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Biofilms could provide favorable conditions for the growth of cells during industrial fermentation. However, biofilm-immobilized fermentation has not yet been reported in Corynebacterium glutamicum ( C. glutamicum ), one of the main strains for amino acid production. This is mainly because C. glutamicum has a poor capability of adsorption onto materials or forming an extracellular polymeric substance (EPS). Here, an engineered strain, C. glutamicum Pro-Δ exeM , was created by removing the extracellular nuclease gene exeM , which effectively increased extracellular DNA (eDNA) in the EPS and cell adhesiveness onto carrier materials. In repeated-batch fermentation using the biofilm, l-proline production increased from 10.2 to 17.1 g/L. In summary, this research demonstrated that a synthetic C. glutamicum biofilm could be favorable for l-proline production, which could be extended to other industrial applications of C. glutamicum , and the strategy may also be applicable to the engineering of other strains.

Efficient Biofilm-Based Fermentation Strategies by eDNA Formation for l-Proline Production with Corynebacterium glutamicum