Engineered Saccharomyces Cerevisiae Research Articles

Enhanced upgrading of lignocellulosic substrates by coculture of Saccharomyces cerevisiae and Acinetobacter baylyi ADP1

BackgroundLignocellulosic biomass as feedstock has a huge potential for biochemical production. Still, efficient utilization of hydrolysates derived from lignocellulose is challenged by their complex and heterogeneous composition and the presence of inhibitory compounds, such as furan aldehydes. Using microbial consortia where two specialized microbes complement each other could serve as a potential approach to improve the efficiency of lignocellulosic biomass upgrading.ResultsThis study describes the simultaneous inhibitor detoxification and production of lactic acid and wax esters from a synthetic lignocellulosic hydrolysate by a defined coculture of engineered Saccharomyces cerevisiae and Acinetobacter baylyi ADP1. A. baylyi ADP1 showed efficient bioconversion of furan aldehydes present in the hydrolysate, namely furfural and 5-hydroxymethylfurfural, and did not compete for substrates with S. cerevisiae, highlighting its potential as a coculture partner. Furthermore, the remaining carbon sources and byproducts of S. cerevisiae were directed to wax ester production by A. baylyi ADP1. The lactic acid productivity of S. cerevisiae was improved approximately 1.5-fold (to 0.41 ± 0.08 g/L/h) in the coculture with A. baylyi ADP1, compared to a monoculture of S. cerevisiae.ConclusionThe coculture of yeast and bacterium was shown to improve the consumption of lignocellulosic substrates and the productivity of lactic acid from a synthetic lignocellulosic hydrolysate. The high detoxification capacity and the ability to produce high-value products by A. baylyi ADP1 demonstrates the strain to be a potential candidate for coculture to increase production efficiency and economics of S. cerevisiae fermentations.

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

β-carotene, a precursor to vitamin A, holds significant promise for health and nutrition applications. This study introduces an optimized approach for β-carotene production in Saccharomyces cerevisiae, leveraging metabolic engineering and a novel use of agricultural waste. The GAL80 gene deletion facilitated efficient β-carotene synthesis from sucrose, avoiding the costly galactose induction, and achieved titers up to 727.8 ± 68.0 mg/L with content levels of 71.8 ± 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 β-carotene titer of 354.9 ± 8.2 mg/L and a content of 60.5 ± 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 β-carotene production.

Application of multiple strategies to enhance oleanolic acid biosynthesis by engineered Saccharomyces cerevisiae

Oleanolic acid and its derivatives are widely used in the pharmaceutical, agricultural, cosmetic and food industries. Previous studies have shown that oleanolic acid production levels in engineered cell factories are low, which is why oleanolic acid is still widely extracted from traditional medicinal plants. To construct a highly efficient oleanolic acid production strain, rate-limiting steps were regulated by inducible promoters and the expression of key genes in the oleanolic acid synthetic pathway was enhanced. Subsequently, precursor pool expansion, pathway refactoring and diploid construction were considered to harmonize cell growth and oleanolic acid production. The multi-strategy combination promoted oleanolic acid production of up to 4.07 g/L in a 100 L bioreactor, which was the highest level reported.

Enhancing xylose-fermentation capacity of engineered Saccharomyces cerevisiae by multistep evolutionary engineering in inhibitor-rich lignocellulose hydrolysate.

Major progress in developing Saccharomyces cerevisiae strains that utilize the pentose sugar xylose has been achieved. However, the high inhibitor content of lignocellulose hydrolysates still hinders efficient xylose fermentation, which remains a major obstacle for commercially viable second-generation bioethanol production. Further improvement of xylose utilization in inhibitor-rich lignocellulose hydrolysates remains highly challenging. In this work, we have developed a robust industrial S. cerevisiae strain able to efficiently ferment xylose in concentrated undetoxified lignocellulose hydrolysates. This was accomplished with novel multistep evolutionary engineering. First, a tetraploid strain was generated and evolved in xylose-enriched pretreated spruce biomass. The best evolved strain was sporulated to obtain a genetically diverse diploid population. The diploid strains were then screened in industrially relevant conditions. The best performing strain, MDS130, showed superior fermentation performance in three different lignocellulose hydrolysates. In concentrated corncob hydrolysate, with initial cell density of 1g DW/l, at 35°C, MDS130 completely coconsumed glucose and xylose, producing±7% v/v ethanol with a yield of 91% of the maximum theoretical value and an overall productivity of 1.22g/l/h. MDS130 has been developed from previous industrial yeast strains without applying external mutagenesis, minimizing the risk of negative side-effects on other commercially important properties and maximizing its potential for industrial application.

Mitochondrial membrane transporters as attractive targets for the fermentative production of succinic acid from glycerol in Saccharomyces cerevisiae.

Previously, we reported an engineered Saccharomyces cerevisiae CEN.PK113-1A derivative able to produce succinic acid (SA) from glycerol with net CO2 fixation. Apart from an engineered glycerol utilization pathway, the strain was equipped with the reductive branch of the TCA cycle (rTCA) and a heterologous SA exporter. However, the results indicated that a significant amount of carbon still entered the CO2-releasing oxidative TCA cycle. The current study aimed to tune down the flux through the oxidative TCA cycle by targeting the mitochondrial uptake of pyruvate and cytosolic intermediates of the rTCA pathway, as well as the succinate dehydrogenase complex. Thus, we tested the effects of deletions of MPC1, MPC3, OAC1, DIC1, SFC1, and SDH1 on SA production. The highest improvement was achieved by the combined deletion of MPC3 and SDH1. The respective strain produced up to 45.5g/L of SA, reached a maximum SA yield of 0.66 gSA/gglycerol, and accumulated the lowest amounts of byproducts. Based on the obtained data, we consider a further reduction of mitochondrial import of pyruvate and rTCA intermediates highly attractive. Moreover, the approaches presented in the current study might also be valuable for improving SA production when sugars (instead of glycerol) are the source of carbon.

Proteomic analysis reveals that the co-ordination of cytosolic and mitochondrial pathways is beneficial for sabinene biosynthesis in engineered Saccharomyces cerevisiae.

Reconstruction and optimization of biosynthetic pathways can help to overproduce target chemicals in microbial cell factories based on genetic engineering. However, the perturbation of biosynthetic pathways on cellular metabolism is not well investigated and profiling the engineered microbes remains challenging. The rapid development of omics tools has the potential to characterize the engineered microbial cell factory. Here, we performed label-free quantitative proteomic analysis and metabolomic analysis of engineered sabinene overproducing Saccharomyces cerevisiae strains. Combined metabolic analysis andproteomic analysis of targeted mevalonate (MVA) pathway showed that co-ordination of cytosolic and mitochondrial pathways had balanced metabolism, and genome integration of biosynthetic genes had higher sabinene production with less MVA enzymes. Furthermore, comparative proteomic analysis showed that compartmentalized mitochondria pathway had perturbation on central cellular metabolism. This study provided an omics analysis example for characterizing engineered cell factory, which can guide future regulation of the cellular metabolism and maintaining optimal protein expression levels for the synthesis of target products.

Whole-cell bioconversion for producing thymoquinone by engineered Saccharomyces cerevisiae

Whole-cell bioconversion for producing thymoquinone by engineered Saccharomyces cerevisiae

β-Dicarbonyls Facilitate Engineered Microbial Bromoform Biosynthesis.

Ruminant livestock produce around 24% of global anthropogenic methane emissions. Methanogenesis in the animal rumen is significantly inhibited by bromoform, which is abundant in seaweeds of the genus Asparagopsis. This has prompted the development of livestock feed additives based on Asparagopsis to mitigate methane emissions, although this approach alone is unlikely to satisfy global demand. Here we engineer a non-native biosynthesis pathway to produce bromoform in vivo with yeast as an alternative biological source that may enable sustainable, scalable production of bromoform by fermentation. β-dicarbonyl compounds with low pKa values were identified as essential substrates for bromoform production and enabled bromoform synthesis in engineered Saccharomyces cerevisiae expressing a vanadate-dependent haloperoxidase gene. In addition to providing a potential route to the sustainable biological production of bromoform at scale, this work advances the development of novel microbial biosynthetic pathways for halogenation.

Engineering Saccharomyces cerevisiae for targeted hydrolysis and fermentation of glucuronoxylan through CRISPR/Cas9 genome editing

BackgroundThe abundance of glucuronoxylan (GX) in agricultural and forestry residual side streams positions it as a promising feedstock for microbial conversion into valuable compounds. By engineering strains of the widely employed cell factory Saccharomyces cerevisiae with the ability to directly hydrolyze and ferment GX polymers, we can avoid the need for harsh chemical pretreatments and costly enzymatic hydrolysis steps prior to fermentation. However, for an economically viable bioproduction process, the engineered strains must efficiently express and secrete enzymes that act in synergy to hydrolyze the targeted polymers.ResultsThe aim of this study was to equip the xylose-fermenting S. cerevisiae strain CEN.PK XXX with xylanolytic enzymes targeting beechwood GX. Using a targeted enzyme approach, we matched hydrolytic enzyme activities to the chemical features of the GX substrate and determined that besides endo-1,4-β-xylanase and β-xylosidase activities, α-methyl-glucuronidase activity was of great importance for GX hydrolysis and yeast growth. We also created a library of strains expressing different combinations of enzymes, and screened for yeast strains that could express and secrete the enzymes and metabolize the GX hydrolysis products efficiently. While strains engineered with BmXyn11A xylanase and XylA β-xylosidase could grow relatively well in beechwood GX, strains further engineered with Agu115 α-methyl-glucuronidase did not display an additional growth benefit, likely due to inefficient expression and secretion of this enzyme. Co-cultures of strains expressing complementary enzymes as well as external enzyme supplementation boosted yeast growth and ethanol fermentation of GX, and ethanol titers reached a maximum of 1.33 g L− 1 after 48 h under oxygen limited condition in bioreactor fermentations.ConclusionThis work underscored the importance of identifying an optimal enzyme combination for successful engineering of S. cerevisiae strains that can hydrolyze and assimilate GX. The enzymes must exhibit high and balanced activities, be compatible with the yeast’s expression and secretion system, and the nature of the hydrolysis products must be such that they can be taken up and metabolized by the yeast. The engineered strains, particularly when co-cultivated, display robust growth and fermentation of GX, and represent a significant step forward towards a sustainable and cost-effective bioprocessing of GX-rich biomass. They also provide valuable insights for future strain and process development targets.

Decolorization and detoxication of malachite green by engineered Saccharomyces cerevisiae expressing novel thermostable laccase from Trametes trogii

Malachite Green (MG) is a widely used industrial dye that is hazardous to health. Herein, the decolourisation and detoxification of MG were achieved using the engineered Saccharomyces cerevisiae expressing novel thermostable laccase lcc1 from Trametes trogii. The engineered strain RCL produced a high laccase activity of 121.83 U L-1. Lcc1 was stable at temperatures ranging from 20 ℃ to 60 ℃ and showed a high tolerance to organic solvents. Moreover, Lcc1 could decolorize different kinds of dyes (azo, anthraquinone and triphenylmethane), among which, the decolorization ability of MG is the highest, reaching 95.10 %, and the decolorization rate of other triphenylmethane dyes also over 50 %. The RCL decolorized about 95 % of 50 mg L-1 of MG dye in 10 h at 30 ℃. The MG degradation products were analyzed. The industrial application potential of the RCL was evaluated by treating industrial wastewater and the decolourisation rates were over 90 %.

Engineered Saccharomyces cerevisiae as a Biosynthetic Platform of Nucleotide Sugars.

Glycosylation of biomolecules can greatly alter their physicochemical properties, cellular recognition, subcellular localization, and immunogenicity. Glycosylation reactions rely on the stepwise addition of sugars using nucleotide diphosphate (NDP)-sugars. Making these substrates readily available will greatly accelerate the characterization of new glycosylation reactions, elucidation of their underlying regulation mechanisms, and production of glycosylated molecules. In this work, we engineered Saccharomyces cerevisiae to heterologously express nucleotide sugar synthases to access a wide variety of uridine diphosphate (UDP)-sugars from simple starting materials (i.e., glucose and galactose). Specifically, activated glucose, uridine diphosphate d-glucose (UDP-d-Glc), can be converted to UDP-d-glucuronic acid (UDP-d-GlcA), UDP-d-xylose (UDP-d-Xyl), UDP-d-apiose (UDP-d-Api), UDP-d-fucose (UDP-d-Fuc), UDP-l-rhamnose (UDP-l-Rha), UDP-l-arabinopyranose (UDP-l-Arap), and UDP-l-arabinofuranose (UDP-l-Araf) using the corresponding nucleotide sugar synthases of plant and microbial origins. We also expressed genes encoding the salvage pathway to directly activate free sugars to achieve the biosynthesis of UDP-l-Arap and UDP-l-Araf. We observed strong inhibition of UDP-d-Glc 6-dehydrogenase (UGD) by the downstream product UDP-d-Xyl, which we circumvented using an induction system (Tet-On) to delay the production of UDP-d-Xyl to maintain the upstream UDP-sugar pool. Finally, we performed a time-course study using strains containing the biosynthetic pathways to produce five non-native UDP-sugars to elucidate their time-dependent interconversion and the role of UDP-d-Xyl in regulating UDP-sugar metabolism. These engineered yeast strains are a robust platform to (i) functionally characterize sugar synthases in vivo, (ii) biosynthesize a diverse selection of UDP-sugars, (iii) examine the regulation of intracellular UDP-sugar interconversions, and (iv) produce glycosylated secondary metabolites and proteins.

Genome-wide identification of resistance genes and response mechanism analysis of key gene knockout strain to catechol in Saccharomyces cerevisiae.

Engineering Saccharomyces cerevisiae for biodegradation and transformation of industrial toxic substances such as catechol (CA) has received widespread attention, but the low tolerance of S. cerevisiae to CA has limited its development. The exploration and modification of genes or pathways related to CA tolerance in S. cerevisiae is an effective way to further improve the utilization efficiency of CA. This study identified 36 genes associated with CA tolerance in S. cerevisiae through genome-wide identification and bioinformatics analysis and the ERG6 knockout strain (ERG6Δ) is the most sensitive to CA. Based on the omics analysis of ERG6Δ under CA stress, it was found that ERG6 knockout affects pathways such as intrinsic component of membrane and pentose phosphate pathway. In addition, the study revealed that 29 genes related to the cell wall-membrane system were up-regulated by more than twice, NADPH and NADP+ were increased by 2.48 and 4.41 times respectively, and spermidine and spermine were increased by 2.85 and 2.14 times, respectively, in ERG6Δ. Overall, the response of cell wall-membrane system, the accumulation of spermidine and NADPH, as well as the increased levels of metabolites in pentose phosphate pathway are important findings in improving the CA resistance. This study provides a theoretical basis for improving the tolerance of strains to CA and reducing the damage caused by CA to the ecological environment and human health.

Engineered Saccharomyces cerevisiae harbors xylose isomerase and xylose transporter improves co-fermentation of xylose and glucose for ethanol production

Saccharomyces cerevisiae cannot assimilate xylose, second to glucose derived from lignocellulosic biomass. Here, the engineered S. cerevisiae strains INVSc-XI and INVSc-XI/XT were constructed using xylA and Xltr1p to co-utilize xylose and glucose, achieving economic viability and sustainable production of fuels. The xylose utilization rate of INVSc-XI/XT was 2.3-fold higher than that of INVSc-XI, indicating that overexpressing Xltr1p could further enhance xylose utilization. In mixed sugar media, a small amount of glucose enhanced the consumption of xylose by INVSc-XI/XT. Transcriptome analysis showed that glucose increased the upregulation of acetate of coenzyme A synthetase (ACS), alcohol dehydrogenase (ADH), and transketolase (TKL) gene expression in INVSc-XI/XT, further promoting xylose utilization and ethanol yield. The highest ethanol titer of 2.91 g/L with a yield of 0.29 g/g at 96 h by INVSc-XI/XT was 56.9% and 63.0% of the theoretical ethanol yield from glucose and xylose, respectively. These results showed overexpression of xylA and Xltr1p is a promising strategy for improving xylose and glucose conversion to ethanol. Although the ability of strain INVSc-XI/XT to produce ethanol was not very satisfactory, glucose was discovered to influence xylose utilization in strain INVSc-XI/XT. Altering the glucose concentration is a promising strategy to improve the xylose and glucose co-utilization.

Characterization of the Cytochrome P450 CYP716C52 in Celastrol Biosynthesis and Its Applications in Engineered Saccharomyces cerevisiae.

Celastrol is a bioactive pentacyclic triterpenoid with promising therapeutic effects that is mainly distributed in Celastraceae plants. Although some enzymes involved in the celastrol biosynthesis pathway have been reported, many biosynthetic steps remain unknown. Herein, transcriptomics and metabolic profiles of multiple species in Celastraceae were integrated to screen for cytochrome P450s (CYPs) that are closely related to celastrol biosynthesis. The CYP716 enzyme, TwCYP716C52, was found to be able to oxidize the C-2 position of polpunonic acid, a precursor of celastrol, to form the wilforic acid C. RNAi-mediated repression of TwCYP716C52 in Tripterygium wilfordii suspension cells further confirmed its involvement in celastrol biosynthesis. The C-2 catalytic mechanisms of TwCYP716C52 were further explored by using molecular docking and site-directed mutagenesis experiments. Moreover, a modular optimization strategy was used to construct an engineered yeast to produce wilforic acid C at a titer of 5.8 mg·L-1. This study elucidates the celastrol biosynthetic pathway and provides important functional genes and sufficient precursors for further enzyme discovery.

Engineering Saccharomyces cerevisiae for high-efficient production of ursolic acid via cofactor engineering and acetyl-CoA optimization

Ursolic acid (UA) is an important plant-derived pentacyclic triterpene with many physiological and pharmacological activities. Although the heterologous biosynthesis of UA in microbes has been achieved, the titer is too low to be applied for commercial industrialization. The low efficiency of key enzymes such as cytochrome P450 enzyme (CYP450) and the imbalance between endogenous metabolism and exogenous pathways are considered key elements. To solve the problem, high-efficient CYP450s and its compatible cytochrome P450 reductases (CPRs) were screened and characterized. Ej8656, a CYP450 from Eriobotrya japonica, combined with LjCPR, a CPR from Lotus japonicus, showed the best performance in an engineered α-amyrin producing Saccharomyces cerevisiae with a UA titer of 43.0 mg/L. Furthermore, combining cofactor engineering of NADH/NADPH and optimization of acetyl-CoA supply, UA production was improved to 61.0 mg/L. In addition, fermentation optimization was carried out using the constructed S. cerevisiae and the titer of UA was increased to 90.0 mg/L. Finally, 2.33 g/L UA and 1.21 g/L α-amyrin were obtained after scale-up experiment in a 5 L fermenter. The UA production was 70.0-fold of the original strain WN1, and is the highest titer reported in S. cerevisiae. Our study provides a combined way to improve the efficiency of heterologous biosynthesis of UA and offers new ideas and methods for the efficient synthesis of other triterpenoids.

Bioconversion of citrus waste into mucic acid by xylose-fermenting Saccharomyces cerevisiae

Mucic acid holds promise as a platform chemical for bio-based nylon synthesis; however, its biological production encounters challenges including low yield and productivity. In this study, an efficient and high-yield method for mucic acid production was developed by employing genetically engineered Saccharomyces cerevisiae expressing the NAD+-dependent uronate dehydrogenase (udh) gene. To overcome the NAD+ dependency for the conversion of pectin to mucic acid, xylose was utilized as a co-substrate. Through optimization of the udh expression system, the engineered strain achieved a notable output, producing 20 g/L mucic acid with a highest reported productivity of 0.83 g/L-h and a theoretical yield of 0.18 g/g when processing pectin-containing citrus peel waste. These results suggest promising industrial applications for the biological production of mucic acid. Additionally, there is potential to establish a viable bioprocess by harnessing pectin-rich fruit waste alongside xylose-rich cellulosic biomass as raw materials.

Improved production of Taxol® precursors in S. cerevisiae using combinatorial in silico design and metabolic engineering

BackgroundIntegrated metabolic engineering approaches that combine system and synthetic biology tools enable the efficient design of microbial cell factories for synthesizing high-value products. In this study, we utilized in silico design algorithms on the yeast genome-scale model to predict genomic modifications that could enhance the production of early-step Taxol® in engineered Saccharomyces cerevisiae cells.ResultsUsing constraint-based reconstruction and analysis (COBRA) methods, we narrowed down the solution set of genomic modification candidates. We screened 17 genomic modifications, including nine gene deletions and eight gene overexpressions, through wet-lab studies to determine their impact on taxadiene production, the first metabolite in the Taxol® biosynthetic pathway. Under different cultivation conditions, most single genomic modifications resulted in increased taxadiene production. The strain named KM32, which contained four overexpressed genes (ILV2, TRR1, ADE13, and ECM31) involved in branched-chain amino acid biosynthesis, the thioredoxin system, de novo purine synthesis, and the pantothenate pathway, respectively, exhibited the best performance. KM32 achieved a 50% increase in taxadiene production, reaching 215 mg/L. Furthermore, KM32 produced the highest reported yields of taxa-4(20),11-dien-5α-ol (T5α-ol) at 43.65 mg/L and taxa-4(20),11-dien-5-α-yl acetate (T5αAc) at 26.2 mg/L among early-step Taxol® metabolites in S. cerevisiae.ConclusionsThis study highlights the effectiveness of computational and integrated approaches in identifying promising genomic modifications that can enhance the performance of yeast cell factories. By employing in silico design algorithms and wet-lab screening, we successfully improved taxadiene production in engineered S. cerevisiae strains. The best-performing strain, KM32, achieved substantial increases in taxadiene as well as production of T5α-ol and T5αAc. These findings emphasize the importance of using systematic and integrated strategies to develop efficient yeast cell factories, providing potential implications for the industrial production of high-value isoprenoids like Taxol®.

Improved titer and stability of selenium nanoparticles produced by engineered Saccharomyces cerevisiae

Selenium nanoparticles (SeNPs) have gained significant attention in the fields of medicine and healthcare products due to their various biological activities and low toxicity. In this study, we focused on genetically modifying the Saccharomyces cerevisiae strain YW16 (CICC 1406), which has the ability to efficiently reduce sodium selenite and produce red SeNPs. By overexpressing genes involved in glutathione production, we successfully increased the glutathione titer of the modified strain YJ003 from 41.0 mg/L to 212.0 mg/L. Moreover, we improved the conversion rate of 2.0 g/L sodium selenite from 49.3% to 59.6%. Furthermore, we identified three surface proteins of SeNPs, and found that overexpression of Act1, one of the identified proteins, led to increased stability of SeNPs across different acid-base and temperature conditions. Through a 135-h feed fermentation process using 5.0 g/L sodium selenite, we achieved an impressive conversion rate of 88.7% for sodium selenite, and each gram of SeNPs contained 195.7 mg of selenium. Overall, our findings present an efficient method for yeast to synthesize SeNPs with high stability. These SeNPs hold great potential for applications in nanomedicine or as nutritional supplements to address selenium deficiency.

Denovo production of resveratrol by engineered Saccharomyces cerevisiae W303-1a using pretreated Gracilaria corticata extracts.

Assembly and construction of resveratrol production pathway in Saccharomyces cerevisiae for denovo production of resveratrol using seaweed extract as fermentation medium. Genes involved in the production of resveratrol from tyrosine pathway, tyrosine ammonia lyase (FTAL) gene from Flavobacterium johnsoniae (FjTAL), the 4-coumarate:CoA ligase gene from Arabidopsis thaliana (4CL1) and the stilbene synthase gene from Vitis vinifera (VvSTS) were introduced into low copy, high copy and integrative vector and transformed into S. cerevisiae W303-1a. The resulting strains W303-1a/pARS-res5, W303-1a/2µ-res1 and W303-1a/IntUra-res9 produced a level of 2.39 ± 0.01, 3.33 ± 0.03 and 8.34 ± 0.03mg resveratrol l-1 respectively. CRISPR mediated integration at the δ locus resulted in 17.13 ± 1.1mg resveratrol l-1. Gracilaria corticata extract was tested as a substrate for the growth of transformant to produce resveratrol. The strain produced a comparable level, 13.6 ± 0.54mg resveratrol l-1 when grown in seaweed extract medium. The strain W303-1a/IntδC-res1 utilized Gracillaria hydrolysate and produced 13.6 ± 0.54mg resveratrol l-1 and further investigations are being carried out focusing on pathway engineering and optimization of process parameters to enhance resveratrol yield.

Improved 2,3-Butanediol Production Rate of Metabolically Engineered Saccharomyces cerevisiae by Deletion of RIM15 and Activation of Pyruvate Consumption Pathway.

Saccharomyces cerevisiae is a promising host for the bioproduction of higher alcohols, such as 2,3-butanediol (2,3-BDO). Metabolically engineered S. cerevisiae strains that produce 2,3-BDO via glycolysis have been constructed. However, the specific 2,3-BDO production rates of engineered strains must be improved. To identify approaches to improving the 2,3-BDO production rate, we investigated the factors contributing to higher ethanol production rates in certain industrial strains of S. cerevisiae compared to laboratory strains. Sequence analysis of 11 industrial strains revealed the accumulation of many nonsynonymous substitutions in RIM15, a negative regulator of high fermentation capability. Comparative metabolome analysis suggested a positive correlation between the rate of ethanol production and the activity of the pyruvate-consuming pathway. Based on these findings, RIM15 was deleted, and the pyruvate-consuming pathway was activated in YHI030, a metabolically engineered S. cerevisiae strain that produces 2,3-BDO. The titer, specific production rate, and yield of 2,3-BDO in the test tube-scale culture using the YMS106 strain reached 66.4 ± 4.4 mM, 1.17 ± 0.017 mmol (g dry cell weight h)-1, and 0.70 ± 0.03 mol (mol glucose consumed)-1. These values were 2.14-, 2.92-, and 1.81-fold higher than those of the vector control, respectively. These results suggest that bioalcohol production via glycolysis can be enhanced in a metabolically engineered S. cerevisiae strain by deleting RIM15 and activating the pyruvate-consuming pathway.