Engineering yeast consortia by partitioning glucose and multiple organic-acid utilization to enhance β-carotene production from simulated distillery wastewater

Synthetic microbial consortia (SMC) have emerged as a promising platform, often outperforming monocultures in metabolic efficiency. Despite this potential, the simultaneous co-utilization of glucose and diverse organic acids in wastewater for value-added chemical production remains a significant technical challenge. In this study, to achieve simultaneous co-utilization of glucose, lactate, and acetate to enable β-carotene production, an engineered Saccharomyces cerevisiae strain Sc -CarLA was first constructed by introducing the lactate utilization pathway from Shewanella oneidensis MR-1 and the acetate metabolism pathway from Escherichia coli into the original S. cerevisiae strain Sc -Car. However, severe competition between glucose and organic acids metabolism significantly hindered fermentation, resulting in reduced carbon source consumption and ultimately yielding only 30.9 ± 2.7 mg/L of β-carotene, a ~32% decrease compared to the original strain Sc -Car. To overcome this bottleneck, a novel SMC was first proposed and constructed, which separates the glucose metabolic pathway from the organic acid utilization pathway into two specialized strains. A glucose-specialist strain Sc -CarG1 was constructed by deleting the ADY2 gene, which encodes a vital organic acid transporter, from strain Sc -Car. Concurrently, an organic acids specialist strain, Sc -CarLA2, was constructed by deleting glucose transporter genes GAL2 and HXT1-7 . Subsequently, the co-culture of these two strains, Sc -CarG1 and Sc -CarLA2, exhibited improved fermentation performance, rapidly depleting glucose within 12 h and completely consuming acetate and lactate within 36 h. A two-parallel regulatory strategy, compositional division of labor (DOL) of microbes and temporal DOL, was then employed to further optimize the fermentation performance. As a result, the β-carotene titer in the compositional DOL system reached 64.0 ± 1.5 mg/L at the optimal cell ratio of 1:2 ( Sc -CarG1: Sc -CarLA2). Finally, the temporal DOL system, characterized by the sequential inoculation of the organic acids-specialist strain Sc -CarLA2 followed by the glucose-specific strain Sc -CarG1, enhanced the β-carotene titer to 71.9 ± 2.8 mg/L. A novel SMC that partitions the glucose metabolism pathway and organic acids utilization pathways into two specialist strains was first constructed, efficiently alleviating the bottleneck caused by carbon catabolite repression between glucose and organic acid metabolism, thereby facilitating rapid utilization of carbon sources and significantly boosting β-carotene production.