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Table 1 Enzyme and strain engineering for efficient expression of Tv DAO in Pichia pastoris.

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

Strain Sequence CN Intracellular activity Biomass
  Codon usage PTS1   [U/g wcw] [g wcw/batch]
Tv5aI native PNL 1 137 220
Tv1aII native SKL 1 90 274
Tv5I optimized PNL 1-2 348 259
Tv1II optimized SKL 1 767 144
Tv1a_mcII optimized SKL 5 550 240
Tv1_mcII optimized SKL 16-17 1283 117
  1. IPNL sequence: 5'-CCA-AAC-CTT-3'; IISKL sequence: 5'-TCC-AAG-CTG-3'
  2. Enzyme activities are reported for P. pastoris cells obtained in 1.5-L bioreactor cultivation employing standard induction with 3 mL/min methanol. Codon usage: native as in the original Tv DAO gene, or optimized for expression using the AOX1 promoter. PTS1 - peroxisomal targeting using the native C-terminal tripeptide PNL or the engineered motif SKL. CN: Number of copies of the expression cassette integrated into the P. pastoris genome. wcw: wet cell weight. Activity measurements were performed three times revealing a standard deviation of < 10%.