Single-chain antibody fragment production in Pichia pastoris: Benefits of prolonged pre-induction glycerol feeding.

Secretory production of a single-chain antibody fragment (scFv) by recombinant Pichia pastoris using the methanol inducible AOX1 promoter is limited biochemically by retarded secretion, and economically by the high demand for pure oxygen. To address the problem, the adaptation phase with growth-limiting feeding of glycerol before the production phase was optimized. In a standard procedure with a short glycerol-feeding phase before induction, scFv accumulated in the supernatant only after 15 h. Conversely, scFv started to appear immediately in the medium upon methanol induction when the glycerol-feeding phase was extended to 18 h. Interestingly, despite a significantly lower cell density in the cultivation with extended glycerol feeding, the same amount of functional product of 300 mg/L was obtained about 30 h after the start of glycerol feeding with both methods. mRNA analysis revealed that the higher and faster production of the product was related to longer lasting induction of the scFv mRNA. Additional effects of a better adaptation of the secretion machinery may be suggested by higher expression of unfolded protein response-related genes KAR2 and PDI. A clear benefit of the longer glycerol-feeding phase was a 75% reduction of the consumption of both pure oxygen and methanol, and a significantly lower cell density, which would be beneficial for down-stream purification of the product.

Thiamin diphosphate in biological chemistry: exploitation of diverse thiamin diphosphate-dependent enzymes for asymmetric chemoenzymatic synthesis.

Thiamin diphosphate-dependent enzymes participate in numerous biosynthetic pathways and catalyse a broad range of reactions, mainly involving the cleavage and formation of C-C bonds. For example, they catalyse the nonoxidative and oxidative decarboxylation of 2-keto acids, produce 2-hydroxy ketones and transfer activated aldehydes to a variety of acceptors. Moreover, they can also catalyse C-N, C-O and C-S bond formation. Because of their substrate spectra and different stereospecificity, these enzymes extend the synthetic potential for asymmetric carboligations appreciably. Different strategies have been developed to identify new members of this promiscuous enzyme class and the reactions they catalyse. This enabled us to introduce solutions for longstanding synthetic problems, such as asymmetric cross-benzoin condensation. Moreover, through a combination of protein structure analysis, enzyme and substrate engineering, and screening methods we explored additional stereochemical routes that have not been described previously for any of these interesting enzymes.

Rational protein design of ThDP-dependent enzymes-engineering stereoselectivity.

Benzoylformate decarboxylase (BFD) from Pseudomonas putida is an exceptional thiamin diphosphate-dependent enzyme, as it catalyzes the formation of (S)-2-hydroxy-1-phenylpropan-1-one from benzaldehyde and acetaldehyde. This is the only currently known S-selective reaction (92 % ee) catalyzed by this otherwise R-selective class of enzymes. Here we describe the molecular basis of the introduction of S selectivity into ThDP-dependent decarboxylases. By shaping the active site of BFD through the use of rational protein design, structural analysis, and molecular modeling, optimal steric stabilization of the acceptor aldehyde in a structural element called the S pocket was identified as the predominant interaction for adjusting stereoselectivity. Our studies revealed Leu461 as a hot spot for stereoselectivity in BFD. Exchange to alanine and glycine resulted in variants that catalyze the S-stereoselective addition of larger acceptor aldehydes, such as propanal with benzaldehyde and its derivatives-a reaction not catalyzed by the wild-type enzyme. Crystal structure analysis of the variant BFDL461A supports the modeling studies.

Structure of the branched-chain keto acid decarboxylase (KdcA) from Lactococcus lactis provides insights into the structural basis for the chemoselective and enantioselective carboligation reaction.

The thiamin diphosphate (ThDP) dependent branched-chain keto acid decarboxylase (KdcA) from Lactococcus lactis catalyzes the decarboxylation of 3-methyl-2-oxobutanoic acid to 3-methylpropanal (isobutyraldehyde) and CO2. The enzyme is also able to catalyze carboligation reactions with an exceptionally broad substrate range, a feature that makes KdcA a potentially valuable biocatalyst for C-C bond formation, in particular for the enzymatic synthesis of diversely substituted 2-hydroxyketones with high enantioselectivity. The crystal structures of recombinant holo-KdcA and of a complex with an inhibitory ThDP analogue mimicking a reaction intermediate have been determined to resolutions of 1.6 and 1.8 A, respectively. KdcA shows the fold and cofactor-protein interactions typical of thiamin-dependent enzymes. In contrast to the tetrameric assembly displayed by most other ThDP-dependent decarboxylases of known structure, KdcA is a homodimer. The crystal structures provide insights into the structural basis of substrate selectivity and stereoselectivity of the enzyme and thus are suitable as a framework for the redesign of the substrate profile in carboligation reactions.

Characterization of phenylpyruvate decarboxylase, involved in auxin production of Azospirillum brasilense.

Azospirillum brasilense belongs to the plant growth-promoting rhizobacteria with direct growth promotion through the production of the phytohormone indole-3-acetic acid (IAA). A key gene in the production of IAA, annotated as indole-3-pyruvate decarboxylase (ipdC), has been isolated from A. brasilense, and its regulation was reported previously (A. Vande Broek, P. Gysegom, O. Ona, N. Hendrickx, E. Prinsen, J. Van Impe, and J. Vanderleyden, Mol. Plant-Microbe Interact. 18:311-323, 2005). An ipdC-knockout mutant was found to produce only 10% (wt/vol) of the wild-type IAA production level. In this study, the encoded enzyme is characterized via a biochemical and phylogenetic analysis. Therefore, the recombinant enzyme was expressed and purified via heterologous overexpression in Escherichia coli and subsequent affinity chromatography. The molecular mass of the holoenzyme was determined by size-exclusion chromatography, suggesting a tetrameric structure, which is typical for 2-keto acid decarboxylases. The enzyme shows the highest kcat value for phenylpyruvate. Comparing values for the specificity constant kcat/Km, indole-3-pyruvate is converted 10-fold less efficiently, while no activity could be detected with benzoylformate. The enzyme shows pronounced substrate activation with indole-3-pyruvate and some other aromatic substrates, while for phenylpyruvate it appears to obey classical Michaelis-Menten kinetics. Based on these data, we propose a reclassification of the ipdC gene product of A. brasilense as a phenylpyruvate decarboxylase (EC 4.1.1.43).