Co-production of l -Lysine and Heterologous Squalene in CRISPR/dCas9-Assisted Corynebacterium glutamicum

Corynebacterium glutamicum is widely used for a large-scale industrial producer of feed additive amino acids, such as l-lysine. Moreover, C. glutamicum has been engineered for producing various non-native chemicals, including terpenes. For the first time, C. glutamicum was engineered for co-production of l-lysine and heterologous squalene. To control metabolic fluxes for either the l-lysine biosynthesis pathway or the squalene biosynthesis pathway, pyruvate, an intermediate in the central metabolism, a node was regulated by a clustered regularly interspaced short palindromic repeat (CRISPR) interference system. Repressing pyc encoding for pyruvate carboxylase in the l-lysine producer (DM1919) and its derivatives resulted in 99.24 ± 7.63 mg/L total squalene and 6.25 ± 0.20 g/L extracellular lysine at 120 h. Furthermore, various oil overlays were tested for efficient co-productions. In situ extraction with corn oil (10%, v/v) exhibited a separation of 99.75% (w/v) of total squalene (intra- and extracellular squalene), while l-lysine can be secreted in the medium. This co-production strategy will help a potential bioprocess of amino acid production with various terpenes.