Expression of lignocellulolytic enzymes in Pichia pastoris

BACKGROUND: Sustainable utilization of plant biomass as renewable source for fuels and chemical building blocks requires a complex mixture of diverse enzymes, including hydrolases which comprise the largest class of lignocellulolytic enzymes. These enzymes need to be available in large amounts at a low price to allow sustainable and economic biotechnological processes. Over the past years Pichia pastoris has become an attractive host for the cost-efficient production and engineering of heterologous (eukaryotic) proteins due to several advantages. RESULTS: In this paper codon optimized genes and synthetic alcohol oxidase 1 promoter variants were used to generate Pichia pastoris strains which individually expressed cellobiohydrolase 1, cellobiohydrolase 2 and beta-mannanase from Trichoderma reesei and xylanase A from Thermomyces lanuginosus. For three of these enzymes we could develop strains capable of secreting gram quantities of enzyme per liter in fed-batch cultivations. Additionally, we compared our achieved yields of secreted enzymes and the corresponding activities to literature data. CONCLUSION: In our experiments we could clearly show the importance of gene optimization and strain characterization for successfully improving secretion levels. We also present a basic guideline how to correctly interpret the interplay of promoter strength and gene dosage for a successful improvement of the secretory production of lignocellulolytic enzymes in Pichia pastoris

Expression of lignocellulolytic enzymes in <it>Pichia pastoris</it>

Abstract Background Sustainable utilization of plant biomass as renewable source for fuels and chemical building blocks requires a complex mixture of diverse enzymes, including hydrolases which comprise the largest class of lignocellulolytic enzymes. These enzymes need to be available in large amounts at a low price to allow sustainable and economic biotechnological processes. Over the past years Pichia pastoris has become an attractive host for the cost-efficient production and engineering of heterologous (eukaryotic) proteins due to several advantages. Results In this paper codon optimized genes and synthetic alcohol oxidase 1 promoter variants were used to generate Pichia pastoris strains which individually expressed cellobiohydrolase 1, cellobiohydrolase 2 and beta-mannanase from Trichoderma reesei and xylanase A from Thermomyces lanuginosus. For three of these enzymes we could develop strains capable of secreting gram quantities of enzyme per liter in fed-batch cultivations. Additionally, we compared our achieved yields of secreted enzymes and the corresponding activities to literature data. Conclusion In our experiments we could clearly show the importance of gene optimization and strain characterization for successfully improving secretion levels. We also present a basic guideline how to correctly interpret the interplay of promoter strength and gene dosage for a successful improvement of the secretory production of lignocellulolytic enzymes in Pichia pastoris.</p

Thermodynamic characterization of the protein-protein interaction in the heteromeric Bacillus subtilis pyridoxalphosphate synthase

Two biosynthetic routes for the synthesis of pyridoxal 5'-phosphate (PLP), the biologically active compound of vitamin B6, have been characterized. The major pathway leads to direct formation of PLP from a pentasaccharide and a trisaccharide and is operative in plants, fungi, protozoa, and bacteria. This reaction is catalyzed by a single glutamine amidotransferase enzyme complex consisting of a pyridoxal synthase, termed Pdx1, and a glutaminase, termed Pdx2. In this complex, Pdx2 generates ammonia from L-glutamine and supplies it to Pdx1 for incorporation into PLP. The glutaminase activity of Pdx2 requires the presence of Pdx1 in a heteromeric complex, previously characterized by a crystallographic three-dimensional (3D) structure determination. Here, we give a thermodynamic description of complex formation of Bacillus subtilis PLP synthase in the absence or presence of L-glutamine. We show that L-glutamine directly affects the tightness of the protein complex, which exhibits dissociation constants of 6.9 and 0.3 microM in its absence and presence, respectively (at 25 degrees C). This result relates to the positioning of L-glutamine on the heterodimer interface as seen in the 3D structure. In an analysis of the temperature dependence of the enthalpy, negative heat capacity changes (deltaCp) agree with a protein interface governed by hydrophobic interactions. The measured heat capacity change is also a function of L-glutamine, with a negative deltaCp in the presence of L-glutamine and a more negative one in its absence. These findings suggest that L-glutamine not only affects the strength of complex formation but also determines the forces involved in complex formation, with regard to different relative contributions of hydrophobic and hydrophilic interactions

Structural and thermodynamic insights into the assembly of the heteromeric pyridoxal phosphate synthase from plasmodium falciparum

Pyridoxal 5'-phosphate (PLP) is required as a cofactor by many enzymes. The predominant de novo biosynthetic route is catalyzed by a heteromeric glutamine amidotransferase consisting of the synthase subunit Pdx1 and the glutaminase subunit Pdx2. Previously, Bacillus subtilis PLP synthase was studied by X-ray crystallography and complex assembly had been characterized by isothermal titration calorimetry. The fully assembled PLP synthase complex contains 12 individual Pdx1/Pdx2 glutamine amidotransferase heterodimers. These studies revealed the occurrence of an encounter complex that is tightened in the Michaelis complex when the substrate l-glutamine binds. In this study, we have characterized complex formation of PLP synthase from the malaria-causing human pathogen Plasmodium falciparum using isothermal titration calorimetry. The presence of l-glutamine increases the tightness of the interaction about 30-fold and alters the thermodynamic signature of complex formation. The thermodynamic data are integrated in a 3D homology model of P. falciparum PLP synthase. The negative experimental heat capacity (C(p)) describes a protein interface that is dominated by hydrophobic interactions. In the absence of l-glutamine, the experimental C(p) is less negative than in its presence, contrasting to the previously characterised bacterial PLP synthase. Thus, while the encounter complexes differ, the Michaelis complexes of plasmodial and bacterial systems have similar characteristics concerning the relative contribution of apolar/polar surface area. In addition, we have verified the role of the N-terminal region of PfPdx1 for complex formation. A "swap mutant" in which the complete alphaN-helix of plasmodial Pdx1 was exchanged with the corresponding segment from B. subtilis shows cross-binding to B. subtilis Pdx2. The swap mutant also partially elicits glutaminase activity in BsPdx2, demonstrating that formation of the protein complex interface via alphaN and catalytic activation of the glutaminase are linked processes