Enhanced production of 3,4-dihydroxybutyrate from xylose by engineered yeast via xylonate re-assimilation under alkaline condition.

To realize lignocellulose-based bioeconomy, efficient conversion of xylose into valuable chemicals by microbes is necessary. Xylose oxidative pathways that oxidize xylose into xylonate can be more advantageous than conventional xylose assimilation pathways because of fewer reaction steps without loss of carbon and ATP. Moreover, commodity chemicals like 3,4-dihydroxybutyrate and 3-hydroxybutyrolactone can be produced from the intermediates of xylose oxidative pathway. However, successful implementations of xylose oxidative pathway in yeast have been hindered because of the secretion and accumulation of xylonate which is a key intermediate of the pathway, leading to low yield of target product. Here, high-yield production of 3,4-dihydroxybutyrate from xylose by engineered yeast was achieved through genetic and environmental perturbations. Specifically, 3,4-dihydroxybutyrate biosynthetic pathway was established in yeast through deletion of ADH6 and overexpression of yneI. Also, inspired by the mismatch of pH between host strain and key enzyme of XylD, alkaline fermentations (pH ≥ 7.0) were performed to minimize xylonate accumulation. Under the alkaline conditions, xylonate was re-assimilated by engineered yeast and combined product yields of 3,4-dihydroxybutyrate and 3-hydroxybutyrolactone resulted in 0.791 mol/mol-xylose, which is highest compared with previous study. These results shed light on the utility of the xylose oxidative pathway in yeast.

L-Fucose is involved in human-gut microbiome interactions.

L-Fucose is one of the key metabolites in human-gut microbiome interactions. It is continuously synthesized by humans in the form of fucosylated glycans and fucosyl-oligosaccharides and delivered into the gut throughout their lifetime. Gut microorganisms metabolize L-fucose and produce short-chain fatty acids, which are absorbed by epithelial cells and used as energy sources or signaling molecules. Recent studies have revealed that the carbon flux in L-fucose metabolism by gut microorganisms is distinct from that in other sugar metabolisms because of cofactor imbalance and low efficiencies in energy synthesis of L-fucose metabolism. The large amounts of short-chain fatty acids produced during microbial L-fucose metabolism are used by epithelial cells to recover most of the energy used up during L-fucose synthesis. In this review, we present a detailed overview of microbial L-fucose metabolism and a potential solution for disease treatment and prevention using genetically engineered probiotics that modulate fucose metabolism. Our review contributes to the understanding of human-gut microbiome interactions through L-fucose metabolism. KEY POINTS: • Fucose-metabolizing microorganisms produce large amounts of short-chain fatty acids • Fucose metabolism differs from other sugar metabolisms by cofactor imbalance • Modulating fucose metabolism is the key to control host-gut microbiome interactions.

Identification of the enantiomeric nature of 2-keto-3-deoxy-galactonate in the catabolic pathway of 3,6-anhydro-L-galactose.

A novel metabolic pathway of 3,6-anhydro-L-galactose (L-AHG), the main sugar component in red macroalgae, was first discovered in the marine bacterium Vibrio sp. EJY3. L-AHG is converted to 2-keto-3-deoxy-galactonate (KDGal) in two metabolic steps. Here, we identified the enantiomeric nature of KDGal in the L-AHG catabolic pathway via stereospecific enzymatic reactions accompanying the biosynthesis of enantiopure L-KDGal and D-KDGal. Enantiopure L-KDGal and D-KDGal were synthesized by enzymatic reactions derived from the fungal galacturonate and bacterial oxidative galactose pathways, respectively. KDGal, which is involved in the L-AHG pathway, was also prepared. The results obtained from the reactions with an L-KDGal aldolase, specifically acting on L-KDGal, showed that KDGal in the L-AHG pathway exists in an L-enantiomeric form. Notably, we demonstrated the utilization of L-KDGal by Escherichia coli for the first time. E. coli cannot utilize L-KDGal as the sole carbon source. However, when a mixture of L-KDGal and D-galacturonate was used, E. coli utilized both. Our study suggests a stereoselective method to determine the absolute configuration of a compound. In addition, our results can be used to explore the novel L-KDGal catabolic pathway in E. coli and to construct an engineered microbial platform that assimilates L-AHG or L-KDGal as substrates. KEY POINTS: • Stereospecific enzyme reactions were used to identify enantiomeric nature of KDGal • KDGal in the L-AHG catabolic pathway exists in an L-enantiomeric form • E. coli can utilize L-KDGal as a carbon source when supplied with D-galacturonate.

Engineering xylose fermentation in an industrial yeast: continuous cultivation as a tool for selecting improved strains.

Production of second-generation ethanol from lignocellulosic residues should be fueling the energy matrix in the near future. Lignocellulosic biomass has received considerable attention as an alternative renewable resource toward reducing the demand for fossil energy sources, contributing to a future sustainable bio-based economy. Fermentation of lignocellulosic hydrolysates poses many scientific and technological challenges as the drawback of Saccharomyces cerevisiae's inability in fermenting pentose sugars (derived from hemicellulose). To overcome the inability of S. cerevisiae to ferment xylose and increase yeast robustness in the presence of inhibitory compound-containing media, the industrial S. cerevisiae strain SA-1 was engineered using CRISPR-Cas9 with the oxidoreductive xylose pathway from Scheffersomyces stipitis (encoded by XYL1, XYL2, and XYL3). The engineered strain was then cultivated in a xylose-limited chemostat under increasing dilution rates (for 64 days) to improve its xylose consumption kinetics under aerobic conditions. The evolved strain (DPY06) and its parental strain (SA-1 XR/XDH) were evaluated under microaerobic in a hemicellulosic hydrolysate-based medium. DPY06 exhibited 35% higher volumetric ethanol productivity compared to its parental strain.

Production of Ethyl-agarobioside, a Novel Skin Moisturizer, by Mimicking the Alcoholysis from the Japanese Sake-Brewing Process.

Agarobiose (AB; d-galactose-ß-1,4-AHG), produced by one-step acid hydrolysis of agarose of red seaweed, is considered a promising cosmetic ingredient due to its skin-moisturizing activity. In this study, the use of AB as a cosmetic ingredient was found to be hampered due to its instability at high temperature and alkaline pH. Therefore, to increase the chemical stability of AB, we devised a novel process to synthesize ethyl-agarobioside (ethyl-AB) from the acid-catalyzed alcoholysis of agarose. This process mimics the generation of ethyl α-glucoside and glyceryl α-glucoside by alcoholysis in the presence of ethanol and glycerol during the traditional Japanese sake-brewing process. Ethyl-AB also showed in vitro skin-moisturizing activity similar to that of AB, but showed higher thermal and pH stability than AB. This is the first report of ethyl-AB, a novel compound produced from red seaweed, as a functional cosmetic ingredient with high chemical stability.

Reshaping the 2-Pyrone Synthase Active Site for Chemoselective Biosynthesis of Polyketides.

Engineering enzymes with novel reactivity and applying them in metabolic pathways to produce valuable products are quite challenging due to the intrinsic complexity of metabolic networks and the need for high in vivo catalytic efficiency. Triacetic acid lactone (TAL), naturally generated by 2-pyrone synthase (2PS), is a platform molecule that can be produced via microbial fermentation and further converted into value-added products. However, these conversions require extra synthetic steps under harsh conditions. We herein report a biocatalytic system for direct generation of TAL derivatives under mild conditions with controlled chemoselectivity by rationally engineering the 2PS active site and then rewiring the biocatalytic pathway in the metabolic network of E. coli to produce high-value products, such as kavalactone precursors, with yields up to 17 mg/L culture. Computer modeling indicates sterics and hydrogen-bond interactions play key roles in tuning the selectivity, efficiency and yield.

Genome-wide transcriptional regulation in Saccharomyces cerevisiae in response to carbon dioxide.

Sugar metabolism by Saccharomyces cerevisiae produces ample amounts of CO2 under both aerobic and anaerobic conditions. High solubility of CO2 in fermentation media, contributing to enjoyable sensory properties of sparkling wine and beers by S. cerevisiae, might affect yeast metabolism. To elucidate the overlooked effects of CO2 on yeast metabolism, we examined glucose fermentation by S. cerevisiae under CO2 as compared to N2 and O2 limited conditions. While both CO2 and N2 conditions are considered anaerobic, less glycerol and acetate but more ethanol were produced under CO2 condition. Transcriptomic analysis revealed that significantly decreased mRNA levels of GPP1 coding for glycerol-3-phosphate phosphatase in glycerol synthesis explained the reduced glycerol production under CO2 condition. Besides, transcriptional regulations in signal transduction, carbohydrate synthesis, heme synthesis, membrane and cell wall metabolism, and respiration were detected in response to CO2. Interestingly, signal transduction was uniquely regulated under CO2 condition, where upregulated genes (STE3, MSB2, WSC3, STE12, and TEC1) in the signal sensors and transcriptional factors suggested that MAPK signaling pathway plays a critical role in CO2 sensing and CO2-induced metabolisms in yeast. Our study identifies CO2 as an external stimulus for modulating metabolic activities in yeast and a transcriptional effector for diverse applications.

Selective production of retinol by engineered Saccharomyces cerevisiae through the expression of retinol dehydrogenase.

Retinol is a fat-soluble vitamin A that is widely used in the food and pharmaceutical industries. Currently, retinol is commercially produced by chemical synthesis. Microbial production of retinol has been alternatively explored but restricted to a mixture of retinoids including retinol, retinal, and retinoic acid. Thus, we introduced heterologous retinol dehydrogenase into retinoids mixture-producing Saccharomyces cerevisiae for the selective production of retinol using xylose. Expression of human RDH10 and Escherichia coli ybbO led to increase in retinol production, but retinal remained as a major product. In contrast, S. cerevisiae harboring human RDH12 produced retinol selectively with negligible production of retinal. The resulting strain (SR8A-RDH12) produced retinol only. However, more glycerol was accumulated due to intracellular redox imbalance. Therefore, Lactococcus lactis noxE coding for H2 O-forming NADH oxidase was additionally introduced to resolve the redox imbalance. The resulting strain produced 52% less glycerol and more retinol with a 30% higher yield than a parental strain. As the produced retinol was not stable, we examined culture and storage conditions including temperature, light, and antioxidants for the optimal production of retinol. In conclusion, we achieved selective production of retinol efficiently from xylose by introducing human RDH12 and NADH oxidase into S. cerevisiae.

Strain engineering and metabolic flux analysis of a probiotic yeast Saccharomyces boulardii for metabolizing L-fucose, a mammalian mucin component.

BACKGROUND: Saccharomyces boulardii is a probiotic yeast that exhibits antimicrobial and anti-toxin activities. Although S. boulardii has been clinically used for decades to treat gastrointestinal disorders, several studies have reported weak or no beneficial effects of S. boulardii administration in some cases. These conflicting results of S. boulardii efficacity may be due to nutrient deficiencies in the intestine that make it difficult for S. boulardii to maintain its metabolic activity. RESULTS: To enable S. boulardii to overcome any nutritional deficiencies in the intestine, we constructed a S. boulardii strain that could metabolize L-fucose, a major component of mucin in the gut epithelium. The fucU, fucI, fucK, and fucA from Escherichia coli and HXT4 from S. cerevisiae were overexpressed in S. boulardii. The engineered S. boulardii metabolized L-fucose and produced 1,2-propanediol under aerobic and anaerobic conditions. It also produced large amounts of 1,2-propanediol under strict anaerobic conditions. An in silico genome-scale metabolic model analysis was performed to simulate the growth of S. boulardii on L-fucose, and elementary flux modes were calculated to identify critical metabolic reactions for assimilating L-fucose. As a result, we found that the engineered S. boulardii consumes L-fucose via (S)-lactaldehyde-(S)-lactate-pyruvate pathway, which is highly oxygen dependent. CONCLUSION: To the best of our knowledge, this is the first study in which S. cerevisiae and S. boulardii strains capable of metabolizing L-fucose have been constructed. This strategy could be used to enhance the metabolic activity of S. boulardii and other probiotic microorganisms in the gut.

System analysis of Lipomyces starkeyi during growth on various plant-based sugars.

Oleaginous yeasts have received significant attention due to their substantial lipid storage capability. The accumulated lipids can be utilized directly or processed into various bioproducts and biofuels. Lipomyces starkeyi is an oleaginous yeast capable of using multiple plant-based sugars, such as glucose, xylose, and cellobiose. It is, however, a relatively unexplored yeast due to limited knowledge about its physiology. In this study, we have evaluated the growth of L. starkeyi on different sugars and performed transcriptomic and metabolomic analyses to understand the underlying mechanisms of sugar metabolism. Principal component analysis showed clear differences resulting from growth on different sugars. We have further reported various metabolic pathways activated during growth on these sugars. We also observed non-specific regulation in L. starkeyi and have updated the gene annotations for the NRRL Y-11557 strain. This analysis provides a foundation for understanding the metabolism of these plant-based sugars and potentially valuable information to guide the metabolic engineering of L. starkeyi to produce bioproducts and biofuels. KEY POINTS: • L. starkeyi metabolism reprograms for consumption of different plant-based sugars. • Non-specific regulation was observed during growth on cellobiose. • L. starkeyi secretes ß-glucosidases for extracellular hydrolysis of cellobiose.

Genome-edited Saccharomyces cerevisiae strains for improving quality, safety, and flavor of fermented foods.

While Cas9-based genome editing enabled precise and sophisticated genetic perturbations in conventional and non-conventional yeast strains, its applications for food fermentations have been extremely limited. In order to improve quality and flavor of various yeast-fermented foods, we isolated and engineered a diploid or polyploid Saccharomyces cerevisiae strain (N1) which exhibits robust sugar fermentation, strong acid tolerance, and rapid gas production from Korean Nuruk. First, RGT2 and SNF3 coding for glucose sensors were deleted to increase respiration. A bread dough fermented with the N1ΔRGT2ΔSNF3 strain showed an 18% increased volume due to higher carbon dioxide production. Second, ASP3 coding for asparaginase was overexpressed and URE2 coding for a transcriptional factor of nitrogen catabolite repression (NCR) was deleted to increase asparagine consumption. When the N1ΔURE2::PGPD-ASP3 strain was applied to a potato dough, asparagine was rapidly depleted in the dough, resulting in potato chips with negligible amounts of acrylamide. Third, the N1ΔURE2 strain was utilized to increase levels of the amino acids which provide a savory taste during rice wine fermentation. The above genome-edited yeast strains contain no heterologous DNA. As such, they can be used to improve fermented foods with no subjection to GM regulation.

A SWEET surprise: Anaerobic fungal sugar transporters and chimeras enhance sugar uptake in yeast.

In the yeast Saccharomyces cerevisiae, microbial fuels and chemicals production on lignocellulosic hydrolysates is constrained by poor sugar transport. For biotechnological applications, it is desirable to source transporters with novel or enhanced function from nonconventional organisms in complement to engineering known transporters. Here, we identified and functionally screened genes from three strains of early-branching anaerobic fungi (Neocallimastigomycota) that encode sugar transporters from the recently discovered Sugars Will Eventually be Exported Transporter (SWEET) superfamily in Saccharomyces cerevisiae. A novel fungal SWEET, NcSWEET1, was identified that localized to the plasma membrane and complemented growth in a hexose transporter-deficient yeast strain. Single cross-over chimeras were constructed from a leading NcSWEET1 expression-enabling domain paired with all other candidate SWEETs to broadly scan the sequence and functional space for enhanced variants. This led to the identification of a chimera, NcSW1/PfSW2:TM5-7, that enhanced the growth rate significantly on glucose, fructose, and mannose. Additional chimeras with varied cross-over junctions identified residues in TM1 that affect substrate selectivity. Furthermore, we demonstrate that NcSWEET1 and the enhanced NcSW1/PfSW2:TM5-7 variant facilitated novel co-consumption of glucose and xylose in S. cerevisiae. NcSWEET1 utilized 40.1% of both sugars, exceeding the 17.3% utilization demonstrated by the control HXT7(F79S) strain. Our results suggest that SWEETs from anaerobic fungi are beneficial tools for enhancing glucose and xylose co-utilization and offers a promising step towards biotechnological application of SWEETs in S. cerevisiae.

Enhancing acid tolerance of Escherichia coli via viroporin-mediated export of protons and its application for efficient whole-cell biotransformation.

Escherichia coli-based whole-cell biocatalysts are widely used for the sustainable production of value-added chemicals. However, weak acids present as substrates and/or products obstruct the growth and fermentation capability of E. coli. Here, we show that a viroporin consisting of the influenza A matrix-2 (M2) protein, is activated by low pH and has proton channel activity in E. coli. The heterologous expression of the M2 protein in E. coli resulted in a significant increase in the intracellular pH and cell viability in the presence of various weak acids with different lengths of carbon chains. In addition, the feasibility of developing a robust and efficient E. coli-based whole-cell biocatalyst via introduction of the proton-selective viroporin was explored by employing (Z)-11-(heptanolyoxy)undec-9-enoic acid (ester) and 2-fucosyllactose (2'-FL) as model products, whose production is hampered by cytosolic acidification. The engineered E. coli strains containing the proton-selective viroporin exhibited approximately 80% and 230% higher concentrations of the ester and 2'-FL, respectively, than the control strains without the M2 protein. The simple and powerful strategy developed in this study can be applied to produce other valuable chemicals whose production involves substrates and/or products that cause cytosolic acidification.

Increased Accumulation of Squalene in Engineered Yarrowia lipolytica through Deletion of PEX10 and URE2.

Squalene is a triterpenoid serving as an ingredient of various products in the food, cosmetic, pharmaceutical industries. The oleaginous yeast Yarrowia lipolytica offers enormous potential as a microbial chassis for the production of terpenoids, such as carotenoid, limonene, linalool, and farnesene, as the yeast provides ample storage space for hydrophobic products. Here, we present a metabolic design that allows the enhanced accumulation of squalene in Y. lipolytica. First, we improved squalene accumulation in Y. lipolytica by overexpressing the genes (ERG and HMG) coding for the mevalonate pathway enzymes. Second, we increased the production of lipid where squalene is accumulated by overexpressing DGA1 (encoding diacylglycerol acyltransferase) and deleting PEX10 (for peroxisomal membrane E3 ubiquitin ligase). Third, we deleted URE2 (coding for a transcriptional regulator in charge of nitrogen catabolite repression [NCR]) to induce lipid accumulation regardless of the carbon-to-nitrogen ratio in culture media. The resulting engineered Y. lipolytica exhibited a 115-fold higher squalene content (22.0 mg/g dry cell weight) than the parental strain. These results suggest that the biological function of Ure2p in Y. lipolytica is similar to that in Saccharomyces cerevisiae, and its deletion can be utilized to enhance the production of hydrophobic target products in oleaginous yeast strains. IMPORTANCE This study demonstrated a novel strategy for increasing squalene production in Y. lipolytica. URE2, a bifunctional protein that is involved in both nitrogen catabolite repression and oxidative stress response, was identified and demonstrated correlation to squalene production. The data suggest that double deletion of PEX10 and URE2 can serve as a positive synergistic effect to help yeast cells in boosting squalene production. This discovery can be combined with other strategies to engineer cell factories to efficiently produce terpenoid in the future.

Domesticating a food spoilage yeast into an organic acid-tolerant metabolic engineering host: Lactic acid production by engineered Zygosaccharomyces bailii.

Lactic acid represents an important class of commodity chemicals, which can be produced by microbial cell factories. However, due to the toxicity of lactic acid at lower pH, microbial production requires the usage of neutralizing agents to maintain neutral pH. Zygosaccharomyces bailii, a food spoilage yeast, can grow under the presence of organic acids used as food preservatives. This unique trait of the yeast might be useful for producing lactic acid. With the goal of domesticating the organic acid-tolerant yeast as a metabolic engineering host, seven Z. bailii strains were screened in a minimal medium with 10 g/L of acetic, or 60 g/L of lactic acid at pH 3. The Z. bailii NRRL Y7239 strain was selected as the most robust strain to be engineered for lactic acid production. By applying a PAN-ARS-based CRISPR-Cas9 system consisting of a transfer RNA promoter and NAT selection, we demonstrated the targeted deletion of ADE2 and site-specific integration of Rhizopus oryzae ldhA coding for lactate dehydrogenase into the PDC1 locus. The resulting pdc1::ldhA strain produced 35 g/L of lactic acid without ethanol production. This study demonstrates the feasibility of the CRISPR-Cas9 system in Z. bailii, which can be applied for a fundamental study of the species.

Production of neoagarooligosaccharides by probiotic yeast Saccharomyces cerevisiae var. boulardii engineered as a microbial cell factory.

BACKGROUND: Saccharomyces cerevisiae var. boulardii is a representative probiotic yeast that has been widely used in the food and pharmaceutical industries. However, S. boulardii has not been studied as a microbial cell factory for producing useful substances. Agarose, a major component of red macroalgae, can be depolymerized into neoagarooligosaccharides (NAOSs) by an endo-type ß-agarase. NAOSs, including neoagarotetraose (NeoDP4), are known to be health-benefiting substances owing to their prebiotic effect. Thus, NAOS production in the gut is required. In this study, the probiotic yeast S. boulardii was engineered to produce NAOSs by expressing an endo-type ß-agarase, BpGH16A, derived from a human gut bacterium Bacteroides plebeius. RESULTS: In total, four different signal peptides were compared in S. boulardii for protein (BpGH16A) secretion for the first time. The SED1 signal peptide derived from Saccharomyces cerevisiae was selected as optimal for extracellular production of NeoDP4 from agarose. Expression of BpGH16A was performed in two ways using the plasmid vector system and the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system. The production of NeoDP4 by engineered S. boulardii was verified and quantified. NeoDP4 was produced by S. boulardii engineered using the plasmid vector system and CRISPR-Cas9 at 1.86 and 0.80 g/L in a 72-h fermentation, respectively. CONCLUSIONS: This is the first report on NAOS production using the probiotic yeast S. boulardii. Our results suggest that S. boulardii can be considered a microbial cell factory to produce health-beneficial substances in the human gut.

In-depth understanding of molecular mechanisms of aldehyde toxicity to engineer robust Saccharomyces cerevisiae.

Aldehydes are ubiquitous electrophilic compounds that ferment microorganisms including Saccharomyces cerevisiae encounter during the fermentation processes to produce food, fuels, chemicals, and pharmaceuticals. Aldehydes pose severe toxicity to the growth and metabolism of the S. cerevisiae through a variety of toxic molecular mechanisms, predominantly via damaging macromolecules and hampering the production of targeted compounds. Compounds with aldehyde functional groups are far more toxic to S. cerevisiae than all other functional classes, and toxic potency depends on physicochemical characteristics of aldehydes. The yeast synthetic biology community established a design-build-test-learn framework to develop S. cerevisiae cell factories to valorize the sustainable and renewable biomass, including the lignin-derived substrates. However, thermochemically pretreated biomass-derived substrate streams contain diverse aldehydes (e.g., glycolaldehyde and furfural), and biological conversions routes of lignocellulosic compounds consist of toxic aldehyde intermediates (e.g., formaldehyde and methylglyoxal), and some of the high-value targeted products have aldehyde functional group (e.g., vanillin and benzaldehyde). Numerous studies comprehensively characterized both single and additive effects of aldehyde toxicity via systems biology investigations, and novel molecular approaches have been discovered to overcome the aldehyde toxicity. Based on those novel approaches, researchers successfully developed synthetic yeast cell factories to convert lignocellulosic substrates to valuable products, including aldehyde compounds. In this mini-review, we highlight the salient relationship of physicochemical characteristics and molecular toxicity of aldehydes, the molecular detoxification and macromolecules protection mechanisms of aldehydes, and the advances of engineering robust S. cerevisiae against complex mixtures of aldehyde inhibitors. KEY POINTS: • We reviewed structure-activity relationships of aldehyde toxicity on S. cerevisiae. • Two-tier protection mechanisms to alleviate aldehyde toxicity are presented. • We highlighted the strategies to overcome the synergistic toxicity of aldehydes.

Metabolic engineering of non-pathogenic microorganisms for 2,3-butanediol production.

2,3-Butanediol (2,3-BDO) is a promising commodity chemical with various industrial applications. While petroleum-based chemical processes currently dominate the industrial production of 2,3-BDO, fermentation-based production of 2,3-BDO provides an attractive alternative to chemical-based processes with regards to economic and environmental sustainability. The achievement of high 2,3-BDO titer, yield, and productivity in microbial fermentation is a prerequisite for the production of 2,3-BDO at large scales. Also, enantiopure production of 2,3-BDO production is desirable because 2,3-BDO stereoisomers have unique physicochemical properties. Pursuant to these goals, many metabolic engineering strategies to improve 2,3-BDO production from inexpensive sugars by Klebsiella oxytoca, Bacillus species, and Saccharomyces cerevisiae have been developed. This review summarizes the recent advances in metabolic engineering of non-pathogenic microorganisms to enable efficient and enantiopure production of 2,3-BDO. KEY POINTS: • K. oxytoca, Bacillus species, and S. cerevisiae have been engineered to achieve efficient 2,3-BDO production. • Metabolic engineering of non-pathogenic microorganisms enabled enantiopure production of 2,3-BDO. • Cost-effective 2,3-BDO production can be feasible by using renewable biomass.

Investigating the role of the transcriptional regulator Ure2 on the metabolism of Saccharomyces cerevisiae: a multi-omics approach.

Ure2 regulates nitrogen catabolite repression in Saccharomyces cerevisiae. Deletion of URE2 induces a physiological state mimicking the nitrogen starvation and autophagic responses. Previous work has shown that deletion of URE2 increases the fermentation rate of some wine-producing strains of S. cerevisiae. In this work, we investigated the effect of URE2 deletion (ΔURE2) on the metabolism of S. cerevisiae. During growth on glucose, the ΔURE2 mutant grew at a 40% slower rate than the wild type; however, it produced ethanol at a 31% higher rate. To better under the behavior of this mutant, we performed transcriptomics and metabolomics. Analysis of the RNA sequencing results and metabolite levels indicates that the mutant strain exhibited characteristics of both nitrogen starvation and autophagy, including the upregulation of allantoin, urea, and amino acid uptake and utilization pathways and selective autophagic machinery. In addition, pyruvate decarboxylase and alcohol dehydrogenase isoforms were expressed at higher rates than the wild type. The mutant also accumulated less trehalose and glycogen, and produced more lipids. The induction of a nitrogen starvation-like state and increase in lipid production in nitrogen-rich conditions suggest that URE2 may be a promising target for metabolic engineering in S. cerevisiae and other yeasts for the production of lipids and lipid-derived compounds. KEY POINTS: • Deletion of URE2 increases ethanol and lipid production in Saccharomyces cerevisiae. • Deletion of URE2 reduces glycogen and trehalose production. • Metabolic changes mimic nitrogen starvation and autophagic response.

Integrating transcriptomic and metabolomic analysis of the oleaginous yeast Rhodosporidium toruloides IFO0880 during growth under different carbon sources.

Rhodosporidium toruloides is an oleaginous yeast capable of producing a variety of biofuels and bioproducts from diverse carbon sources. Despite numerous studies showing its promise as a platform microorganism, little is known about its metabolism and physiology. In this work, we investigated the central carbon metabolism in R. toruloides IFO0880 using transcriptomics and metabolomics during growth on glucose, xylose, acetate, or soybean oil. These substrates were chosen because they can be derived from plants. Significant changes in gene expression and metabolite concentrations were observed during growth on these four substrates. We mapped these changes onto the governing metabolic pathways to better understand how R. toruloides reprograms its metabolism to enable growth on these substrates. One notable finding concerns xylose metabolism, where poor expression of xylulokinase induces a bypass leading to arabitol production. Collectively, these results further our understanding of central carbon metabolism in R. toruloides during growth on different substrates. They may also help guide the metabolic engineering and development of better models of metabolism for R. toruloides.Key points• Gene expression and metabolite concentrations were significantly changed.• Reduced expression of xylulokinase induces a bypass leading to arabitol production.• R. toruloides reprograms its metabolism to allow growth on different substrates.