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

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

Strain	Sequence		CN	Intracellular activity	Biomass
	Codon usage	PTS1		[U/g wcw]	[g wcw/batch]
Tv5a^I	native	PNL	1	137	220
Tv1a^II	native	SKL	1	90	274
Tv5^I	optimized	PNL	1-2	348	259
Tv1^II	optimized	SKL	1	767	144
Tv1a_mc^II	optimized	SKL	5	550	240
Tv1_mc^II	optimized	SKL	16-17	1283	117

^IPNL sequence: 5'-CCA-AAC-CTT-3'; ^IISKL sequence: 5'-TCC-AAG-CTG-3'
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%.

ISSN: 1475-2859