Exploring the dual roles of tRNAs in Komagataella phaffii production hosts

tRNAs play an essential role in protein synthesis by decoding mRNA and delivering amino acids to ribosomes during translation. Beyond this, tRNA genes (tDNAs) contribute to genome organisation by acting as chromatin barriers by maintaining the boundaries between active and repressive chromatin using a property known as barrier activity. Exploiting these dual roles of tRNAs could be a promising approach to enhancing recombinant protein production. Here, we explore tRNA-based strategies in the yeast Komagataella phaffii, an emerging production platform.

Our findings show that K. phaffii tDNAs exhibit barrier activity in the baker’s yeast, S. cerevisiae, outperforming native barriers in a mating screen. Notably, the AOX1 locus, a popular integration site in K. phaffii expression systems, lacks protection from active barrier tDNAs, suggesting potential susceptibility to chromatin-based repression. Introducing a K. phaffii tDNA expectedly increased chromatin accessibility at this locus but paradoxically suppressed transgene transcription, highlighting complex roles of tDNAs in regulating expression.

Assessing downstream applications of tRNA-based interventions, this thesis identifies an anticodon missing from the K. phaffii repertoire, for which the cognate codon is highly prevalent amongst endogenous proteins, thereby implying a significant demand on wobble decoding in K. phaffii cells. Wobble decoding, a mechanism that allows a single tRNA to recognise multiple codons through flexible base-pairing at the third codon position, is found not to be a bottleneck in the translation of recombinant proteins, nor does it impede translation of endogenous proteins. Yet, introduction of an exogenous S. cerevisiae tRNA gene carrying the missing anticodon was proven to have potential in being an effective intervention for recombinant protein production, suggesting a bottleneck in tRNA supply.

In summary, this thesis highlights the potential of leveraging the diverse and complex roles of tRNAs to enhance recombinant protein production.