Poster Presentation 1B-33
Expression of fungal Lignin Peroxidase in Pichia Pastoris
Abhijeet P. Borole, Tanya Kuritz¶,
Miguel Rodriguez, Jr. and Brian H. Davison
Life Sciences Division
Phone: (865)576-7421; Fax: (865)574-6442; E-mail: borolea@ornl.gov
¶ Chemical Sciences Division
Lignin peroxidase is an enzyme
which is potentially useful for processing of renewable resources in the pulp
and paper industry, as well as for enzymatic transformations of polyaromatic hydrocarbons. The current cost of its production (via fungal
fermentations) is, however, prohibitive. This work describes cloning and expression of the
lignin peroxidase gene in Pichia
pastoris and the use of directed evolution to
improve expression. The lipH2
gene of Phanerocheate chrysosporium
was mutagenized by PCR and subcloned
into a series of P. pastoris vectors (Invitrogen) harboring a combination of native fungal and/or
yeast promoters, consensus sequences and leader sequences. The vectors were transformed into several
different strains of P. pastoris. The recombinant clones were screened for
activity using a standard 2,2’ azino-bis-(3-ethylbenzothiazole-6-sulfonate)
(ABTS) assay. We observed
methanol-inducible expression in the host P. pastoris
X33 strain transformed with the construct that contained yeast promoter,
engineered yeast consensus sequence and native leader sequence. The activity and the amount of enzyme secreted
into the medium was, however, very low and not sufficient for production
purposes. Attempts to improve expression
via directed evolution, by error-prone PCR, resulted in marginal improvements,
suggesting that more fundamental research is needed to enable genetic
compatibility of fungal and yeast systems.