The secretory production of cutinase ICCG-A2 in Corynebacterium glutamicum using acetate as the sole carbon source

The utilization of edible biomass as feedstocks poses sustainability concerns, prompting the exploration of affordable alternative carbon sources. Acetate is a promising candidate due to its high availability and low cost. This study developed an engineered platform for the secretory production of the cutinase ICCG-A2 in Corynebacterium glutamicum using high-concentration acetate as the sole carbon source. When cultured on sodium acetate (equimolar carbon content relative to glucose) as the sole carbon source, the engineered strain showed 0.67‑fold maximum cell density but slightly higher extracellular enzyme activity (1.17‑fold) relative to glucose. Next, combinatorial optimization of signal peptides and promoters identified the WapA-P_A256 pair as optimal, boosting the per-OD₆₀₀ specific enzyme activity to 2.57-fold relative to the control. Co-expressing the cgl2496 and acs gene enhanced acetate tolerance and assimilation, shortening fermentation time by approximately 25% and raising per-OD₆₀₀ specific enzyme activity to 1.71-fold relative to the starting strain in strain Cg-ACE, despite minor growth impairment. Medium optimization (C/N ratio and ions) alleviated this defect, increasing maximum cell density and extracellular enzyme activity to 1.25-fold and 1.72-fold relative to the initial medium. Scale-up to 1-L flasks further improved performance: extracellular enzyme activity reached 2.02-fold versus 125-mL flasks and 4.27-fold versus glucose. This work provides a systematic strategy for acetate-based biomanufacturing, supporting efficient conversion of acetate to high-value biochemicals.