Expanding the Genetic Repertoire of the Methylotrophic Yeast <i>Pichia pastoris</i>

Abstract

To increase the utility of protein mutagenesis with unnatural amino acids, a recombinant expression system in the methylotrophic yeast <i>Pichia pastoris</i> was developed. Aminoacyl-tRNA synthetase/suppressor tRNA (aaRS/tRNA_CUA) pairs previously evolved in <i>Saccharomyces cerevisiae</i> to be specific for unnatural amino acids were inserted between eukaryotic transcriptional control elements and stably incorporated into the <i>P. pastoris</i> genome. Both the <i>Escherichia coli</i> tyrosyl- and leucyl-RS/tRNA_CUA pairs were shown to be orthogonal in <i>P. pastoris</i> and used to incorporate eight unnatural amino acids in response to an amber codon with high yields and fidelities. In one example, we show that a recombinant human serum albumin mutant containing a keto amino acid (<i>p</i>-acetylphenylalanine) can be efficiently expressed in this system and selectively conjugated via oxime ligation to a therapeutic peptide mimetic containing an ϵ-(2-(aminooxy)acetyl)-l-lysine residue. Moreover, unnatural amino acid expression in the methylotrophic host was systematically optimized by modulation of aaRS levels to express mutant human serum albumin in excess of 150 mg/L in shake flasks, more than an order of magnitude better than that reported in <i>S. cerevisiae</i>. This methodology should allow the production of high yields of complex proteins containing unnatural amino acids whose expression is not practical in existing systems.