(48g) Expression and Characterization of Three Trichoderma Reesei Cellulose Hydrolases in Kluyveromyces Lactis

The recalcitrance of cellulose to hydrolysis is a major technological barrier to commercial implementation of products based on cellulose degradation.  Enzymatic hydrolysis is seen as an environmentally friendly way of breaking down cellulose into its constituent glucose molecules for fermentation to fuels and other fine chemicals.  However, the enzymes which are currently available to hydrolyze cellulose have low specific activities and high production costs.  Expression in yeast expression hosts is a promising method for doing fundamental research on protein activity, with the goal of increasing enzyme specific activity and lowering production costs.  The first step in heterologous enzyme production is characterization of the enzyme when produced in a foreign host.

In this research genes for two exoglucanases and one endoglucanase were isolated from Trichoderma reesei strain QM9414 (ATCC #26921) and inserted into Kluyveromyces lactis GG799 (New England Biolabs) for expression and characterization.  Messenger RNA (mRNA) was isolated using the RNeasy kit, and cDNA synthesized with the Omniscript Reverse Transcription kit (Qiagen) using a 15 residue oligo-dT primer.  Genes for Cel7A (CBH I), Cel6A (CBH II), and Cel7B (EG I) were amplified from the cDNA by polymerase chain reaction (PCR) using primers designed to eliminate the native T. reesei secretory signal sequence and fuse to the α-mating factor secretory signal sequence of K. lactis on the pKLAC1 vector plasmid (NEB) under control of the inducible P_LAC4-PBI promoter.  After transformation and selection, K. lactis transformants were grown on rich media containing 10 g/L yeast extract, 20 g/L peptone, and 20 g/L galactose (YPGal) to induce heterologous protein production.

Cell culture supernatant from K. lactis strains containing each of the three genes was spotted onto Petri dishes containing 50 mM citrate buffer pH 4.5, 0.5% carboxymethyl cellulose (CMC), 0.7% agar, and allowed to incubate overnight at room temperature.  Plates were stained with 0.3% Congo Red and destained with 1 M NaCl.  Clearing zones around the areas spotted with recombinant Cel7B indicated active endoglucanase enzyme.

Next, cell culture supernatant containing rCel7B was desalted by size exclusion chromatography (SEC) over Toyopearl HW-40F resin (Tosoh Biosciences) with a size exclusion limit of 10 kDa.  Protein concentration was performed by anion exchange chromatography (AEX) using Toyopearl DEAE-650M (Tosoh Biosciences) and eluted with 0.5 M NaCl.  Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie Brilliant Blue staining of the rCel7B showed a tight but continuous distribution of molecular weights centered at an approximate value of 67 kDa.  This is slightly higher than the value of 54 kDa reported literature for the native protein¹.  Protein concentration was determined by the Bradford method, and activity assays of the rCel7B were performed using 1% CMC as the substrate in 50 mM citrate buffer, pH 4.5, at 40°C, and the dinitro-salicylic acid (DNS) method was used to detect reducing end formation.  Specific activity of the rCel7B was determined to be approximately 0.5 M glucose/(g enzyme-hr), compared to a glucose standard curve, or about one-fifth of reported values for the native enzyme².

SDS-PAGE of cell culture supernatant containing rCel6A showed three distinct bands of differing molecular weights at approximately 50, 60, and 65 kDa.  This is comparable to the reported value of 58 kDa for the native Cel6A³.  SDS-PAGE following SEC and AEX chromatography of rCel6A in the same manner as for rCel7B discovered that the largest molecular weight form of rCel6A was not bound to the AEX resin, and was therefore not concentrated.  Exoglucanase activity assays were performed on the concentrated rCel6A fraction by incubating 0.1 mg/mL enzyme with 1 mg/mL Avicel PH-101 powdered cellulose (Sigma-Aldrich) in 50 mM citrate, pH 4.5, at 50°C with 250 RPM orbital shaking for three days.  Samples were taken every 24 hours and analyzed by HPLC for glucose production.  No significant exoglucanase activity was detected.  Ultrafiltration was then performed using a Vivacell 70 protein concentrator (Sartorius Stedim) with a 10 kDa cut-off membrane, on the crude rCel6A cell culture supernatant, and on the flowthrough from SEC and AEX chromatography.  Exoglucanase activity assays detected glucose production both in the concentrated supernatant, and in the concentrated flowthrough, indicating that rCel6A is produced in both active and inactive forms by K. lactis, and activity is found only in the largest 65 kDa form of the enzyme.  Furthermore, glucose production from rCel6A on Avicel was found to increase with the addition of 0.01 mg/mL of rCel7B, even though no glucose production was detected from rCel7B acting on Avicel alone.  This result indicates that the recombinant forms of the enzymes retain their specificity for endo- or exo- activity, and can function together synergistically to increase cellulose hydrolysis rate.

SDS-PAGE of rCel7A showed a similar continuous distribution of molecular weights, much like rCel7B.  Concentration was performed by ultrafiltration as for rCel6A.  No detectable exo- or endo-glucanase activity was detected for rCel7A either alone or in combination with other enzymes.  Post-processing, likely glycosylation, is apparently incompatible with producing active recombinant forms of this enzyme in the K. lactis expression system.  DNA sequencing is being performed to verify that complete copies of the gene sequences were correctly inserted into the yeast genome.

References

1.         Shoemaker, S.; Watt, K.; Tsitovsky, G.; Cox, R., Characterization and properties of cellulases purified from Trichoderma reesei strain L27. Nature Biotechnology 1983, 1, 687-690.

2.         Karlsson, J.; Saloheimo, M.; Siika-aho, M.; Tenkanen, M.; Penttilä, M.; Tjerneld, F., Homologous expression and characterization of Cel61A (EG IV) of Trichoderma reesei. European Journal of Biochemistry 2001, 268, 6498-6507.

3.         Tomme, P.; Van Tilbeurgh, H.; Pettersson, G.; Van Damme, J.; Vandekerckhove, J., Studies of the cellulolytic system of Trichoderma reesei QM9414. European Journal of Biochemistry 1988, 170, 575-581.