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The feasibility of using a single vector to clone a cytochrome P450 monooxygenase (P450) in different yeasts and then compare whole‐cell hydroxylase activity was investigated. A broad‐range yeast expression vector using the ylTEFp to drive expression of the cloned gene and the scTEFp to drive the hygromycin resistance marker gene was used to clone the genes encoding two self‐sufficient P450s, CYP102A1 and CYP505A1. Both genes were cloned into Saccharomyces cerevisiae, Kluyveromyces marxianus, Yarrowia lipolytica (two strains) and Arxula adeninivorans. 4‐Hexylbenzoic acid (HBA), which is subterminally hydroxylated by both CYP102A1 and CYP505A1, was used to compare whole‐cell hydroxylase activity of transformants. Kluyveromyces marxianus and A. adeninivorans exhibited activity with both CYP102A1 and CYP505A1, while S. cerevisiae only displayed CYP102A1 activity and Y. lipolytica only CYP505A1 activity. The highest CYP102A1 activity (0.8 mM HBA converted in 24 h) was observed with concentrated resting‐cell suspensions of S. cerevisiae. The CYP505A1 activity observed with growing cultures of A. adeninivorans was however at least 12 times higher than the CYP102A1 activity of S. cerevisiae with up to 2 mM HBA converted within 6 h. The use of K. marxianus and A. adeninivorans for P450 expression has not previously been reported.

The cytochrome P450 monooxygenase (CYP) superfamily comprises hemethiolate enzymes that perform remarkable regio- and stereospecific oxidative chemistry. As such, CYPs are key agents for the structural and functional tailoring of triterpenoids, one of the largest classes of plant natural products with widespread applications in pharmaceuticals, food, cosmetics, and agricultural industries. In this review, we provide a full overview of 149 functionally characterised CYPs involved in the biosynthesis of triterpenoids and steroids in primary as well as in specialised metabolism. We describe the phylogenetic distribution of triterpenoid- and steroid-modifying CYPs across the plant CYPome, present a structure-based summary of their reactions, and highlight recent examples of particular interest to the field. Our review therefore provides a comprehensive up-to-date picture of CYPs involved in the biosynthesis of triterpenoids and steroids in plants as a starting point for future research.

Overuse of acetolactate synthase (ALS)–inhibiting herbicides in rice has led to the evolution of halosulfuron-resistant rice flatsedge in Arkansas and Mississippi. Resistant accessions were cross-resistant to labeled field rates of ALS-inhibiting herbicides from four different families, in comparison to a susceptible (SUS) biotype. Resistance index of Arkansas and Mississippi accessions based on an R/S ratio of the lethal dose required for 50% plant mortality (LD50) to bispyribac-sodium, halosulfuron, imazamox, and penoxsulam was ≥ 21-fold. Control of Arkansas, Mississippi, and SUS accessions with labeled field rates of 2,4-D, bentazon, and propanil was ≥ 93%. An enzyme assay revealed that an R/S ratio for 50% inhibition (I50) of ALS for halosulfuron was 2,600 and 200 in Arkansas and Mississippi, respectively. Malathion studies did not reveal enhanced herbicide metabolism in resistant plants. The ALS enzyme assay and cross-resistance studies point toward altered a target site as the potential mechanism of resistance. Trp574–Leu amino acid substitution within the ALS gene was found in both Arkansas and Mississippi rice flatsedge accessions using the Illumina HiSeq platform, which corresponds to the mechanism of resistance found in many weed species. Field-rate applications of 2,4-D, bentazon, and propanil can be used to control these ALS-resistant rice flatsedge accessions. Nomenclature: 2,4-D; acetolactate synthase; bentazon; bispyribac-sodium; halosulfuron; imazamox; malathion; propanil; rice flatsedge, Cyperus iria L; rice, Oryza sativa L.

C–H bonds are ubiquitous structural units of organic molecules. Although these bonds are generally considered to be chemically inert, the recent emergence of methods for C–H functionalization promises to transform the way synthetic chemistry is performed. The intermolecular amination of C–H bonds represents a particularly desirable and challenging transformation for which no efficient, highly selective, and renewable catalysts exist. Here we report the directed evolution of an iron-containing enzymatic catalyst—based on a cytochrome P450 monooxygenase—for the highly enantioselective intermolecular amination of benzylic C–H bonds. The biocatalyst is capable of up to 1,300 turnovers, exhibits excellent enantioselectivities, and provides access to valuable benzylic amines. Iron complexes are generally poor catalysts for C–H amination: in this catalyst, the enzyme's protein framework confers activity on an otherwise unreactive iron-haem cofactor. The intermolecular amination of C–H bonds is an enabling transformation for the synthesis of nitrogen-containing molecules; however, developing catalysts for this class of reactions is very challenging. Now, an iron-based enzyme for this reaction has been engineered, demonstrating that a protein can confer a difficult new function upon an otherwise unreactive base metal.

Fruit growth and ripening are controlled by multiple phytohormones. How these hormones coordinate and interact with each other to control these processes at the molecular level is unclear. We found in the early stages of Fragaria vesca (woodland strawberry) fruit development, auxin increases both widths and lengths of fruits, while gibberellin [gibberellic acid (GA)] mainly promotes their longitudinal elongation. Auxin promoted GA biosynthesis and signaling by activating GA biosynthetic and signaling genes, suggesting auxin function is partially dependent on GA function. To prevent the repressive effect of abscisic acid (ABA) on fruit growth, auxin and GA suppressed ABA accumulation during early fruit development by activating the expression of FveCYP707A4a encoding cytochrome P450 monooxygenase that catalyzes ABA catabolism. At the onset of fruit ripening, both auxin and GA levels decreased, leading to a steep increase in the endogenous level of ABA that drives fruit ripening. ABA repressed the expression of FveCYP707A4a but promoted that of FveNCED, a rate-limiting step in ABA biosynthesis. Accordingly, altering FveCYP707A4a expression changed the endogenous ABA levels and affected FveNCED expression. Hence, ABA catabolism and biosynthesis are tightly linked by feedback and feedforward loops to limit ABA contents for fruit growth and to quickly increase ABA contents for the onset of fruit ripening. These results indicate that FveCYP707A4a not only regulates ABA accumulation but also provides a hub to coordinate fruit size and ripening times by relaying auxin, GA, and ABA signals.

Multidimensional fitness landscapes provide insights into the molecular basis of laboratory and natural evolution. To date, such efforts usually focus on limited protein families and a single enzyme trait, with little concern about the relationship between protein epistasis and conformational dynamics. Here, we report a multiparametric fitness landscape for a cytochrome P450 monooxygenase that was engineered for the regio- and stereoselective hydroxylation of a steroid. We develop a computational program to automatically quantify non-additive effects among all possible mutational pathways, finding pervasive cooperative signs and magnitude epistasis on multiple catalytic traits. By using quantum mechanics and molecular dynamics simulations, we show that these effects are modulated by long-range interactions in loops, helices and β-strands that gate the substrate access channel allowing for optimal catalysis. Our work highlights the importance of conformational dynamics on epistasis in an enzyme involved in secondary metabolism and offers insights for engineering P450s. Connecting conformational dynamics and epistasis has so far been limited to a few proteins and a single fitness trait. Here, the authors provide evidence of positive epistasis on multiple catalytic traits in the evolution and dynamics of engineered cytochrome P450 monooxygenase, offering insights for in silico protein design.

Summary As one of the largest gene families in plants, the cytochrome P450 monooxygenase genes (CYPs) are involved in diverse biological processes including biotic and abiotic stress response. Moreover, P450 genes are prone to expanding due to gene tandem duplication during evolution, resulting in generations of novel alleles with the neo‐function or enhanced function. Here, the bread wheat (Triticum aestivum) gene TaCYP81D5 was found to lie within a cluster of five tandemly arranged CYP81D genes, although only a single such gene (BdCYP81D1) was present in the equivalent genomic region of the wheat relative Brachypodium distachyon. The imposition of salinity stress could up‐regulate TaCYP81D5, but the effect was abolished in plants treated with an inhibitor of reactive oxygen species synthesis. In SR3, a wheat cultivar with an elevated ROS content, the higher expression and the rapider response to salinity of TaCYP81D5 were related to the chromatin modification. Constitutively expressing TaCYP81D5 enhanced the salinity tolerance both at seedling and reproductive stages of wheat via accelerating ROS scavenging. Moreover, an important component of ROS signal transduction, Zat12, was proven crucial in this process. Though knockout of solely TaCYP81D5 showed no effect on salinity tolerance, knockdown of BdCYP81D1 or all TaCYP81D members in the cluster caused the sensitivity to salt stress. Our results provide the direct evidence that TaCYP81D5 confers salinity tolerance in bread wheat and this gene is prospective for crop improvement.

The genetic manipulation of basidiomycete mushrooms is notoriously difficult and immature, and there is a lack of research reports on clustered regularly interspaced short palindromic repeat (CRISPR) based gene editing of functional genes in mushrooms. In this work, Ganoderma lucidum, a famous traditional medicinal basidiomycete mushroom, which produces a type of unique triterpenoid-anti-tumor ganoderic acids (GAs), was used, and a CRISPR/CRISPR-associated protein-9 nuclease (Cas9) editing system for functional genes of GA biosynthesis was constructed in the mushroom. As proof of concept, the effect of different gRNA constructs with endogenous u6 promoter and self-cleaving ribozyme HDV on ura3 disruption efficiency was investigated at first. The established system was applied to edit a cytochrome P450 monooxygenase (CYP450) gene cyp5150l8, which is responsible for a three-step biotransformation of lanosterol at C-26 to ganoderic acid 3-hydroxy-lanosta-8, 24-dien-26 oic acid. As a result, precisely edited cyp5150l8 disruptants were obtained after sequencing confirmation. The fermentation products of the wild type (WT) and cyp5150l8 disruptant were analyzed, and a significant decrease in the titer of four identified GAs was found in the mutant compared to WT. Another CYP gene involved in the biosynthesis of squalene-type triterpenoid 2, 3; 22, 23-squalene dioxide, cyp505d13, was also disrupted using the established CRISPR-Cas9 based gene editing platform of G. lucidum. The work will be helpful to strain molecular breeding and biotechnological applications of G. lucidum and other basidiomycete mushrooms.

Cinnamate 4-hydroxylase (C4H) is the first key cytochrome P450 monooxygenase (P450) enzyme in the phenylpropanoid pathway. It belongs to the CYP73 family of P450 superfamily, and catalyzes the conversion of trans -cinnamic acid to p -coumaric acid. Since p -coumaric acid serves as the precursor for the synthesis of a wide variety of metabolites involved in plant development and stress resistance, alteration in the expression of soybean C4H genes is expected to affect the downstream metabolite levels, and its ability to respond to stress. In this study, we identified four C4H genes in the soybean genome that are distributed into both class I and class II CYP73 family. GmC4H2 , GmC4H14 and GmC4H20 displayed tissue- and developmental stage-specific gene expression patterns with their transcript accumulation at the highest level in root tissues. GmC4H10 appears to be a pseudogene as its transcript was not detected in any soybean tissues. Furthermore, protein homology modelling revealed substrate docking only for GmC4H2, GmC4H14 and GmC4H20. To demonstrate the function of GmC4Hs, we modified a cloning vector for the heterologous expression of P450s in yeast, and used it for microsomal protein production and enzyme assay. Our results confirmed that GmC4H2, GmC4H14 and GmC4H20 contain the ability to hydroxylate trans -cinnamic acid with varying efficiencies.

The polyphagous corn earworm Helicoverpa zea relies on cytochrome P450 monooxygenases with broad substrate specificities to cope with the wide diversity of phytochemicals it encounters among its numerous host plants. These enzymes also contribute to the ability of this insect to tolerate toxins from sources other than its hosts, including microbial and synthetic toxins. Although upregulation of xenobiotic-metabolizing P450s in some herbivorous insects is closely linked to host plant toxins, transcriptional and/or post-transcriptional regulation of detoxification in this polyphagous species also appears to be relatively unspecialized. Reverse transcription polymerase chain reaction and metabolic analyses indicate that rare and infrequently encountered phytochemicals, as well as synthetic substances, can enhance metabolic activity in an adaptive fashion against both natural and synthetic toxins.