The transition from 2G to 3G-feedstocks enabled efficient production of fuels and chemicals

For decades micoorganisms have been engineered for the utilization of lignocellulose-based second-generation (2G) feedstocks, but with the concerns of increased levels of atmospheric CO2 causing global warming there is an emergent need to transition from the utilization of 2G feedstocks to third-generation (3G) feedstocks such as CO2 and its derivatives. Here, we established a yeast platform that is capable of simultaneously converting 2G and 3G feedstocks into bulk and value-added chemicals. We demonstrated that by adopting 3G substrates such as CO2 and formate, the conversion of 2G feedstocks could be substantially improved. Specifically, formate could provide reducing power and energy for xylose conversion into valuable chemicals. Simultaneously, it can form a concentrated CO2 pool inside the cell, providing thermodynamically and kinetically favoured amounts of precursors for CO2 fixation pathways, e.g., the Calvin–Benson–Bassham (CBB) cycle. Furthermore, we demonstrated that formate could directly be utilized as a carbon source by yeast to synthesize endogenous amino acids. The engineered strain achieved a one-carbon (C1) assimilation efficiency of 9.2%, which was the highest efficiency observed in the co-utilization of 2G and 3G feedstocks. We applied this strategy for productions of both bulk and value-added chemicals, including ethanol, free fatty acids (FFAs), and longifolene, resulting in yield enhancements of 18.4%, 49.0%, and ∼100%, respectively. The strategy demonstrated here for co-utilization of 2G and 3G feedstocks sheds lights on both basic and applied research for the up-coming establishment of 3G biorefineries. Herein, a yeast platform has been established that is capable of simultaneously utilizing 2G and 3G feedstocks. Specifically, the utilization of CO2 and formate enhances the conversion of xylose, and the production of bulk and value-added chemicals. [Display omitted] •Developed dual-function yeast: improved xylose conversion and product yield via cofactor regulation.•Introduced carbon fixation pathways to boost one-carbon assimilation efficiency, reaching 9.2%.•Transcriptomic analysis identified metabolic targets for advanced 2G and 3G feedstock conversion.