Establishment of the CRISPR-Cpf1 gene editing system in and multiplexed gene knockout.

is a significant industrial microorganism. Traditional gene editing techniques relying on homologous recombination often exhibit low efficiency due to their reliance on resistance genes. Additionally, the established CRISPR gene editing technology, utilizing Cas9 endonuclease, faces challenges in achieving simultaneous knockout of multiple genes. To address this limitation, the CRISPR-Cpf1 system has been developed, enabling multiplexed gene editing across various microorganisms. Key to the efficient gene editing capability of this system is the rigorous screening of highly effective expression elements to achieve conditional expression of protein Cpf1. In this study, we employed mCherry as a reporter gene and harnessed P for regulating the expression of Cpf1 to establish the CRISPR-Cpf1 gene editing system in . Our system achieved a 100 % knockout efficiency for the single gene and up to 80 % for simultaneous knockout of the double genes and . Furthermore, the culture of a series of protease-deficient strains revealed that the protease encoded by contributed significantly to extracellular enzyme activity (approximately 80 %), whereas proteases encoded by , , and genes contributed to a smaller proportion of extracellular enzyme activity. These findings provide support for effective molecular modification and metabolic regulation in industrial organisms.