. In addition, K94R/S365P was 3.1-fold higher activity than the wild type at 42.5 °C, which is the optimal temperature of the wild type. K94R/S365P showed also stimulated in 2.5-fold lower concentration of CaCl2 and delayed aggregation temperature in the presence of CaCl2 compared to the wild type. In pH stability, K94R/S365P was not influenced, but the optimum pH was transferred from pH 7 to pH 6. In long-term hydrolysis, K94R/S365P reduced the newly released reducing sugar yields after 12h reaction; however, the yields consistently increased until 72h. Finally, the total reducing sugar of K94R/S365P was 5.0-fold higher than the wild type at 50 °C, pH6. EngZ (K94R/S365P) can support information to develop thermostability of GH9 endoglucanase with a high catalytic efficiency as the potential industrial bioprocess candidate.