Mutant huntingtin impairs neurodevelopment in human brain organoids through CHCHD2-mediated neurometabolic failure.

Expansion of the glutamine tract (poly-Q) in the protein huntingtin (HTT) causes the neurodegenerative disorder Huntington's disease (HD). Emerging evidence suggests that mutant HTT (mHTT) disrupts brain development. To gain mechanistic insights into the neurodevelopmental impact of human mHTT, we engineered male induced pluripotent stem cells to introduce a biallelic or monoallelic mutant 70Q expansion or to remove the poly-Q tract of HTT. The introduction of a 70Q mutation caused aberrant development of cerebral organoids with loss of neural progenitor organization. The early neurodevelopmental signature of mHTT highlighted the dysregulation of the protein coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2), a transcription factor involved in mitochondrial integrated stress response. CHCHD2 repression was associated with abnormal mitochondrial morpho-dynamics that was reverted upon overexpression of CHCHD2. Removing the poly-Q tract from HTT normalized CHCHD2 levels and corrected key mitochondrial defects. Hence, mHTT-mediated disruption of human neurodevelopment is paralleled by aberrant neurometabolic programming mediated by dysregulation of CHCHD2, which could then serve as an early interventional target for HD.

Modular segmentation, spatial analysis and visualization of volume electron microscopy datasets.

Volume electron microscopy is the method of choice for the in situ interrogation of cellular ultrastructure at the nanometer scale, and with the increase in large raw image datasets generated, improving computational strategies for image segmentation and spatial analysis is necessary. Here we describe a practical and annotation-efficient pipeline for organelle-specific segmentation, spatial analysis and visualization of large volume electron microscopy datasets using freely available, user-friendly software tools that can be run on a single standard workstation. The procedures are aimed at researchers in the life sciences with modest computational expertise, who use volume electron microscopy and need to generate three-dimensional (3D) segmentation labels for different types of cell organelles while minimizing manual annotation efforts, to analyze the spatial interactions between organelle instances and to visualize the 3D segmentation results. We provide detailed guidelines for choosing well-suited segmentation tools for specific cell organelles, and to bridge compatibility issues between freely available open-source tools, we distribute the critical steps as easily installable Album solutions for deep learning segmentation, spatial analysis and 3D rendering. Our detailed description can serve as a reference for similar projects requiring particular strategies for single- or multiple-organelle analysis, which can be achieved with computational resources commonly available to single-user setups.

Saccharomyces cerevisiae as a Heterologous Host for Natural Products.

Cell factories can provide a sustainable supply of natural products with applications as pharmaceuticals, food-additives or biofuels. Besides being an important model organism for eukaryotic systems, Saccharomyces cerevisiae is used as a chassis for the heterologous production of natural products. Its success as a cell factory can be attributed to the vast knowledge accumulated over decades of research, its overall ease of engineering and its robustness. Many methods and toolkits have been developed by the yeast metabolic engineering community with the aim of simplifying and accelerating the engineering process.In this chapter, a range of methodologies are highlighted, which can be used to develop novel natural product cell factories or to improve titer, rate and yields of an existing cell factory with the goal of developing an industrially relevant strain. The addressed topics are applicable for different stages of a cell factory engineering project and include the choice of a natural product platform strain, expression cassette design for heterologous or native genes, basic and advanced genetic engineering strategies, and library screening methods using biosensors. The many engineering methods available and the examples of yeast cell factories underline the importance and future potential of this host for industrial production of natural products.

Expansion of the Yeast Modular Cloning Toolkit for CRISPR-Based Applications, Genomic Integrations and Combinatorial Libraries.

Standardisation of genetic parts has become a topic of increasing interest over the last decades. The promise of simplifying molecular cloning procedures, while at the same time making them more predictable and reproducible has led to the design of several biological standards, one of which is modular cloning (MoClo). The Yeast MoClo toolkit provides a large library of characterised genetic parts combined with a comprehensive and flexible assembly strategy. Here we aimed to (1) simplify the adoption of the standard by providing a simple design tool for including new parts in the MoClo library, (2) characterise the toolkit further by demonstrating the impact of a BglII site in promoter parts on protein expression, and (3) expand the toolkit to enable efficient construction of gRNA arrays, marker-less integration cassettes and combinatorial libraries. These additions make the toolkit more applicable for common engineering tasks and will further promote its adoption in the yeast biological engineering community.

Integration of a multi-step heterologous pathway in Saccharomyces cerevisiae for the production of abscisic acid.

BACKGROUND: The sesquiterpenoid abscisic acid (ABA) is mostly known for regulating developmental processes and abiotic stress responses in higher plants. Recent studies show that ABA also exhibits a variety of pharmacological activities. Affordable and sustainable production will be required to utilize the compound in agriculture and as a potential pharmaceutical. Saccharomyces cerevisiae is an established workhorse for the biotechnological production of chemicals. In this study, we constructed and characterised an ABA-producing S. cerevisiae strain using the ABA biosynthetic pathway from Botrytis cinerea. RESULTS: Expression of the B. cinerea genes bcaba1, bcaba2, bcaba3 and bcaba4 was sufficient to establish ABA production in the heterologous host. We characterised the ABA-producing strain further by monitoring ABA production over time and, since the pathway contains two cytochrome P450 enzymes, by investigating the effects of overexpressing the native S. cerevisiae or the B. cinerea cytochrome P450 reductase. Both, overexpression of the native or heterologous cytochrome P450 reductase, led to increased ABA titres. We were able to show that ABA production was not affected by precursor or NADPH supply, which suggested that the heterologous enzymes were limiting the flux towards the product. The B. cinerea cytochrome P450 monooxygenases BcABA1 and BcABA2 were identified as pathway bottlenecks and balancing the expression levels of the pathway enzymes resulted in 4.1-fold increased ABA titres while reducing by-product formation. CONCLUSION: This work represents the first step towards a heterologous ABA cell factory for the commercially relevant sesquiterpenoid.