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.

A de novo Genome of a Chinese Radish Cultivar

Here, we report a high-quality draft genome of a Chinese radish(Raphanus sativus) cultivar. This draft contains 387.73 Mb of assembled scaffolds, 83.93% of the scaffolds were anchored onto nine pseudochromosomes and 95.09% of 43 240 protein-coding genes were functionally annotated. 184.75 Mb(47.65%) of repeat sequences was identified in the assembled genome. By comparative analyses of the radish genome against 10 other plant genomes, 2 275 genes in 780 gene families were found unique to R. sativus. This genome is a good reference for genomic study and of great value for genetic improvement of radish.

OBV(obscure vein),a C_(2)H_(2)zinc finger transcription factor,positively regulates chloroplast development and bundle sheath extension formation in tomato(Solanum lycopersicum)leaf veins

Leaf veins play an important role in plant growth and development,and the bundle sheath(BS)is believed to greatly improve the photosynthetic efficiency of C_(4) plants.The OBV mutation in tomato(Solanum lycopersicum)results in dark veins and has been used widely in processing tomato varieties.However,physiological performance has difficulty explaining fitness in production.In this study,we confirmed that this mutation was caused by both the increased chlorophyll content and the absence of bundle sheath extension(BSE)in the veins.Using genome-wide association analysis and map-based cloning,we revealed that OBV encoded a C_(2)H_(2) L domain class transcription factor.It was localized in the nucleus and presented cell type-specific gene expression in the leaf veins.Furthermore,we verified the gene function by generating CRISPR/Cas9 knockout and overexpression mutants of the tomato gene.RNA sequencing analysis revealed that OBV was involved in regulating chloroplast development and photosynthesis,which greatly supported the change in chlorophyll content by mutation.Taken together,these findings demonstrated that OBV affected the growth and development of tomato by regulating chloroplast development in leaf veins.This study also provides a solid foundation to further decipher the mechanism of BSEs and to understand the evolution of photosynthesis in land plants.

Rare SNP Identified a TCP Transcription Factor Essential for Tendril Development in Cucumber

Rare genetic variants are abundant in genomes but less tractable in genome-wide association study. Here we exploit a strategy of rare variation mapping to discover a gene essential for tendril development in cucumber （Cucumis sativus L.）. In a collection of 〉3000 lines, we discovered a unique tendril-less line that forms branches instead of tendrils and, therefore, loses its climbing ability. We hypothesized that this unusual phenotype was caused by a rare variation and subsequently identified the causative single nucleotide poly- morphism. The affected gene TEN encodes a TCP transcription factor conserved within the cucurbits and is expressed specifically in tendrils, representing a new organ identity gene. The variation occurs within a pro- tein motif unique to the cucurbits and impairs its function as a transcriptional activator. Analyses of transcrip- tomes from near-isogenic lines identified downstream genes required for the tendril＇s capability to sense and climb a support. This study provides an example to explore rare functional variants in plant genomes.