Designing high-performance yeast factories for the production of high-value aromatics based on a novel species and its consortia
Due to their broad applications in the field of polymers, cosmetics, nutraceuticals and pharmaceuticals, aromatic compounds have garnered multitudinous attentions. Microbial production of aromatic compounds from simple building blocks serves as a promising approach toward sustainable and scalable production of value-added chemicals. Owning to its well understood physiology, metabolism, and genetics, ease of manipulation, and capability of functionally expressing challenging endoplasmic reticulum-associated P450s, Saccharomyces cerevisiae has been overwhelmingly employed for the production of aromatic compounds, especially those plant sourced aromatic natural products with complex structures, albeit at low titers. Although numerous endeavors have been devoted to enhancing the production of aromatic compounds in S. cerevisiae, the final titers remain low and need further improvement for industrial commercialization. The insufficient production is ascribed to two aspects: one is limited precursor availability caused by tight regulations imposed on central metabolism and the other is the stepwise loss resulted from non-balanced and low throughput metabolic pathways.
Looking beyond S. cerevisiae, many non-conventional yeasts possess highly desirable traits that are obtained through long term evolution in particular environment but challenging to be horizontally transferred to model hosts without compromise. However, due to the lack of the available synthetic biology elements (e. g., promoters, terminators, and vectors) and efficient genome editing method, a majority of the described non-conventional yeasts are underexploited. Unlike the model host, Scheffersomyces stipitis natively assimilates xylose that can be integrated into central metabolism exclusively through pentose phosphate pathway; thus S. stipitis has evolved highly active pentose phosphate pathway, which would be beneficial for providing the precursor, erythrose-4-phosphate, for the first committed step of the biosynthesis of aromatic compounds. Transcriptomic analysis of S. stipitis grown in various sugar-containing cultures facilitated the discovery of strong and constitutive promoters and terminators, and the identification of the rate-limiting steps in mixed-sugar utilization. The constitutive expression of genes involved in intracellular xylose metabolization together with a novel xylose-specific transporter that is free from glucose inhibition resulted in efficient simultaneous mixed-sugar utilization in S. stipitis. Further introduction of a minimal shikimate-accumulating pathway resulted in the highest shikimate titer (4.5 g/L) and yield (90.5 mg/g sugar) ever reported in yeasts. Synthetic yeast consortia were developed for the production of precursors of benzyisoquinoline alkaloids by leveraging the active upstream module from S. stipitis and the extensively optimized downstream module in S. cerevisiae. The transfer of the connecting molecule, shikimate, in the yeast consortia was empowered by two novel shikimate transporters. Through independently optimizing the two modules and tuning the cell ratio of the two specialists, the final titer of the exemplary molecule, norcoclaurine, was 160 μg/L, higher than the best titer achieved in S. cerevisiae monoculture. Future efforts should focus on ameliorating both upstream and downstream modules of the yeast consortia through genome scale engineering.