Yeast flocculation is a community-building cell aggregation trait that is an important mechanism of stress resistance and a useful phenotype for brewers; however, it is also a nuisance in many industrial processes, in clinical settings, and in the laboratory. Chemostat-based evolution experiments are impaired by inadvertent selection for aggregation, which we observe in 35% of populations. These populations provide a testing ground for understanding the breadth of genetic mechanisms Saccharomyces cerevisiae uses to flocculate, and which of those mechanisms provide the biggest adaptive advantages. In this study, we employed experimental evolution as a tool to ask whether one or many routes to flocculation are favored, and to engineer a strain with reduced flocculation potential. Using a combination of whole genome sequencing and bulk segregant analysis, we identified causal mutations in 23 independent clones that had evolved cell aggregation during hundreds of generations of chemostat growth. In 12 of those clones, we identified a transposable element insertion in the promoter region of known flocculation gene FLO1, and, in an additional five clones, we recovered loss-of-function mutations in transcriptional repressor TUP1, which regulates FLO1 and other related genes. Other causal mutations were found in genes that have not been previously connected to flocculation. Evolving a flo1 deletion strain revealed that this single deletion reduces flocculation occurrences to 3%, and demonstrated the efficacy of using experimental evolution as a tool to identify and eliminate the primary adaptive routes for undesirable traits.

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CYP4Z1 is an "orphan" cytochrome P450 (P450) enzyme that has provoked interest because of its hypothesized role in breast cancer through formation of the signaling molecule 20-hydroxyeicosatetraenoic acid (20-HETE). We expressed human CYP4Z1 in and evaluated its catalytic capabilities toward arachidonic and lauric acids (AA and LA). Specific and sensitive mass spectrometry assays enabled discrimination of the regioselectivity of hydroxylation of these two fatty acids. CYP4Z1 generated 7-, 8-, 9-, 10-, and 11-hydroxy LA, whereas the 12-hydroxy metabolite was not detected. HET0016, the prototypic CYP4 inhibitor, only weakly inhibited laurate metabolite formation (IC ∼15 M). CYP4Z1 preferentially oxidized AA to the 14(S),15(R)-epoxide with high regioselectivity and stereoselectivity, a reaction that was also insensitive to HET0016, but neither 20-HETE nor 20-carboxy-AA were detectable metabolites. Docking of LA and AA into a CYP4Z1 homology model was consistent with this preference for internal fatty acid oxidation. Thus, human CYP4Z1 has an inhibitor profile and product regioselectivity distinct from most other CYP4 enzymes, consistent with CYP4Z1's lack of a covalently linked heme. These data suggest that, if CYP4Z1 modulates breast cancer progression, it does so by a mechanism other than direct production of 20-HETE.

CYP2C9 encodes a cytochrome P450 enzyme responsible for metabolizing up to 15% of small molecule drugs, and CYP2C9 variants can alter the safety and efficacy of these therapeutics. In particular, the anti-coagulant warfarin is prescribed to over 15 million people annually and polymorphisms in CYP2C9 can affect patient response leading to an increased risk of hemorrhage. We developed Click-seq, a pooled yeast-based activity assay to test thousands of variants. Using Click-seq, we measured the activity of 6,142 missense variants expressed in yeast. We also measured the steady-state cellular abundance of 6,370 missense variants expressed in a human cell line using Variant Abundance by Massively Parallel sequencing (VAMP-seq). These data revealed that almost two-thirds of CYP2C9 variants showed decreased activity, and that protein abundance accounted for half of the variation in CYP2C9 function. We also measured activity scores for 319 previously unannotated human variants, many of which may have clinical relevance.

As costs of next-generation sequencing decrease, identification of genetic variants has far outpaced our ability to understand their functional consequences. This lack of understanding is a central challenge to a key promise of pharmacogenomics: using genetic information to guide drug selection and dosing. Recently developed multiplexed assays of variant effect enable experimental measurement of the function of thousands of variants simultaneously. Here, we describe multiplexed assays that have been performed on nearly 25,000 variants in eight key pharmacogenes ( ADRB2, CYP2C9, CYP2C19, NUDT15, SLCO1B1, TMPT, VKORC1, and the LDLR promoter), discuss advances in experimental design, and explore key challenges that must be overcome to maximize the utility of multiplexed functional data. Expected final online publication date for the Annual Review of Pharmacology and Toxicology, Volume 62 is January 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.