We report on the generation of artificial metalloenzymes based on the noncovalent incorporation of biotinylated rhodium-diphosphine complexes in (strept)avidin as host proteins. A chemogenetic optimization procedure allows one to optimize the enantioselectivity for the reduction of acetamidoacrylic acid (up to 96% ee (R) in streptavidin S112G and up to 80% ee (S) in WT avidin). The association constant between a prototypical cationic biotinylated rhodium-diphosphine catalyst precursor and the host proteins was determined at neutral pH: log K(a) = 7.7 for avidin (pI = 10.4) and log K(a) = 7.1 for streptavidin (pI = 6.4). It is shown that the optimal operating conditions for the enantioselective reduction are 5 bar at 30 degrees C with a 1% catalyst loading.

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A recombinant avidin-producing Mut+ Pichia pastoris strain was used as a model organism to study the influence of the methanol feeding strategy on the specific product productivity (q(p)) and protein glycosylation. Fed-batch cultivations performed at various specific growth rates (micro) and residual methanol concentrations showed that the specific avidin productivity is growth-dependent. The specific productivity increases strongly with the specific growth rate for micro ranging from 0 to 0.02 h(-1), and increases only slightly with the specific growth rate above this limit. N-terminal glycosylation was also found to be influenced by the specific growth rate, since 9-mannose glycans were the most abundant form at low growth rates, whereas 10-mannose carbohydrate chains were favored at higher micro. These results show that culture parameters, such as the specific growth rate, may significantly affect the activity of glycoproteins produced in Pichia pastoris. In terms of process optimization, this suggests that a compromise on the specific growth rate may have to be found, in certain cases, to work with an acceptable productivity while avoiding the addition of many mannoses.

A recombinant glycosylated avidin (recGAvi) with an acidic isoelectric point was expressed and secreted by the methylotrophic yeast Pichia pastoris. The coding sequence for recGAvi was de novo synthesized based on the codon usage of P. pastoris. RecGAvi is secreted at approximately 330 mg/L of culture supernatant. RecGAvi monomer displays a molecular weight of 16.5 kDa, as assessed by ESI mass spectrometry. N-terminal amino acid sequencing indicates the presence of three additional amino acids (E-A-E), which contribute to further lowering the isoelectric point to 5.4. The data presented here demonstrate that the P. pastoris system is suitable for the production of recGAvi and that the recombinant avidin displays biotin-binding properties similar to those of the hen-egg white protein.

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