Adaptively evolved chitin overproduction in Saccharomyces cerevisiae

Biopolymer chitin has properties such as biocompatibility, strength, chemical functionality, and biodegradability that are attractive for applications. It could be biotechnologically produced using fungi, but the economical feasibility depends on the chitin content in cells. However, chitin content in cells is governed by 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. After ∼200 generations of independent asexual adaptive evolution of S. cerevisiae lineages originating from wild type (BY4742), kre6Δ0 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 88 unique single-nucleotide variants affecting 43 coding sequences. High-frequency variants detected in the evolved population with the highest chitin content (i.e., Fks1 p. Pro647Arg and p. Arg1357Cys, Fks2 p. Arg1203∗, and Ecm21 p. Glu642∗) 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 a specific variant combination (i.e., Fks1 p. Pro647Arg, Fks2 p. Arg1203∗, and Ecm21 p. Glu642∗). Interestingly, they did not improve CAS tolerance. Biotechnological production requires industrial compatibility which has been proven for S. cerevisiae. The variants identified here form a basis for developing an industrial S. cerevisiae strain for biotechnological production of animal-free chitin.