Biology subjects

Bouwmeester, H. J.

Publications and source records attributed to Bouwmeester, H. J..

4 recordsLinked to original sources

bioRxiv2026

Metabolic Engineering Boosts Strigolactone Production in Nicotiana benthamiana and Uncovers a Novel P450 Function

Strigolactones (SLs) are plant hormones regulating shoot branching and symbiotic interactions, but their trace-level abundance limits research and applications. Here, we optimized a Nicotiana benthamiana transient expression system for SL production by tuning agroinfiltration parameters and co-expressing rate-limiting carotenoid biosynthetic genes. Overexpression of Zea mays PSY1 or an Arabidopsis PSY-GGPS11 fusion increased carlactone production over 2-fold and enhanced downstream SL accumulation. Using this platform, we discovered that sorghum cytochrome P450 SbCYP728B35 catalyzes conversion of 5-deoxystrigol to sorgolactone, revealing a previously unknown function. These results establish metabolic engineering of precursor supply as an effective strategy for boosting SL production and demonstrate N. benthamiana as a robust system for pathway elucidation and biotechnological synthesis of bioactive strigolactones.

plant biology↗

bioRxiv2020

Integrating structure-based machine learning and co-evolution to investigate specificity in plant sesquiterpene synthases

Sesquiterpene synthases (STSs) catalyze the formation of a large class of plant volatiles called sesquiterpenes. While thousands of putative STS sequences from diverse plant species are available, only a small number of them have been functionally characterized. Sequence identity-based screening for desired enzymes, often used in biotechnological applications, is difficult to apply here as STS sequence similarity is strongly affected by species. This calls for more sophisticated computational methods for functionality prediction. We investigate the specificity of precursor cation formation in these elusive enzymes. By inspecting multi-product STSs, we demonstrate that STSs have a strong selectivity towards one precursor cation. We use a machine learning approach combining sequence and structure information to accurately predict precursor cation specificity for STSs across all plant species. We combine this with a co-evolutionary analysis on the wealth of uncharacterized putative STS sequences, to pinpoint residues and distant functional contacts influencing cation formation and reaction pathway selection. These structural factors can be used to predict and engineer enzymes with specific functions, as we demonstrate by predicting and characterizing two novel STSs from Citrus bergamia. Author summaryPredicting enzyme function is a popular problem in the bioinformatics field that grows more pressing with the increase in protein sequences, and more attainable with the increase in experimentally characterized enzymes. Terpenes and terpenoids form the largest classes of natural products and find use in many drugs, flavouring agents, and perfumes. Terpene synthases catalyze the biosynthesis of terpenes via multiple cyclizations and carbocation rearrangements, generating a vast array of product skeletons. In this work, we present a three-pronged computational approach to predict carbocation specificity in sesquiterpene synthases, a subset of terpene synthases with one of the highest diversities of products. Using homology modelling, machine learning and co-evolutionary analysis, our approach combines sparse structural data, large amounts of uncharacterized sequence data, and the current set of experimentally characterized enzymes to provide insight into residues and structural regions that likely play a role in determining product specifcity. Similar techniques can be repurposed for function prediction and enzyme engineering in many other classes of enzymes.

bioinformatics↗

bioRxiv2020

Strigolactone defective mutants of Arabidopsis exhibit delayed sepal senescence

Flower sepals are critical for flower development and vary greatly in lifespan depending on their function postpollination. However, very little is known on what controls sepal longevity. Using a sepal senescence mutant screen, we directly connected strigolactones (SL) with sepal longevity. We identified two Arabidopsis mutants that harbour novel mutations in the SL biosynthetic gene MORE AXILLARY GROWTH1 (MAX1) and receptor DWARF14 (AtD14). The mutation in AtD14 caused a substitution of the catalytic Ser-97 to Phe in the enzyme active site. The lesion in MAX1 changed a highly conserved Gly-469 to Arg in the haem-iron ligand signature of the cytochrome P450 protein, which caused loss-of-function of MAX1. nCounter-based transcriptional analysis suggested an interaction between SL and sugar signalling in controlling dark-induced inflorescence senescence. The results uncover an important function for SL in regulating floral organ senescence in addition to its other diverse functions in plant development and stress response. One-sentence summaryTwo novel mutants in the strigolactone pathway demonstrate a role for the hormone in sepal senescence, and transcriptional analysis highlights interaction between strigolactones and sugar signalling.

plant biology↗

bioRxiv2018

An Analysis of Characterized Plant Sesquiterpene Synthases

Plants exhibit a vast array of sesquiterpenes, C15 hydrocarbons which often function as herbivore-repellents or pollinator-attractants. These in turn are produced by a diverse range of sesquiterpene synthases. A comprehensive analysis of these enzymes in terms of product specificity has been hampered by the lack of a centralized resource of sufficient functionally annotated sequence data. To address this, we have gathered 262 plant sesquiterpene synthase sequences with experimentally characterized products. The annotated enzyme sequences allowed for an analysis of terpene synthase motifs, leading to the extension of one motif and recognition of a variant of another. In addition, putative terpene synthase sequences were obtained from various resources and compared with the annotated sesquiterpene synthases. This analysis indicated regions of terpene synthase sequence space which so far are unexplored experimentally. Finally, we present a case describing mutational studies on residues altering product specificity, for which we analyzed conservation in our database. This demonstrates an application of our database in choosing likely-functional residues for mutagenesis studies aimed at understanding or changing sesquiterpene synthase product specificity.

bioinformatics↗