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

Abstract

Background: Trigonopsis 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.

Results: As 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 x 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 >or= 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.

Conclusions: Stepwise 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.

Figure 1

Industrial applications of TvDAO. A: Chemo-enzymatic conversion of cephalosporin C to 7-aminocephalosporanic acid. Spontaneous decarboxylation of ketoadipyl-7-ACA (amino cephalosporanic acid) is promoted by the H₂O₂ formed in the oxidase reaction of TvDAO. B: Chemoenzymatic production of pure enantiomers of amino acids by amino acid oxidases in combination with imine reduction benefits from the additional presence of a catalase that destroys H₂O₂.

Figure 2

High-cell-density bioreactor cultivation of P. pastoris strain Tv1_mc using induction with a mix-feed strategy. Values are referenced to oxidase activity obtained using the standard methanol feed and set as 100%. The fed-batch induction phase lasted 90 h in each case. MeOH: 3 mL/h methanol. MeOH/2: 1.5 mL/h methanol, Glu/MeOH: 3 mL/h glucose-methanol mixture, Gly/MeOH: 3 mL/h glycerol-methanol mixture. Activity measurements were performed three times, resulting in standard deviations of < 14%.

Figure 3

Effect of biomass concentration on permeabilization of recombinant Pichia pastoris cells expressing TvDAO. Cells of Tv1_mc cultivated in a bioreactor were treated with isopropanol for 20 min and 20 h. The activity of permeabilized cells is given as a percentage of the total intracellular activity (1283 U g/wcw) measured after cell lysis. Activity measurements were performed three times resulting in standard deviations of < 5%.