Rapid Screening of Diverse Biotransformations for Enzyme Evolution.
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ABSTRACT:
SUBMITTER: Kempa EE
PROVIDER: S-EPMC8154213 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:A wide range of organisms use sex pheromones to communicate with each other and to identify appropriate mating partners. While the evolution of chemical communication has been suggested to cause sexual isolation and speciation, the mechanisms that govern evolutionary transitions in sex pheromone production are poorly understood. Here, we decipher the molecular mechanisms underlying the rapid evolution in the expression of a gene involved in sex pheromone production in Drosophilid flies. Long-chain cuticular hydrocarbons (e.g., dienes) are produced female-specifically, notably via the activity of the desaturase DESAT-F, and are potent pheromones for male courtship behavior in Drosophila melanogaster. We show that across the genus Drosophila, the expression of this enzyme is correlated with long-chain diene production and has undergone an extraordinary number of evolutionary transitions, including six independent gene inactivations, three losses of expression without gene loss, and two transitions in sex-specificity. Furthermore, we show that evolutionary transitions from monomorphism to dimorphism (and its reversion) in desatF expression involved the gain (and the inactivation) of a binding-site for the sex-determination transcription factor, DOUBLESEX. In addition, we documented a surprising example of the gain of particular cis-regulatory motifs of the desatF locus via a set of small deletions. Together, our results suggest that frequent changes in the expression of pheromone-producing enzymes underlie evolutionary transitions in chemical communication, and reflect changing regimes of sexual selection, which may have contributed to speciation among Drosophila.
| S-EPMC2711336 | biostudies-literature
Project description:BackgroundEnzymes for plant cell wall deconstruction are a major cost in the production of ethanol from lignocellulosic biomass. The goal of this research was to develop optimized synthetic mixtures of enzymes for multiple pretreatment/substrate combinations using our high-throughput biomass digestion platform, GENPLAT, which combines robotic liquid handling, statistical experimental design and automated Glc and Xyl assays. Proportions of six core fungal enzymes (CBH1, CBH2, EG1, ?-glucosidase, a GH10 endo-?1,4-xylanase, and ?-xylosidase) were optimized at a fixed enzyme loading of 15 mg/g glucan for release of Glc and Xyl from all combinations of five biomass feedstocks (corn stover, switchgrass, Miscanthus, dried distillers' grains plus solubles [DDGS] and poplar) subjected to three alkaline pretreatments (AFEX, dilute base [0.25% NaOH] and alkaline peroxide [AP]). A 16-component mixture comprising the core set plus 10 accessory enzymes was optimized for three pretreatment/substrate combinations. Results were compared to the performance of two commercial enzymes (Accellerase 1000 and Spezyme CP) at the same protein loadings.ResultsWhen analyzed with GENPLAT, corn stover gave the highest yields of Glc with commercial enzymes and with the core set with all pretreatments, whereas corn stover, switchgrass and Miscanthus gave comparable Xyl yields. With commercial enzymes and with the core set, yields of Glc and Xyl were highest for grass stovers pretreated by AP compared to AFEX or dilute base. Corn stover, switchgrass and DDGS pretreated with AFEX and digested with the core set required a higher proportion of endo-?1,4-xylanase (EX3) and a lower proportion of endo-?1,4-glucanase (EG1) compared to the same materials pretreated with dilute base or AP. An optimized enzyme mixture containing 16 components (by addition of ?-glucuronidase, a GH11 endoxylanase [EX2], Cel5A, Cel61A, Cip1, Cip2, ?-mannanase, amyloglucosidase, ?-arabinosidase, and Cel12A to the core set) was determined for AFEX-pretreated corn stover, DDGS, and AP-pretreated corn stover. The optimized mixture for AP-corn stover contained more exo-?1,4-glucanase (i.e., the sum of CBH1 + CBH2) and less endo-?1,4-glucanase (EG1 + Cel5A) than the optimal mixture for AFEX-corn stover. Amyloglucosidase and ?-mannanase were the two most important enzymes for release of Glc from DDGS but were not required (i.e., 0% optimum) for corn stover subjected to AP or AFEX. As a function of enzyme loading over the range 0 to 30 mg/g glucan, Glc release from AP-corn stover reached a plateau of 60-70% Glc yield at a lower enzyme loading (5-10 mg/g glucan) than AFEX-corn stover. Accellerase 1000 was superior to Spezyme CP, the core set or the 16-component mixture for Glc yield at 12 h, but the 16-component set was as effective as the commercial enzyme mixtures at 48 h.ConclusionThe results in this paper demonstrate that GENPLAT can be used to rapidly produce enzyme cocktails for specific pretreatment/biomass combinations. Pretreatment conditions and feedstock source both influence the Glc and Xyl yields as well as optimal enzyme proportions. It is predicted that it will be possible to improve synthetic enzyme mixtures further by the addition of additional accessory enzymes.
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Project description:In functionally diverse enzyme superfamilies (SFs), conserved structural and active site features reflect catalytic capabilities 'hard-wired' in each SF architecture. Overlaid on this foundation, evolutionary changes in active site machinery, structural topology and other aspects of structural organization and interactions support the emergence of new reactions, mechanisms, and substrate specificity. This review connects topological with functional variation in each of the haloalkanoic acid dehalogenase (HAD) and vicinal oxygen chelate fold (VOC) SFs and a set of redox-active thioredoxin (Trx)-fold SFs to illustrate a few of the varied themes nature has used to evolve new functions from a limited set of structural scaffolds.
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Project description:Enzyme orthologs sharing identical primary functions can have different promiscuous activities. While it is possible to mine this natural diversity to obtain useful biocatalysts, generating comparably rich ortholog diversity is difficult, as it is the product of deep evolutionary processes occurring in a multitude of separate species and populations. Here, we take a first step in recapitulating the depth and scale of natural ortholog evolution on laboratory timescales. Using a continuous directed evolution platform called OrthoRep, we rapidly evolve the Thermotoga maritima tryptophan synthase β-subunit (TmTrpB) through multi-mutation pathways in many independent replicates, selecting only on TmTrpB's primary activity of synthesizing L-tryptophan from indole and L-serine. We find that the resulting sequence-diverse TmTrpB variants span a range of substrate profiles useful in industrial biocatalysis and suggest that the depth and scale of evolution that OrthoRep affords will be generally valuable in enzyme engineering and the evolution of biomolecular functions.
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Project description:Successful evolutionary enzyme engineering requires a high throughput screening or selection method, which considerably increases the chance of obtaining desired properties and reduces the time and cost. In this review, a series of high throughput screening and selection methods are illustrated with significant and recent examples. These high throughput strategies are also discussed with an emphasis on compatibility with phenotypic analysis during directed enzyme evolution. Lastly, certain limitations of current methods, as well as future developments, are briefly summarized.
| S-EPMC4461044 | biostudies-other
Project description:The mechanisms underlying the rapid evolution of novel enzymatic activities from promiscuous side activities are poorly understood. Recently emerged enzymes catalyzing the catabolic degradation of xenobiotic substances that have been spread out into the environment during the last decades provide an exquisite opportunity to study these mechanisms. A prominent example is the herbicide atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine), which is degraded through a number of enzymatic reactions constituting the Atz pathway. Here, we analyzed the evolution of the hydroxyatrazine ethylaminohydrolase AtzB, a Zn(II)-dependent metalloenzyme that adopts the amidohydrolase fold and catalyzes the second step of the Atz pathway. We searched for promiscuous side activities of AtzB, which might point to the identity of its progenitor. These investigations revealed that AtzB has low promiscuous guanine deaminase activity. Furthermore, we found that the two closest AtzB homologues, which have not been functionally annotated up to now, are guanine deaminases with modest promiscuous hydroxyatrazine hydrolase activity. Based on sequence comparisons with the closest AtzB homologues, the guanine deaminase activity of AtzB could be increased by three orders of magnitude through the introduction of only four active site mutations. Interestingly, introducing the inverse four mutations into the AtzB homologues significantly enhanced their hydroxyatrazine hydrolase activity, and in one case is even equivalent to that of wild-type AtzB. Molecular dynamics simulations elucidated the structural and molecular basis for the mutation-induced activity changes. The example of AtzB highlights how novel enzymes with high catalytic proficiency can evolve from low promiscuous side activities by only few mutational events within a short period of time.
| S-EPMC7615792 | biostudies-literature
Project description:Here, we present the results of an integrative study that leverages naturally segregating variation and a recent, adaptive divergence event affecting seasonal timing to identify developmental mechanisms underlying diapause progression in a tephritid fly, Rhagoletis pomonella. Also called the apple maggot fly, R. pomonella is native to North America, where it infests fruits of native Crataegus (hawthorn) species throughout its range. Derived populations of the fly infest apples (Malus domesticus), and thus have evolved in the last ~250 years since apples were introduced. Many molecular studies in conjunction with mark-recapture experiments document that the populations, or host races, remain genetically distinct despite ongoing gene flow, making R. pomonella a textbook example of incipient speciation with gene flow and host associated divergence. Strong natural selection on two primary traits, host finding behavior and seasonal timing, maintain genetic divergence. Both populations (hereafter apple and haw flies) have one generation per year, with a functionally obligate pupal diapause, overwintering in the soil. Adults must emerge coincident with host fruit availability, typically a period of only a few weeks, in order to successfully oviposit into fruits. Apple flies have evolved an earlier (~3 week) emergence timing to synchronize with apples, which fruit about 3 weeks earlier than hawthorn at a typical, sympatric site in the Midwest (e.g., Michigan or Illinois, USA). We combined RNA sequencing (RNAseq), phenotyping of emergence timing and brain morphology, and whole genome pooled resequencing (Poolseq) to infer mechanisms underlying segregating variation for diapause phenology in each Rhagoletic pomonella host race, and the evolved difference in phenology between host races.
2020-08-19 | GSE140473 | GEO
Project description:A highly sensitive chemiluminescent enzyme immunoassay (CLEIA) method was developed in this study for efficient screening of triazophos residues in a large number of samples. Based on the maximum residue limits (MRLs) set by China and CAC for triazophos in different agro-products, the representative apple, orange, cabbage, zucchini, and rice samples were selected as spiked samples, and the triazophos at the concentrations of the MRL values were spiked to blank samples. Subsequently, the five samples with the spiked triazophos standard were measured by CLEIA 100 times, and the detection results indicated that the correction factors of the apple, orange, cabbage, zucchini, and rice were determined as 0.79, 0.66, 0.85, 0.76, and 0.91, respectively. In this experiment, 1500 real samples were detected by both the CLEIA and the GC-MS methods. With the GC-MS method, 1462 samples were identified as negative samples and 38 samples as positive samples. Based on the correction factors, the false positive rate of the CLEIA method was 0.13%, and false negative rate was 0. The results showed that the established CLEIA method could be used to screen a large number of real samples.
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Project description:Microbes play a critical role in the global arsenic biogeocycle. Most studies have focused on redox cycling of inorganic arsenic in bacteria and archaea. The parallel cycles of organoarsenical biotransformations are less well characterized. Here we describe organoarsenical biotransformations in the environmental microbe Shewanella putrefaciens. Under aerobic growth conditions, S. putrefaciens reduced the herbicide MSMA (methylarsenate or MAs(V)) to methylarsenite (MAs(III)). Even though it does not contain an arsI gene, which encodes the ArsI C-As lyase, S. putrefaciens demethylated MAs(III) to As(III). It cleaved the C-As bond in aromatic arsenicals such as the trivalent forms of the antimicrobial agents roxarsone (Rox(III)), nitarsone (Nit(III)) and phenylarsenite (PhAs(III)), which have been used as growth promoters for poultry and swine. S. putrefaciens thiolated methylated arsenicals, converting MAs(V) into the more toxic metabolite monomethyl monothioarsenate (MMMTAs(V)), and transformed dimethylarsenate (DMAs(V)) into dimethylmonothioarsenate (DMMTAs(V)). It also reduced the nitro groups of Nit(V), forming p-aminophenyl arsenate (p-arsanilic acid or p-AsA(V)), and Rox(III), forming 3-amino-4-hydroxybenzylarsonate (3A4HBzAs(V)). Elucidation of organoarsenical biotransformations by S. putrefaciens provides a holistic appreciation of how these environmental pollutants are degraded.
| S-EPMC4984541 | biostudies-literature
Project description:The highly potent vasoconstrictor peptide endothelin (ET) is generated from an inactive precursor, big endothelin (bET), by endothelin-converting enzyme (ECE). ECE is a phosphoramidon-sensitive zinc metallopeptidase, which is closely related to neprilysin (neutral endopeptidase). It is possible that compounds which inhibit the formation of ET may be used as new drugs for the treatment of cardiovascular diseases. Such an approach requires a fast, simple and selective assay to measure ECE activity, allowing rapid screening of inhibitors. We describe here two new ECE substrates based on the concept of 'intramolecularly quenched fluorescence' which may fulfill this aim. One, S(1) [Pya(21)-Nop(22)-bET-1(19--35)], is the (19--35) fragment of the natural peptide big-ET-1(1--38), which is modified by introducing the fluorescent amino acid, pyrenylalanine (Pya), in position 21 and a quencher, p-nitrophenylalanine (Nop), in position 22. The second substrate (S(2)) is a small peptide, Ac-Ser-Gly-Pya-Lys-Ala-Phe-Ala-Nop-Gly-Lys-NH(2), from a biased substrate peptide library. The recombinant, hECE-1c, cleaved both Pya(21)-Nop(22)-bET-1(19--35) and the natural substrate selectively between residues 21 and 22, whereas cleavage occurred between alanine and phenylalanine in the small peptide. In both cases, this generated intense fluorescence emission. The synthesis and kinetic parameters of these substrates are described. These assays, which can be used directly on tissue homogenates, are the most sensitive and selective described to date for ECE, and are easily automated for a high-throughput screening of inhibitors.
| S-EPMC1221908 | biostudies-other