RAtional REdesign of Phenylalanine Ammonia-Lyases for reversing their natural selectivity (RARE-PAL)

OBJECTIVES

OBJECTIVE 1.

Rational design driven site-directed mutagenesis for identification of D-selectivity modulating residues:

Related work-packages:

  • WP1. Site-directed mutagenesis driven by rational design concepts
  • WP2. Ala-scan of active site residues combined with monodental substrate fixation
  • WP3. D-selectivity measurements

OBJECTIVE 2.

Iterative saturation mutagenesis – CASTing at D-selectivity modulating sites:

Related work-packages:

  • WP1. Saturation mutagenesis
  • WP2. Activity screens of transformants from saturation mutagenesis

OBJECTIVE 3.

Functional and structural characterization of novel PALs of reversed D-selectivity:

Related work-packages:

  • WP1. Expression, isolation and purification of the PALs of D-activity
  • WP2. Functional characterization of PALs of enhanced D-selectivity
  • WP3. Structural characterization of the novel recombinant PALs of enhanced D-selectivity

OBJECTIVE 4.

Development of PAL-based biotechnologies for valuable D-phenylalalanines:

Related work-packages:

  • WP1. Reaction engineering using D-PALs as whole cell biocatalysts
  • WP2. Engineering of biotransformations with purified, immobilized D-PALs