Enhanced inhibitor tolerance and increased lipid productivity through adaptive laboratory evolution in the oleaginous yeast Metshnikowia pulcherrima

Microbial lipid production from second generation feedstocks presents a sustainable route to future fuels, foods and bulk chemicals, and the oleaginous yeast Metshnikowia pulcherrima has previously shown potential as platform organism due to its ability to be grown in non-sterile conditions and metabolising a wide range carbon sources in lignocellulosic hydrolysates. However, the generation of inhibitors from depolymerisation causes downstream bioprocessing complications and inhibitors remains deleterious to both biomass and lipid formation. Using either a single inhibitor (formic acid) or an inhibitor cocktail (formic acid, acetic acid, fufural and HMF), two strategies of adaptive laboratory evolution were performed to improve fermentation inhibitor tolerance in M. pulcherrima. Both strategies had increased growth rates and reduced lag times under inhibiting conditions versus the progenitor. Interestingly, the lipid production of the inhibitor cocktail evolved strains markedly increased, with one strain producing 41% lipid by dry weight compared to 22% of the progenitor. The evolved lineage accumulated lipid rapidly, yielding 6.1 g/L of lipid (35% cell dry weight) within 48 hours (0.128 g L-1 h-1) when grown in a non-sterile 2L stirred tank bioreactor. Furthermore, the lipid profile was analogous to palm oil, consisting of 39% C16:0 and 56% C18:1 after 48 hours.