Adaptive laboratory evolution of Bacillus subtilis to overcome toxicity of lignocellulosic hydrolysate derived from Distiller's dried grains with solubles (DDGS)

Jasper L S P Driessen; Josefin Johnsen; Ivan Pogrebnyakov; Elsayed T T Mohamed; Solange I Mussatto; Adam M Feist; Sheila I Jensen; Alex T Nielsen

doi:10.1016/j.mec.2023.e00223

Adaptive laboratory evolution of Bacillus subtilis to overcome toxicity of lignocellulosic hydrolysate derived from Distiller's dried grains with solubles (DDGS)

Metab Eng Commun. 2023 May 1:16:e00223. doi: 10.1016/j.mec.2023.e00223. eCollection 2023 Jun.

Authors

Jasper L S P Driessen¹, Josefin Johnsen¹, Ivan Pogrebnyakov¹, Elsayed T T Mohamed¹, Solange I Mussatto², Adam M Feist^{1

3}, Sheila I Jensen¹, Alex T Nielsen¹

Affiliations

¹ Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet, 220, 2800, Kongens Lyngby, Denmark.
² Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads 223, 2800, Kongens Lyngby, Denmark.
³ Department of Bioengineering, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0412, USA.

Abstract

Microbial tolerance to toxic compounds formed during biomass pretreatment is a significant challenge to produce bio-based products from lignocellulose cost effectively. Rational engineering can be problematic due to insufficient prerequisite knowledge of tolerance mechanisms. Therefore, adaptive laboratory evolution was applied to obtain 20 tolerant lineages of Bacillus subtilis strains able to utilize Distiller's Dried Grains with Solubles-derived (DDGS) hydrolysate. Evolved strains showed both improved growth performance and retained heterologous enzyme production using 100% hydrolysate-based medium, whereas growth of the starting strains was essentially absent. Whole-genome resequencing revealed that evolved isolates acquired mutations in the global regulator codY in 15 of the 19 sequenced isolates. Furthermore, mutations in genes related to oxidative stress (katA, perR) and flagella function appeared in both tolerance and control evolution experiments without toxic compounds. Overall, tolerance adaptive laboratory evolution yielded strains able to utilize DDGS-hydrolysate to produce enzymes and hence proved to be a valuable tool for the valorization of lignocellulose.

Keywords: Adaptive laboratory evolution; Bacillus subtilis; Enzyme production; Lignocellulose; Tolerance.