Accurate phenotype-to-genotype mapping of high-diversity yeast libraries by heat-shock-electroporation (HEEL).

High-throughput DNA transformation techniques are invaluable when generating high-diversity mutant libraries, a cornerstone of successful protein engineering. However, transformation efficiencies have a direct correlation with the probability of introducing multiple DNA molecules into each cell, although reliable library screenings require cells that contain a single unique genotype. Thus, transformation methods that yield a high multiplicity of transformations are unsuitable for high-diversity library screenings.

Harnessing the spatial and transcriptional regulation of monoterpenoid indole alkaloid metabolism in Alstonia scholaris leads to the identification of broad geissoschizine cyclase activities.

Monoterpene indole alkaloids (MIAs) are valuable metabolites produced in numerous medicinal plants from the Apocynaceae family such as Alstonia scholaris, which synthesizes strictamine, a MIA displaying neuropharmacological properties of a potential importance. To get insights into the MIA metabolism in A. scholaris, we studied here both the spatial and transcriptional regulations of MIA genes by performing a robust transcriptomics analysis of the main plant organs, leaf epidermis but also by sequencing RNA from leaves transiently overexpressing the master transcriptional regulator MYC2.

Debottlenecking cytochrome P450-dependent metabolic pathways for the biosynthesis of commercial natural products.

Covering: 2016 to the end of 2024This highlight article aims to provide a perspective on the challenges that novel biotechnological processes face in the biomanufacturing of natural products (NPs) whose biosynthesis pathways rely on cytochrome P450 monooxygenases. This enzyme superfamily is one of the most versatile in the biosynthesis of a plethora of NPs finding use across the food, nutrition, medicine, chemical and cosmetics industries. These enzymes often exhibit excellent regio- and stereoselectivity, but they can suffer from low activity and instability, which are serious issues impairing the development of high performing bioprocesses.

High-throughput G protein-coupled receptor-based autocrine screening for secondary metabolite production in yeast.

Biosensors are valuable tools in accelerating the test phase of the design-build-test-learn cycle of cell factory development, as well as in bioprocess monitoring and control. G protein-coupled receptor (GPCR)-based biosensors enable cells to sense a wide array of molecules and environmental conditions in a specific manner. Due to the extracellular nature of their sensing, GPCR-based biosensors require compartmentalization of distinct genotypes when screening production levels of a strain library to ensure that detected levels originate exclusively from the strain under assessment.

The Madagascar palm genome provides new insights on the evolution of Apocynaceae specialized metabolism.

Specialized metabolites possess diverse interesting biological activities and some cardenolides- and monoterpene indole alkaloids- (MIAs) derived pharmaceuticals are currently used to treat human diseases such as cancers or hypertension. While these two families of biocompounds are produced by specific subfamilies of , one member of this medicinal plant family, the succulent tree Drake (also known as Madagascar palm), does not produce such specialized metabolites. To explore the evolutionary paths that have led to the emergence and loss of cardenolide and MIA biosynthesis in , we sequenced and assembled the genome by combining Oxford Nanopore Technologies long-reads and Illumina short-reads.

Coupling High-Throughput and Targeted Screening for Identification of Nonobvious Metabolic Engineering Targets.

Identification of metabolic engineering targets is a fundamental challenge in strain development programs. While high-throughput (HTP) genetic engineering methodologies capable of generating vast diversity are being developed at a rapid rate, a majority of industrially interesting molecules cannot be screened at sufficient throughput to leverage these techniques. We propose a workflow that couples HTP screening of common precursors (e.

The Rauvolfia tetraphylla genome suggests multiple distinct biosynthetic routes for yohimbane monoterpene indole alkaloids.

Monoterpene indole alkaloids (MIAs) are a structurally diverse family of specialized metabolites mainly produced in Gentianales to cope with environmental challenges. Due to their pharmacological properties, the biosynthetic modalities of several MIA types have been elucidated but not that of the yohimbanes. Here, we combine metabolomics, proteomics, transcriptomics and genome sequencing of Rauvolfia tetraphylla with machine learning to discover the unexpected multiple actors of this natural product synthesis.

Early-stage economic and environmental impact assessment for optimized bioprocess development: Monoterpenoid indole alkaloids.

Microbial refactoring offers sustainable production of plant-sourced pharmaceuticals associated with high production costs, ecological harms, and supply chain dependencies. Here, microbial tabersonine production in Saccharomyces cerevisiae is modeled during early-stage development (TRL: 3-5), guiding decisions for process-scale economic and environmental optimization. The base-case 0.

An updated version of the Madagascar periwinkle genome.

The Madagascar periwinkle, , belongs to the family. This medicinal plant, endemic to Madagascar, produces many important drugs including the monoterpene indole alkaloids (MIA) vincristine and vinblastine used to treat cancer worldwide. Here, we provide a new version of the genome sequence obtained through the combination of Oxford Nanopore Technologies long-reads and Illumina short-reads.

Genome Assembly of the Medicinal Plant Voacanga thouarsii.

The Apocynaceae tree Voacanga thouarsii, native to southern Africa and Madagascar, produces monoterpene indole alkaloids (MIA), which are specialized metabolites with a wide range of bioactive properties. Voacanga species mainly accumulates tabersonine in seeds making these species valuable medicinal plants currently used for industrial MIA production. Despite their importance, the MIA biosynthesis in Voacanga species remains poorly studied.

The Vinca minor genome highlights conserved evolutionary traits in monoterpene indole alkaloid synthesis.

Vinca minor, also known as the lesser periwinkle, is a well-known species from the Apocynaceae, native to central and southern Europe. This plant synthesizes monoterpene indole alkaloids, which are a class of specialized metabolites displaying a wide range of bioactive- and pharmacologically important properties. Within the almost 50 monoterpene indole alkaloids it produces, V.

Burden Imposed by Heterologous Protein Production in Two Major Industrial Yeast Cell Factories: Identifying Sources and Mitigation Strategies.

Production of heterologous proteins, especially biopharmaceuticals and industrial enzymes, in living cell factories consumes cellular resources. Such resources are reallocated from normal cellular processes toward production of the heterologous protein that is often of no benefit to the host cell. This competition for resources is a burden to host cells, has a negative impact on cell fitness, and may consequently trigger stress responses.

Transportome-wide engineering of Saccharomyces cerevisiae.

Synthetic biology enables the production of small molecules by recombinant microbes for pharma, food, and materials applications. The secretion of products reduces the cost of separation and purification, but it is challenging to engineer due to the limited understanding of the transporter proteins' functions. Here we describe a method for genome-wide transporter disruption that, in combination with a metabolite biosensor, enables the identification of transporters impacting the production of a given target metabolite in yeast Saccharomyces cerevisiae.

Exploring small-scale chemostats to scale up microbial processes: 3-hydroxypropionic acid production in S. cerevisiae.

Background: The physiological characterization of microorganisms provides valuable information for bioprocess development. Chemostat cultivations are a powerful tool for this purpose, as they allow defined changes to one single parameter at a time, which is most commonly the growth rate. The subsequent establishment of a steady state then permits constant variables enabling the acquisition of reproducible data sets for comparing microbial performance under different conditions.

Matching NLR Immune Receptors to Autoimmunity in camta3 Mutants Using Antimorphic NLR Alleles.

To establish infection, pathogens deploy effectors to modify or remove host proteins. Plant immune receptors with nucleotide-binding, leucine-rich repeat domains (NLRs) detect these modifications and trigger immunity. Plant NLRs thus guard host "guardees.

CrEdit: CRISPR mediated multi-loci gene integration in Saccharomyces cerevisiae.

Background: One of the bottlenecks in production of biochemicals and pharmaceuticals in Saccharomyces cerevisiae is stable and homogeneous expression of pathway genes. Integration of genes into the genome of the production organism is often a preferred option when compared to expression from episomal vectors. Existing approaches for achieving stable simultaneous genome integrations of multiple DNA fragments often result in relatively low integration efficiencies and furthermore rely on the use of selection markers.

Transcriptome and genome size analysis of the Venus flytrap.

The insectivorous Venus flytrap (Dionaea muscipula) is renowned from Darwin's studies of plant carnivory and the origins of species. To provide tools to analyze the evolution and functional genomics of D. muscipula, we sequenced a normalized cDNA library synthesized from mRNA isolated from D.

ATAF1 transcription factor directly regulates abscisic acid biosynthetic gene NCED3 in Arabidopsis thaliana.

ATAF1, an Arabidopsis thaliana NAC transcription factor, plays important roles in plant adaptation to environmental stress and development. To search for ATAF1 target genes, we used protein binding microarrays and chromatin-immunoprecipitation (ChIP). This identified T[A,C,G]CGT[A,G] and TT[A,C,G]CGT as ATAF1 consensus binding sequences.

Structure, function and networks of transcription factors involved in abiotic stress responses.

Transcription factors (TFs) are master regulators of abiotic stress responses in plants. This review focuses on TFs from seven major TF families, known to play functional roles in response to abiotic stresses, including drought, high salinity, high osmolarity, temperature extremes and the phytohormone ABA. Although ectopic expression of several TFs has improved abiotic stress tolerance in plants, fine-tuning of TF expression and protein levels remains a challenge to avoid crop yield loss.

Order by disorder in plant signaling.

Protein intrinsic disorder (ID), referring to the lack of a fixed tertiary structure, is an emerging topic in plant science. Proteins with ID challenge our perception of protein interactions because of their malleable behavior. They are abundant in highly regulated processes such as cellular signaling and transcription, where they exploit the flexibility of ID.

NAC genes: time-specific regulators of hormonal signaling in Arabidopsis.

Environmental stresses on both animals and plants impose massive transcriptional perturbations. Successful adaptations to such stresses are being orchestrated by both activating and repressing effects of transcription factors on specific target genes. We have recently published a systematic characterization of members of the large NAC gene transcription factor family in the model weed Arabidopsis thaliana.

The HvNAC6 transcription factor: a positive regulator of penetration resistance in barley and Arabidopsis.

Pathogens induce the expression of many genes encoding plant transcription factors, though specific knowledge of the biological function of individual transcription factors remains scarce. NAC transcription factors are encoded in plants by a gene family with proposed functions in both abiotic and biotic stress adaptation, as well as in developmental processes. In this paper, we provide convincing evidence that a barley NAC transcription factor has a direct role in regulating basal defence.