Whole-genome sequencing of parental populations and populations adaptively evolved in the presence of caspofungin

Biopolymer chitin has ideal properties for applications such as biocompatibility, strength, chemical functionality, and biodegradability. Biotechnological production of chitin using fungal cells could be feasible if the chitin content of the cells was enhanced. However, the chitin content is dependent on several cellular regulatory pathways and has complex genetic underpinnings. Here, adaptive laboratory evolution (ALE) in presence of caspofungin (CAS) was successfully devised for selecting chitin overaccumulation in Saccharomyces cerevisiae coupled with cells' fitness. After ~200 generations of independent asexual adaptive evolution of S. cerevisiae lineages originating from wild type (BY4742), kre6?0 strain and an UV treated population of wild type cells, all lineages tolerated the highest introduced concentration of CAS and most of them overproduced chitin. The highest specific chitin content was found in the ALE mid-point population of a wild type -based lineage. Whole genome sequencing of parental and evolved populations revealed solutions involving 88 unique single-nucleotide variants affecting 43 coding sequences likely underlying the acquired CAS tolerance. High-frequency variants detected in the evolved population with the highest chitin content were re-engineered into the wild type strain. The re-engineering revealed that chitin content in cells could be increased up to 7-fold with specific variant combinations. Interestingly, the variants leading into the highest chitin content did not improve CAS tolerance. Biotechnological chitin production calls for compatibility with industrial settings which has been proven for S. cerevisiae. The variants identified here form a basis for developing an industrial S. cerevisiae strain for high-grade, animal-free, chitin extraction.