Inhibition Control by Continuous Extractive Fermentation Enhances De Novo 2‐Phenylethanol Production by Yeast

Current microbial cell factory methods for producing chemicals from renewable resources primarily rely on (fed-)batch production systems, leading to the accumulation of the desired product. Industrially relevant chemicals like 2-phenylethanol (2PE), a flavor and fragrance compound, can exhibit toxicity at low concentrations, inhibit the host activity, and negatively impact titer, rate, and yield. Batch liquid-liquid (L-L) In Situ Product Removal (ISPR) was employed to mitigate inhibition effects, but was not found sufficient for industrial-scale application. Here, we demonstrated that continuous selective L-L ISPR provides the solution for maintaining the productivity of de novo produced 2PE at an industrial pilot scale. A unique bioreactor concept called "Fermentation Accelerated by Separation Technology" (FAST) utilizes hydrostatic pressure differences to separate aqueous- and extractant streams within one unit operation, where both production and product extraction take place - allowing for the control of the concentration of the inhibiting compound. Controlled aqueous 2PE levels (0.43 ± 0.02 g kg^-1) and extended production times (>100 h) were obtained and co-inhibiting by-product formation was reduced, resulting in a twofold increase of the final product output of batch L-L ISPR approaches. This study establishes that continuous selective L-L ISPR, enabled by FAST, can be applied for more economically viable production of inhibiting products.