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.

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.

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.

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.

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 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.

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.

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.

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.

D-Lactic Acid Production from Sugarcane Bagasse by Genetically Engineered Saccharomyces cerevisiae.

Lactic acid (LA) is a promising bio-based chemical that has broad applications in food, nutraceutical, and bioplastic industries. However, production of the D-form of LA (D-LA) from fermentative organisms is lacking. In this study, Saccharomyces cerevisiae harboring the D-lactate dehydrogenase (DLDH) gene from Leuconostoc mesenteroides was constructed (CEN.PK2_DLDH). To increase D-LA production, the CRISPR/Cas12a system was used for the deletion of gpd1, gpd2, and adh1 to minimize glycerol and ethanol production. Although an improved D-LA titer was observed for both CEN.PK2_DLDHΔgpd and CEN.PK2_DLDHΔgpdΔadh1, growth impairment was observed. To enhance the D-LA productivity, CEN.PK2_DLDHΔgpd was crossed with the weak acid-tolerant S. cerevisiae BCC39850. The isolated hybrid2 showed a maximum D-LA concentration of 23.41 ± 1.65 g/L, equivalent to the improvement in productivity and yield by 2.2 and 1.5 folds, respectively. The simultaneous saccharification and fermentation using alkaline pretreated sugarcane bagasse by the hybrid2 led to an improved D-LA conversion yield on both the washed solid and whole slurry (0.33 and 0.24 g/g glucan). Our findings show the exploitation of natural yeast diversity and the potential strategy of gene editing combined with conventional breeding on improving the performance of S. cerevisiae for the production of industrially potent 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.

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.

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.

Quantification of Branched-Chain Alcohol-Based Biofuels and Other Fermentation Metabolites via High-Performance Liquid Chromatography.

As the consequences 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 bio-gasoline and other biofuels from renewable feedstocks can significantly reduce dependence on fossil fuels as well as lower the emissions of greenhouse gases. A significant amount of research over the past two decades has led to the development of microbial strains for the production of advanced fuel compounds. Crucial to these efforts are robust methods to quantify the amount of the biofuel compound being produced as well as the other metabolites that might be present during fermentation. Here, we provide a protocol for the quantification of branched-chain alcohols, specifically isobutanol and isopropanol, using high-performance liquid chromatography (HPLC).

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.

Metabolic engineering of Pichia pastoris for production of isopentanol (3-Methyl-1-butanol).

In recent years, the increasingly serious and clear effects of climate change have increased interest in renewable fuels and platform chemicals. Microbial platforms that can produce these compounds in an economically efficient way have emerged as an attractive alternative to the traditional production approaches. Here, we engineered the industrially-relevant yeast Pichia pastoris to produce the platform chemical 3-methyl-1-butanol (3M1B, isopentanol) directly from the renewable carbon source glucose. Specifically, we overexpressed the endogenous valine and leucine biosynthetic pathways to increase the production of the key pathway intermediate, 2-ketoisocaproate (2-KIC). Overexpression of the artificial keto-acid degradation pathway converted 2-KIC into 3M1B. Down-regulation of the side-product ethanol production using the CRISPR/Cas9 system led to a strain that is able to produce 3M1B at a titer of 191.0 ±â€¯9.6 mg/L, the highest titer reported to date in a non-conventional yeast. We envision that our yeast system will pave the way for an efficient production system for this important class of platform compounds.

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.

Systematic improvement of isobutanol production from D-xylose in engineered Saccharomyces cerevisiae.

As the importance of reducing carbon emissions as a means to limit the serious effects of global climate change becomes apparent, synthetic biologists and metabolic engineers are looking to develop renewable sources for transportation fuels and petroleum-derived chemicals. In recent years, microbial production of high-energy fuels has emerged as an attractive alternative to the traditional production of transportation fuels. In particular, the Baker's yeast Saccharomyces cerevisiae, a highly versatile microbial chassis, has been engineered to produce a wide array of biofuels. Nevertheless, a key limitation of S. cerevisiae is its inability to utilize xylose, the second most abundant sugar in lignocellulosic biomass, for both growth and chemical production. Therefore, the development of a robust S. cerevisiae strain that is able to use xylose is of great importance. Here, we engineered S. cerevisiae to efficiently utilize xylose as a carbon source and produce the advanced biofuel isobutanol. Specifically, we screened xylose reductase (XR) and xylose dehydrogenase (XDH) variants from different xylose-metabolizing yeast strains to identify the XR-XDH combination with the highest activity. Overexpression of the selected XR-XDH variants, a xylose-specific sugar transporter, xylulokinase, and isobutanol pathway enzymes in conjunction with the deletions of PHO13 and GRE3 resulted in an engineered strain that is capable of producing isobutanol at a titer of 48.4 ± 2.0 mg/L (yield of 7.0 mg/g D-xylose). This is a 36-fold increase from the previous report by Brat and Boles and, to our knowledge, is the highest isobutanol yield from D-xylose in a microbial system. We hope that our work will set the stage for an economic route for the production of advanced biofuel isobutanol and enable efficient utilization of lignocellulosic biomass.