New Yarrowia lipolytica chassis strains for industrial enzyme production

Background: Yarrowia lipolytica has become an established platform for producing a wide variety of biomolecules, including recombinant proteins (rProts). Its robust metabolism and resistance to multiple environmental stress factors make it specifically well-suited for the role of microbial cell factory. Yet, an adequate physiological background still requires some adjustments to make it a true ‘industrial workhorse’. Over years of continuous evolution, Y. lipolytica-based expression system has been engineered to establish auxotrophy markers selection, relieve the secretory pathway by deletion of multiple native secretory proteins, abolish filamentation, and create an inducible transcription system. Results: In this study, built on previous work, and continued fine-tuning of Y. lipolytica for rProts synthesis. We i) introduced another auxotrophy to facilitate more complex engineering strategies, ii) eliminated bacterial cloning elements previously introduced with a shuttle vector to eradicate antibiotic-resistance genes, and iii) conducted massive deletion of extracellular proteases and a peroxidase. The newly constructed chassis strains JMY9438 and JMY9451/9452, both bearing triple auxotrophy, differ in proteolytic activity (eliminated in the latter). Taking advantage of the new quality, the strains were tested for their efficiency in rProt production, cloned in 1, 2, or 3 copies. Titration of rProts amounts was possible only up to 2 copies of the heterologous gene. Considering the absolute amounts of rProt, strain maintaining the proteases with 2 copies of the rProt-coding gene turned out to be the most efficient. However, it is the strain deleted in five extracellular proteases and only 1 copy of the heterologous gene was the most efficient producer when the measure was expressed per cell. Conclusions: We provide a new generation of Y. lipolyticachassis strain, specifically designed for rProts production. The study demonstrated that the massive deletion of extracellular proteases offered a background for high-level rProt production without the necessity of burdening it with multi-copy transformations.