Multi-step pathway engineering in probiotic Saccharomyces boulardii for abscisic acid production in the gut

The plant hormone abscisic acid (ABA) has gained attention for its role in animals and humans, particularly due to its protective effects in various immune and inflammatory disorders. Given its high concentrations in fruits like figs, bilberries and apricots, ABA shows promise as a nutraceutical. However scalability, short half-life and cost limit the use of ABA-enriched fruit extracts and synthetic supplements. In this study, we propose an alternative ABA administration method to overcome these challenges. We genetically engineered a strain of the probiotic <i>Saccharomyces boulardii to produce and deliver ABA directly to the gut of mice. Using t</i>he biosynthesis pathway from <i>Botrytis cinerea</i>, four genes (<i>bcaba1-4</i>) were integrated into <i>S. boulardii</i>, enabling ABA production at 30 °C, as previously described in <i>Saccharomyces cerevisiae</i>. Introducing an additional cytochrome P450 reductase gene resulted in a 7-fold increase in ABA titers, surpassing previous ABA-producing <i>S. cerevisiae</i> strains. Supplementation of the ABA-producing <i>S. boulardii</i> in the diet of mice (at a concentration of 5 × 10⁸ CFU/g) led to effective gut colonization but resulted in low serum ABA levels (approximately 1.8 ng/mL). The absence of detectable serum ABA after administration of the ABA-producing probiotic through oral gavage, prompted further investigation to determine the underlying cause. The physiological body temperature (37 °C) was identified as a major bottleneck for ABA production. Modifications to enhance the mevalonate pathway flux improved ABA levels at 37 °C. However, additional modifications are needed to optimize ABA production before testing this probiotic in disease contexts in mice.