Stepwise engineering of a Pichia pastoris D-amino acid oxidase whole cell catalyst

Abstract

BackgroundTrigonopsis variabilis D-amino acid oxidase (TvDAO) is a well characterized enzyme used for cephalosporin C conversion on industrial scale. However, the demands on the enzyme with respect to activity, operational stability and costs also vary with the field of application. Processes that use the soluble enzyme suffer from fast inactivation of TvDAO while immobilized oxidase preparations raise issues related to expensive carriers and catalyst efficiency. Therefore, oxidase preparations that are more robust and active than those currently available would enable a much broader range of economically viable applications of this enzyme in fine chemical syntheses. A multi-step engineering approach was chosen here to develop a robust and highly active Pichia pastoris TvDAO whole-cell biocatalyst.ResultsAs compared to the native T. variabilis host, a more than seven-fold enhancement of the intracellular level of oxidase activity was achieved in P. pastoris through expression optimization by codon redesign as well as efficient subcellular targeting of the enzyme to peroxisomes. Multi copy integration further doubled expression and the specific activity of the whole cell catalyst. From a multicopy production strain, about 1.3 × 103 U/g wet cell weight (wcw) were derived by standard induction conditions feeding pure methanol. A fed-batch cultivation protocol using a mixture of methanol and glycerol in the induction phase attenuated the apparent toxicity of the recombinant oxidase to yield final biomass concentrations in the bioreactor of ≥ 200 g/L compared to only 117 g/L using the standard methanol feed. Permeabilization of P. pastoris using 10% isopropanol yielded a whole-cell enzyme preparation that showed 49% of the total available intracellular oxidase activity and was notably stabilized (by three times compared to a widely used TvDAO expressing Escherichia coli strain) under conditions of D-methionine conversion using vigorous aeration.ConclusionsStepwise optimization using a multi-level engineering approach has delivered a new P. pastoris whole cell TvDAO biocatalyst showing substantially enhanced specific activity and stability under operational conditions as compared to previously reported preparations of the enzyme. The production of the oxidase through fed-batch bioreactor culture and subsequent cell permeabilization is high-yielding and efficient. Therefore this P. pastoris catalyst has been evaluated for industrial purposes.

Glyco-variant library of the versatile enzyme horseradish peroxidase

“…The codon table described in Abad et al (2010) was applied for codon optimization. Secretion of HRP C1A to the cultivation broth was facilitated by an N-terminally fused prepro-signal sequence of the S. cerevisiae alpha-factor.…”

Glucose-methanol co-utilization in Pichia pastoris studied by metabolomics and instationary 13C flux analysis

“…Pichia pastoris has become an important yeast cell factory platform for the production of recombinant proteins [1-3] and has additional potential applications for whole cell biocatalysis [4,5]. Moreover, the development of systems biotechnology tools specific for this cell factory [6-11] has increased the knowledge for rational, model based strain improvements [12], as well as optimization of media composition and culture conditions.…”

Expression of lignocellulolytic enzymes in Pichia pastoris

“…Codon pair optimized sequence in black (CP), wt sequence in grey, high CAI codons for methanol induced gene expression in white (HM) [11]. Released cellobiose concentration is represented in bars.…”

“…Gene sequences were optimized by applying the high CAI codons for methanol induced genes (HM) [11]. Copy numbers (CN) are specified in the legend.…”