The increasing demand for natural resources and concerns about climate change have fueled interest in producing biofuels and chemicals through microbial fermentation using renewable biomass. Lignocellulosic plant materials offer a sustainable alternative to petrochemical processes. However, efficient bioconversion is hindered by acetate, a fermentation inhibitor present in cellulosic hydrolysates due to hemicellulose and lignin acetylation. Acetate's toxicity significantly impacts bioconversion efficiency. While acetate generation can be reduced through genetic modifications of plant cell walls or optimized depolymerization processes, complete elimination is impossible. Pre-fermentation acetate removal methods, though available, increase costs. Engineering acetate tolerance in yeast has been attempted, but complete elimination of inhibitory effects remains elusive. Previous work demonstrated an NADH-dependent acetate reduction pathway in engineered yeast, improving ethanol production from xylose and acetate mixtures under anaerobic conditions. However, this pathway's capacity is limited by enzyme activity, ATP supply, and NADH levels. Furthermore, coupled acetate reduction and xylose assimilation limit its application to cellulosic ethanol production. Efficient acetate detoxification and valorization by *S. cerevisiae*, a versatile and robust industrial production platform, is crucial for industrial cellulosic biorefineries. Conversion of acetate to acetyl-CoA, and subsequent production of valuable acetyl-CoA-derived molecules (fatty acids, sterols, polyketides, isoprenoids), has been shown in oleaginous yeasts. While *S. cerevisiae* can produce acetyl-CoA from acetate, producing acetyl-CoA derivatives from acetate hasn't been reported due to acetate toxicity and slow metabolism. *S. cerevisiae*'s use in fermentative ethanol production from glucose overshadows its ability to grow on acetate as a sole carbon source under aerobic, neutral pH conditions. However, respiratory acetate metabolism as a sole carbon source leads to unfavorable growth kinetics due to reactive oxygen species (ROS) and ATP imbalance. Acetate is also toxic at low pH. Crucially, glucose inhibits acetate transport and metabolism, trapping it as a fermentation inhibitor. This research focuses on detoxifying acetate in cellulosic hydrolysates and utilizing its consumption to enhance acetyl-CoA supply in *S. cerevisiae* for value-added product production. The study investigates whether xylose, unlike glucose, can enable efficient co-consumption of xylose and acetate under aerobic conditions, potentially improving acetyl-CoA supply for increased production of acetyl-CoA-derived chemicals such as triacetic acid lactone (TAL) and vitamin A. The proposed metabolic design leverages xylose metabolism to generate NADPH and ATP for cell growth and acetate assimilation, directing acetate toward acetyl-CoA production, potentially mitigating the negative effects of respiratory acetate metabolism.

The existing literature extensively covers the challenges of using lignocellulosic biomass for biofuel and bioproduct production. Numerous studies highlight the inhibitory effects of acetate, a byproduct of the pretreatment and hydrolysis processes, on microbial fermentation. Various strategies for acetate removal or tolerance enhancement have been explored, including physical, chemical, and biological methods. The limitations of these approaches are well-documented, including increased processing costs and incomplete removal. Studies on engineering yeast strains for improved acetate tolerance have also been undertaken, with some success in mitigating but not eliminating the inhibitory effects. Previous research by the authors themselves demonstrated the successful implementation of an NADH-dependent acetate reduction pathway in *S. cerevisiae*, resulting in improved ethanol production under anaerobic conditions. However, this approach suffers from limitations in efficiency and applicability to other bioproducts. Research on acetyl-CoA metabolism in various microorganisms, including oleaginous yeasts, has revealed the potential for redirecting acetate towards the production of high-value chemicals. The use of xylose as a carbon source in yeast fermentation has also received attention, with some studies demonstrating improved performance compared to glucose. The literature comprehensively shows the need for more efficient and cost-effective strategies for the utilization of acetate and xylose from lignocellulosic biomass for the production of biofuels and bioproducts.

The study employed several key methodologies. Plasmid and strain construction involved codon optimization of the *Gerbera hybrida* 2-pyrone synthase gene (2PS) for TAL production. This gene was cloned into various yeast integrative plasmids to create strains with differing 2PS gene copy numbers. Cas9-based genetic modification was used to disrupt the *TRPI* gene, creating an additional auxotrophic marker. The resulting strains were used for fermentation experiments. Media and culture conditions for shake flask and bioreactor fermentations were meticulously controlled. Modified Verduyn medium was utilized, with glucose, xylose, acetate, or ethanol as carbon sources. Media pH was maintained at 5.5. Fed-batch fermentation in a 3-liter bioreactor involved co-feeding of xylose and acetate. Switchgrass hemicellulose hydrolysate, prepared through dilute acid hydrolysis and neutralization, was used as a substrate to assess the bioconversion efficiency in shake flask fermentations, both in concentrated and unconcentrated forms. RNA sequencing (RNA-seq) was performed using the SR7 strain (parent of SR8) grown on glucose and xylose to study gene expression patterns related to acetate assimilation. Quantitative analysis involved measuring cell growth (OD600), glucose, xylose, acetate, glycerol, and ethanol concentrations using HPLC, lipid content via a chloroform/methanol extraction method, ergosterol content through saponification and n-heptane extraction followed by HPLC, and TAL concentration using HPLC with UV detection. For the bioreactor fermentation with TAL precipitation, serial dilutions were employed for accurate measurements. β-carotene and vitamin A were also quantified using HPLC with UV detection in experiments using the SR8A strain.

The study demonstrated that xylose, unlike glucose, does not inhibit acetate consumption in engineered *S. cerevisiae* under aerobic conditions, enabling efficient co-consumption. RNA-seq analysis revealed that xylose increased the expression of genes involved in acetate transport (ADY2, JEN1) and acetyl-CoA synthesis (ACS1), while decreasing the expression of PMA1 (proton-translocating ATPase). Co-consumption of xylose and acetate significantly improved cell growth, lipid, and ergosterol accumulation, suggesting enhanced acetyl-CoA supply. The engineered strain Tal4, with four copies of the 2PS gene, produced significantly more TAL from xylose than glucose. Co-feeding xylose and acetate in a 4:1 ratio resulted in a remarkable 23.89 g/L TAL titer and 0.29 g/L/h productivity in bioreactor fermentation. The Tal4 strain successfully converted switchgrass hemicellulose hydrolysate, without any detoxification steps, producing 3.55 g/L TAL. Moreover, increased vitamin A production was observed in the SR8A strain through xylose and acetate co-utilization. The study showed that the co-consumption of xylose and acetate enhances acetyl-CoA production in the yeast, which significantly increases the production of TAL and vitamin A. The experiments revealed that up to 12.38 g/L of acetate could be co-assimilated with 40 g/L of xylose at a rate of 0.23 g/L/h. This capacity and rate are significantly higher than those reported in previous anaerobic acetate reduction studies.

The findings address the research question by demonstrating that acetate, a common inhibitor in lignocellulosic hydrolysates, can be effectively utilized as a substrate for high-value bioproduct production when co-fed with xylose in engineered *S. cerevisiae*. The synergistic effect of xylose and acetate metabolism enhances acetyl-CoA supply, boosting the production of acetyl-CoA-derived chemicals. The results highlight the potential of this strategy for economic and sustainable bioconversion of plant cell wall biomass. The significant improvement in TAL titer and productivity compared to previous reports underscores the efficacy of the approach. The successful conversion of switchgrass hemicellulose hydrolysate further demonstrates the feasibility of integrating this strategy into industrial biorefineries. The successful demonstration of enhanced vitamin A production suggests the broad applicability of this approach to other acetyl-CoA-derived bioproducts. These results indicate the feasibility of coupling sugar metabolism with organic acid assimilation in *S. cerevisiae*, potentially extending applications to other substrates like acid whey.

This study presents a novel and highly effective strategy for converting plant cell wall hemicellulose into valuable bioproducts by utilizing acetate, a common inhibitor, as a substrate in conjunction with xylose. The engineered *S. cerevisiae* strains achieved exceptionally high TAL titers and productivity. The success with switchgrass hydrolysate demonstrates the applicability of this strategy for sustainable biorefineries. Future research could explore the optimization of this strategy for other acetyl-CoA derived bioproducts and explore the potential of this strategy for other mixed-substrate fermentations.

The study primarily focused on TAL and vitamin A production. While the results suggest broader applicability, further research is needed to validate this for other bioproducts. The high concentration of TAL produced in the bioreactor resulted in precipitation, which may affect the accuracy of measurements and requires further optimization. The use of switchgrass hydrolysate, while relevant to biorefineries, might not represent all lignocellulosic feedstocks. Further research is needed to test the versatility of the approach with diverse feedstocks. The genetic background of the yeast strains was also limited and only one type of yeast was used.