Rational genome and metabolic engineering of Candida viswanathii by split CRISPR to produce hundred grams of dodecanedioic acid

Candida viswanathii is a promising cell factory for producing dodecanedioic acid (DDA) and other long chain dicarboxylic acids. However, metabolic engineering of C. viswanathii is difficult partly due to the lack of synthetic biology toolkits. Here we developed CRISPR-based approaches for rational genome and metabolic engineering of C. viswanathii. We first optimized the CRISPR system and protocol to promote the homozygous gene integration efficiency to >60%. We also designed a split CRISPR system for one-step integration of multiple genes into C. viswanathii. We uncovered that co-expression of CYP52A19, CPRb and FAO2 that catalyze different steps in the biotransformation enhances DDA production and abolishes accumulation of intermediates. We also unveiled that co-expression of additional enzyme POS5 further promotes DDA production and augments cell growth. We harnessed the split CRISPR system to co-integrate these 4 genes (13.6 kb) into C. viswanathii and generated a stable strain that doubles the DDA titer (224 g/L), molar conversion (83%) and productivity (1.87 g/L/h) when compared with the parent strain. This study altogether identifies appropriate enzymes/promoters to augment dodecane conversion to DDA and implicates the potential of split CRISPR system for metabolic engineering of C. viswanathii.