Heterologous Production of Squalene from Glucose in Engineered Corynebacterium glutamicum Using Multiplex CRISPR Interference and High-Throughput Fermentation

The sustainable production of squalene has driven the development of microbial cell factories due to the limitation of low-yielding bioprocesses from plants and illegal harvesting shark liver. We report the metabolic engineering of Corynebacterium glutamicum to produce squalene from glucose. Combinatorial metabolic engineering strategies for precursor rebalancing, redox balancing, and blocking the competing pathway for the isopentenyl diphosphate availabilities were applied by repressing the target genes using the CRISPR interference. The best engineered strain using high-throughput fermentation produced squalene from glucose at 5.4 ± 0.3 mg/g dry cell weight (DCW) and 105.3 ± 3.0 mg/L, which was a 5.2-fold increase over the parental strain. In addition, flask cultivation of C. glutamicum overexpressing the dxs and idi genes with squalene synthase gene and repressing the idsA gene resulted in production of squalene at 5.8 ± 0.4 mg/g DCW and 82.8 ± 6.2 mg/L, which was a 3.4-fold increase over the parental strain.