UC Irvine

In vivo hypermutation holds great promise for engineering new biomolecular functions and enabling the study of biomolecular evolution. In this work, we improve the mutagenesis capabilities of our lab’s in vivo hypermutation system, OrthoRep, and apply it to the evolution of the tryptophan synthase β-subunit TrpB. We first demonstrate that OrthoRep can be used to rapidly evolve this enzyme toward L-tryptophan production in yeast independent of the tryptophan synthase α-subunit. We find that a randomly sampled panel of the resulting enzymes exhibits a broad range of substrate promiscuities, recapitulating the cryptic genetic variation often found in natural protein orthologs.

To facilitate more rapid biomolecular evolution, we next engineered the OrthoRep error prone DNA polymerase toward increased mutation rates and reduced mutation bias, culminating in a set of polymerases that exhibit a mutation rate of 10^-4 substitutions per base per generation, 1-million-fold higher than the native yeast genomic mutation rate. Application of this accelerated in vivo hypermutation to TrpB evolution, coupled with a computational pipeline for analysis of the resulting mutation-rich high throughput sequencing datasets, uncovered evidence for structurally distributed interdependence of mutations, as well as indirect evolutionary forces shaping outcomes. Finally, we developed a genetic circuit that enables direct selection for the production of noncanonical amino acids by TrpB. This approach, when combined with in vivo hypermutation by OrthoRep and a wealth of engineered TrpBs with altered substrate preferences, has potential in greatly improving the enzymatic noncanonical amino acid production platform of Trp.