Optimizing expression of Nanobody® molecules in Pichia pastoris through co-expression of auxiliary proteins under methanol and methanol-free conditions.

BACKGROUND: Ablynx NV, a subsidiary of Sanofi, has a long-standing focus on the development of Nanobody® molecules as biopharmaceuticals (Nanobody® is a registered trademark of Ablynx NV). Nanobody molecules are single variable domains, and they have been met with great success part due to their favorable expression properties in several microbial systems. Nevertheless, the search for the host of the future is an ongoing and challenging process. Komagataella phaffi (Pichia pastoris) is one of the most suitable organisms to produce Nanobody molecules. In addition, genetic engineering of Pichia is easy and an effective approach to improve titers. RESULTS: Here we report that P. pastoris engineered to co-express genes encoding four auxiliary proteins (HAC1, KAR2, PDI and RPP0), leads to a marked improvement in the expression of Nanobody molecules using the AOX1 methanol induction system. Titer improvement is mainly attributed to HAC1, and its beneficial effect was also observed in a methanol-free expression system. CONCLUSION: Our findings are based on over a thousand fed-batch fermentations and offer a valuable guide to produce Nanobody molecules in P. pastoris. The presented differences in expressability between types of Nanobody molecules will be helpful for researchers to select both the type of Nanobody molecule and Pichia strain and may stimulate further the development of a more ecological methanol-free expression platform.

Pichia pastoris Mut(S) strains are prone to misincorporation of O-methyl-L-homoserine at methionine residues when methanol is used as the sole carbon source.

BACKGROUND: Over the last few decades the methylotrophic yeast Pichia pastoris has become a popular host for a wide range of products such as vaccines and therapeutic proteins. Several P. pastoris engineered strains and mutants have been developed to improve the performance of the expression system. Yield and quality of a recombinant product are important parameters to monitor during the host selection and development process but little information is published regarding quality differences of a product produced by different P. pastoris strains. RESULTS: We compared titer and quality of several Nanobodies(®) produced in wild type and Mut(S) strains. Titer in fed-batch fermentation was comparable between all strains for each Nanobody but a significant difference in quality was observed. Nanobodies expressed in Mut(S) strains contained a product variant with a Δ-16 Da mass difference that was not observed in wild type strains. This variant showed substitution of methionine residues due to misincorporation of O-methyl-L-homoserine, also called methoxine. Methoxine is likely synthesized by the enzymatic action of O-acetyl homoserine sulfhydrylase and we confirmed that Nanobodies produced in the corresponding knock-out strain contained no methoxine variants. We could show the incorporation of methoxine during biosynthesis by its addition to the culture medium. CONCLUSION: We showed that misincorporation of methoxine occurs particularly in P. pastoris Mut(S) strains. This reduction in product quality could outweigh the advantages of using Mut strains, such as lower oxygen and methanol demand, heat formation and in some cases improved expression. Methoxine incorporation in recombinant proteins is likely to occur when an excess of methanol is present during fermentation but can be avoided when the methanol feed rate protocol is carefully designed.

Construction of cellobiose phosphorylase variants with broadened acceptor specificity towards anomerically substituted glucosides.

The general application of glycoside phosphorylases such as cellobiose phosphorylase (CP) for glycoside synthesis is hindered by their relatively narrow substrate specificity. We have previously reported on the creation of Cellulomonas uda CP enzyme variants with either modified donor or acceptor specificity. Remarkably, in this study it was found that the donor mutant also displays broadened acceptor specificity towards several beta-glucosides. Triple mutants containing donor (T508I/N667A) as well as acceptor mutations (E649C or E649G) also display a broader acceptor specificity than any of the parent enzymes. Moreover, further broadening of the acceptor specificity has been achieved by site-saturation mutagenesis of residues near the active site entrance. The best enzyme variant contains the additional N156D and N163D mutations and is active towards various alkyl beta-glucosides, methyl alpha-glucoside and cellobiose. In comparison with the wild-type C. uda CP enzyme, which cannot accept anomerically substituted glucosides at all, the obtained increase in substrate specificity is significant. The described CP enzyme variants should be useful for the synthesis of cellobiosides and other glycosides with prebiotic and pharmaceutical properties.

Crystallization and X-ray diffraction studies of cellobiose phosphorylase from Cellulomonas uda.

Disaccharide phosphorylases are able to catalyze both the synthesis and the breakdown of disaccharides and have thus emerged as attractive platforms for tailor-made sugar synthesis. Cellobiose phosphorylase from Cellulomonas uda (CPCuda) is an enzyme that belongs to glycoside hydrolase family 94 and catalyzes the reversible breakdown of cellobiose [beta-D-glucopyranosyl-(1,4)-D-glucopyranose] to alpha-D-glucose-1-phosphate and D-glucose. Crystals of ligand-free recombinant CPCuda and of its complexes with substrates and reaction products yielded complete X-ray diffraction data sets to high resolution using synchrotron radiation but suffered from significant variability in diffraction quality. In at least one case an intriguing space-group transition from a primitive monoclinic to a primitive orthorhombic lattice was observed during data collection. The structure of CPCuda was determined by maximum-likelihood molecular replacement, thus establishing a starting point for an investigation of the structural and mechanistic determinants of disaccharide phosphorylase activity.

Enzymatic production of alpha-D-galactose 1-phosphate by lactose phosphorolysis.

alpha-D-Galactose 1-phosphate (alphaGal1P) is an important building block for the synthesis of nucleotide sugars that are substrates for glycosyltransferases. We have previously reported the creation of novel lactose phosphorylase enzymes that are useful for the synthesis of alphaGal1P from the cheap and abundant lactose. Here we describe the application of such a lactose phosphorylase in a production system using permeabilized Escherichia coli cells. After purification of the product from the reaction mixture by anion-exchange chromatography and ethanol precipitation, 9.5 grams of highly pure alphaGal1P were obtained from a 1 l reaction volume.

Cloning, sequence analysis and heterologous expression of the Myrothecium gramineum orotidine-5'-monophosphate decarboxylase gene.

A 2918 bp sequence coding for the orotidine-5'-monophosphate decarboxylase enzyme (OMPD) was isolated from the genome of Myrothecium gramineum. This sequence was analysed and, remarkably, it is the first OMPD gene of a Sordariomycete that has an intron. The gene codes for an enzyme of 282 amino acids. The nucleotide sequence and the amino acid sequence were compared with fungal OMPD sequences. They show the highest similarity to OMPD genes and enzymes of Aspergillus sp., Penicillium sp. and Cladosporium fulvum. The functionality of the gene as a selection marker was proven by complementation of the uracil auxotrophy of Aspergillus nidulans FGSC A722.

Engineering of cellobiose phosphorylase for glycoside synthesis.

Disaccharide phosphorylases are increasingly applied for glycoside synthesis, since they are very regiospecific and use cheap and easy to obtain donor substrates. A promising enzyme is cellobiose phosphorylase (CP), which was discovered more than 50 years ago. Many other bacterial CP enzymes have since then been characterized, cloned and applied for glycoside synthesis. However, the general application of wild-type CP for glycoside synthesis is hampered by its relatively narrow substrate specificity. Recently we have taken some successful efforts to broaden the substrate specificity of Cellulomonas uda CP by directed evolution and protein engineering. This review will give an overview of the obtained results and address the applicability of the engineered CP enzymes for glycoside synthesis. CP is the first example of an extensively engineered disaccharide phosphorylase, and may provide valuable information for protein engineering of other phosphorylase enzymes.

Probing the active site of cellodextrin phosphorylase from Clostridium stercorarium: kinetic characterization, ligand docking, and site-directed mutagenesis.

Cellodextrin phosphorylase from Clostridium stercorarium has been recombinantly expressed in Escherichia coli for the first time. Kinetic characterization of the purified enzyme has revealed that aryl and alkyl ß-glucosides can be efficiently glycosylated, an activity that has not yet been described for this enzyme class. To obtain a better understanding of the factors that determine the enzyme's specificity, homology modeling and ligand docking were applied. Residue W168 has been found to form a hydrophobic stacking interaction with the substrate in subsite +2, and its importance has been examined by means of site-directed mutagenesis. The mutant W168A retains about half of its catalytic activity, indicating that other residues also contribute to the binding affinity of subsite +2. Finally, residue D474 has been identified as the catalytic acid, interacting with the glycosidic oxygen between subsites -1 and +1. Mutating this residue results in complete loss of activity. These results, for the first time, provide an insight in the enzyme-substrate interactions that determine the activity and specificity of cellodextrin phosphorylases.

High throughput calorimetry for evaluating enzymatic reactions generating phosphate.

A calorimetric assay is described for the high-throughput screening of enzymes that produce inorganic phosphate. In the current example, cellobiose phosphorylase (EC 2.4.1.20) is tested for its ability to synthesise rare disaccharides. The generated phosphate is measured in a high-throughput calorimeter by coupling the reaction to pyruvate oxidase and catalase. This procedure allows for the simultaneous analysis of 48 reactions in microtiter plate format and has been validated by comparison with a colorimetric phosphate assay. The proposed assay has a coefficient of variation of 3.14% and is useful for screening enzyme libraries for enhanced activity and substrate libraries for enzyme promiscuity.

Creating lactose phosphorylase enzymes by directed evolution of cellobiose phosphorylase.

Disaccharide phosphorylases are interesting enzymes for the production of sugar phosphates from cheap starting materials and for the synthesis of novel glycosides. Cellobiose phosphorylase (CP) from Cellulomonas uda was subjected to directed evolution in order to create enzyme variants with significantly increased lactose phosphorylase (LP) activity, useful for the production of alpha-D-galactose 1-phosphate. In a first round, random mutagenesis was performed on part of the CP gene and the resultant library was selected on minimal lactose medium. One clone containing six amino acid mutations was found with increased LP activity compared with the wild-type CP enzyme. The negative and neutral mutations were eliminated by site-directed mutagenesis and the resultant enzyme variant containing two amino acid substitutions (T508A/N667T) showed more LP activity than the parent mutant. Saturation mutagenesis of the beneficial sites and screening for improved mutants allowed us to identify the T508I/N667A mutant which has 7.5 times higher specific activity on lactose than the wild-type. The kinetic parameters of the mutants were determined and showed that the increased LP activity was caused by a higher k(cat) value. This is the first report of an engineered CP with modified substrate specificity.