Thermobifida Fusca Research Articles

Cutinases catalyze polyacrylate hydrolysis and prevent their aggregation

During the recycling of waste paper, the accumulation of polyacrylates present in the waste paper causes the formation of tacky substances known as stickies. Deposition of these stickies on the machinery decreases the quality of the recycled paper and increases the usage of circulating water. Enzymes that hydrolyze these polyacrylates can minimize or eliminate the deposition of stickies. In initial experiments, the abilities of the cutinases from Humicola insolens, Fusarium solani and Thermobifida fusca to hydrolyze poly (methyl acrylate) (PMA) and poly (ethyl acrylate) (PEA) within a macroporous resin were compared. Then, to simulate the environment encountered during paper recycling, PMA and PEA dispersions were used as substrates. The decrease in turbidity was measured at a concentration of 0.5 mg mL−1. When used at pH 8.0 and 30–50 °C, T. fusca cutinase limited the turbidity decrease to about 1.0% and favored the hydrolysis of PEA over PMA. F. solani and H. insolens cutinases performed best at pH 8.5 and temperatures of 35 and 50 °C, respectively. At a polyacrylate concentration of 0.1 mg mL−1, the optimal temperatures of these cutinases decreased. The optimal T. fusca cutinase dosage was lower than those of F. solani and H. insolens cutinases.

Assembly and Translocation of a CRISPR-Cas Primed Acquisition Complex

CRISPR-Cas systems confer an adaptive immunity against viruses. Following viral injection, Cas1-Cas2 integrates segments of the viral genome (spacers) into the CRISPR locus. In type I CRISPR-Cas systems, efficient "primed" spacer acquisition and viral degradation (interference) require both the Cascade complex and the Cas3 helicase/nuclease. Here, we present single-molecule characterization of the Thermobifida fusca (Tfu) primed acquisition complex (PAC). We show that TfuCascade rapidly samples non-specific DNA via facilitated one-dimensional diffusion. Cas3 loads at target-bound Cascade and the Cascade/Cas3 complex translocates via a looped DNA intermediate. Cascade/Cas3 complexes stall at diverse protein roadblocks, resulting in a double strand break at the stall site. In contrast, Cas1-Cas2 samples DNA transiently via 3D collisions. Moreover, Cas1-Cas2 associates with Cascade and translocates with Cascade/Cas3, forming the PAC. PACs can displace different protein roadblocks, suggesting a mechanism for long-range spacer acquisition. This work provides a molecular basis for the coordinated steps in CRISPR-based adaptive immunity.

Continuous production of d-lactic acid from cellobiose in cell recycle fermentation using β-glucosidase-displaying Escherichia coli

The present study demonstrates continuous production of d-lactic acid from cellobiose in a cell recycle fermentation with a hollow fiber membrane using recombinant Escherichia coli constructed by deleting its pyruvate formate-lyase activating enzyme gene pflA and expressing a heterologous β-glucosidase on its cell surface. The β-glucosidase gene bglC from Thermobifida fusca YX was cloned into a cell surface display vector pGV3, resulting in pGV3-bglC. Recombinant E.coli JM109 harboring the pGV3-bglC showed β-glucosidase activity (18.9 ± 5.7 U/OD600), indicating the cell surface functioning of mutant β-glucosidase. pH-stat cultivation using d-lactic acid producer E.coli BW25113 (ΔpflA) harboring pGV3-bglC in minimum medium with 10g/L cellobiose in a jar fermentor under anaerobic condition resulted in 5.2 ± 0.1g/L of d-lactic acid was obtained after 84h cultivation, indicating that the engineered E.coli produced d-lactic acid directly from cellobiose. For continuous d-lactic acid production, cell recycle fermentation was conducted under anaerobic condition and the culture was continuously ultrafiltrated with a hollow fiber cartridge. The permeate was drawn to the reservoir and a minimum medium containing 10g/L cellobiose was fed to the fermentor at the same rate (dilution rate, 0.05 h-1). Thus, this system maintained the d-lactic acid production (4.3-5.0g/L), d-lactic acid production rate (0.22-0.25g/L/h), and showed no residual cellobiose in the culture during 72h operation. Interestingly, the d-lactic acid production rate in cell recycle fermentation was more than 3 times higher than that in the batch operation (0.06 ± 0.00g/L/h).

Cold-Adaptation Signatures in the Ligand Rebinding Kinetics to the Truncated Hemoglobin of the Antarctic Bacterium Pseudoalteromonas haloplanktis TAC125.

Cold-adapted organisms have evolved proteins endowed with higher flexibility and lower stability in comparison to their thermophilic homologues, resulting in enhanced reaction rates at low temperatures. In this context, protein-bound water molecules were suggested to play a major role, and their weaker interactions at protein active sites have been associated with cold adaptation. In this work, we tested this hypothesis on truncated hemoglobins (a family of microbial heme-proteins of yet-unclear function) applying molecular dynamics simulations and ligand-rebinding kinetics on a protein from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 in comparison with its thermophilic Thermobifida fusca homologue. The CO rebinding kinetics of the former highlight several geminate phases, with an unusually long-lived geminate intermediate. An articulated tunnel with at least two distinct docking sites was identified by analysis of molecular dynamics simulations and was suggested to be at the origin of the unusual geminate rebinding phase. Water molecules are present in the distal pocket, but their stabilization by TrpG8, TyrB10, and HisCD1 is much weaker than in thermophilic Thermobifida fusca truncated hemoglobin, resulting in a faster geminate rebinding. Our results support the hypothesis that weaker water-molecule interactions at the reaction site are associated with cold adaptation.

Immobilization of β‐Glucosidase BglC on Decanedioic Acid‐Modified Magnetic Nanoparticles

Abstractβ‐Glucosidase BglC from Thermobifida fusca was efficiently immobilized on decanedioic acid‐modified magnetic nanoparticles (DMNP). Interestingly, the immobilized BglC showed much higher organic solvent stability than the free enzyme, indicating that this enzyme immobilized on DMNP should be especially suitable for catalyzing reactions in organic solvents. Additionally, the immobilized BglC also showed better thermal stability, storage stability, and mechanical stability than the free counterpart. Moreover, the reuse times were satisfactory and even crude plant materials (e.g., rice husk and corn cob) were used as substrates, which should be valuable for the decomposition of natural organic materials and the full utilization of bioresources.

Engineering Escherichia coli for Glutarate Production as the C5 Platform Backbone.

Glutarate is a linear-chain dicarboxylic acid with wide applications in the production of polyesters and polyamides such as nylon-4,5 and nylon-5,5. Previous studies focused on the biological production of glutarate from lysine with low yields and titers. Here, we report on glutarate production by Escherichia coli using a five-step reverse adipate degradation pathway (RADP) identified in Thermobifida fusca By expressing the enzymes of RADP, the glutarate was detected by strain Bgl146 in shaken flasks. After fermentation optimization, the titer of glutarate by Bgl146 was increased to 4.7 ± 0.2 mM in shaken flasks. We further eliminated pathways for the major metabolites competing for carbon flux by CRISPR/Cas9 (ΔarcA, ΔldhA, ΔatoB, and ΔpflB). Moreover, the final strain Bgl4146 produced 36.5 ± 0.3 mM glutarate by fed-batch fermentation. These results constitute the highest glutarate titer reported in E. coliIMPORTANCE Glutarate is an important C5 linear-chain dicarboxylic acid, which is widely used in polyesters and polyamides such as nylon-4,5 and nylon-5,5 in the chemical industry. Glutarate is currently produced from the feedstocks derived from petroleum, specifically by oxidation of a mixture of cyclohexanone and cyclohexanol catalyzed by nitric acid. However, the chemical synthesis results in high pollution and dramatic greenhouse gas emission. Thus, the biological production of glutarate directly from the substrate is of great importance. Although there have been reports using Corynebacterium glutamicum to produce glutarate, it has serious limitations due to the limited lysine supply and long fermentation time. To solve this problem, a novel synthetic pathway was constructed in this study, and the highest glutarate titer was reported in Escherichia coli using a short fermentation time without lysine addition, making bio-based glutarate production much more feasible.

Coevolution of both Thermostability and Activity of Polyphosphate Glucokinase from Thermobifida fusca YX.

Thermostability and specific activity of enzymes are two of the most important properties for industrial biocatalysts. Here, we developed a petri dish-based double-layer high-throughput screening (HTS) strategy for rapid identification of desired mutants of polyphosphate glucokinase (PPGK) from a thermophilic actinobacterium, Thermobifida fusca YX, with both enhanced thermostability and activity. Escherichia coli colonies representing a PPGK mutant library were grown on the first-layer Phytagel-based plates, which can remain solid for 1 h, even at heat treatment temperatures of more than 100°C. The second layer that was poured on the first layer contained agarose, substrates, glucose 6-phosphate dehydrogenase (G6PDH), the redox dye tetranitroblue tetrazolium (TNBT), and phenazine methosulfate. G6PDH was able to oxidize the product from the PPGK-catalyzed reaction and generate NADH, which can be easily examined by a TNBT-based colorimetric assay. The best mutant obtained after four rounds of directed evolution had a 7,200-fold longer half-life at 55°C, 19.8°C higher midpoint of unfolding temperature (Tm ), and a nearly 3-fold enhancement in specific activities compared to those of the wild-type PPGK. The best mutant was used to produce 9.98 g/liter myo-inositol from 10 g/liter glucose, with a theoretical yield of 99.8%, along with two other hyperthermophilic enzymes at 70°C. This PPGK mutant featuring both great thermostability and high activity would be useful for ATP-free production of glucose 6-phosphate or its derived products.IMPORTANCE Polyphosphate glucokinase (PPGK) is an enzyme that transfers a terminal phosphate group from polyphosphate to glucose, producing glucose 6-phosphate. A petri dish-based double-layer high-throughput screening strategy was developed by using ultrathermostable Phytagel as the first layer instead of agar or agarose, followed by a redox dye-based assay for rapid identification of ultrathermostable PPGK mutants. The best mutant featuring both great thermostability and high activity could produce glucose 6-phosphate from glucose and polyphosphate without in vitro ATP regeneration.

Cloning and characterization of a chitinase from Thermobifida fusca reveals Tfu_0580 as a thermostable and acidic endochitinase

Being capable of hydrolyzing chitin, chitinases have various applications such as production of N-acetylchitooligosaccharides (COSs) and N-acetylglucosamine (GlcNAc), degrading chitin as a consolidated bioprocessing, and bio-control of fungal phytopathogens. Here, a putative chitinase in Thermobifida fusca, Tfu_0580, is characterized. Tfu_0580 was purified by homogeneity with a molecular weight of 44.9 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Tfu_0580 displayed a clear activity against colloidal chitin, which is comparable to a commercial Streptomyces griseus chitinase. Enzyme activities against p-nitrophenyl β-D-N,N′,N′′-triacetylchitotriose (p-NP-(GlcNAc)3), N,N′-diacetyl-β-D-chitobioside (p-NP-(GlcNAc)2) and p-nitrophenyl N-acetyl-β-D-glucosaminide (p-NP-(GlcNAc)) showed that Tfu_0580 exhibited highest activity against p-NP-(GlcNAc)3. Further optimization of the enzyme activity conditions showed: 1) an optimum catalytic activity at pH 6.0 and 30 °C; 2) activity over broad pH (4.8–7.5) and temperature (20–55 °C); 3) stimulation of activity by the metallic ions Ca2+ and Mn2+.

Refactoring the upper sugar metabolism of Pseudomonas putida for co-utilization of cellobiose, xylose, and glucose

Given its capacity to tolerate stress, NAD(P)H/ NAD(P) balance, and increased ATP levels, the platform strain Pseudomonas putida EM42, a genome-edited derivative of the soil bacterium P. putida KT2440, can efficiently host a suite of harsh reactions of biotechnological interest. Because of the lifestyle of the original isolate, however, the nutritional repertoire of P. putida EM42 is centered largely on organic acids, aromatic compounds and some hexoses (glucose and fructose). To enlarge the biochemical network of P. putida EM42 to include disaccharides and pentoses, we implanted heterologous genetic modules for D-cellobiose and D-xylose metabolism into the enzymatic complement of this strain. Cellobiose was actively transported into the cells through the ABC complex formed by native proteins PP1015-PP1018. The knocked-in β-glucosidase BglC from Thermobifida fusca catalyzed intracellular cleavage of the disaccharide to D-glucose, which was then channelled to the default central metabolism. Xylose oxidation to the dead end product D-xylonate was prevented by deleting the gcd gene that encodes the broad substrate range quinone-dependent glucose dehydrogenase. Intracellular intake was then engineered by expressing the Escherichia coli proton-coupled symporter XylE. The sugar was further metabolized by the products of E. coli xylA (xylose isomerase) and xylB (xylulokinase) towards the pentose phosphate pathway. The resulting P. putida strain co-utilized xylose with glucose or cellobiose to complete depletion of the sugars. These results not only show the broadening of the metabolic capacity of a soil bacterium towards new substrates, but also promote P. putida EM42 as a platform for plug-in of new biochemical pathways for utilization and valorization of carbohydrate mixtures from lignocellulose processing.

A downstream box fusion allows stable accumulation of a bacterial cellulase in Chlamydomonas reinhardtii chloroplasts

BackgroundWe investigated strategies to improve foreign protein accumulation in the chloroplasts of the model algae Chlamydomonas reinhardtii and tested the outcome in both standard culture conditions as well as one pertinent to algal biofuel production. The downstream box (DB) of the TetC or NPTII genes, the first 15 codons following the start codon, was N-terminally fused to the coding region of cel6A, an endoglucanase from Thermobifida fusca. We also employed a chimeric regulatory element, consisting of the 16S rRNA promoter and the atpA 5′UTR, previously reported to enhance protein expression, to regulate the expression of the TetC-cel6A gene. We further investigated the accumulation of TetC-Cel6A under N-deplete growth conditions.ResultsBoth of the DB fusions improved intracellular accumulation of Cel6A in transplastomic C. reinhardtii strains though the TetC DB was much more effective than the NPTII DB. Furthermore, using the chimeric regulatory element, the TetC-Cel6A protein accumulation displayed a significant increase to 0.3% total soluble protein (TSP), whereas NPTII-Cel6A remained too low to quantify. Comparable levels of TetC- and NPTII-cel6A transcripts were observed, which suggests that factors other than transcript abundance mediate the greater TetC-Cel6A accumulation. The TetC-Cel6A accumulation was stable regardless of the growth stage, and the transplastomic strain growth rate was not altered. When transplastomic cells were suspended in N-deplete medium, cellular levels of TetC-Cel6A increased over time along with TSP, and were greater than those in cells suspended in N-replete medium.ConclusionsThe DB fusion holds great value as a tool to enhance foreign protein accumulation in C. reinhardtii chloroplasts and its influence is related to translation or other post-transcriptional processes. Our results also suggest that transplastomic protein production can be compatible with algal biofuel production strategies. Cells displayed a consistent accumulation of recombinant protein throughout the growth phase and nitrogen starvation, a strategy used to induce lipid production in algae, led to higher cellular heterologous protein content. The latter result is contrary to what might have been expected a priori and is an important result for the development of future algal biofuel systems, which will likely require co-products for economic sustainability.

Studies on Degradation of 7-ketocholesterol by Environmental Bacterial Isolates

Medical bioremediation is a unique strategy of targeting pathogenic compounds with an exogenous enzyme of microbial origin. The objective of this study was to isolate and screen the microorganisms from diverse environmental samples for their ability to catabolize 7-ketocholesterol. Isolation of bacterial strains was performed and molecular identification was carried out by amplification and sequencing of 16S rDNA for 4 the best degrader isolates. Degradation was confirmed on the basis of UV spectrophotometric and HPLC analysis. Four bacterial isolates, showing high catabolic activity towards 7-ketocholesterol were isolated: Alcanivorax jadensis IP4 (accession number KP309836; sea water sediment), Streptomyces auratus IP2 (accession number KP309837; soil), Serratia marcescens IP3 (accession number KP309838; soil) and Thermobifida fusca IP1 (accession number KM677184; manure piles). All the isolates were capable of utilizing 7-ketocholesterol as the sole organic substrate, resulting in its mineralisation. The most rapid degradation was observed with A. jadensis IP4 followed by T. fusca IP1. The degradation was followed and analyzed by HPLC. A. jadensis IP4 removed 7-ketocholesterol below detection levels within 8 days.

Metabolic engineering of Escherichia coli for producing adipic acid through the reverse adipate-degradation pathway

Adipic acid is an important dicarboxylic acid mainly used for the production of nylon 6–6 fibers and resins. Previous studies focused on the biological production of adipic acid directly from different substrates, resulting in low yields and titers. In this study, a five-step reverse adipate-degradation pathway (RADP) identified in Thermobifida fusca has been reconstructed in Escherichia coli BL21 (DE3). The resulting strain (Mad136) produced 0.3 g L−1 adipic acid with a 11.1% theoretical yield in shaken flasks, and we confirmed that the step catalyzed by 5-Carboxy-2-pentenoyl-CoA reductase (Tfu_1647) as the rate-limiting step of the RADP. Overexpression of Tfu_1647 by pTrc99A carried by strain Mad146 produced with a 49.5% theoretical yield in shaken flasks. We further eliminated pathways for major metabolites competing for carbon flux by CRISPR/Cas9 and deleted the succinate-CoA ligase gene to promote accumulation of succinyl-CoA, which is the precursor for adipic acid synthesis. The final engineered strain Mad123146, which could achieve 93.1% of the theoretical yield in the shaken flask, was able to produce 68.0 g L−1 adipic acid by fed-batch fermentation. To the best of our knowledge, these results constitute the highest adipic acid titer reported in E. coli.

Structural determinants of ligand binding in truncated hemoglobins: Resonance Raman spectroscopy of the native states and their carbon monoxide and hydroxide complexes

The ligand binding characteristics of heme-containing proteins are determined by a number of factors, including the nature and conformation of the distal residues and their capability to stabilize the heme-bound ligand via hydrogen-bonding and electrostatic interactions. In this regard, the heme pockets of truncated hemoglobins (TrHbs) constitute an interesting case study as they share many common features, including a number of polar cavity residues. In this review, we will focus on three proteins of group II TrHbs, from Thermobifida fusca (Tf-HbO) and Pseudoalteromonas haloplanktis TAC125 (Ph-HbO). Although the residues in positions G8 (Trp) and B10 (Tyr) are conserved in all three proteins, the CD1 residue is a Tyr in T. fusca and a His in P. haloplanktis. Comparison of the ligand binding characteristics of these proteins, in particular the hydroxo and CO ligands by means of resonance Raman spectroscopy, reveals that this single difference in the key heme cavity residues markedly affects their ligand binding capability and conformation. Furthermore, although the two Ph-HbOs (Ph-HbO-2217 and Ph-HbO-0030) have identical key cavity residues, they display distinct ligand binding properties.

Locust bean gum galactomannan hydrolyzed by thermostable β-d-mannanase may reduce the secretion of pro-inflammatory factors and the release of granule constituents

Locust bean gum (LBG) galactomannan has been claimed to have applications in the biopharmaceutical field. However, the effects of LBG galactomannan on immunomodulatory aspects are not yet clear. The purpose of this study was to over-express thermostable β-d-mannanase from the thermophilic actinomycete Thermobifida fusca BCRC 19214 using a Pichia pastoris expression system. The maximum intracellular β-d-mannanase activity obtained from the cell-free extract was approximately 40.0U/mL after 72h of cultivating a P. pastoris transformant (pPICZ-man) induced with methanol. Hydrolysis of native LBG galactomannan with 8U/mL β-d-mannanase for 24h significantly decreased the weight-average molecular weight of LBG galactomannan from 5,580,010 to 3188. Native and hydrolyzed LBG galactomannan in a range of 0–0.2% did not trigger significant cytotoxicity after 24h of treatment compared with the control. The native LBG galactomannan stimulated RAW 264.7 cells to produce cytokine TNF-α dose-dependently, but there was no significant IL-1β or nitric oxide production. The native LBG galactomannan also stimulated β-hexosaminidase secretion in RBL-2H3 cells. After the native LBG galactomannan was hydrolyzed with β-d-mannanase, all of the immunological properties disappeared. These results suggest the possible immunomodulatory effects of native LBG galactomannan.

Substituting Both the N-Terminal and "Cord" Regions of a Xylanase from Aspergillus oryzae to Improve Its Temperature Characteristics.

To improve the temperature characteristics of AoXyn11A, a mesophilic glycoside hydrolase family (GHF) 11 xylanase from Aspergillus oryzae CICC40186, its N-terminal and "cord" regions were selected to be substituted by means of the computer-aided analysis and calculation. In brief, one mutant, named ATX11A41, possessing the lowest root-mean-square deviation (RMSD) value was designed based on the molecular dynamics (MD) simulation by substituting the N-terminal 41 amino acids of AoXyn11A with the corresponding 42 ones of pXYL11, a thermophilic GHF11 xylanase from Thermobifida fusca. On the basis of the primary structure alignment of pXYL11 with ATX11A41 (or AoXyn11A), another mutant, named ATX11A41/cord, was designed by substituting the cord region (93GTYNPGSGG101) of ATX11A41 with the corresponding one (93GTYRPTG99) of pXYL11. Both mutant-encoding genes, ATx11A41 and ATx11A41/cord, were constructed as designed theoretically by a megaprimer PCR technique and were expressed in Pichia pastoris GS115. The specific activities of recombinant (re) AoXyn11A, ATX11A41, and ATX11A41/cord were 2916.7, 2667.6, and 2457.0U/mg, respectively. The analysis of temperature characteristics displayed that the temperature optimum (Topt) of reATX11A41 or reATX11A41/cord was 65°C, which was 15°C higher than that of reAoXyn11A. The thermal inactivation half-life (t1/2) values of reATX11A41 and reATX11A41/cord at 60°C were 55 and 83min, respectively, whereas that of reAoXyn11A was only 18min at 50°C. The melting temperature (Tm) values of reAoXyn11A, reATX11A41, and reATX11A41/cord were 54.2, 66.7, and 71.9°C, respectively. In conclusion, the above findings indicated that the substitution of both the N-terminal and cord regions of a mesophilic AoXyn11A greatly contributed to its improved temperature characteristics.

Characterization of CYP154F1 from Thermobifida fusca YX and Extension of Its Substrate Spectrum by Site-Directed Mutagenesis.

Previous studies on cytochrome P450 monooxygenases (CYP) from family 154 reported their substrate promiscuity and high activity. Hence, herein, the uncharacterized family member CYP154F1 is described. Screening of more than 100 organic compounds revealed that CYP154F1 preferably accepts small linear molecules with a carbon chain length of 8-10 atoms. In contrast to thoroughly characterized CYP154E1, CYP154F1 has a much narrower substrate spectrum and lower activity. A structural alignment of homology models of CYP154F1 and CYP154E1 revealed few differences in the active sites of both family members. By gradual mutagenesis of the CYP154F1 active site towards those of CYP154E1, a key residue accounting for the different activities of both enzymes was identified at position 234. Substitution of T234 for large hydrophobic amino acids led to up to tenfold higher conversion rates of small substrates, such as geraniol. Replacement of T234 by small hydrophobic amino acids, valine or alanine, resulted in mutants with extended substrate spectra. These mutants are able to convert some of the larger substrates of CYP154E1, such as (E)-stilbene and (+)-nootkatone.

Over-Expression of the Thermobifida fusca β-Glucosidase in a Yarrowia lipolytica Transformant to Degrade Soybean Isoflavones

A gene (bgl) encoding a β-glucosidase in thermophilic actinomycete Thermobifida fusca NTU 22 was cloned into a Yarrowia lipolytica expression system. Heterologous expression resulted in extracellular β-glucosidase production with activity as high as 630 U/mL in a Hinton flask culture filtrate. This recombinant β-glucosidase was purified 9.2-fold from crude culture filtrate by DEAE-Sepharose FF column chromatography as measured by its increase in specific activity. The overall yield of the purified enzyme was 47.5%. The molecular weight of the purified β-glucosidase estimated by SDS-PAGE was 45 kDa, which agreed with the predicted molecular weight based on the nucleotide sequence. About 15% enzyme activity loss was observed after the enzyme was heat-treated at 50 °C for 180 min. It was also found that the activity of the enzyme was inhibited by Hg2+, Cu2+, Ba2+, Ag+, p-chloromercuribenzene, and iodoacetate. The β-glucosidase from T. fusca had the most activity for daidzein-7-glucoside and genistein-7-glucoside among the tested flavonoid glycosides, but there was moderate or little activity for luteolin-7-glucoside, cyanidine-3-glucoside, and quercetin-3-glucoside. These properties are important for the soybean isoflavone applications of this β-glucosidase.

Casein-based scaffold for artificial cellulosome design

Abstract Cellulosomal systems are known as highly efficient biocatalysts in the degradation of lignocellulosic biomass in nature, but they remain unsuitable for industrial applications. In seeking alternatives to natural cellulosomes, casein was chosen as a scaffold for cellulase clustering. Casein is recognized as an excellent substrate for microbial transglutaminase (MTG) because it contains naturally reactive glutamine and lysine residues. A substrate peptide containing an MTG-reactive lysine residue was inserted into the C-terminus of the endoglucanase Cel5A and Cel6A from Thermobifida fusca using genetic engineering. The engineered cellulases, EG(Cel5A) and EG(Cel6A), were conjugated onto casein in different ratios by an MTG-mediated site-specific protein crosslinking reaction. Overall, a more than two-fold increase was observed when EG(Cel5A) was conjugated onto N,N-dimethylcasein, but a small or no change was observed for EG(Cel6A).

Development of a novel cellulase biosensor that detects crystalline cellulose hydrolysis using a transcriptional regulator

Successful utilization of cellulose as renewable biomass depends on the development of economically feasible technologies, which can aid in enzymatic hydrolysis. In this study, we developed a whole-cell biosensor for detecting cellulolytic activity that relies on the recognition of cellobiose using the transcriptional factor CelR from Thermobifida fusca and transcriptional activation of its downstream gfp reporter gene. The fluorescence intensity of whole-cell biosensor, which was named as cellobiose-detectible genetic enzyme screening system (CBGESS), was directly proportional to the concentration of cellobiose. The strong fluorescence intensity of CBGESS demonstrated the ability to detect cellulolytic activity with two cellulosic substrates, carboxymethyl cellulose and p-nitrophenyl β-D-cellobioside in cellulase-expressing Escherichia coli. In addition, CBGESS easily sensed crystalline cellulolytic activity when commercial Celluclast 1.5L was dropped on an Avicel plate. Therefore, CBGESS is a powerful tool for detecting cellulolytic activity with high sensitivity in the presence of soluble or insoluble cellulosic substrates. CBGESS may be further applied to excavate novel cellulases or microbes from both genetic libraries and various environments.

Metabolic Profile of the Cellulolytic Industrial Actinomycete Thermobifida fusca.

Actinomycetes have a long history of being the source of numerous valuable natural products and medicinals. To expedite product discovery and optimization of biochemical production, high-throughput technologies can now be used to screen the library of compounds present (or produced) at a given time in an organism. This not only facilitates chemical product screening, but also provides a comprehensive methodology to the study cellular metabolic networks to inform cellular engineering. Here, we present some of the first metabolomic data of the industrial cellulolytic actinomycete Thermobifida fusca generated using LC-MS/MS. The underlying objective of conducting global metabolite profiling was to gain better insight on the innate capabilities of T. fusca, with a long-term goal of facilitating T. fusca-based bioprocesses. The T. fusca metabolome was characterized for growth on two cellulose-relevant carbon sources, cellobiose and Avicel. Furthermore, the comprehensive list of measured metabolites was computationally integrated into a metabolic model of T. fusca, to study metabolic shifts in the network flux associated with carbohydrate and amino acid metabolism.