Enhancing cell density and recombinant protein production through the control of acetate accumulation.

is widely used in biopharmaceutical production due to its ability to grow aerobically and produce proteins intracellularly. However, the limitation of the fermentation process is acetate accumulation, a by-product of overflow metabolism during high-glucose aerobic growth, which negatively impacts cell growth and protein expression. Traditional strategies to mitigate this include genetic modifications or low-density fermentation, which have significant limitations. In the present study, a novel fed-batch fermentation strategy was developed to reduce acetate accumulation and enhance the production of recombinant pneumococcal surface adhesin A (PsaA). A design of experiments (DOE) was conducted to optimize the culture media and develop a real-time, feedback-controlled feeding strategy that prevents acetate accumulation without requiring genetic alterations. Initial runs with 20 g/L glucose resulted in acetate accumulation of 7-8 g/L and limited biomass growth. By lowering glucose concentration to 10 g/L and inducing a carbon-limited phase via controlled feeding, cells switched from acetate production to consumption through the reverse Pta-AckA pathway. This shift led to an over 80% reduction in acetate levels. Optimized conditions consistently yielded higher cell densities. OD₆₀₀ values of 100-120 were achieved. The desired yield of the protein pneumococcal surface adhesin A (PsaA) was 3.0 g/L, representing a 2.0-fold increase over unoptimized runs. SDS-PAGE and quantitative analyses confirmed consistent robust protein expression. The strategy was validated across multiple batches, proving reproducible, scalable, and regulatory friendly. This approach offers a cost-effective and efficient alternative to genetic modification for controlling overflow metabolism and enhancing recombinant protein yields in .