Enhanced production of recombinant calf chymosin in Kluyveromyces lactis via CRISPR-Cas9 engineering.

As an important industrial enzyme, chymosin has been widely used in cheese manufacturing. Fermentation with Kluyveromyces lactis has allowed recombinant chymosin production to fit the growing global demand for cheese consumption; yet improvements can be made to allow for stable and larger-scale production. In this work, various chymosin producing (CP) strains were constructed via targeted chromosomal integration of various copies of a prochymosin expression cassette (PEC) using a CRISPR-Cas9 platform optimized for K. lactis. It enabled the demonstration that chymosin yields could be increased along with gradual chromosomal accumulation of PEC inserts within up to 3 copies. Finally, an optimal CP3i strain was constructed, and with which high yields of recombinant chymosin were attained, reaching ca. 1,200 SU/mL in shake-flask fermentation and ca. 28,000 SU/mL in batch-mode bioreaction, respectively. The activity of the product in milk-curding was observed. These findings provide direction to apply K. lactis-based platforms in the subsequent industrial-scale production of recombinant chymosin.