Enhanced chemoselectivity of a plant cytochrome P450 through protein engineering of surface and catalytic residues

Cytochrome P450s(P450s)are the most versatile catalysts utilized by plants to produce structurally and functionally diverse metabolites.Given the high degree of gene redundancy and challenge to functionally characterize plant P450s,protein engineering is used as a complementarystrategy to study the mechanisms of P450-mediated reactions,or to alter their functions.We previously proposed an approach of engineering plant P450s based on combining high accuracy homology models generated by Rosetta combined with data-driven design using evoluti onary information of these enzymes.With this strategy,we repurposed a multi-functional P450(CYP87D20)into a monooxygenase after red esigning its active site.Since most plant P450s are membrane-anchored proteins that are adapted to the micro-environments of plant cells,expressing them in heterologous hosts usually results in problems of expression or activity.Here,we applied compu-tational design to tackle these issues by simultaneous optimization of the protein surface and active site.After screening 17 variants,effective su bstitutions of surface residues were observed to improve both expression and activity of CYP87D20.In addition,the identified substitutions were additive and by com-bining them a highly eficient C11 hydroxylase of cucurbitadienol was created to participate in the mogrol biosynthesis.This study shows the importance of considering the interplay between surface and active site residues for P450 engineering.Our integrated strategy also opens an avenue to create more tai loring enzymes with desired functions for the metabolic engineering of high-valued compounds like mogrol,the precursor of natural sweetener mogrosides.

The domestication-associated L1 gene encodes a eucomic acid synthase pleiotropically modulating pod pigmentation and shattering in soybean

Pod coloration is a domestication-related trait in soybean,with modern cultivars typically displaying brown or tan pods,while their wild relative,Glycine soja,possesses black pods.However,the factors regulating this color variation remain unknown.In this study,we cloned and characterized L1,the classical locus responsible for black pods in soybean.By using map-based cloning and genetic analyses,we identified the causal gene of L1 and revealed that it encodes a hydroxymethylglutaryl-coenzyme A(CoA)lyase-like(HMGL-like)domain protein.Biochemical assays showed that L1 functions as a eucomic acid synthase and facilitates the synthesis of eucomic acid and piscidic acid,both of which contribute to coloration of pods and seed coats in soybean.Interestingly,we found that L1 plants are more prone to pod shattering under light exposure than l1 null mutants because dark pigmentation increases photothermal efficiency.Hence,pleiotropic effects of L1 on pod color and shattering,as well as seed pigmentation,likely contributed to the preference forl1 alleles during soybean domestication and improvement.Collectively,our study provides new insights into the mechanism of pod coloration and identifies a new target for future de novo domestication oflegume crops.

A structural and data-driven approach to engineering a plant cytochrome P450 enzyme

Functional manipulation of biosynthetic enzymes such as cytochrome P450 s(or P450 s) has attracted great interest in metabolic engineering of plant natural products.Cucurbitacins and mogrosides are plant triterpenoids that share the same backbone but display contrasting bioactivities.This structural and functional diversity of the two metabolites can be manipulated by engineering P450 s.However,the functional redesign of P450 s through directed evolution(DE) or structure-guided protein engineering is time consuming and challenging,often because of a lack of high-throughput screening methods and crystal structures of P450 s.In this study,we used an integrated approach combining computational protein design,evolutionary information,and experimental data-driven optimization to alter the substrate specificity of a multifunctional P450(CYP87 D20)from cucumber.After three rounds of iterative design and evaluation of 96 protein variants,CYP87 D20,which is involved in the cucurbitacin C biosynthetic pathway,was successfully transformed into a P450 mono-oxygenase that performs a single specific hydroxylation at C11 of cucurbitadienol.This integrated P450-engineering approach can be further applied to create a de novo pathway to produce mogrol,the precursor of the natural sweetener mogroside,or to alter the structural diversity of plant triterpenoids by functionally manipulating other P450 s.

Developmentally Regulated Glucosylation of Bitter Triterpenoid in Cucumber by the UDP-Glucosyltransferase UGT73AM3

Dear Editor,Plants have evolved great plasticity to adapt to external environments. A huge number of structurally diverse metabolites gener- ated through the glycosylation process is one potential mechanism that contributes to this plasticity （Bowles et al., 2005）.