Carbon dioxide valorization into resveratrol via lithoautotrophic fermentation using engineered Cupriavidus necator H16.

BACKGROUND: Industrial biomanufacturing of value-added products using CO2 as a carbon source is considered more sustainable, cost-effective and resource-efficient than using common carbohydrate feedstocks. Cupriavidus necator H16 is a representative H2-oxidizing lithoautotrophic bacterium that can be utilized to valorize CO2 into valuable chemicals and has recently gained much attention as a promising platform host for versatile C1-based biomanufacturing. Since this microbial platform is genetically tractable and has a high-flux carbon storage pathway, it has been engineered to produce a variety of valuable compounds from renewable carbon sources. In this study, the bacterium was engineered to produce resveratrol autotrophically using an artificial phenylpropanoid pathway. RESULTS: The heterologous genes involved in the resveratrol biosynthetic pathway-tyrosine ammonia lyase (TAL), 4-coumaroyl CoA ligase (4CL), and stilbene synthase (STS) -were implemented in C. necator H16. The overexpression of acetyl-CoA carboxylase (ACC), disruption of the PHB synthetic pathway, and an increase in the copy number of STS genes enhanced resveratrol production. In particular, the increased copies of VvSTS derived from Vitis vinifera resulted a 2-fold improvement in resveratrol synthesis from fructose. The final engineered CR-5 strain produced 1.9 mg/L of resveratrol from CO2 and tyrosine via lithoautotrophic fermentation. CONCLUSIONS: To the best of our knowledge, this study is the first to describe the valorization of CO2 into polyphenolic compounds by engineering a phenylpropanoid pathway using the lithoautotrophic bacterium C. necator H16, demonstrating the potential of this strain a platform for sustainable chemical production.

De novo biosynthesis of 2'-fucosyllactose by bioengineered Corynebacterium glutamicum.

2'-Fucosyllactose (2'-FL) which is well-known human milk oligosaccharide was biotechnologically synthesized using engineered Corynebacterium glutamicum, a GRAS microbial workhorse. By construction of the complete de novo pathway for GDP-L-fucose supply and heterologous expression of Escherichia coli lactose permease and Helicobacter pylori α-1,2-fucosyltransferase, bioengineered C. glutamicum BCGW_TL successfully biosynthesized 0.25 g L-1 2'-FL from glucose. The additional genetic perturbations including the expression of a putative 2'-FL exporter and disruption of the chromosomal pfkA gene allowed C. glutamicum BCGW_cTTLEΔP to produce 2.5 g L-1 2'-FL batchwise. Finally, optimized fed-batch cultivation of the BCGW_cTTLEΔP using glucose, fructose, and lactose resulted in 21.5 g L-1 2'-FL production with a productivity of 0.12 g L-1 •h, which were more than 3.3 times higher value relative to the batch culture of the BCGW_TL. Conclusively, it would be a groundwork to adopt C. glutamicum for biotechnological production of other food additives including human milk oligosaccharides.

Adaptive laboratory evolution and transcriptomics-guided engineering of Escherichia coli for increased isobutanol tolerance.

As a renewable energy from biomass, isobutanol is considered as a promising alternative to fossil fuels. To biotechnologically produce isobutanol, strain development using industrial microbial hosts, such as Escherichia coli, has been conducted by introducing a heterologous isobutanol synthetic pathway. However, the toxicity of produced isobutanol inhibits cell growth, thereby restricting improvements in isobutanol titer, yield, and productivity. Therefore, the development of robust microbial strains tolerant to isobutanol is required. In this study, isobutanol-tolerant mutants were isolated from two E. coli parental strains, E. coli BL21(DE3) and MG1655(DE3), through adaptive laboratory evolution (ALE) under high isobutanol concentrations. Subsequently, 16 putative genes responsible for isobutanol tolerance were identified by transcriptomic analysis. When overexpressed in E. coli, four genes (fadB, dppC, acs, and csiD) conferred isobutanol tolerance. A fermentation study with a reverse engineered isobutanol-producing E. coli JK209 strain showed that fadB or dppC overexpression improved isobutanol titers by 1.5 times, compared to the control strain. Through coupling adaptive evolution with transcriptomic analysis, new genetic targets utilizable were identified as the basis for the development of an isobutanol-tolerant strain. Thus, these new findings will be helpful not only for a fundamental understanding of microbial isobutanol tolerance but also for facilitating industrially feasible isobutanol production.

Characterization and structural analysis of the endo-1,4-ß-xylanase GH11 from the hemicellulose-degrading Thermoanaerobacterium saccharolyticum useful for lignocellulose saccharification.

Xylanases are important for the enzymatic breakdown of lignocellulose-based biomass to produce biofuels and other value-added products. We report functional and structural analyses of TsaGH11, an endo-1,4-ß-xylanase from the hemicellulose-degrading bacterium, Thermoanaerobacterium saccharolyticum. TsaGH11 was shown to be a thermophilic enzyme that favors acidic conditions with maximum activity at pH 5.0 and 70 °C. It decomposes xylans from beechwood and oat spelts to xylose-containing oligosaccharides with specific activities of 5622.0 and 3959.3 U mg-1, respectively. The kinetic parameters, Km and kcat towards beechwood xylan, are 12.9 mg mL-1 and 34,015.3 s-1, respectively, resulting in kcat/Km value of 2658.7 mL mg-1 s-1, higher by 102-103 orders of magnitude compared to other reported GH11s investigated with the same substrate, demonstrating its superior catalytic performance. Crystal structures of TsaGH11 revealed a ß-jelly roll fold, exhibiting open and close conformations of the substrate-binding site by distinct conformational flexibility to the thumb region of TsaGH11. In the room-temperature structure of TsaGH11 determined by serial synchrotron crystallography, the electron density map of the thumb domain of the TsaGH11 molecule, which does not affect crystal packing, is disordered, indicating that the thumb domain of TsaGH11 has high structural flexibility at room temperature, with the water molecules in the substrate-binding cleft being more disordered than those in the cryogenic structure. These results expand our knowledge of GH11 structural flexibility at room temperature and pave the way for its application in industrial biomass degradation.

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.

Anti-Menopausal Effect of Soybean Germ Extract and Lactobacillus gasseri in the Ovariectomized Rat Model.

Menopause is a significant phase in a woman's life. Menopausal symptoms can affect overall well-being and quality of life. Conventionally, hormone replacement therapy (HRT) is used to alleviate menopausal symptoms; however, depending on the conditions, HRT may lead to side effects, necessitating the exploration of alternative therapies with fewer side effects. In this study, we investigated the effects of a combination of soybean germ extract (S30) containing 30% (w/w) isoflavone and a probiotic, Lactobacillus gasseri (LGA1), on menopausal conditions in an ovariectomized (OVX) rat model. We evaluated the impact of S30+LGA on body weight, estrogen markers, uterine and bone health, vascular markers, and neurotransmitter levels. The results revealed that treatment with S30+LGA1 significantly improved body weight and uterine and bone health. Moreover, S30+LGA1 demonstrated promising effects on lipid profile, liver function, and vascular markers and positively impacted serotonin and norepinephrine levels, indicating potential mood-enhancing effects. In conclusion, S30+LGA1, possessing anti-menopausal effects in vitro and in vivo, can be recommended as a soy-based diet, which offers various health benefits, especially for menopausal women.

Microbiome Engineering Using Probiotic Yeast: Saccharomyces boulardii and the Secreted Human Lysozyme Lead to Changes in the Gut Microbiome and Metabolome of Mice.

The probiotic yeast Saccharomyces boulardii has great potential for use as a chassis for microbiome engineering because of its high resistance to environmental stress, well-developed genetic tools, and the ability to secrete recombinant proteins in the intestine. As oral feeding of lysozyme has been reported to change the gut microbiome and fecal metabolites, we engineered S. boulardii to secrete human lysozyme, and investigated the changes in the microbiome and fecal metabolites in response to the administration of the engineered probiotic yeast into mice. Administration of S. boulardii changed the structure of the gut microbiome by promoting the growth of clostridia and increasing the diversity of strains. The human lysozyme secreted by S. boulardii in the intestine resulted in a unique gut microbiome structure through selective growth. In addition, the administration of probiotic yeast S. boulardii affected host energy metabolism and decreased blood urea and fructose levels, suggesting a mechanism of health benefits in mice. IMPORTANCE Our study identified changes in the microbiome by administering wild-type S. boulardii in mice to healthy mice based on long-read sequencing and demonstrated that a recombinant protein secreted by engineered S. boulardii in the intestine could change the microbiome. Our results provide valuable information for the development of therapeutics using engineered S. boulardii that changes the gut microbiome and host physiology.

Enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with modulated 3-hydroxyvalerate fraction by overexpressing acetolactate synthase in Cupriavidus necator H16.

The elastomeric properties of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a biodegradable copolymer, strongly depend on the molar composition of 3-hydroxyvalerate (3HV). This paper reports an improved artificial pathway for enhancing the 3HV component during PHBV biosynthesis from a structurally unrelated carbon source by Cupriavidus necator H16. To increase the intracellular accumulation of propionyl-CoA, a key precursor of the 3HV monomer, we developed a recombinant strain by genetically manipulating the branched-chain amino acid (e.g., valine, isoleucine) pathways. Overexpression of the heterologous feedback-resistant acetolactate synthase (alsS), (R)-citramalate synthase (leuA), homologous 3-ketothiolase (bktB), and the deletion of 2-methylcitrate synthase (prpC) resulted in biosynthesis of 42.5 % (g PHBV/g dry cell weight) PHBV with 64.9 mol% 3HV monomer from fructose as the sole carbon source. This recombinant strain also accumulated the highest PHBV content of 54.5 % dry cell weight (DCW) with 24 mol% 3HV monomer from CO2 ever reported. The lithoautotrophic cell growth and PHBV production by the recombinant C. necator were promoted by oxygen stress. The thermal properties of PHBV showed a decreasing trend of the glass transition and melting temperatures with increasing 3HV fraction. The average molecular weights of PHBV with modulated 3HV fractions were between 20 and 26 × 104 g/mol.

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.

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.

Optimal ß-galactosidases for producing high-titer 3,6-anhydro-L-galactose from red-algal agarobiose.

3,6-Anhydro-L-galactose (L-AHG) is a monomeric sugar in agarose derived from red macroalgae. Owing to its various physiological activities such as anti-inflammation, moisturizing, skin whitening, anti-colon cancer, and anti-cariogenicity, L-AHG is a potential functional ingredient. In our previous study, a simple and efficient two-step L-AHG production process was designed for high-titer L-AHG production, where a single enzyme was used after the liquefaction of agarose by acid prehydrolysis. However, the enzyme used did not completely hydrolyze agarobiose (AB). Therefore, in this study, for the efficient hydrolysis of AB and the high-titer production of L-AHG, various ß-galactosidases belonging to glycoside hydrolase families 1, 2, 35, and 42 were compared by testing their substrate specificities and kinetic parameters. Among the five ß-galactosidases, Bga42A, originating from Bifidobacterium longum ssp. infantis ATCC 15,697, showed the highest substrate specificity. Consequently, the two-step process utilizing Bga42A as a single enzyme resulted in a high-titer production of L-AHG at 85.9 g/L, demonstrating the feasibility of producing L-AHG from agarose. KEY POINTS: • L-AHG derived from red macroalgae has various physiological activities. • Various ß-galactosidases were evaluated to efficiently hydrolyze agarobiose. • Bga42A showed the highest substrate specificity against agarobiose. • The highest amount of L-AHG with 85.9 g/L was simply produced.

Engineering Cupriavidus necator H16 for enhanced lithoautotrophic poly(3-hydroxybutyrate) production from CO2.

BACKGROUND: A representative hydrogen-oxidizing bacterium Cupriavidus necator H16 has attracted much attention as hosts to recycle carbon dioxide (CO2) into a biodegradable polymer, poly(R)-3-hydroxybutyrate (PHB). Although C. necator H16 has been used as a model PHB producer, the PHB production rate from CO2 is still too low for commercialization. RESULTS: Here, we engineer the carbon fixation metabolism to improve CO2 utilization and increase PHB production. We explore the possibilities to enhance the lithoautotrophic cell growth and PHB production by introducing additional copies of transcriptional regulators involved in Calvin Benson Bassham (CBB) cycle. Both cbbR and regA-overexpressing strains showed the positive phenotypes for 11% increased biomass accumulation and 28% increased PHB production. The transcriptional changes of key genes involved in CO2-fixing metabolism and PHB production were investigated. CONCLUSIONS: The global transcriptional regulator RegA plays an important role in the regulation of carbon fixation and shows the possibility to improve autotrophic cell growth and PHB accumulation by increasing its expression level. This work represents another step forward in better understanding and improving the lithoautotrophic PHB production by C. necator H16.

Elucidation of the fucose metabolism of probiotic Lactobacillus rhamnosus GG by metabolomic and flux balance analyses.

Lactobacillus rhamnosus GG (LGG) is one of the most widely used probiotics because of its health benefits and safety. Fucose is among the most abundant hexoses in the human intestine, and LGG consumes fucose to produce energy or proliferate. However, no study has elucidated the metabolism by which LGG metabolizes fucose to produce energy, biomass, and extracellular metabolites. We used metabolomics and flux balance analysis to elucidate these mechanisms and highlight how they might affect the host. We found three different metabolic flux modes by which LGG anaerobically metabolizes fucose to produce energy and biomass. These metabolic flux modes differ from homolactic or heterolactic fermentation and account for the production of lactic acid, 1,2-propanediol, acetic acid, formic acid, and carbon dioxide as a result of fucose metabolism in LGG. We also used gas chromatography/time-of-flight mass spectrometry to identify a variety of short-chain fatty acids and organic acids secreted during fucose metabolism by LGG. Our study is the first to elucidate the unique fucose metabolism of LGG in anaerobic condition.

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.

GC-TOF/MS-Based Metabolomics for Comparison of Volar and Non-Volar Skin Types.

Skin has heterogenous identities on different body sites despite similar cellular compositions. There are two types of skin, volar (palmoplantar) and non-volar (dorsal), which are characterized by epidermal thickness, pigmentation, and presence of hair follicles. However, the mechanisms underlying the development of these different skin types remain unclear. To investigate these, we profiled the cellular metabolites of volar and non-volar skin in mice using gas chromatography-time-of-flight/mass spectrometry (GC-TOF/MS), and further assessed the metabolic differences between them. In total, 96 metabolites from both volar and non-volar skin of mice were identified using the BinBase database system. Metabolomics analysis revealed important differences associated with amino acid metabolism (phenylalanine, tyrosine, and tryptophan biosynthesis; aspartate and glutamate metabolism), sugar metabolism (pentose phosphate pathway), and nucleotide metabolism (pyrimidine metabolism) in volar skin. Fifty metabolites were identified as potential biomarkers differentiating the physiological characteristics of these skin types. Of these, nine were highly increased whereas 41 were significantly decreased in volar skin compared with those in non-volar skin. Overall, these results provide valuable information for understanding the metabolic differences between volar and non-volar skin.

Non-Targeted Metabolomics Approach Revealed Significant Changes in Metabolic Pathways in Patients with Chronic Traumatic Encephalopathy.

Chronic traumatic encephalopathy (CTE) is a neurodegenerative disease that is frequently found in athletes and those who have experienced repetitive head traumas. CTE is associated with a variety of neuropathologies, which cause cognitive and behavioral impairments in CTE patients. However, currently, CTE can only be diagnosed after death via brain autopsy, and it is challenging to distinguish it from other neurodegenerative diseases with similar clinical features. To better understand this multifaceted disease and identify metabolic differences in the postmortem brain tissues of CTE patients and control subjects, we performed ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS)-based non-targeted metabolomics. Through multivariate and pathway analysis, we found that the brains of CTE patients had significant changes in the metabolites involved in astrocyte activation, phenylalanine, and tyrosine metabolism. The unique metabolic characteristics of CTE identified in this study were associated with cognitive dysfunction, amyloid-beta deposition, and neuroinflammation. Altogether, this study provided new insights into the pathogenesis of CTE and suggested appealing targets for both diagnosis and treatment for the disease.

Metabolic discrimination of synovial fluid between rheumatoid arthritis and osteoarthritis using gas chromatography/time-of-flight mass spectrometry.

INTRODUCTION: Rheumatoid arthritis (RA) and osteoarthritis (OA) are clinicopathologically different. OBJECTIVES: We aimed to assess the feasibility of metabolomics in differentiating the metabolite profiles of synovial fluid between RA and OA using gas chromatography/time-of-flight mass spectrometry. METHODS: We first compared the global metabolomic changes in the synovial fluid of 19 patients with RA and OA. Partial least squares-discriminant, hierarchical clustering, and univariate analyses were performed to distinguish metabolites of RA and OA. These findings were then validated using synovial fluid samples from another set of 15 patients with RA and OA. RESULTS: We identified 121 metabolites in the synovial fluid of the first 19 samples. The score plot of PLS-DA showed a clear separation between RA and OA. Twenty-eight crucial metabolites, including hypoxanthine, xanthine, adenosine, citrulline, histidine, and tryptophan, were identified to be capable of distinguishing RA metabolism from that of OA; these were found to be associated with purine and amino acid metabolism. CONCLUSION: Our results demonstrated that metabolite profiling of synovial fluid could clearly discriminate between RA and OA, suggesting that metabolomics may be a feasible tool to assist in the diagnosis and advance the comprehension of pathological processes for diseases.

Multi-Step Enzymatic Production and Purification of 2-Keto-3-Deoxy-Galactonate from Red-Macroalgae-Derived Agarose.

2-keto-3-deoxy sugar acids, which have potential as precursors in medicinal compound production, have gained attention in various fields. Among these acids, 2-keto-3-deoxy-l-galactonate (KDGal) has been biologically produced from D-galacturonate originating from plant-derived pectin. KDGal is also found in the catabolic pathway of 3,6-anhydro-l-galactose (AHG), the main component of red-algae-derived agarose. AHG is converted to 3,6-anhydrogalactonate by AHG dehydrogenase and subsequently isomerized to KDGal by 3,6-anhydrogalactonate cycloisomerase. Therefore, we used the above-described pathway to produce KDGal from agarose. Agarose was depolymerized to AHG and to agarotriose (AgaDP3) and agaropentaose (AgaDP5), both of which have significantly higher molecular weights than AHG. When only AHG was converted to KDGal, AgaDP3 and AgaDP5 remained unreacted. Finally, KDGal was effectively purified from the enzymatic products by size-exclusion chromatography based on the differences in molecular weights. These results show that KDGal can be enzymatically produced and purified from agarose for use as a precursor to high-value products.

Evaluation and optimization of quantitative analysis of cofactors from yeast by liquid chromatography/mass spectrometry.

Cofactors play pivotal roles in catabolism and anabolism in all living organisms. Many studies have investigated the concentration of cofactors in living organisms to understand their metabolic status, which can be used to produce valuable chemicals or to understand the pathophysiology of diseases. Among various analytical platforms, liquid chromatography/mass spectrometry (LC/MS) is the most frequently used method for the quantification of cofactors. Several studies have reported various analytical methods for cofactors using LC/MS. However, the lack of optimal LC/MS methods makes it challenging to analyze various cofactors simultaneously. In addition, the method of extracting cofactors from cells needs to be optimized because conventional protocols probably have low extraction efficiency, which makes it difficult to reflect the actual concentration of cofactors in cells. In this study, we systematically compared various analytical methods and suggested optimal methods for the analysis of cofactors using LC/MS. In addition, we systematically compared quenching methods and extraction solvents and suggested optimal methods for the extraction of cofactors from Saccharomyces cerevisiae. The optimized methods can be used as standard protocols for LC/MS analysis and the extraction of cofactors from S. cerevisiae.

Isomer-Specific Monitoring of Sialylated N-Glycans Reveals Association of α2,3-Linked Sialic Acid Epitope With Behcet's Disease.

Behcet's disease (BD) is an immune disease characterized by chronic and relapsing systemic vasculitis of unknown etiology, which can lead to blindness and even death. Despite continuous efforts to discover biomarkers for accurate and rapid diagnosis and optimal treatment of BD, there is still no signature marker with high sensitivity and high specificity. As the link between glycosylation and the immune system has been revealed, research on the immunological function of glycans is being actively conducted. In particular, sialic acids at the terminus of glycoconjugates are directly implicated in immune responses, cell-cell/pathogen interactions, and tumor progression. Therefore, changes in sialic acid epitope in the human body are spotlighted as a new indicator to monitor the onset and progression of immune diseases. Here, we performed global profiling of N-glycan compositions derived from the sera of 47 healthy donors and 47 BD patients using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to preferentially determine BD target glycans. Then, three sialylated biantennary N-glycans were further subjected to the separation of linkage isomers and quantification using porous graphitized carbon-liquid chromatography (PGC-LC)/multiple reaction monitoring (MRM)-MS. We were able to successfully identify 11 isomers with sialic acid epitopes from the three glycan compositions consisting of Hex5HexNAc4NeuAc1, Hex5HexNAc4Fuc1NeuAc1, and Hex5HexNAc4NeuAc2. Among them, three isomers almost completely distinguished BD from control with high sensitivity and specificity with an area under the curve (AUC) of 0.945, suggesting the potential as novel BD biomarkers. In particular, it was confirmed that α2,3-sialic acid at the terminus of biantennary N-glycan was the epitope associated with BD. In this study, we present a novel approach to elucidating the association between BD and glycosylation by tracing isomeric structures containing sialic acid epitopes. Isomer-specific glycan profiling is suitable for analysis of large clinical cohorts and may facilitate the introduction of diagnostic assays for other immune diseases.