Biology subjects

Bathe, U.

Publications and source records attributed to Bathe, U..

5 recordsLinked to original sources

bioRxiv2025

Harnessing Mass Spectrometry-Based Proteomics for Continuous Directed Evolution

Continuous directed evolution is a powerful Synthetic Biology tool to engineer proteins with desired functions in vivo. Mimicking natural evolution, it involves repeated cycles of high-frequency mutagenesis, selection, and replication within platform cells, where the function of the target gene is tightly linked to the host cells fitness. However, cells might escape the selection pressure due to the inherent flexibility of their metabolism, which allows for adaptation. Whole-proteome analysis as well as targeted proteomics offer valuable insights into global and specific cellular changes. They can identify modifications in the target protein and its interactors to help understand its evolution and network integration. Using the continuous evolution of the Arabidopsis methionine synthases AtMS1 and AtMS2 as an example, we demonstrate how mass spectrometry-based proteomics can be applied in CDE, propose specific checkpoints for its integration and illustrate its role in informed decision making.

synthetic biology↗

bioRxiv2023

Metabolic arms race between a plant and a fungal pathogen

In this work, we uncover a metabolite interaction between barley and the fungal pathogen Bipolaris sorokiniana (Bs), involving hordedanes, a previously undescribed set of labdane-related diterpenoids with antimicrobial properties. Bs infection of barley roots elicits hordedane synthesis from a 600-kb gene cluster. Heterologous reconstruction of the synthesis pathway in yeast produced several hordedanes, including one of the most advanced products 19-b-hydroxy-hordetrienoic acid (19-OH-HTA). Barley mutants in the diterpene synthase genes of the cluster are unable to produce hordedanes but, unexpectedly, show reduced Bs colonization. Accordingly, 19-OH-HTA enhances both germination and growth of Bs, while it inhibits other fungi, and Bs chemically modifies 19-OH-HTA. Thus, plant and pathogen molecular interactions extend beyond protein-protein recognition and the simple detoxification of plant antimicrobial metabolites. One-Sentence SummaryA fungal pathogen uses barley diterpenoid phytoalexins to facilitate root colonization.

plant biology↗

bioRxiv2023

Production of 130 diterpenoids by combinatorial biosynthesis in yeast

Diterpenoids form a diverse group of natural products, many of which are or could become pharmaceuticals or industrial chemicals. However, low concentration, presence in complex mixtures and challenging synthesis often limit their exploitation. The modular character of diterpene biosynthesis and the substrate flexibility of the enzymes involved make combinatorial biosynthesis a promising approach. Here, we report on the assembly in yeast of 130 diterpenoids by pairwise combinations of ten diterpene synthases producing (+)-copalyl diphosphate-derived backbones and four cytochrome P450 enzymes (CYPs); 80 of these diterpenoids have not yet been reported. The CYPs accepted the majority of substrates they were given but remained regioselective. Our results bode well for the systematic exploration of diterpenoid chemical space using combinatorial assembly in yeast.

synthetic biology↗

bioRxiv2021

Using Continuous Directed Evolution to Improve Enzymes for Plant Applications

Continuous directed evolution of enzymes and other proteins in microbial hosts is capable of outperforming classical directed evolution by executing hypermutation and selection concurrently in vivo, at scale, with minimal manual input. Provided that a target enzymes activity can be coupled to growth of the host cells, the activity can be improved simply by selecting for growth. Like all directed evolution, the continuous version requires no prior mechanistic knowledge of the target. Continuous directed evolution is thus a powerful new way to modify plant or non-plant enzymes for use in plant metabolic research and engineering. Here, we first describe the basic features of the Saccharomyces cerevisiae OrthoRep system for continuous directed evolution and compare it briefly with other systems. We then give a step-by-step account of three ways in which OrthoRep can be deployed to evolve primary metabolic enzymes, using a THI4 thiazole synthase as an example and illustrating the mutational outcomes obtained. We close by outlining applications of OrthoRep that serve growing demands (i) to change the characteristics of plant enzymes destined for return to plants, and (ii) to adapt ( plantize) enzymes from prokaryotes - especially exotic prokaryotes - to function well in mild, plant-like conditions. One-sentence summaryContinuous directed evolution using the yeast OrthoRep system is a powerful new way to improve enzymes for use in plant engineering as illustrated by plantizing a bacterial thiamin synthesis enzyme..

synthetic biology↗

bioRxiv2021

A barley gene cluster for the biosynthesis of diterpenoid phytoalexins

Phytoalexins are specialized metabolites that are induced upon pathogen infection and contribute to the defense arsenal of plants. Maize and rice produce multiple diterpenoid phytoalexins and there is evidence from genomic sequences that other monocots may also produce diterpenoid phytoalexins. Here we report on the identification and characterization of a gene cluster in barley (Hordeum vulgare cv. Golden Promise) that is involved in the production of a set of labdane-related diterpenoids upon infection of roots by the fungal pathogen Bipolaris sorokiniana. The cluster is localized on chromosome 2, covers over 600 kb and comprises genes coding for a (+)-copalyl diphosphate synthase (HvCPS2), a kaurene synthase like (HvKSL4) and several cytochrome P450 oxygenases (CYPs). Expression of HvCPS2 and HvKSL4 in yeast and Nicotiana benthamiana resulted in the production of a single major product, whose structure was determined to be of the cleistanthane type and was named hordediene. Co-expression of HvCPS2, HvKSL4 and one of the CYPs from the cluster (CYP89E31) afforded two additional products, hordetriene and 11-hydroxy-hordetriene. Both of these compounds could be detected in extracts of barley roots infected by B. sorokiniana, validating the function of these genes in planta. Furthermore, diterpenoids with multiple oxidations and with molecular masses of 316, 318 and 332 were induced in infected barley roots and secreted in the medium, indicating that additional oxidases, possibly from the same genomic cluster are involved in the production of these phytoalexins. Our results provide the basis for further investigation of the role of this gene cluster in the defense of barley against pathogens and more generally in the interaction with the microbiome.

plant biology↗