Metabolic recycling of storage lipids promotes squalene biosynthesis in yeast.

BACKGROUND: Metabolic rewiring in microbes is an economical and sustainable strategy for synthesizing valuable natural terpenes. Terpenes are the largest class of nature-derived specialized metabolites, and many have valuable pharmaceutical or biological activity. Squalene, a medicinal terpene, is used as a vaccine adjuvant to improve the efficacy of vaccines, including pandemic coronavirus disease 2019 (COVID-19) vaccines, and plays diverse biological roles as an antioxidant and anticancer agent. However, metabolic rewiring interferes with inherent metabolic pathways, often in a way that impairs the cellular growth and fitness of the microbial host. In particular, as the key starting molecule for producing various compounds including squalene, acetyl-CoA is involved in numerous biological processes with tight regulation to maintain metabolic homeostasis, which limits redirection of metabolic fluxes toward desired products. RESULTS: In this study, focusing on the recycling of surplus metabolic energy stored in lipid droplets, we show that the metabolic recycling of the surplus energy to acetyl-CoA can increase squalene production in yeast, concomitant with minimizing the metabolic interferences in inherent pathways. Moreover, by integrating multiple copies of the rate-limiting enzyme and implementing N-degron-dependent protein degradation to downregulate the competing pathway, we systematically rewired the metabolic flux toward squalene, enabling remarkable squalene production (1024.88 mg/L in a shake flask). Ultimately, further optimization of the fed-batch fermentation process enabled remarkable squalene production of 6.53 g/L. CONCLUSIONS: Our demonstration of squalene production via engineered yeast suggests that plant- or animal-based supplies of medicinal squalene can potentially be complemented or replaced by industrial fermentation. This approach will also provide a universal strategy for the more stable and sustainable production of high-value terpenes.

Enhanced Production of Bacterial Cellulose from Miscanthus as Sustainable Feedstock through Statistical Optimization of Culture Conditions.

Biorefineries are attracting attention as an alternative to the petroleum industry to reduce carbon emissions and achieve sustainable development. In particular, because forests play an important role in potentially reducing greenhouse gas emissions to net zero, alternatives to cellulose produced by plants are required. Bacterial cellulose (BC) can prevent deforestation and has a high potential for use as a biomaterial in various industries such as food, cosmetics, and pharmaceuticals. This study aimed to improve BC production from lignocellulose, a sustainable feedstock, and to optimize the culture conditions for Gluconacetobacter xylinus using Miscanthus hydrolysates as a medium. The productivity of BC was improved using statistical optimization of the major culture parameters which were as follows: temperature, 29 °C; initial pH, 5.1; and sodium alginate concentration, 0.09% (w/v). The predicted and actual values of BC production in the optimal conditions were 14.07 g/L and 14.88 g/L, respectively, confirming that our prediction model was statistically significant. Additionally, BC production using Miscanthus hydrolysates was 1.12-fold higher than in the control group (commercial glucose). Our result indicate that lignocellulose can be used in the BC production processes in the near future.

Development of Colorimetric Whole-Cell Biosensor for Detection of Heavy Metals in Environment for Public Health.

Heavy metals cause various fetal diseases in humans. Heavy metals from factory wastewater can contaminate drinking water, fish, and crops. Inductively coupled plasma-mass spectrometry (ICP-MS) and atomic absorption spectrometry (AAS) are commonly used to analyze heavy metal contents; however, these methods require pre-treatment processes and are expensive and complex. To overcome these limitations, three metal-sensing materials using a whole-cell biosensor in Escherichia coli (E. coli) were developed. Strains were engineered to harbor three kinds of plasmids containing the copA, zntA, and mer promoters for sensing copper, cadmium, and mercury, respectively. The luciferase (lux) gene was inserted as a reporter into the plasmid, which was later replaced with a fused protein sequence containing OmpA (1-159) and mCherry for optical detection. The constructed strains could detect mercury, cadmium, and copper at 0.1-0.75 ppm, 0.2-0.75 ppm, and 2-7.5 ppm, respectively, with linearity values of 0.99030, 0.99676, and 0.95933, respectively. The immobilization linearity value was 0.99765. Notably, these three heavy metals could be detected by visual analysis of the strains. Overall, these findings establish this novel sensor as a potential approach for heavy metal detection in biological samples and foods.

Effective Cryopreservation of a Bioluminescent Auxotrophic Escherichia coli-Based Amino Acid Array to Enable Long-Term Ready-to-Use Applications.

Amino acid arrays comprising bioluminescent amino acid auxotrophic Escherichia coli are effective systems to quantitatively determine multiple amino acids. However, there is a need to develop a method for convenient long-term preservation of the array to enable its practical applications. Here, we reported a potential strategy to efficiently maintain cell viability within the portable array. The method involves immobilization of cells within agarose gel supplemented with an appropriate cryoprotectant in individual wells of a 96-well plate, followed by storage under freezing conditions. Six cryoprotectants, namely dimethyl sulfoxide, glycerol, ethylene glycol, polyethylene glycol, sucrose, and trehalose, were tested in the methionine (Met) auxotroph-based array. Carbohydrate-type cryoprotectants (glycerol, sucrose, and trehalose) efficiently preserved the linearity of determination of Met concentration. In particular, the array with 5% trehalose exhibited the best performance. The Met array with 5% trehalose could determine Met concentration with high linearity (R2 value = approximately 0.99) even after storage at -20 °C for up to 3 months. The clinical utilities of the Met and Leu array, preserved at -20 °C for 3 months, were also verified by successfully quantifying Met and Leu in spiked blood serum samples for the diagnosis of the corresponding metabolic diseases. This long-term preservation protocol enables the development of a ready-to-use bioluminescent E. coli-based amino acid array to quantify multiple amino acids and can replace the currently used laborious analytical methods.

Co3O4/Au Hybrid Nanostructures as Efficient Peroxidase Mimics for Colorimetric Biosensing.

Nanomaterials with enzyme-like characteristics (nanozymes) have emerged as potential replacements for natural enzymes due to their potential to overcome several critical limitations of natural enzymes, including low stability as well as high costs in preparation and purification. Herein, we have developed hybrid nanostructures that incorporate cobalt oxide nanoparticles (Co3O4 NPs) and gold nanoclusters (AuNCs) through electrostatic attraction induced by simple incubation in an aqueous buffer for 2 hours. Owing to the synergistic effect of Co3O4 NPs and AuNCs, the constructed Co3O4/Au hybrid nanostructures yielded highly enhanced peroxidase-like activity and enabled rapid catalytic oxidation of a chromogenic substrate, 3,3',5,5'-tetramethylbenzidine (TMB), producing a blue colored solution depending on the amount of H2O2. Moreover, we observed catalytic activity of the Co3O4/Au hybrid over a broad pH range, especially at physiologically relevant pH in the range of 5.0-7.4, which is advantageous for applications in biological systems. Using the hybrid as peroxidase mimic, we successfully determined the level of target H2O2 or glucose by coupling with glucose oxidase with excellent specificity and sensitivity. Based on this study, we expect that Co3O4/Au hybrid nanostructures can serve as potent peroxidase mimics for the detection of clinically important target molecules.

Heterologous Production of Squalene from Glucose in Engineered Corynebacterium glutamicum Using Multiplex CRISPR Interference and High-Throughput Fermentation.

The sustainable production of squalene has driven the development of microbial cell factories due to the limitation of low-yielding bioprocesses from plants and illegal harvesting shark liver. We report the metabolic engineering of Corynebacterium glutamicum to produce squalene from glucose. Combinatorial metabolic engineering strategies for precursor rebalancing, redox balancing, and blocking the competing pathway for the isopentenyl diphosphate availabilities were applied by repressing the target genes using the CRISPR interference. The best engineered strain using high-throughput fermentation produced squalene from glucose at 5.4 ± 0.3 mg/g dry cell weight (DCW) and 105.3 ± 3.0 mg/L, which was a 5.2-fold increase over the parental strain. In addition, flask cultivation of C. glutamicum overexpressing the dxs and idi genes with squalene synthase gene and repressing the idsA gene resulted in production of squalene at 5.8 ± 0.4 mg/g DCW and 82.8 ± 6.2 mg/L, which was a 3.4-fold increase over the parental strain.

Optimization of Antioxidant and Skin-Whitening Compounds Extraction Condition from Tenebrio molitor Larvae (Mealworm).

Skin-whitening ingredients are a very important part of the development of functional cosmetics and a wide variety of raw materials are used. Tyrosinase is a key enzyme in the animal melanogenic pathway that is the rate-limiting step for the production of melanin. Several synthetic and naturally occurring tyrosinase inhibitors have been studied for skin-whitening. The development of natural agents is becoming more important due to the disadvantages of synthetics such as high cytotoxicity, insufficient penetration power, and low activity. The purpose of this study was to evaluate the total phenol content (TPC), antioxidant, and tyrosinase inhibition activity of mealworm (Tenebrio molitor larvae) extract, and the subsequent optimization of the extraction condition using statistically-based optimization. The major extraction variables extraction temperature, time, and ethanol concentration were optimized using response surface methodology (RSM). The results showed that optimum extraction temperature of 88.1 °C, extraction time of 43.7 min, and ethanol concentration of 72.0 v/v%, provided the predicted maximum levels of total phenolic compounds (TPC) of 5.41 mg GAE/g dry weight (DW) and tyrosinase inhibition activity (TIA) of 82.4%. From the validation experiment, 5.61 ± 0.2 mg GAE/g dry weight (DW), tyrosinase inhibition of 79.6 ± 3.3%, and radical scavenging activity of 91.8 ± 5.1 µg/mL were found and showed to be very similar to the predicted values. These results suggest that mealworm has great potential as a source of bioactive compounds which could be used as cosmetics, food, and pharmaceutical agents.

Convenient Colorimetric Detection of Thrombin via Aptamer-Mediated Inhibition and Restoration of the Oxidase Activity of Nanoceria.

Cerium oxide nanoparticles, also called nanoceria, have recently gained much attention as oxidase-mimicking nanozymes that catalyze the oxidation of chromogenic substrates for color generation without the addition of H2O2. Herein, we have developed a unique colorimetric biosensor for thrombin in human blood plasma, which relies on thrombin-binding aptamer (TBA)-mediated inhibition of the oxidase activity of nanoceria and its restoration by very selective interactions of TBA with target thrombin. In this system, nanoceria were first incubated with TBA, resulting in quick reduction of the oxidase activity of nanoceria via the adsorption of single-stranded (ss)DNA-type TBA on nanoceria. By the addition of sample solutions containing target thrombin, TBA bound on the nanoceria would strongly interact with free thrombin and be detached from the nanoceria, thereby increasing the available surface area of the nanoceria and consequently enhancing the oxidase activity. Using this strategy, target thrombin was successfully detected at concentrations as low as 100 pM over a wide linear range from 0.1 to 10 nM. The diagnostic capability of this method has been demonstrated by detecting thrombin in human blood plasma, showing its great potential in the practical applications.

Improving the efficiency of homologous recombination by chemical and biological approaches in Yarrowia lipolytica.

Gene targeting is a challenge in Yarrowia lipolytica (Y. lipolytica) where non-homologous end-joining (NHEJ) is predominant over homologous recombination (HR). To improve the frequency and efficiency of HR in Y. lipolytica, the ku70 gene responsible for a double stand break (DSB) repair in the NHEJ pathway was disrupted, and the cell cycle was synchronized to the S-phase with hydroxyurea, respectively. Consequently, the HR frequency was over 46% with very short homology regions (50 bp): the pex10 gene was accurately deleted at a frequency of 60% and the ß-carotene biosynthetic genes were integrated at the correct locus at an average frequency of 53%. For repeated use, the URA3 marker gene was also excised and deleted at a frequency of 100% by HR between the 100 bp homology regions flanking the URA3 gene. It was shown that appropriate combination of these chemical and biological approaches was very effective to promote HR and construct genetically modified Y. lipolytica strains for biotechnological applications.

Lactic acid production from cellobiose and xylose by engineered Saccharomyces cerevisiae.

Efficient and rapid production of value-added chemicals from lignocellulosic biomass is an important step toward a sustainable society. Lactic acid, used for synthesizing the bioplastic polylactide, has been produced by microbial fermentation using primarily glucose. Lignocellulosic hydrolysates contain high concentrations of cellobiose and xylose. Here, we constructed a recombinant Saccharomyces cerevisiae strain capable of fermenting cellobiose and xylose into lactic acid. Specifically, genes (cdt-1, gh1-1, XYL1, XYL2, XYL3, and ldhA) coding for cellobiose transporter, ß-glucosidase, xylose reductase, xylitol dehydrogenase, xylulokinase, and lactate dehydrogenase were integrated into the S. cerevisiae chromosomes. The resulting strain produced lactic acid from cellobiose or xylose with high yields. When fermenting a cellulosic sugar mixture containing 10 g/L glucose, 40 g/L xylose, and 80 g/L cellobiose, the engineered strain produced 83 g/L of lactic acid with a yield of 0.66 g lactic acid/g sugar (66% theoretical maximum). This study demonstrates initial steps toward the feasibility of sustainable production of lactic acid from lignocellulosic sugars by engineered yeast.

Planococcus faecalis sp. nov., a carotenoid-producing species isolated from stools of Antarctic penguins.

Taxonomic studies were performed on a novel carotenoid-producing strain, designated AJ003T, isolated from faeces of Antarctic penguins. Cells of strain AJ003T were aerobic, Gram-stain-positive, cocci-shaped and orange. Strain AJ003T was capable of growing in a broad temperature range, including sub-zero growth (below − 20 to 30 °C). 16S rRNA gene sequence analysis revealed that strain AJ003T was closely related to Planococcus halocryophilus Or1T (97.4 % similarity), Planococcus antarcticus DSM 14505T (97.3 %), Planococcus kocurii NCIMB 629T (97.3 %), and Planococcus donghaensis JH1T (97.1 %). The predominant cellular fatty acids were anteiso-C15 : 0, and iso-C16 : 0.MK-7 and MK-8 were the quinones identified, and the major pigment was glycosyl-4,4'-diaponeurosporen-4'-ol-4-oic acid. The major polar lipid was phosphatidylglycerol. DNA­DNA relatedness of strain AJ003T with respect to its closest phylogenetic neighbours was 38.2 ± 0.5 % for Planococcus halocryophilus DSM 24743T, 32.2 ± 0.2 % for Planococcus antarcticus DSM 14505T, 21.0 ± 0.3 % for Planococcus kocurii DSM 20747T and 18.6 ± 1.4 % for Planococcus donghaensis KCTC 13050T. The DNA G+C content of strain AJ003T was 40.0 ± 0.6 mol%. Based on the phenotypic, chemotaxonomic and phylogenetic data, strain AJ003T is concluded to represent a novel species of the genus Planococcus, for which the name Planococcus faecalis sp. nov. is proposed. The type strain is AJ003T ( = KCTC 33580T = CECT 8759T).

Lactic acid production from xylose by engineered Saccharomyces cerevisiae without PDC or ADH deletion.

Production of lactic acid from renewable sugars has received growing attention as lactic acid can be used for making renewable and bio-based plastics. However, most prior studies have focused on production of lactic acid from glucose despite that cellulosic hydrolysates contain xylose as well as glucose. Microbial strains capable of fermenting both glucose and xylose into lactic acid are needed for sustainable and economic lactic acid production. In this study, we introduced a lactic acid-producing pathway into an engineered Saccharomyces cerevisiae capable of fermenting xylose. Specifically, ldhA from the fungi Rhizopus oryzae was overexpressed under the control of the PGK1 promoter through integration of the expression cassette in the chromosome. The resulting strain exhibited a high lactate dehydrogenase activity and produced lactic acid from glucose or xylose. Interestingly, we observed that the engineered strain exhibited substrate-dependent product formation. When the engineered yeast was cultured on glucose, the major fermentation product was ethanol while lactic acid was a minor product. In contrast, the engineered yeast produced lactic acid almost exclusively when cultured on xylose under oxygen-limited conditions. The yields of ethanol and lactic acid from glucose were 0.31 g ethanol/g glucose and 0.22 g lactic acid/g glucose, respectively. On xylose, the yields of ethanol and lactic acid were <0.01 g ethanol/g xylose and 0.69 g lactic acid/g xylose, respectively. These results demonstrate that lactic acid can be produced from xylose with a high yield by S. cerevisiae without deleting pyruvate decarboxylase, and the formation patterns of fermentations can be altered by substrates.

Enhancement of ß-Glucosidase Activity from a Brown Rot Fungus Fomitopsis pinicola KCTC 6208 by Medium Optimization.

ß-Glucosidase, which hydrolyzes cellobiose into two glucoses, plays an important role in the process of saccharification of the lignocellulosic biomass. In this study, we optimized the activity of ß-glucosidase of brown-rot fungus Fomitopsis pinicola KCTC 6208 using the response surface methodology (RSM) with various concentrations of glucose, yeast extract and ascorbic acid, which are the most significant nutrients for activity of ß-glucosidase. The highest activity of ß-glucosidase was achieved 3.02% of glucose, 4.35% of yeast extract, and 7.41% ascorbic acid where ascorbic acid was most effective. The maximum activity of ß-glucosidase predicted by the RSM was 15.34 U/mg, which was similar to the experimental value 14.90 U/mg at the 16th day of incubation. This optimized activity of ß-glucosidase was 23.6 times higher than the preliminary activity value, 0.63 U/mg, and was also much higher than previous values reported in other fungi strains. Therefore, a simplified medium supplemented with a cheap vitamin source, such as ascorbic acid, could be a cost effective mean of increasing ß-glucosidase activity.

Evidence of syntrophic acetate oxidation by Spirochaetes during anaerobic methane production.

To search for evidence of syntrophic acetate oxidation by cluster II Spirochaetes with hydrogenotrophic methanogens, batch reactors seeded with five different anaerobic sludge samples supplemented with acetate as the sole carbon source were operated anaerobically. The changes in abundance of the cluster II Spirochaetes, two groups of acetoclastic methanogens (Methanosaetaceae and Methanosarcinaceae), and two groups of hydrogenotrophic methanogens (Methanomicrobiales and Methanobacteriales) in the reactors were assessed using qPCR targeting the 16S rRNA genes of each group. Increase in the cluster II Spirochaetes (9.0±0.4-fold) was positively correlated with increase in hydrogenotrophic methanogens, especially Methanomicrobiales (5.6±1.0-fold), but not with acetoclastic methanogens. In addition, the activity of the cluster II Spirochaetes decreased (4.6±0.1-fold) in response to high hydrogen partial pressure, but their activity was restored after consumption of hydrogen by the hydrogenotrophic methanogens. These results strongly suggest that the cluster II Spirochaetes are involved in syntrophic acetate oxidation in anaerobic digesters.

The acetylproteome of Gram-positive model bacterium Bacillus subtilis.

N(ε) -lysine acetylation, a reversible and highly regulated PTM, has been shown to occur in the model Gram-negative bacteria Escherichia coli and Salmonella enterica. Here, we extend this acetylproteome analysis to Bacillus subtilis, a model Gram-positive bacterium. Through anti-acetyllysine antibody-based immunoseparation of acetylpeptides followed by nano-HPLC/MS/MS analysis, we identified 332 unique lysine-acetylated sites on 185 proteins. These proteins are mainly involved in cellular housekeeping functions such as central metabolism and protein synthesis. Fifity-nine of the lysine-acetylated proteins showed homology with lysine-acetylated proteins previously identified in E. coli, suggesting that acetylated proteins are more conserved. Notably, acetylation was found at or near the active sites predicted by Prosite signature, including SdhA, RocA, Kbl, YwjH, and YfmT, indicating that lysine acetylation may affect their activities. In 2-amino-3-ketobutyrate CoA ligase Kbl, a class II aminotransferase, a lysine residue involved in pyridoxal phosphate attachment was found to be acetylated. This data set provides evidence for the generality of lysine acetylation in eubacteria and opens opportunities to explore the consequences of acetylation modification on the molecular physiology of B. subtilis.

Characterization of Saccharomyces cerevisiae promoters for heterologous gene expression in Kluyveromyces marxianus.

Kluyveromyces marxianus is now considered one of the best choices of option for industrial applications of yeast because the strain is able to grow at high temperature, utilizes various carbon sources, and grows fast. However, the use of K. marxianus as a host for industrial applications is still limited. This limitation is largely due to a lack of knowledge on the characteristics of the promoters since the time and amount of protein expression is strongly dependent on the promoter employed. In this study, four well-known constitutive promoters (P(CYC), P(TEF), P(GPD), and P(ADH)) of Saccharomyces cerevisiae were characterized in K. marxianus in terms of protein expression level and their stochastic behavior. After constructing five URA3-auxotrophic K. marxianus strains and a plasmid vector, four cassettes each comprising one of the promoters--the gene for the green fluorescence protein (GFP)--CYC1 terminator (T(CYC)) were inserted into the vector. GFP expression under the control of each one of the promoters was analyzed by reverse transcription PCR, fluorescence microscopy, and flow cytometer. Using these combined methods, the promoter strength was determined to be in the order of P(GPD) > P(ADH) ∼ P(TEF) >> P(CYC). All promoters except for the P(CYC) exhibited three distinctive populations, including non-expressing cells, weakly expressing cells, and strongly expressing cells. The relative ratios between populations were strongly dependent on the promoter and culture time. Forward scattering was independent of GFP fluorescence intensity, indicating that the different fluorescence intensities were not just due to different cell sizes derived from budding. It also excluded the possibility that the non-expressing cells resulted from plasmid loss because plasmid stability was maintained at almost 100 % over the culture time. The same cassettes, cloned into a single copy plasmid pRS416 and transformed into S. cerevisiae, showed only one population. When the cassettes were integrated into the chromosome, the stochastic behavior was markedly reduced. These combined results imply that the gene expression stochasticity should be overcome in order to use this strain for delicate metabolic engineering, which would require the co-expression of several genes.

Genome-wide enrichment screening reveals multiple targets and resistance genes for triclosan in Escherichia coli.

Triclosan is a widely used biocide effective against different microorganisms. At bactericidal concentrations, triclosan appears to affect multiple targets, while at bacteriostatic concentrations, triclosan targets FabI. The site-specific antibiotic-like mode-of-action and a widespread use of triclosan in household products claimed to possibly induce cross-resistance to other antibiotics. Thus, we set out to define more systematically the genes conferring resistance to triclosan; A genomic library of Escherichia coli strain W3110 was constructed and enriched in a selective medium containing a lethal concentration of triclosan. The genes enabling growth in the presence of triclosan were identified by using a DNA microarray and confirmed consequently by ASKA clones overexpressing the selected 62 candidate genes. Among these, forty-seven genes were further confirmed to enhance the resistance to triclosan; these genes, including the FabI target, were involved in inner or outer membrane synthesis, cell-surface material synthesis, transcriptional activation, sugar phosphotransferase (PTS) systems, various transporter systems, cell division, and ATPase and reductase/dehydrogenase reactions. In particular, overexpression of pgsA, rcsA, or gapC conferred to E. coli cells a similar level of triclosan resistance induced by fabI overexpression. These results indicate that triclosan may have multiple targets other than well-known FabI and that there are several undefined novel mechanisms for the resistance development to triclosan, thus probably inducing cross antibiotic resistance.

Cell-based quantification of homocysteine utilizing bioluminescent Escherichia coli auxotrophs.

A cell-based quantitative assay system for Hcy has been developed by utilizing two Escherichia coli auxotrophs that grow in the presence of methionine (Met) and either homocysteine (Hcy) or Met, respectively. A bioluminescent reporter gene, which produces luminescence as cells grow, was inserted into the auxotrophs, so that cell growth can be readily determined. When the relative luminescence unit (RLU) values from the two auxotrophs immobilized within agarose gels arrayed on a well plate were measured, the amount of Hcy was quantitatively determined on the basis of differences between two RLU values corresponding to cell growth of two auxotrophs with excellent levels of precision and reproducibility. Finally, the diagnostic utility of this assay system was verified by its employment in reliably determining different stages of hyperhomocysteinemia in human plasma samples providing CVs of within and between assays that are less than 2.9% and 7.1%, respectively, and recovery rates of within and between assays that are in the range of 99.1-103.5% and 97.5-105.5%, respectively. In contrast to existing conventional methods, the new system developed in this effort is simple, rapid, and cost-effective. As a result, it has great potential to serve as a viable alternative for Hcy quantification in the diagnosis of hyperhomocysteinemia.

Improved galactose fermentation of Saccharomyces cerevisiae through inverse metabolic engineering.

Although Saccharomyces cerevisiae is capable of fermenting galactose into ethanol, ethanol yield and productivity from galactose are significantly lower than those from glucose. An inverse metabolic engineering approach was undertaken to improve ethanol yield and productivity from galactose in S. cerevisiae. A genome-wide perturbation library was introduced into S. cerevisiae, and then fast galactose-fermenting transformants were screened using three different enrichment methods. The characterization of genetic perturbations in the isolated transformants revealed three target genes whose overexpression elicited enhanced galactose utilization. One confirmatory (SEC53 coding for phosphomannomutase) and two novel targets (SNR84 coding for a small nuclear RNA and a truncated form of TUP1 coding for a general repressor of transcription) were identified as overexpression targets that potentially improve galactose fermentation. Beneficial effects of overexpression of SEC53 may be similar to the mechanisms exerted by overexpression of PGM2 coding for phosphoglucomutase. While the mechanism is largely unknown, overexpression of SNR84, improved both growth and ethanol production from galactose. The most remarkable improvement of galactose fermentation was achieved by overexpression of the truncated TUP1 (tTUP1) gene, resulting in unrivalled galactose fermentation capability, that is 250% higher in both galactose consumption rate and ethanol productivity compared to the control strain. Moreover, the overexpression of tTUP1 significantly shortened lag periods that occurs when substrate is changed from glucose to galactose. Based on these results we proposed a hypothesis that the mutant Tup1 without C-terminal repression domain might bring in earlier and higher expression of GAL genes through partial alleviation of glucose repression. mRNA levels of GAL genes (GAL1, GAL4, and GAL80) indeed increased upon overexpression of tTUP. The results presented in this study illustrate that alteration of global regulatory networks through overexpression of the identified targets (SNR84 and tTUP1) is as effective as overexpression of a rate limiting metabolic gene (PGM2) in the galactose assimilation pathway for efficient galactose fermentation in S. cerevisiae. In addition, these results will be industrially useful in the biofuels area as galactose is one of the abundant sugars in marine plant biomass such as red seaweed as well as cheese whey and molasses.

Identification of gene targets eliciting improved alcohol tolerance in Saccharomyces cerevisiae through inverse metabolic engineering.

The economic production of biofuels from renewable biomass using Saccharomyces cerevisiae requires tolerance to high concentrations of sugar and alcohol. Here we applied an inverse metabolic engineering approach to identify endogenous gene targets conferring improved alcohol tolerance in S. cerevisiae. After transformation with a S. cerevisiae genomic library, enrichment of the transformants exhibiting improved tolerance was performed by serial subculture in the presence of iso-butanol (1%). Through sequence analysis of the isolated plasmids from the selected transformants, four endogenous S. cerevisiae genes were identified as overexpression targets eliciting improved tolerance to both iso-butanol and ethanol. Overexpression of INO1, DOG1, HAL1 or a truncated form of MSN2 resulted in remarkably increased tolerance to high concentrations of iso-butanol and ethanol. Overexpression of INO1 elicited the highest ethanol tolerance, resulting in higher titers and volumetric productivities in the fermentation experiments performed with high glucose concentrations. In addition, the INO1-overexpressing strain showed a threefold increase in the specific growth rate as compared to that of the control strain under conditions of high levels of glucose (10%) and ethanol (5%). Although alcohol tolerance in yeast is a complex trait affected by simultaneous interactions of many genes, our results using a genomic library reveal potential target genes for better understanding and possible engineering of metabolic pathways underlying alcohol tolerance phenotypes.