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Since the mid-1980s, our understanding of avian reproductive strategies has shifted as new molecular technologies have revealed how common promiscuity is in many monogamous avian species. However, the prevalence and importance of extra-pair paternity in polygynous species is less well studied. We used microsatellite loci from related icterid species to identify the extra-pair paternity rate in a polygynous population of Bobolinks (Dolichonyx oryzivorus) that bred in the Champlain Valley of Vermont from 2002 to 2018. We assigned paternity for 120 nestlings and found that 42% of nestlings were sired by extra-pair males. These results highlight the importance of extra-pair paternity in the reproductive behavior of Bobolinks that breed in agricultural habitats. Received 29 March 2020. Accepted I October 2021.

The ability of a small molecule to interact with multiple target proteins provides the molecular basis of polypharmacology. So-defined compound promiscuity is intensely investigated in drug discovery. For example, for kinase inhibitors, the interplay between target selectivity and promiscuity plays a decisive role for different therapeutic applications. The “promiscuity cliff” (PC) concept was introduced previously to aid in promiscuity analysis. A PC is defined as a pair of structurally similar compounds with a large difference in promiscuity. Accordingly, PCs can reveal small structural modifications that might be responsible for selectivity or multi-target activity. In network representations, PCs form clusters of varying size and complexity that are difficult to analyze interactively. Herein, we introduce a computational method to systematically identify PC pathways, which are particularly rich in structure-promiscuity information, and extract them from PC clusters. PC pathways provide informative templates for experimental design. In a proof-of-concept investigation, we have applied the new computational approach to systematically identify pathways in more than 600 PC clusters formed by inhibitors of the human kinome, demonstrating the utility of the method and revealing many interesting promiscuity patterns.

Females of many species mate with multiple males, thereby inciting competition among ejaculates from rival males for fertilization. In response to increasing sperm competition, males are predicted to enhance their investment in sperm production. This prediction is so widespread that testes size (correcting for body size) is commonly used as a proxy of sperm competition, even in the absence of any other information about a species' reproductive behaviour. By contrast, a debate about whether sperm competition selects for smaller or larger sperm has persisted for nearly three decades, with empirical studies demonstrating every possible response. Here, we synthesize nearly 40 years of sperm competition research in a meta-analytical framework to determine how the evolution of sperm number (i.e. testes size) and sperm size (i.e. sperm head, midpiece, flagellum and total length) is influenced by varying levels of sperm competition across species. Our findings support the long-held assumption that higher levels of sperm competition are associated with relatively larger testes. We also find clear evidence that sperm competition is associated with increases in all components of sperm length. We discuss these results in the context of different theoretical predictions and general patterns in the breeding biology and selective environment of sperm. This article is part of the theme issue ‘Fifty years of sperm competition’.

Multidrug transporters (MDTs) are major contributors to microbial drug resistance and are further utilized for improving host phenotypes in biotechnological applications. Therefore, the identification of these MDTs and the understanding of their mechanisms of action in vivo are of great importance. However, their promiscuity and functional redundancy represent a major challenge towards their identification. Here, a multistep tolerance adaptive laboratory evolution (TALE) approach was leveraged to achieve this goal. Specifically, a wild-type E. coli K-12-MG1655 and its cognate knockout individual mutants ΔemrE, ΔtolC, and ΔacrB were evolved separately under increasing concentrations of two lipophilic cations, tetraphenylphosphonium (TPP[sup.+]), and methyltriphenylphosphonium (MTPP[sup.+]). The evolved strains showed a significant increase in MIC values of both cations and an apparent cross-cation resistance. Sequencing of all evolved mutants highlighted diverse mutational mechanisms that affect the activity of nine MDTs including acrB, mdtK, mdfA, acrE, emrD, tolC, acrA, mdtL, and mdtP. Besides regulatory mutations, several structural mutations were recognized in the proximal binding domain of acrB and the permeation pathways of both mdtK and mdfA. These details can aid in the rational design of MDT inhibitors to efficiently combat efflux-based drug resistance. Additionally, the TALE approach can be scaled to different microbes and molecules of medical and biotechnological relevance.

Abstract A key step in metabolic pathway evolution is the recruitment of promiscuous enzymes to perform new functions. Despite the recognition that promiscuity is widespread in biology, factors dictating the preferential recruitment of one promiscuous enzyme over other candidates are unknown. Escherichia coli contains four sugar kinases that are candidates for recruitment when the native glucokinase machinery is deleted—allokinase (AlsK), manno(fructo)kinase (Mak), N-acetylmannosamine kinase (NanK), and N-acetylglucosamine kinase (NagK). The catalytic efficiencies of these enzymes are 103- to 105-fold lower than native glucokinases, ranging from 2,400 M−1 s−1 for the most active candidate, NagK, to 15 M−1 s−1 for the least active candidate, AlsK. To investigate the relationship between catalytic activities of promiscuous enzymes and their recruitment, we performed adaptive evolution of a glucokinase-deficient E. coli strain to restore glycolytic metabolism. We observed preferential recruitment of NanK via a trajectory involving early mutations that facilitate glucose uptake and amplify nanK transcription, followed by nonsynonymous substitutions in NanK that enhance the enzyme's promiscuous glucokinase activity. These substitutions reduced the native activity of NanK and reduced organismal fitness during growth on an N-acetylated carbon source, indicating that enzyme recruitment comes at a cost for growth on other substrates. Notably, the two most active candidates, NagK and Mak, were not recruited, suggesting that catalytic activity alone does not dictate evolutionary outcomes. The results highlight our lack of knowledge regarding biological drivers of enzyme recruitment and emphasize the need for a systems-wide approach to identify factors facilitating or constraining this important adaptive process.

Hydroxytyrosol, a valuable plant-derived phenolic compound, is increasingly produced from microbial fermentation. However, the promiscuity of the key enzyme HpaBC, the two-component flavin-dependent monooxygenase from Escherichia coli, often leads to low yields. To address this limitation, we developed a novel strategy utilizing microbial consortia catalysis for hydroxytyrosol production. We designed a biosynthetic pathway using tyrosine as the substrate and selected enzymes and overexpressing glutamate dehydrogenase GdhA to realize the cofactor cycling by coupling reactions catalyzed by the transaminase and the reductase. Additionally, the biosynthetic pathway was divided into two parts and performed by separate E. coli strains. Furthermore, we optimized the inoculation time, strain ratio, and pH to maximize the hydroxytyrosol yield. Glycerol and ascorbic acid were added to the co-culture, resulting in a 92% increase in hydroxytyrosol yield. Using this approach, the production of 9.2 mM hydroxytyrosol was achieved from 10 mM tyrosine. This study presents a practical approach for the microbial production of hydroxytyrosol that can be promoted to produce other value-added compounds.

Cannabis sativa L. has been cultivated and used around the globe for its medicinal properties for millennia 1 . Some cannabinoids, the hallmark constituents of Cannabis , and their analogues have been investigated extensively for their potential medical applications 2 . Certain cannabinoid formulations have been approved as prescription drugs in several countries for the treatment of a range of human ailments 3 . However, the study and medicinal use of cannabinoids has been hampered by the legal scheduling of Cannabis , the low in planta abundances of nearly all of the dozens of known cannabinoids 4 , and their structural complexity, which limits bulk chemical synthesis. Here we report the complete biosynthesis of the major cannabinoids cannabigerolic acid, Δ 9 -tetrahydrocannabinolic acid, cannabidiolic acid, Δ 9 -tetrahydrocannabivarinic acid and cannabidivarinic acid in Saccharomyces cerevisiae , from the simple sugar galactose. To accomplish this, we engineered the native mevalonate pathway to provide a high flux of geranyl pyrophosphate and introduced a heterologous, multi-organism-derived hexanoyl-CoA biosynthetic pathway 5 . We also introduced the Cannabis genes that encode the enzymes involved in the biosynthesis of olivetolic acid 6 , as well as the gene for a previously undiscovered enzyme with geranylpyrophosphate:olivetolate geranyltransferase activity and the genes for corresponding cannabinoid synthases 7 , 8 . Furthermore, we established a biosynthetic approach that harnessed the promiscuity of several pathway genes to produce cannabinoid analogues. Feeding different fatty acids to our engineered strains yielded cannabinoid analogues with modifications in the part of the molecule that is known to alter receptor binding affinity and potency 9 . We also demonstrated that our biological system could be complemented by simple synthetic chemistry to further expand the accessible chemical space. Our work presents a platform for the production of natural and unnatural cannabinoids that will allow for more rigorous study of these compounds and could be used in the development of treatments for a variety of human health problems. Genetic engineering of yeast enables the production of cannabinoids and cannabinoid analogues from the simple sugar galactose, without the need to cultivate Cannabis .

Small molecules with multi-target activity, also termed promiscuous compounds, are increasingly considered for pharmaceutical applications. The use of promiscuous chemical entities represents a departure from the compound specificity paradigm, one of the pillars of modern drug discovery. The popularity of promiscuous compounds is due to the concept of polypharmacology; another more recent drug discovery paradigm. It refers to insights that the efficacy of drugs often depends on interactions with multiple targets. Views concerning the extent to which small molecules might form well-defined interactions with multiple targets often differ, but comprehensive experimental investigations of promiscuity are currently rare. On the other hand, large volumes of active compounds and experimental measurements are becoming available and enable data-driven analyses of compound selectivity versus promiscuity. In this perspective, we discuss computational methods and data structures designed for promiscuity analysis. In addition, findings from large-scale exploration of activity profiles of inhibitors covering the human kinome are summarized. Although many kinase inhibitors are expected to be promiscuous, they are frequently found to be selective, which provides opportunities for target-directed drug discovery (rather than polypharmacology). We also discuss that machine learning yields evidence for the existence of structure–promiscuity relationships.