# EASY-C: Extraction and Analysis of Small Yeast Chromosomes—A rapid and universal platform for recovering artificial mini-chromosomes from synthetic Sc2.0 yeast and large plasmids from Saccharomyces cerevisiae and nonconventional yeast species

## TL;DR

EASY-C is a fast and simple method for isolating synthetic mini-chromosomes and large plasmids from various yeast species, including synthetic and nonconventional strains.

## Contribution

EASY-C introduces a universal and efficient protocol for extracting and analyzing small yeast chromosomes and plasmids across diverse yeast species.

## Key findings

- EASY-C successfully isolates synthetic mini-chromosomes and plasmids in under 2.5 hours using small volumes.
- The method works for a range of constructs, including centromeric vectors and high-copy plasmids.
- EASY-C is applicable to nonconventional yeast species like Starmerella sp., Maudiozyma sp., and Kazachstania sp.

## Abstract

The protocol for Extraction and Analysis of Small Yeast-Chromosomes (EASY-C) is a transformative, rapid, cost-effective, and user-friendly method designed for the efficient isolation of artificial synthetic mini-chromosomes (~42–52 kb) and large plasmids (~12 kb) from Saccharomyces cerevisiae wild-type and synthetic yeast strains (Sc2.0), and a range of nonconventional yeast (NCY) species. In this two-step workflow, the DNA from yeast is first extracted and transferred into bacteria, and then the circular DNA is recovered from the bacteria and subjected to downstream analysis, including long-read sequencing. The EASY-C protocol operates at small volumes (~1 mL) and requires less than 2.5 hours, allowing the use of standard commercial plasmid purification kits for bacterial plasmids. Under the tested conditions, the EASY-C methodology yielded clean DNA that could be digested and linearized prior to sequencing, resulting in a higher number of high-quality reads (~2000). The EASY-C protocol worked successfully for the extraction of a variety of constructs, including low-copy centromeric vectors (CEN/ARS), high-copy plasmids (pan/ARS), and artificial mini-chromosomes harbouring (CEN/ARS). It is also applicable to a variety of yeast species, including NCY such as Starmerella sp., Maudiozyma sp., and Kazachstania sp. Thanks to its precision, robustness, and simplicity, EASY-C equips researchers with a powerful, time-saving tool and cost-effective approach to accelerate the validation of a wide array of synthetic genetic and metabolic constructs engineered in vivo across diverse yeast species.

Graphical Abstract