Optimal phylogenetic reconstruction of insertion and deletion events.

MOTIVATION: Insertions and deletions (indels) influence the genetic code in fundamentally distinct ways from substitutions, significantly impacting gene product structure and function. Despite their influence, the evolutionary history of indels is often neglected in phylogenetic tree inference and ancestral sequence reconstruction, hindering efforts to comprehend biological diversity determinants and engineer variants for medical and industrial applications. RESULTS: We frame determining the optimal history of indel events as a single Mixed-Integer Programming (MIP) problem, across all branch points in a phylogenetic tree adhering to topological constraints, and all sites implied by a given set of aligned, extant sequences. By disentangling the impact on ancestral sequences at each branch point, this approach identifies the minimal indel events that jointly explain the diversity in sequences mapped to the tips of that tree. MIP can recover alternate optimal indel histories, if available. We evaluated MIP for indel inference on a dataset comprising 15 real phylogenetic trees associated with protein families ranging from 165 to 2000 extant sequences, and on 60 synthetic trees at comparable scales of data and reflecting realistic rates of mutation. Across relevant metrics, MIP outperformed alternative parsimony-based approaches and reported the fewest indel events, on par or below their occurrence in synthetic datasets. MIP offers a rational justification for indel patterns in extant sequences; importantly, it uniquely identifies global optima on complex protein data sets without making unrealistic assumptions of independence or evolutionary underpinnings, promising a deeper understanding of molecular evolution and aiding novel protein design. AVAILABILITY AND IMPLEMENTATION: The implementation is available via GitHub at https://github.com/santule/indelmip.

Engineering Biocatalysts for the C-H Activation of Fatty Acids by Ancestral Sequence Reconstruction.

Selective, one-step C-H activation of fatty acids from biomass is an attractive concept in sustainable chemistry. Biocatalysis has shown promise for generating high-value hydroxy acids, but to date enzyme discovery has relied on laborious screening and produced limited hits, which predominantly oxidise the subterminal positions of fatty acids. Herein we show that ancestral sequence reconstruction (ASR) is an effective tool to explore the sequence-activity landscape of a family of multidomain, self-sufficient P450 monooxygenases. We resurrected 11 catalytically active CYP116B ancestors, each with a unique regioselectivity fingerprint that varied from subterminal in the older ancestors to mid-chain in the lineage leading to the extant, P450-TT. In lineages leading to extant enzymes in thermophiles, thermostability increased from ancestral to extant forms, as expected if thermophily had arisen de novo. Our studies show that ASR can be applied to multidomain enzymes to develop active, self-sufficient monooxygenases as regioselective biocatalysts for fatty acid hydroxylation.