A modular cloning (MoClo) toolkit for reliable intracellular protein targeting in the yeast Saccharomyces cerevisiae

Abstract

Modular Cloning (MoClo) allows the combinatorial assembly of plasmids from standardized genetic parts without the need of error-prone PCR reactions. It is a very powerful strategy which enables highly flexible expression patterns without the need of repetitive cloning procedures. In this study, we describe an advanced MoClo toolkit that is designed for the baker’s yeast Saccharomyces cerevisiae and optimized for the targeting of proteins of interest to specific cellular compartments. Comparing different targeting sequences, we developed signals to direct proteins with high specificity to the different mitochondrial subcompartments, such as the matrix and the intermembrane space (IMS). Furthermore, we optimized the subcellular targeting by controlling expression levels using a collection of different promoter cassettes; the MoClo strategy allows it to generate arrays of expression plasmids in parallel to optimize gene expression levels and reliable targeting for each given protein and cellular compartment. Thus, the Mo-Clo strategy enables the generation of protein-expressing yeast plasmids that accurately target proteins of interest to various cellular compartments.

A Yeast Modular Cloning (MoClo) Toolkit Expansion for Optimization of Heterologous Protein Secretion and Surface Display in Saccharomyces cerevisiae

“…It was originally designed to permit facile genome editing, integration, and CRISPR applications and has since been expanded to achieve a wide range of further functionalities. Expansions include further CRISPR and genomic integration capacities, the capability to make combinatorial libraries, greater multiplexing capacity with more selectable markers, genomic integration sites and inducible promoters, and specific subcellular targeting of proteins . The YSD toolkit presented here constitutes another expansion of the MoClo-YTK, which enables the rapid evaluation of panels of SPs and TFPs and/or anchor proteins in order to optimize the secretion and/or surface display of a protein of interest.…”

“…Expansions include further CRISPR and genomic integration capacities, the capability to make combinatorial libraries, 50 greater multiplexing capacity with more selectable markers, genomic integration sites and inducible promoters, 49 and specific subcellular targeting of proteins. 51 The YSD toolkit presented here constitutes another expansion of the MoClo-YTK, which enables the rapid evaluation of panels of SPs and TFPs and/or anchor proteins in order to optimize the secretion and/or surface display of a protein of interest. The secretion promoting sequences and anchor proteins have been curated from the most promising candidates in the literature on these topics.…”

Engineering the Maize Root Microbiome: A Rapid MoClo Toolkit and Identification of Potential Bacterial Chassis for Studying Plant–Microbe Interactions

“…Toolkits in the advanced application group are diverse but enable researchers to perform more complex tasks than expression of recombinant proteins such as genome editing, genome integration, creation of gene circuits, directed evolution, etc. For model organisms (e.g., Escherichia coli, − Pseudomonas putida, − Saccharomyces cerevisiae, − etc. ), a plethora of toolkits in the advanced application group exist that standardize and streamline more complex operations like the ones mentioned earlier.…”

Engineering the Maize Root Microbiome: A Rapid MoClo Toolkit and Identification of Potential Bacterial Chassis for studying Plant-Microbe Interactions

“…For model organisms (e.g. E. coli (11)(12)(13)(14)(15), P. putida (16)(17)(18)(19), Saccharomyces cerevisiae (20)(21)(22)(23), etc. ), a plethora of toolkits in the advanced application group exist that standardize and streamline more complex operations such as genome editing, genome integration, creating gene circuits, directed evolution, etc.…”