Increased production of isobutanol from xylose through metabolic engineering of Saccharomyces cerevisiae overexpressing transcription factor Znf1 and exogenous genes.

Only trace amount of isobutanol is produced by the native Saccharomyces cerevisiae via degradation of amino acids. Despite several attempts using engineered yeast strains expressing exogenous genes, catabolite repression of glucose must be maintained together with high activity of downstream enzymes, involving iron-sulfur assimilation and isobutanol production. Here, we examined novel roles of nonfermentable carbon transcription factor Znf1 in isobutanol production during xylose utilization. RNA-seq analysis showed that Znf1 activates genes in valine biosynthesis, Ehrlich pathway and iron-sulfur assimilation while coupled deletion or downregulated expression of BUD21 further increased isobutanol biosynthesis from xylose. Overexpression of ZNF1 and xylose-reductase/dehydrogenase (XR-XDH) variants, a xylose-specific sugar transporter, xylulokinase, and enzymes of isobutanol pathway in the engineered S. cerevisiae pho13gre3Δ strain resulted in the superb ZNXISO strain, capable of producing high levels of isobutanol from xylose. The isobutanol titer of 14.809 ± 0.400 g/L was achieved, following addition of 0.05 g/L FeSO4.7H2O in 5 L bioreactor. It corresponded to 155.88 mg/g xylose consumed and + 264.75% improvement in isobutanol yield. This work highlights a new regulatory control of alternative carbon sources by Znf1 on various metabolic pathways. Importantly, we provide a foundational step toward more sustainable production of advanced biofuels from the second most abundant carbon source xylose.

Genes controlling hydrolysate toxin tolerance identified by QTL analysis of the natural Saccharomyces cerevisiae BCC39850.

Lignocellulosic material can be converted to valorized products such as fuels. Pretreatment is an essential step in conversion, which is needed to increase the digestibility of the raw material for microbial fermentation. However, pretreatment generates by-products (hydrolysate toxins) that are detrimental to microbial growth. In this study, natural Saccharomyces strains isolated from habitats in Thailand were screened for their tolerance to synthetic hydrolysate toxins (synHTs). The Saccharomyces cerevisiae natural strain BCC39850 (toxin-tolerant) was crossed with the laboratory strain CEN.PK2-1C (toxin-sensitive), and quantitative trait locus (QTL) analysis was performed on the segregants using phenotypic scores of growth (OD600) and glucose consumption. VMS1, DET1, KCS1, MRH1, YOS9, SYO1, and YDR042C were identified from QTLs as candidate genes associated with the tolerance trait. CEN.PK2-1C knockouts of the VMS1, YOS9, KCS1, and MRH1 genes exhibited significantly greater hydrolysate toxin sensitivity to growth, whereas CEN.PK2-1C knock-ins with replacement of VMS1 and MRH1 genes from the BCC39850 alleles showed significant increased ethanol production titers compared with the CEN.PK2-1C parental strain in the presence of synHTs. The discovery of VMS1, YOS9, MRH1, and KCS1 genes associated with hydrolysate toxin tolerance in S. cerevisiae indicates the roles of the endoplasmic-reticulum-associated protein degradation pathway, plasma membrane protein association, and the phosphatidylinositol signaling system in this trait. KEY POINTS: • QTL analysis was conducted using a hydrolysate toxin-tolerant S. cerevisiae natural strain • Deletion of VMS1, YOS9, MRH1, and KCS1 genes associated with hydrolysate toxin-sensitivity • Replacement of VMS1 and MRH1 with natural strain alleles increased ethanol production titers in the presence of hydrolysate toxins.

Red yeasts and their carotenogenic enzymes for microbial carotenoid production.

Carotenoids are C40 isoprene-based compounds with significant commercial interests that harbor diverse bioactivities. Prominent examples of carotenoids are beta-carotene, a precursor to vitamin A essential for proper eye health, and lycopene and astaxanthin, powerful antioxidants implicated in preventing cancers and atherosclerosis. Due to their benefits to human health, the market value for carotenoids is rapidly increasing and is projected to reach USD 1.7 billion by 2025. However, their production now relies on chemical synthesis and extraction from plants that pose risks to food management and numerous biological safety issues. Thus, carotenoid production from microbes is considered a promising strategy for achieving a healthy society with more sustainability. Red yeast is a heterogeneous group of basidiomycetous fungi capable of producing carotenoids. It is a critical source of microbial carotenoids from low-cost substrates. Carotenogenic enzymes from red yeasts have also been highly efficient, invaluable biological resources for biotechnological applications. In this minireview, we focus on red yeast as a promising source for microbial carotenoids, strain engineering strategies for improving carotenoid production in red yeasts, and potential applications of carotenogenic enzymes from red yeasts in conventional and nonconventional yeasts.

Novel carotenogenic gene combinations from red yeasts enhanced lycopene and beta-carotene production in Saccharomyces cerevisiae from the low-cost substrate sucrose.

Carotenoids (C40H56) including lycopene and beta-carotene are relatively strong antioxidants that provide benefits to human health. Here, we screened highly efficient crt variants from red yeasts to improve lycopene and beta-carotene production in Saccharomyces cerevisiae. We identified that crt variants from Sporidiobolus pararoseus TBRC-BCC 63403 isolated from rice leaf in Thailand exhibited the highest activity in term of lycopene and beta-carotene production in the context of yeast. Specifically, the phytoene desaturase SpCrtI possessed up to 4-fold higher in vivo activity based on lycopene content than the benchmark enzyme BtCrtI from Blakeslea trispora in our engineered WWY005 strain. Also, the geranylgeranyl pyrophosphate (GGPP) synthase SpCrtE, the bifunctional phytoene synthase-lycopene cyclase SpCrtYB, and SpCrtI when combined led to 7-fold improvement in beta-carotene content over the benchmark enzymes from Xanthophyllomyces dendrorhous in the laboratory strain CEN.PK2-1C. Sucrose as an alternative to glucose was found to enhance lycopene production in cells lacking GAL80. Lastly, we demonstrated a step-wise improvement in lycopene production from shake-flasks to a 5-L fermenter using the strain with GAL80 intact. Altogether, our study represents novel findings on more effective crt genes from Sp. pararoseus over the previously reported benchmark genes and their potential applications in scale-up lycopene production.

Systematic engineering of Saccharomyces cerevisiae for D-lactic acid production with near theoretical yield.

D-lactic acid is a chiral three-carbon organic acid that can improve the thermostability of polylactic acid. Here, we systematically engineered Saccharomyces cerevisiae to produce D-lactic acid from glucose, a renewable carbon source, at near theoretical yield. Specifically, we screened D-lactate dehydrogenase (DLDH) variants from lactic acid bacteria in three different genera and identified the Leuconostoc pseudomesenteroides variant (LpDLDH) as having the highest activity in yeast. We then screened single-gene deletions to minimize the production of the side products ethanol and glycerol as well as prevent the conversion of D-lactic acid back to pyruvate. Based on the results of the DLDH screening and the single-gene deletions, we created a strain called ASc-d789M which overexpresses LpDLDH and contains deletions in glycerol pathway genes GPD1 and GPD2 and lactate dehydrogenase gene DLD1, as well as downregulation of ethanol pathway gene ADH1 using the L-methionine repressible promoter to minimize impact on growth. ASc-d789M produces D-lactic acid at a titer of 17.09 g/L in shake-flasks (yield of 0.89 g/g glucose consumed or 89% of the theoretical yield). Fed-batch fermentation resulted in D-lactic acid titer of 40.03 g/L (yield of 0.81 g/g glucose consumed). Altogether, our work represents progress towards efficient microbial production of D-lactic acid.

Recent advances in the microbial production of isopentanol (3-Methyl-1-butanol).

As the effects of climate change become increasingly severe, metabolic engineers and synthetic biologists are looking towards greener sources for transportation fuels. The design and optimization of microorganisms to produce gasoline, diesel, and jet fuel compounds from renewable feedstocks can significantly reduce dependence on fossil fuels and thereby produce fewer emissions. Over the past two decades, a tremendous amount of research has contributed to the development of microbial strains to produce advanced fuel compounds, including branched-chain higher alcohols (BCHAs) such as isopentanol (3-methyl-1-butanol; 3M1B) and isobutanol (2-methyl-1-propanol). In this review, we provide an overview of recent advances in the development of microbial strains for the production of isopentanol in both conventional and non-conventional hosts. We also highlight metabolic engineering strategies that may be employed to enhance product titers, reduce end-product toxicity, and broaden the substrate range to non-sugar carbon sources. Finally, we offer glimpses into some promising future directions in the development of isopentanol producing microbial strains.

Improvement in D-xylose utilization and isobutanol production in S. cerevisiae by adaptive laboratory evolution and rational engineering.

As the effects of climate change become apparent, metabolic engineers and synthetic biologists are exploring sustainable sources for transportation fuels. The design and engineering of microorganisms to produce gasoline, diesel, and jet fuel compounds from renewable feedstocks can significantly reduce our dependence on fossil fuels as well as lower the emissions of greenhouse gases. Over the past 2 decades, a considerable amount of work has led to the development of microbial strains for the production of advanced fuel compounds from both C5 and C6 sugars. In this work, we combined two strategies-adaptive laboratory evolution and rational metabolic engineering-to improve the yeast Saccharomyces cerevisiae's ability to utilize D-xylose, a major C5 sugar in biomass, and produce the advanced biofuel isobutanol. Whole genome resequencing of several evolved strains followed by reverse engineering identified two single nucleotide mutations, one in CCR4 and another in TIF1, that improved the yeast's specific growth rate by 23% and 14%, respectively. Neither one of these genes has previously been implicated to play a role in utilization of D-xylose. Fine-tuning the expression levels of the bottleneck enzymes in the isobutanol pathway further improved the evolved strain's isobutanol titer to 92.9 ± 4.4 mg/L (specific isobutanol production of 50.2 ± 2.6 mg/g DCW), a 90% improvement in titer and a 110% improvement in specific production over the non-evolved strain. We hope that our work will set the stage for an economic route to the advanced biofuel isobutanol and enable efficient utilization of xylose-containing biomass.

Engineering high-level production of fatty alcohols by Saccharomyces cerevisiae from lignocellulosic feedstocks.

Fatty alcohols in the C12-C18 range are used in personal care products, lubricants, and potentially biofuels. These compounds can be produced from the fatty acid pathway by a fatty acid reductase (FAR), yet yields from the preferred industrial host Saccharomyces cerevisiae remain under 2% of the theoretical maximum from glucose. Here we improved titer and yield of fatty alcohols using an approach involving quantitative analysis of protein levels and metabolic flux, engineering enzyme level and localization, pull-push-block engineering of carbon flux, and cofactor balancing. We compared four heterologous FARs, finding highest activity and endoplasmic reticulum localization from a Mus musculus FAR. After screening an additional twenty-one single-gene edits, we identified increasing FAR expression; deleting competing reactions encoded by DGA1, HFD1, and ADH6; overexpressing a mutant acetyl-CoA carboxylase; limiting NADPH and carbon usage by the glutamate dehydrogenase encoded by GDH1; and overexpressing the Δ9-desaturase encoded by OLE1 as successful strategies to improve titer. Our final strain produced 1.2g/L fatty alcohols in shake flasks, and 6.0g/L in fed-batch fermentation, corresponding to ~ 20% of the maximum theoretical yield from glucose, the highest titers and yields reported to date in S. cerevisiae. We further demonstrate high-level production from lignocellulosic feedstocks derived from ionic-liquid treated switchgrass and sorghum, reaching 0.7g/L in shake flasks. Altogether, our work represents progress towards efficient and renewable microbial production of fatty acid-derived products.

Integrating carbon-halogen bond formation into medicinal plant metabolism.

Halogenation, which was once considered a rare occurrence in nature, has now been observed in many natural product biosynthetic pathways. However, only a small fraction of halogenated compounds have been isolated from terrestrial plants. Given the impact that halogenation can have on the biological activity of natural products, we reasoned that the introduction of halides into medicinal plant metabolism would provide the opportunity to rationally bioengineer a broad variety of novel plant products with altered, and perhaps improved, pharmacological properties. Here we report that chlorination biosynthetic machinery from soil bacteria can be successfully introduced into the medicinal plant Catharanthus roseus (Madagascar periwinkle). These prokaryotic halogenases function within the context of the plant cell to generate chlorinated tryptophan, which is then shuttled into monoterpene indole alkaloid metabolism to yield chlorinated alkaloids. A new functional group-a halide-is thereby introduced into the complex metabolism of C. roseus, and is incorporated in a predictable and regioselective manner onto the plant alkaloid products. Medicinal plants, despite their genetic and developmental complexity, therefore seem to be a viable platform for synthetic biology efforts.

Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals.

As the serious effects of global climate change become apparent and access to fossil fuels becomes more limited, metabolic engineers and synthetic biologists are looking towards greener sources for transportation fuels. In recent years, microbial production of high-energy fuels by economically efficient bioprocesses has emerged as an attractive alternative to the traditional production of transportation fuels. Here, we engineered the budding yeast Saccharomyces cerevisiae to produce fatty acid-derived biofuels and chemicals from simple sugars. Specifically, we overexpressed all three fatty acid biosynthesis genes, namely acetyl-CoA carboxylase (ACC1), fatty acid synthase 1 (FAS1) and fatty acid synthase 2 (FAS2), in S. cerevisiae. When coupled to triacylglycerol (TAG) production, the engineered strain accumulated lipid to more than 17% of its dry cell weight, a four-fold improvement over the control strain. Understanding that TAG cannot be used directly as fuels, we also engineered S. cerevisiae to produce drop-in fuels and chemicals. Altering the terminal "converting enzyme" in the engineered strain led to the production of free fatty acids at a titer of approximately 400 mg/L, fatty alcohols at approximately 100mg/L and fatty acid ethyl esters (biodiesel) at approximately 5 mg/L directly from simple sugars. We envision that our approach will provide a scalable, controllable and economic route to this important class of chemicals.

Efficient ß-carotene production in engineered Saccharomyces cerevisiae using simple sugars and agricultural waste-based carbon and nitrogen sources.

ß-carotene, a precursor to vitamin A, holds significant promise for health and nutrition applications. This study introduces an optimized approach for ß-carotene production in Saccharomyces cerevisiae, leveraging metabolic engineering and a novel use of agricultural waste. The GAL80 gene deletion facilitated efficient ß-carotene synthesis from sucrose, avoiding the costly galactose induction, and achieved titers up to 727.8 ±â€¯68.0 mg/L with content levels of 71.8 ±â€¯0.4 mg/g dry cell weight (DCW). Furthermore, the application of agricultural by-products, specifically molasses and fish meal as carbon and nitrogen sources, was investigated. This approach yielded a substantial ß-carotene titer of 354.9 ±â€¯8.2 mg/L and a content of 60.5 ±â€¯4.3 mg/g DCW, showcasing the potential of these sustainable substrates for industrial-scale production. This study sets a new benchmark for cost-effective, green manufacturing of vital nutrients, demonstrating a scalable, eco-friendly alternative for ß-carotene production.

Metabolic Engineering of Saccharomyces cerevisiae for Production of Canthaxanthin, Zeaxanthin, and Astaxanthin.

The sustainable production of natural compounds is increasingly important in today's industrial landscape. This study investigates the metabolic engineering of Saccharomyces cerevisiae for the efficient biosynthesis of valuable carotenoids: canthaxanthin, zeaxanthin, and astaxanthin. Utilizing a tailored parental yeast strain, Sp_Bc, we optimized the carotenoid pathway by screening and identifying CrtW and CrtZ enzymatic variants. The CrtW variant from Bradyrhizobium sp. achieved a canthaxanthin titer of 425.1 ± 69.1 µg/L, while the CrtZ variant from Pantoea ananatis achieved a zeaxanthin titer of 70.5 ± 10.8 µg/L. Additionally, we optimized carotenoid production by exploring enzyme fusion strategies for all three studied carotenoids and organelle compartmentalization specifically for enhancing astaxanthin synthesis. We further improved carotenoid production by integrating the optimal gene constructs into the yeast genome and deleting the GAL80 gene, enabling the use of sucrose as a carbon source. The engineered strain Sp_Bc-Can001 ∆gal80 was evaluated in a 5 L bioreactor fermentation, achieving a notable canthaxanthin titer of 60.36 ± 1.51 mg/L using sucrose. This research conclusively establishes S. cerevisiae as a viable platform for efficient carotenoid biosynthesis and, for the first time in this yeast system, illustrates sucrose's viability as a carbon source for canthaxanthin production. These findings pave the way for sustainable, cost-effective carotenoid production at an industrial scale.

Development of a Novel D-Lactic Acid Production Platform Based on Lactobacillus saerimneri TBRC 5746.

D-Lactic acid is a chiral, three-carbon organic acid, that bolsters the thermostability of polylactic acid. In this study, we developed a microbial production platform for the high-titer production of D-lactic acid. We screened 600 isolates of lactic acid bacteria (LAB) and identified twelve strains that exclusively produced D-lactic acid in high titers. Of these strains, Lactobacillus saerimneri TBRC 5746 was selected for further development because of its homofermentative metabolism. We investigated the effects of high temperature and the use of cheap, renewable carbon sources on lactic acid production and observed a titer of 99.4 g/L and a yield of 0.90 g/g glucose (90% of the theoretical yield). However, we also observed L-lactic acid production, which reduced the product's optical purity. We then used CRISPR/dCas9-assisted transcriptional repression to repress the two Lldh genes in the genome of L. saerimneri TBRC 5746, resulting in a 38% increase in D-lactic acid production and an improvement in optical purity. This is the first demonstration of CRISPR/dCas9-assisted transcriptional repression in this microbial host and represents progress toward efficient microbial production of D-lactic acid.

Engineering yeast cell factories to produce biodegradable plastics and their monomers: Current status and prospects.

Traditional plastic products have caused serious environmental pollution due to difficulty to be degraded in the natural environment. In the recent years, biodegradable plastics are receiving increasing attention due to advantages in natural degradability and environmental friendliness. Biodegradable plastics have potential to be used in food, agriculture, industry, medicine and other fields. However, the high production cost of such plastics is the bottleneck that limits their commercialization and application. Yeasts, including budding yeast and non-conventional yeasts, are widely studied to produce biodegradable plastics and their organic acid monomers. Compared to bacteria, yeast strains are more tolerable to multiple stress conditions including low pH and high temperature, and also have other advantages such as generally regarded as safe, and no phage infection. In addition, synthetic biology and metabolic engineering of yeast have enabled its rapid and efficient engineering for bioproduction using various renewable feedstocks, especially lignocellulosic biomass. This review focuses on the recent progress in biosynthesis technology and strategies of monomeric organic acids for biodegradable polymers, including polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), and polybutylene adipate terephthalate (PBAT) using yeast cell factories. Improving the performance of yeast as a cell factory and strategies to improve yeast acid stress tolerance are also discussed. In addition, the critical challenges and future prospects for the production of biodegradable plastic monomer using yeast are also discussed.

Optimizing Ethanol Production in Saccharomyces cerevisiae at Ambient and Elevated Temperatures through Machine Learning-Guided Combinatorial Promoter Modifications.

Bioethanol has gained popularity in recent decades as an ecofriendly alternative to fossil fuels due to increasing concerns about global climate change. However, economically viable ethanol fermentation remains a challenge. High-temperature fermentation can reduce production costs, but Saccharomyces cerevisiae yeast strains normally ferment poorly under high temperatures. In this study, we present a machine learning (ML) approach to optimize bioethanol production in S. cerevisiae by fine-tuning the promoter activities of three endogenous genes. We created 216 combinatorial strains of S. cerevisiae by replacing native promoters with five promoters of varying strengths to regulate ethanol production. Promoter replacement resulted in a 63% improvement in ethanol production at 30 °C. We created an ML-guided workflow by utilizing XGBoost to train high-performance models based on promoter strengths and cellular metabolite concentrations obtained from ethanol production of 216 combinatorial strains at 30 °C. This strategy was then applied to optimize ethanol production at 40 °C, where we selected 31 strains for experimental fermentation. This reduced experimental load led to a 7.4% increase in ethanol production in the second round of the ML-guided workflow. Our study offers a comprehensive library of promoter strength modifications for key ethanol production enzymes, showcasing how machine learning can guide yeast strain optimization and make bioethanol production more cost-effective and efficient. Furthermore, we demonstrate that metabolic engineering processes can be accelerated and optimized through this approach.

Engineering Flocculation for Improved Tolerance and Production of d-Lactic Acid in Pichia pastoris.

d-lactic acid, a chiral organic acid, can enhance the thermal stability of polylactic acid plastics. Microorganisms such as the yeast Pichia pastoris, which lack the natural ability to produce or accumulate high amounts of d-lactic acid, have been metabolically engineered to produce it in high titers. However, tolerance to d-lactic acid remains a challenge. In this study, we demonstrate that cell flocculation improves tolerance to d-lactic acid and increases d-lactic acid production in Pichia pastoris. By incorporating a flocculation gene from Saccharomyces cerevisiae (ScFLO1) into P. pastoris KM71, we created a strain (KM71-ScFlo1) that demonstrated up to a 1.6-fold improvement in specific growth rate at high d-lactic acid concentrations. Furthermore, integrating a d-lactate dehydrogenase gene from Leuconostoc pseudomesenteroides (LpDLDH) into KM71-ScFlo1 resulted in an engineered strain (KM71-ScFlo1-LpDLDH) that could produce d-lactic acid at a titer of 5.12 ± 0.35 g/L in 48 h, a 2.6-fold improvement over the control strain lacking ScFLO1 expression. Transcriptomics analysis of this strain provided insights into the mechanism of increased tolerance to d-lactic acid, including the upregulations of genes involved in lactate transport and iron metabolism. Overall, our work represents an advancement in the efficient microbial production of d-lactic acid by manipulating yeast flocculation.

Developing high-dimensional machine learning models to improve generalization ability and overcome data insufficiency for mixed sugar fermentation simulation.

Biorefinery can be promoted by building accurate machine learning models. This work proposed a strategy to enhance model's generalization ability and overcome insufficient data conditions for mixed sugar fermentation simulation. Multiple inputs single output models, using initial glucose, initial xylose, and time together as inputs, have higher generalization ability than single input single output models with time as sole input in predicting glucose, xylose, ethanol, or biomass separately. Multiple inputs multiple outputs models, integrating outputs, enhanced model accuracy and resulted in an average R2 at 0.99. To overcome data insufficiency conditions, consensus yeast (CY) model, through consolidating data from 4 yeasts, obtained R2 at 0.90. By adjusting the pretrained CY model, the model can save more than 50% data and get R2 at 0.95 and 0.93 for yeast and bacterial fermentation simulation. The strategy can expand the application range and save costs of data curation for ANN models.

Silencing of tryptamine biosynthesis for production of nonnatural alkaloids in plant culture.

Natural products have long served as both a source and inspiration for pharmaceuticals. Modifying the structure of a natural product often improves the biological activity of the compound. Metabolic engineering strategies to ferment "unnatural" products have been enormously successful in microbial organisms. However, despite the importance of plant derived natural products, metabolic engineering strategies to yield unnatural products from complex, lengthy plant pathways have not been widely explored. Here, we show that RNA mediated suppression of tryptamine biosynthesis in Catharanthus roseus hairy root culture eliminates all production of monoterpene indole alkaloids, a class of natural products derived from two starting substrates, tryptamine and secologanin. To exploit this chemically silent background, we introduced an unnatural tryptamine analog to the production media and demonstrated that the silenced plant culture could produce a variety of novel products derived from this unnatural starting substrate. The novel alkaloids were not contaminated by the presence of the natural alkaloids normally present in C. roseus. Suppression of tryptamine biosynthesis therefore did not appear to adversely affect expression of downstream biosynthetic enzymes. Targeted suppression of substrate biosynthesis therefore appears to be a viable strategy for programming a plant alkaloid pathway to more effectively produce desirable unnatural products. Moreover, although tryptamine is widely found among plants, this silenced line demonstrates that tryptamine does not play an essential role in growth or development in C. roseus root culture. Silencing the biosynthesis of an early starting substrate enhances our ability to harness the rich diversity of plant based natural products.

Engineered Production of Isobutanol from Sugarcane Trash Hydrolysates in Pichia pastoris.

Concerns over climate change have led to increased interest in renewable fuels in recent years. Microbial production of advanced fuels from renewable and readily available carbon sources has emerged as an attractive alternative to the traditional production of transportation fuels. Here, we engineered the yeast Pichia pastoris, an industrial powerhouse in heterologous enzyme production, to produce the advanced biofuel isobutanol from sugarcane trash hydrolysates. Our strategy involved overexpressing a heterologous xylose isomerase and the endogenous xylulokinase to enable the yeast to consume both C5 and C6 sugars in biomass. To enable the yeast to produce isobutanol, we then overexpressed the endogenous amino acid biosynthetic pathway and the 2-keto acid degradation pathway. The engineered strains produced isobutanol at a titer of up to 48.2 ± 1.7 mg/L directly from a minimal medium containing sugarcane trash hydrolysates as the sole carbon source. To our knowledge, this is the first demonstration of advanced biofuel production using agricultural waste-derived hydrolysates in the yeast P. pastoris. We envision that our work will pave the way for a scalable route to this advanced biofuel and further establish P. pastoris as a versatile production platform for fuels and high-value chemicals.