Publications by authors named "Kyoung Heon Kim"

178 Publications

Characterization of BpGH16A of Bacteroides plebeius, a key enzyme initiating the depolymerization of agarose in the human gut.

Appl Microbiol Biotechnol 2021 Jan 6;105(2):617-625. Epub 2021 Jan 6.

Department of Biotechnology, Graduate School, Korea University, Seoul, 02841, South Korea.

Seaweeds have received considerable attention as sources of dietary fiber and biomass for manufacturing valuable products. The major polysaccharides of red seaweeds include agar and porphyran. In a marine environment, marine bacteria utilize agar and porphyran through the agarase and porphyranase genes encoded in their genomes. Most of these enzymes identified and characterized so far originate from marine bacteria. Recently, Bacteroides plebeius, a human gut bacterium isolated from seaweed-eating Japanese individuals, was revealed to contain a polysaccharide utilization locus (PUL) targeting the porphyran and agarose of red seaweeds. For example, B. plebeius contains an endo-type β-agarase, BpGH16A, belonging to glycoside hydrolase family 16. BpGH16A cleaves the β-1,4-glycosidic linkages of agarose and produces neoagarooligosccharides from agarose. Since it is crucial to study the characteristics of BpGH16A to understand the depolymerization pathway of red seaweed polysaccharides by B. plebeius in the human gut and to industrially apply the enzyme for the depolymerization of agar, we characterized BpGH16A for the first time. According to our results, BpGH16A is an extracellular endo-type β-agarase with an optimal temperature of 40 °C and an optimal pH of 7.0, which correspond to the temperature and pH of the human colon. BpGH16A depolymerizes agarose into neoagarotetraose (as the main product) and neoagarobiose (as the minor product). Thus, BpGH16A is suggested to be an important enzyme that initiates the depolymerization of red seaweed agarose or agar in the human gut by B. plebeius. KEY POINTS: • Bacteroides plebeius is a human gut bacterium isolated from seaweed-eating humans. • BpGH16A is an extracellular endo-type β-agarase with optimal conditions of 40 °C and pH 7.0. • BpGH16A depolymerizes agarose into neoagarotetraose and neoagarobiose.
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http://dx.doi.org/10.1007/s00253-020-11039-3DOI Listing
January 2021

Metabolomic Elucidation of the Effect of Sucrose on the Secondary Metabolite Profiles in by Ultraperformance Liquid Chromatography-Mass Spectrometry.

ACS Omega 2020 Dec 15;5(51):33186-33195. Epub 2020 Dec 15.

Department of Biotechnology, Graduate School, Korea University, Seoul 02841, South Korea.

Sucrose induces flavonoid accumulation in plants as a defense mechanism against various stresses. However, the relationship between the biosynthesis of flavonoids as secondary metabolites and sucrose levels remains unknown. To understand the change in flavonoid biosynthesis by sucrose, we conducted secondary metabolite profiling in treated with different levels of sucrose using ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry. The partial least squares-discriminant and hierarchical clustering analyses showed significant differences in secondary metabolite profiles in at 50, 150, and 300 mM sucrose levels. The levels of 3 flavonoids such as quercetin 3--β-d-glucosyl-(1→2)-β-d-glucoside, 6-methoxyaromadendrin 3--acetate, and 3-hydroxycoumarin and 19 flavonoids including 6-methoxyaromadendrin 3--acetate, aureusidin, iridin, flavonol 3--(6--malonyl-β-d-glucoside) quercetin 3--glucoside, and rutin increased at 150 and 300 mM sucrose, respectively, compared to 50 mM sucrose, indicating that the flavonoids were accumulated in by a higher concentration of sucrose. This is the first investigation of the change in individual flavonoids as secondary metabolites in by varying sucrose levels, and the results demonstrate that the sucrose causes the accumulation of certain flavonoids as a defense mechanism against osmotic stress.
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http://dx.doi.org/10.1021/acsomega.0c04745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7774254PMC
December 2020

Characterization of an Antibacterial Agent Targeting Ferrous Iron Transport Protein FeoB against and Gram-Positive Bacteria.

ACS Chem Biol 2021 01 30;16(1):136-149. Epub 2020 Dec 30.

Department of Agricultural Biotechnology, Research Institute of Agriculture and Life Science, Seoul National University, Seoul, 08826, Republic of Korea.

The emergence of multidrug-resistant strains has become a serious clinical problem. Iron is absolutely required for the bacterial growth, virulence associated with colonization, and survival from the host immune system. The FeoB protein is a major iron permease in bacterial ferrous iron transport systems (Feo) that has been shown to play a crucial role in virulence of some pathogenic bacteria. However, FeoB is still uncharacterized in Gram-positive pathogens, and its effects on pathogenesis are unknown. In this study, we identified a novel inhibitor, GW3965·HCl, that targets FeoB in . The molecule effectively inhibited FeoB enzyme activity, bacterial growth, and virulence factor expression. Genome-editing and metabolomic analyses revealed that GW3965·HCl inhibited FeoB function and affected the associated mechanisms with reduced iron availability in . Gentamicin resistance and infection assays further demonstrated the power of GW3965·HCl as a safe and efficient antibacterial agent. In addition to , GW3965·HCl also presented its effectiveness on inhibition of the FeoB activity and growth of Gram-positive bacteria. This novel inhibitor will provide new insight for developing a next-generation antibacterial therapy.
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http://dx.doi.org/10.1021/acschembio.0c00842DOI Listing
January 2021

Enhanced 2'-Fucosyllactose production by engineered Saccharomyces cerevisiae using xylose as a co-substrate.

Metab Eng 2020 11 22;62:322-329. Epub 2020 Oct 22.

Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA; Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA. Electronic address:

2'-Fucosyllactose (2'-FL), a human milk oligosaccharide with confirmed benefits for infant health, is a promising infant formula ingredient. Although Escherichia coli, Saccharomyces cerevisiae, Corynebacterium glutamicum, and Bacillus subtilis have been engineered to produce 2'-FL, their titers and productivities need be improved for economic production. Glucose along with lactose have been used as substrates for producing 2'-FL, but accumulation of by-products due to overflow metabolism of glucose hampered efficient production of 2'-FL regardless of a host strain. To circumvent this problem, we used xylose, which is the second most abundant sugar in plant cell wall hydrolysates and is metabolized through oxidative metabolism, for the production of 2'-FL by engineered yeast. Specifically, we modified an engineered S. cerevisiae strain capable of assimilating xylose to produce 2'-FL from a mixture of xylose and lactose. First, a lactose transporter (Lac12) from Kluyveromyces lactis was introduced. Second, a heterologous 2'-FL biosynthetic pathway consisting of enzymes Gmd, WcaG, and WbgL from Escherichia coli was introduced. Third, we adjusted expression levels of the heterologous genes to maximize 2'-FL production. The resulting engineered yeast produced 25.5 g/L of 2'-FL with a volumetric productivity of 0.35 g/L∙h in a fed-batch fermentation with lactose and xylose feeding to mitigate the glucose repression. Interestingly, the major location of produced 2'-FL by the engineered yeast can be changed using different culture media. While 72% of the produced 2'-FL was secreted when a complex medium was used, 82% of the produced 2'-FL remained inside the cells when a minimal medium was used. As yeast extract is already used as food and animal feed ingredients, 2'-FL enriched yeast extract can be produced cost-effectively using the 2'-FL-accumulating yeast cells.
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http://dx.doi.org/10.1016/j.ymben.2020.10.003DOI Listing
November 2020

Zmo0994, a novel LEA-like protein from , increases multi-abiotic stress tolerance in .

Biotechnol Biofuels 2020 26;13:151. Epub 2020 Aug 26.

Department of Biotechnology, Graduate School, Korea University, Seoul, 02841 Republic of Korea.

Background: Pretreatment processes and subsequent enzymatic hydrolysis are prerequisites to utilize lignocellulosic sugar for fermentation. However, the resulting hydrolysate frequently hinders fermentation processes due to the presence of inhibitors and toxic products (e.g., ethanol). Thus, it is crucial to develop robust microbes conferring multi-stress tolerance.

Results: Zmo0994, a functionally uncharacterized protein from , was identified and characterized for the first time. A major effect of Zmo0994 was a significant enhancement in the tolerance to abiotic stresses such as ethanol, furfural, 5'-hydroxymethylfurfural and high temperature, when expressed in . Through transcriptome analysis and in vivo experiments, the cellular mechanism of this protein was revealed as due to its ability to trigger genes, involved in aerobic respiration for ATP synthesis.

Conclusions: These findings have significant implications that might lead to the development of robust microbes for the highly efficient industrial fermentation processes.
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http://dx.doi.org/10.1186/s13068-020-01790-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7448490PMC
August 2020

Overproduction of Exopolysaccharide Colanic Acid by Escherichia coli by Strain Engineering and Media Optimization.

Appl Biochem Biotechnol 2021 Jan 21;193(1):111-127. Epub 2020 Aug 21.

Department of Biotechnology, Korea University Graduate School, Seoul, 02841, South Korea.

Colanic acid (CA) is one of the major bacterial exopolysaccharides. Due to its biological activities, CA has a significant commercial value. However, the cultivation conditions have not been optimized for the large-scale production of CA. Here, we constructed a CA-overproducing Escherichia coli strain (ΔwaaF) and statistically optimized its culture media for maximum CA production. Glucose and tryptone were found the optimal carbon and nitrogen sources, respectively. Fractional factorial design indicated tryptone and NaHPO as the critical nutrients for CA production. Through further optimization, we achieved a maximum CA production of 1910.0 mg/L, which is approximately 12-fold higher than the amount obtained using the non-optimized medium initially used. The predicted value of CA production was comparable with experimental value (2052.8 mg/L) under the optimized conditions. This study constitutes a successful demonstration of media optimization for increased CA production, and paves the way for future research for achieving large-scale CA production.
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http://dx.doi.org/10.1007/s12010-020-03409-4DOI Listing
January 2021

Biochemical characterization of bacterial FeoBs: A perspective on nucleotide specificity.

Arch Biochem Biophys 2020 05 24;685:108350. Epub 2020 Mar 24.

Department of Biotechnology, Graduate School, Korea University, Seoul, 02841, Republic of Korea. Electronic address:

Iron is an essential requirement for the survival and virulence of most bacteria. The bacterial ferrous iron transporter protein FeoB functions as a major reduced iron transporter in prokaryotes, but its biochemical mechanism has not been fully elucidated. In the present study, we compared enzymatic properties of the cytosolic portions of pathogenic bacterial FeoBs to elucidate each bacterial strain-specific characteristic of the Feo system. We show that bacterial FeoBs are classified into two distinct groups that possess either a sole GTPase or an NTPase with a substrate promiscuity. This difference in nucleotide preference alters cellular requirements for monovalent and divalent cations. While the hydrolytic activity of the GTP-dependent FeoBs was stimulated by potassium, the action of the NTP-dependent FeoBs was not significantly affected by the presence of monovalent cations. Mutation of Asn11, having a role in potassium-dependent GTP hydrolysis, changed nucleotide specificity of the NTP-dependent FeoB, resulting in loss of ATPase activity. Sequence analysis suggested a possible association of alanine in the G5 motif for the NTP-dependent activity in FeoBs. This demonstration of the distinct enzymatic properties of bacterial FeoBs provides important insights into mechanistic details of Feo iron transport processes, as well as offers a promising species-specific anti-virulence target.
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http://dx.doi.org/10.1016/j.abb.2020.108350DOI Listing
May 2020

Systematic re-evaluation of the long-used standard protocol of urease-dependent metabolome sample preparation.

PLoS One 2020 17;15(3):e0230072. Epub 2020 Mar 17.

Department of Biotechnology, Graduate School, Korea University, Seoul, Republic of Korea.

In the urinary metabolomics for finding biomarkers in urine, owing to high concentrations of urea, for chromatography-based metabolomic analysis, urea needed to be degraded by urease. This urease pretreatment has been the key step of sample preparation for standard urinary metabolomics until today even for mass spectrometry-based analysis. The urease pretreatment involving incubation of urine with urease contradicts the concept of metabolome sampling, which should immediately arrest metabolic reactions to prevent alterations of a metabolite profile. Nonetheless, the impact of urease pretreatment has not been clearly elucidated yet. We found that activities of urease and endogenous urinary enzymes and metabolite contaminants from the urease preparations introduce artefacts into metabolite profiles, thus leading to misinterpretation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0230072PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7077817PMC
June 2020

Dual Agarolytic Pathways in a Marine Bacterium, sp. Strain EJY3: Molecular and Enzymatic Verification.

Appl Environ Microbiol 2020 03 2;86(6). Epub 2020 Mar 2.

Department of Biotechnology, Graduate School, Korea University, Seoul, South Korea

sp. strain EJY3 is an agarolytic marine bacterium that catabolizes 3,6-anhydro-l-galactose (AHG), a monomeric sugar unit of agarose. While the AHG catabolic pathway in EJY3 has been discovered recently, the complete agarolytic system of EJY3 remains unclear. We have identified five enzymes, namely, the β-agarases GH50A, GH50B, GH50C, and GH50D and the α-neoagarooligosaccharide (NAOS) hydrolase GH117, involved in the agarolytic system of EJY3. Based on the characterization of recombinant enzymes and intracellular metabolite analysis, we found that EJY3 catabolizes agarose via two different agarolytic pathways. Among the four β-agarases of EJY3, GH50A, GH50B, and GH50C were found to be extracellular agarases, producing mainly neoagarotetraose (NeoDP4) and neoagarobiose. By detecting intracellular NeoDP4 in EJY3 grown on agarose, NeoDP4 was observed being taken up by cells. Intriguingly, intracellular NeoDP4 acted as a branching point for the two different downstream agarolytic pathways. First, via the well-known agarolytic pathway, NeoDP4 was depolymerized into monomeric sugars by the exo-type β-agarase GH50D and the α-NAOS hydrolase GH117. Second, via the newly found alternative agarolytic pathway, NeoDP4 was depolymerized into AHG and agarotriose (AgaDP3) by GH117, and AgaDP3 then was completely depolymerized into monomeric sugars by sequential reactions of the agarolytic β-galactosidases (ABG) ABG and GH117. Therefore, by experimentally verifying agarolytic enzymatic activity and transport of NeoDP4 into EJY3 cells, we revealed that EJY3 possesses both the known pathway and the newly discovered alternative pathway that involves α-NAOS hydrolase and ABG. Agarose is the main polysaccharide of red macroalgae and is composed of galactose and 3,6-anhydro-l-galactose. Many marine bacteria possess enzymes capable of depolymerizing agarose into oligomers and then depolymerizing the oligomers into monomers. Here, we experimentally verified that both a well-known agarolytic pathway and a novel agarolytic pathway exist in a marine bacterium, sp. strain EJY3. In agarolytic pathways, agarose is depolymerized mainly into 4-sugar-unit oligomers by extracellular enzymes, which are then transported into cells. The imported oligomers are intracellularly depolymerized into galactose and 3,6-anhydro-l-galactose by two different agarolytic pathways, using different combinations of intracellular enzymes. These results elucidate the depolymerization routes of red macroalgal biomass in the ocean by marine bacteria and provide clues for developing industrial processes for efficiently producing sugars from red macroalgae.
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http://dx.doi.org/10.1128/AEM.02724-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054104PMC
March 2020

Enzymatic synthesis of l-fucose from l-fuculose using a fucose isomerase from sp. and the biochemical and structural analyses of the enzyme.

Biotechnol Biofuels 2019 5;12:282. Epub 2019 Dec 5.

1Department of Biotechnology, Korea University Graduate School, Seoul, 02841 South Korea.

Background: l-Fucose is a rare sugar with potential uses in the pharmaceutical, cosmetic, and food industries. The enzymatic approach using l-fucose isomerase, which interconverts l-fucose and l-fuculose, can be an efficient way of producing l-fucose for industrial applications. Here, we performed biochemical and structural analyses of l-fucose isomerase identified from a novel species of (FucI).

Results: FucI exhibited higher enzymatic activity for l-fuculose than for l-fucose, and the rate for the reverse reaction of converting l-fuculose to l-fucose was higher than that for the forward reaction of converting l-fucose to l-fuculose. In the equilibrium mixture, a much higher proportion of l-fucose (~ ninefold) was achieved at 30 °C and pH 7, indicating that the enzyme-catalyzed reaction favors the formation of l-fucose from l-fuculose. When biochemical analysis was conducted using l-fuculose as the substrate, the optimal conditions for FucI activity were determined to be 40 °C and pH 10. However, the equilibrium composition was not affected by reaction temperature in the range of 30 to 50 °C. Furthermore, FucI was found to be a metalloenzyme requiring Mn as a cofactor. The comparative crystal structural analysis of FucI revealed the distinct conformation of α7-α8 loop of FucI. The loop is present at the entry of the substrate binding pocket and may affect the catalytic activity.

Conclusions: FucI-catalyzed isomerization favored the reaction from l-fuculose to l-fucose. The biochemical and structural data of FucI will be helpful for the better understanding of the molecular mechanism of l-FucIs and the industrial production of l-fucose.
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http://dx.doi.org/10.1186/s13068-019-1619-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6894278PMC
December 2019

3,6-Anhydro-L-galactose increases hyaluronic acid production via the EGFR and AMPKα signaling pathway in HaCaT keratinocytes.

J Dermatol Sci 2019 Nov 2;96(2):90-98. Epub 2019 Nov 2.

School of Food Science and Biotechnology, Kyungpook National University, Daegu 41566, Republic of Korea. Electronic address:

Background: Hyaluronic acid (HA) is an important factor in skin hydration maintenance. In mammalian keratinocytes, hyaluronan synthase 2 (HAS2) is a critical enzyme in HA production. Therefore, the promotion of HAS2 expression in keratinocytes may be a strategy for maintaining skin moisture.

Objective: The aim was to determine the skin hydration effect and regulatory mechanisms of 3,6-anhydro-L-galactose (L-AHG), a main component of red macroalgal carbohydrates in human keratinocytes.

Methods: L-AHG was applied to an immortalized human epidermal keratinocyte cell line (HaCaT cells). HA production, HAS2 protein and mRNA levels, and the activation of the signaling pathways involved in HAS2 expression were measured. HA levels were also evaluated for three dimensional (3D) reconstructed human skin.

Results: Our results suggest that L-AHG upregulates HA production and may enhance HAS2 expression by activating EGFR-mediated ERK, PI3K/Akt, and STAT3 signaling pathways. We confirmed that L-AHG activated the AMPKα signaling pathway which in turn could regulate HAS2 expression in HaCaT cells. The effects of L-AHG on HA production were observed in the 3D reconstructed human skin model.

Conclusion: Our results suggest that L-AHG may enhance skin moisture retention by increasing HA synthesis in human epidermal keratinocytes.
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http://dx.doi.org/10.1016/j.jdermsci.2019.10.005DOI Listing
November 2019

Long-Living Budding Yeast Cell Subpopulation Induced by Ethanol/Acetate and Respiration.

J Gerontol A Biol Sci Med Sci 2020 07;75(8):1448-1456

Institute of Animal Molecular Biotechnology and Korea University, Seoul, Republic of Korea.

Budding yeast generate heterogeneous cells that can be separated into two distinctive cell types: short-living low-density and long-living high-density (HD) cells by density gradient centrifugation. We found that ethanol and acetate induce formation of HD cells, and mitochondrial respiration is required. From their transcriptomes and metabolomes, we found upregulated differentially expressed genes in HD cells involved in the RGT2/RGT1 glucose sensing pathway and its downstream genes encoding hexose transporters. For HD cells, we determined an abundance of various carbon sources including glucose, lactate, pyruvate, trehalose, mannitol, mannose, and galactose. Other upregulated differentially expressed genes in HD cells were involved in the TORC1-SCH9 signaling pathway and its downstream genes involved in cytoplasmic translation. We also measured an abundance of free amino acids in HD cells including valine, proline, isoleucine, and glutamine. These characteristics of the HD cell transcriptome and metabolome may be important conditions for maintaining a long-living phenotype.
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http://dx.doi.org/10.1093/gerona/glz202DOI Listing
July 2020

Deletion of PHO13 improves aerobic L-arabinose fermentation in engineered Saccharomyces cerevisiae.

J Ind Microbiol Biotechnol 2019 Dec 9;46(12):1725-1731. Epub 2019 Sep 9.

School of Food Science and Biotechnology, Kyungpook National University, Daegu, Republic of Korea.

Pentose sugars are increasingly being used in industrial applications of Saccharomyces cerevisiae. Although L-arabinose is a highlighted pentose that has been identified as next-generation biomass, arabinose fermentation has not yet undergone extensive development for industrial utilization. In this study, we integrated a heterologous fungal arabinose pathway with a deletion of PHO13 phosphatase gene. PHO13 deletion increased arabinose consumption rate and specific ethanol productivity under aerobic conditions and consequently depleted sedoheptulose by activation of the TAL1 gene. Global metabolite profiling indicated upregulation of the pentose phosphate pathway and downstream effects such as trehalose accumulation and downregulation of the TCA cycle. Our results suggest that engineering of PHO13 has ample potential for arabinose conversion to ethanol as an industrial source for biofuels.
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http://dx.doi.org/10.1007/s10295-019-02233-yDOI Listing
December 2019

Variation in the synovial fluid metabolome according to disease activity of rheumatoid arthritis.

Clin Exp Rheumatol 2020 May-Jun;38(3):500-507. Epub 2019 Aug 27.

Division of Rheumatology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea.

Objectives: Because genetic and environmental factors both contribute to rheumatoid arthritis (RA), metabolomics could be a very useful tool to elucidate the pathophysiology of RA, and to predict response to treatment. This study was carried out to investigate synovial fluid (SF) metabolic perturbation in RA patients according to the degree of disease activity using gas chromatography/time-of-flight mass spectrometry (GC/TOF MS).

Methods: SF samples were obtained from 48 RA patients. Disease activity was assessed using DAS28-ESR(3). SF metabolomics profiling was performed using GC/TOF-MS, in conjunction with multivariate statistical analyses and pathway analyses.

Results: Significant discrimination of metabolite profiles between moderate and high disease activity groups was shown by PLS-DA, which provided evidence that SF metabolic profiles predicted disease activity. We found the significant correlation between DAS28-ESR(3) value and the intensities of 12 metabolites. The intensities of glycocyamine and indol-3-lactate positively correlated with DAS28-ESR(3) value. On the other hand, β-alanine, asparagine, citrate, cyano-L-alanine, leucine, nicotinamide, citrulline, methionine, oxoproline, and salicylaldehyde negatively correlated with DAS28-ESR(3) value. We found fifteen pathways that were significantly associated with disease activity in RA and that the higher the disease activity, the more amino acid metabolic processes were affected.

Conclusions: We found the SF metabolic alterations in RA patients according to disease activity by using GC/TOF MS and identified 12 candidate metabolic biomarkers that may well reflect the disease activity of RA. SF metabolomic approaches based on GC/TOF MS might provide additional information relating to monitoring disease activity in RA.
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September 2020

Comprehensive genomic and transcriptomic analysis of polycyclic aromatic hydrocarbon degradation by a mycoremediation fungus, Dentipellis sp. KUC8613.

Appl Microbiol Biotechnol 2019 Oct 3;103(19):8145-8155. Epub 2019 Sep 3.

Department of Biotechnology, College of Life Sciences and Biotechnology and Graduate School, Korea University, Seoul, 02841, South Korea.

The environmental accumulation of polycyclic aromatic hydrocarbons (PAHs) is of great concern due to potential carcinogenic and mutagenic risks, as well as their resistance to remediation. While many fungi have been reported to break down PAHs in environments, the details of gene-based metabolic pathways are not yet comprehensively understood. Specifically, the genome-scale transcriptional responses of fungal PAH degradation have rarely been reported. In this study, we report the genomic and transcriptomic basis of PAH bioremediation by a potent fungal degrader, Dentipellis sp. KUC8613. The genome size of this fungus was 36.71 Mbp long encoding 14,320 putative protein-coding genes. The strain efficiently removed more than 90% of 100 mg/l concentration of PAHs within 10 days. The genomic and transcriptomic analysis of this white rot fungus highlights that the strain primarily utilized non-ligninolytic enzymes to remove various PAHs, rather than typical ligninolytic enzymes known for playing important roles in PAH degradation. PAH removal by non-ligninolytic enzymes was initiated by both different PAH-specific and common upregulation of P450s, followed by downstream PAH-transforming enzymes such as epoxide hydrolases, dehydrogenases, FAD-dependent monooxygenases, dioxygenases, and glycosyl- or glutathione transferases. Among the various PAHs, phenanthrene induced a more dynamic transcriptomic response possibly due to its greater cytotoxicity, leading to highly upregulated genes involved in the translocation of PAHs, a defense system against reactive oxygen species, and ATP synthesis. Our genomic and transcriptomic data provide a foundation of understanding regarding the mycoremediation of PAHs and the application of this strain for polluted environments.
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http://dx.doi.org/10.1007/s00253-019-10089-6DOI Listing
October 2019

Redirection of the Glycolytic Flux Enhances Isoprenoid Production in Saccharomyces cerevisiae.

Biotechnol J 2020 Feb 17;15(2):e1900173. Epub 2019 Sep 17.

Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA.

Sufficient supply of reduced nicotinamide adenine dinucleotide phosphate (NADPH) is a prerequisite of the overproduction of isoprenoids and related bioproducts in Saccharomyces cerevisiae. Although S. cerevisiae highly depends on the oxidative pentose phosphate (PP) pathway to produce NADPH, its metabolic flux toward the oxidative PP pathway is limited due to the rigid glycolysis flux. To maximize NADPH supply for the isoprenoid production in yeast, upper glycolytic metabolic fluxes are reduced by introducing mutations into phosphofructokinase (PFK) along with overexpression of ZWF1 encoding glucose-6-phosphate (G6P) dehydrogenase. The PFK mutations (Pfk1 S724D and Pfk2 S718D) result in less glycerol production and more accumulation of G6P, which is a gateway metabolite toward the oxidative PP pathway. When combined with the PFK mutations, overexpression of ZWF1 caused substantial increases of [NADPH]/[NADP ] ratios whereas the effect of ZWF1 overexpression alone in the wild-type strain is not noticeable. Also, the introduction of ZWF1 overexpression and the PFK mutations into engineered yeast overexpressing acetyl-CoA C-acetyltransferase (ERG10), truncated HMG-CoA reductase isozyme 1 (tHMG1), and amorphadiene synthase (ADS) leads to a titer of 497 mg L of amorphadiene (3.7-fold over the parental strain). These results suggest that perturbation of upper glycolytic fluxes, in addition to ZWF1 overexpression, is necessary for efficient NADPH supply through the oxidative PP pathway and enhanced production of isoprenoids by engineered S. cerevisiae.
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http://dx.doi.org/10.1002/biot.201900173DOI Listing
February 2020

Metabolomic Analysis Identifies Alterations of Amino Acid Metabolome Signatures in the Postmortem Brain of Alzheimer's Disease.

Exp Neurobiol 2019 Jun 26;28(3):376-389. Epub 2019 Jun 26.

MolecularRecognition Research Center, Korea Institute of Science and Technology, Seoul 02792, Korea.

Despite significant advances in neuroscience research over the past several decades, the exact cause of AD has not yet fully understood. The metabolic hypothesis as well as the amyloid and tau hypotheses have been proposed to be associated with AD pathogenesis. In order to identify metabolome signatures from the postmortem brains of sporadic AD patients and control subjects, we performed ultra performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometer (UPLC-LTQ-Orbitrap-MS). Not only our study identified new metabolome signatures but also verified previously known metabolome profiles in the brain. Statistical modeling of the analytical data and validation of the structural assignments discovered metabolic biomarkers associated with the AD pathogenesis. Interestingly, hypotaurin, myo-inositol and oxo-proline levels were markedly elevated in AD while lutamate and N-acetyl-aspartate were decreased in the postmortem brain tissue of AD patients. In addition, neurosteroid level such as cortisol was significantly increased in AD. Together, our data indicate that impaired amino acid metabolism is associated with AD pathogenesis and the altered amino acid signatures can be useful diagnostic biomarkers of AD. Thus, modulation of amino acid metabolism may be a possible therapeutic approach to treat AD.
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http://dx.doi.org/10.5607/en.2019.28.3.376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6614073PMC
June 2019

Anticariogenic Activity of Agarobiose and Agarooligosaccharides Derived from Red Macroalgae.

J Agric Food Chem 2019 Jul 24;67(26):7297-7303. Epub 2019 Jun 24.

Department of Biotechnology, Graduate School , Korea University , Seoul 02841 , South Korea.

3,6-Anhydro-l-galactose (AHG) produced from agarose in red macroalgae was recently suggested as an anticariogenic sugar to replace widely used xylitol. However, the multi-step process for obtaining monomeric sugar AHG from agarose may be expensive. Generally, it is easier to obtain oligosaccharides than monosaccharides from polysaccharides. Therefore, a one-step process to obtain agarobiose (AB) from agarose was recently developed, and here, we suggest AB as a new anticariogenic agent, owing to its anticariogenic activity against Streptococcus mutans. Among AHG-containing oligosaccharides, AB, neoagarobiose (NAB), agarooligosaccharides (AOSs), and neoagarooligosaccharides (NAOSs), AB showed higher inhibitory activity than AOSs against the growth and lactic acid production of S. mutans; no such inhibitory activity was observed for NAB and NAOSs. This inhibitory effect of AB was comparable to the previously reported inhibitory activity of AHG against S. mutans. These results suggest that AB, which can be more economically and simply produced than AHG, may serve as an anticariogenic sugar.
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http://dx.doi.org/10.1021/acs.jafc.9b01245DOI Listing
July 2019

Cellulase recycling in high-solids enzymatic hydrolysis of pretreated empty fruit bunches.

Biotechnol Biofuels 2019 6;12:138. Epub 2019 Jun 6.

1Department of Biotechnology, Graduate School, Korea University, Seoul, 02841 South Korea.

Background: The lignocellulosic biomass feedstocks such as empty fruit bunches (EFBs) prove to be potential renewable resources owing to their abundance, low prices, and high carbohydrate contents. Generally, the conversion of lignocellulosic biomass into chemicals, fuels, and materials mainly includes pretreatment, enzymatic hydrolysis, fermentation, and recovery of final products. To increase the economic viability of such processes, the cost of cellulase production and enzymatic hydrolysis should be reduced. For this, recycling cellulase can be considered for reducing the saccharification cost of lignocellulose. In this study, cellulase recycling for high-solids enzymatic hydrolysis (i.e., 20%) was evaluated in saccharification of hydrothermally-pretreated EFBs.

Results: High-solids (20%) enzymatic hydrolysis of hydrothermally-pretreated empty fruit bunches with 40 FPU of Cellic CTec3/g glucan was carried out for cellulase recycling. In the second round of hydrolysis using a recycled enzyme, only 19.3% of glucose yield was obtained. The most important limiting factors for cellulase recycling of this study were identified as the enzyme inhibition by glucose, the loss of enzyme activities, and the non-productive binding of enzymes to insoluble biomass solids. To overcome these limitations, PEG was added prior to the first-round hydrolysis to reduce non-productive enzyme binding, glucose was removed from the enzyme fraction to be reused in the second-round hydrolysis, and EFB solids from the first-round hydrolysis were used in the second-round hydrolysis. These three additional measures with cellulase recycling resulted in a 3.5 times higher glucose yield (i.e., 68.0%) at the second round than that of the control, the second-round hydrolysis with cellulase recycling but without these measures.

Conclusions: Because of the high obstacles found in this  study in achieving high saccharification yields in the high-solids saccharification of high-lignin lignocellulose with cellulase recycling, effective measures for improving enzymatic saccharification yields need to be accompanied with cellulase recycling.
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http://dx.doi.org/10.1186/s13068-019-1476-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6551891PMC
June 2019

Metabolite profile changes and increased antioxidative and antiinflammatory activities of mixed vegetables after fermentation by Lactobacillus plantarum.

PLoS One 2019 22;14(5):e0217180. Epub 2019 May 22.

Department of Biotechnology, Graduate School, Korea University, Seoul, South Korea.

Fermented vegetables have emerged as prebiotics with various health benefits. However, the possible mechanisms behind their health benefits are unclear. To relate the metabolite profile changes in fermented mixed vegetables with associated health benefits of fermented vegetables, we analyzed the metabolite profiles of mixed vegetables, before and after fermentation by Lactobacillus plantarum, using gas chromatography/time-of-flight-mass spectrometry (GC/TOF-MS). To analyze health benefits of fermented vegetables, antioxidative and antiinflammatory activities were measured using RAW 264.7 cells. Among 78 metabolites identified by GC/TOF-MS in this study, those significantly increased after fermentation include antioxidative and/or antiinflammatory agents such as lactate, 3-phennyllactate, indole-3-lactate, β-hydroxybutyrate, γ-aminobutyrate, and glycerol. These metabolites may have been either newly synthesized or depolymerized from high molecular weight polymers from vegetables during fermentation. This is the first metabolomics study to relate metabolite profile changes with increased health benefits of fermented vegetables.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0217180PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6530839PMC
January 2020

Comparative global metabolite profiling of xylose-fermenting Saccharomyces cerevisiae SR8 and Scheffersomyces stipitis.

Appl Microbiol Biotechnol 2019 Jul 19;103(13):5435-5446. Epub 2019 Apr 19.

School of Food Science and Biotechnology, Kyungpook National University, Daegu, Korea.

Bioconversion of lignocellulosic biomass into ethanol requires efficient xylose fermentation. Previously, we developed an engineered Saccharomyces cerevisiae strain, named SR8, through rational and inverse metabolic engineering strategies, thereby improving its xylose fermentation and ethanol production. However, its fermentation characteristics have not yet been fully evaluated. In this study, we investigated the xylose fermentation and metabolic profiles for ethanol production in the SR8 strain compared with native Scheffersomyces stipitis. The SR8 strain showed a higher maximum ethanol titer and xylose consumption rate when cultured with a high concentration of xylose, mixed sugars, and under anaerobic conditions than Sch. stipitis. However, its ethanol productivity was less on 40 g/L xylose as the sole carbon source, mainly due to the formation of xylitol and glycerol. Global metabolite profiling indicated different intracellular production rates of xylulose and glycerol-3-phosphate in the two strains. In addition, compared with Sch. stipitis, SR8 had increased abundances of metabolites from sugar metabolism and decreased abundances of metabolites from energy metabolism and free fatty acids. These results provide insights into how to control and balance redox cofactors for the production of fuels and chemicals from xylose by the engineered S. cerevisiae.
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http://dx.doi.org/10.1007/s00253-019-09829-5DOI Listing
July 2019

Pretreatment and enzymatic saccharification of oak at high solids loadings to obtain high titers and high yields of sugars.

Bioresour Technol 2019 Jul 28;284:391-397. Epub 2019 Mar 28.

Department of Biotechnology, Graduate School, Korea University, Seoul 02841, South Korea. Electronic address:

Production of high-titer sugar from lignocellulose is important in terms of process economics of bio-based product industry. In this study, to obtain high titers and yields of sugars, we combined pretreatment and saccharification steps, both at high solids loadings. First, pretreatment of oak was optimized at a 30% (w/w) solids loading. The whole slurry of the pretreated oak was subjected to a fed-batch saccharification step at the final solids loading of 30%, to minimize loss of fermentable sugars and simplify the processes. As a result, high-titer sugars (157.5 g/L) consisting of 120.2 g/L of glucose and 37.3 g/L of xylose were obtained at 75.9% and 58.6%, respectively, of theoretical maximum yields, based on the initial glucan and xylan contents. Thus, through proper optimization processes of oak, the combination of pretreatment and saccharification at high solids loadings was effective in obtaining both high titers and high yields of sugars from lignocellulose.
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http://dx.doi.org/10.1016/j.biortech.2019.03.134DOI Listing
July 2019

Integrative metabolomics reveals unique metabolic traits in Guillain-Barré Syndrome and its variants.

Sci Rep 2019 01 31;9(1):1077. Epub 2019 Jan 31.

The Department of Bio and Fermentation Convergence Technology, BK21 PLUS Program, Kookmin University, Seoul, 02707, Republic of Korea.

Guillain-Barré syndrome (GBS) is an acute fatal progressive disease caused by autoimmune mechanism mainly affecting peripheral nervous system. Although the syndrome is clinically sub-classified into several variants, specific biomarker and exact pathomechanism of each subtypes are not well elucidated yet. In current study, integrative metabolomic and lipidomic profiles were acquisitioned from cerebrospinal fluid samples of 86 GBS from three variants and 20 disease controls. And the data were systematically compared to our previous result on inflammatory demyelination disorders of central nervous system (IDDs) and healthy controls. Primary metabolite profiles revealed unique metabolic traits in which 9 and 7 compounds were specifically changed in GBS and IDD, respectively. Next, the biomarker panel with 10 primary metabolites showed a fairly good discrimination power among 3 GBS subtypes, healthy controls, and disease controls (AUCs ranged 0.849-0.999). The robustness of the biomarker panel was vigorously validated by multi-step statistical evaluation. Subsequent lipidomics revealed GBS variant-specific alteration where the significant elevations of lyso-phosphatidylcholines and sphingomyelins were unique to AIDP (acute inflammatory demyelinating polyneuropathy) and AMAN (acute motor axonal neuropathy), respectively. And metabolome-wide multivariate correlation analysis identified potential clinical association between GBS disability scale (Hughes score) and CSF lipids (monoacylglycerols, and sphingomyelins). Finally, Bayesian network analysis of covarianced structures of primary metabolites and lipids proposed metabolic hub and potential biochemical linkage associated with the pathology.
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http://dx.doi.org/10.1038/s41598-018-37572-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6355784PMC
January 2019

Metabolomic and Transcriptomic Analyses of for Efficient Fermentation of L-Fucose.

Mar Drugs 2019 Jan 29;17(2). Epub 2019 Jan 29.

Department of Biotechnology, Graduate School, Korea University, Seoul 02841, Korea.

L-Fucose, one of the major monomeric sugars in brown algae, possesses high potential for use in the large-scale production of bio-based products. Although fucose catabolic pathways have been enzymatically evaluated, the effects of fucose as a carbon source on intracellular metabolism in industrial microorganisms such as are still not identified. To elucidate the effects of fucose on cellular metabolism and to find clues for efficient conversion of fucose into bio-based products, comparative metabolomic and transcriptomic analyses were performed on on L-fucose and on D-glucose as a control. When fucose was the carbon source for , integration of the two omics analyses revealed that excess gluconeogenesis and quorum sensing led to severe depletion of ATP, resulting in accumulation and export of fucose extracellularly. Therefore, metabolic engineering and optimization are needed for to more efficiently ferment fucose. This is the first multi-omics study investigating the effects of fucose on cellular metabolism in . These omics data and their biological interpretation could be used to assist metabolic engineering of producing bio-based products using fucose-containing brown macroalgae.
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http://dx.doi.org/10.3390/md17020082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6410053PMC
January 2019

Biosynthetic Routes for Producing Various Fucosyl-Oligosaccharides.

ACS Synth Biol 2019 02 5;8(2):415-424. Epub 2019 Feb 5.

Carl R. Woese Institute for Genomic Biology , University of Illinois at Urbana-Champaign , Urbana , Illinois 61801 , United States.

Fucosyl-oligosaccharides (FOSs) play physiologically important roles as prebiotics, neuronal growth factors, and inhibitors of enteropathogens. However, challenges in designed synthesis and mass production of FOSs hamper their industrial applications. Here, we report flexible biosynthetic routes to produce various FOSs, including unnatural ones, through in vitro enzymatic reactions of various sugar acceptors, such as glucose, cellobiose, and agarobiose, and GDP-l-fucose as the fucose donor by using α1,2-fucosyltransferase (FucT2). Also, the whole-cell conversion for fucosylation of various sugar acceptors by overexpressing the genes associated with GDP-l-fucose production and fucT2 gene in Escherichia coli was demonstrated by producing 17.74 g/L of 2'-fucosylgalactose (2'-FG). Prebiotic effects of 2'-FG were verified on the basis of selective fermentability of 2'-FG by probiotic bifidobacteria. These biosynthetic routes can be used to engineer industrial microorganisms for more economical, more flexible, and safer production of FOSs than chemical synthesis of FOSs.
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http://dx.doi.org/10.1021/acssynbio.8b00436DOI Listing
February 2019

Beneficial Effects of Marine Algae-Derived Carbohydrates for Skin Health.

Mar Drugs 2018 Nov 21;16(11). Epub 2018 Nov 21.

School of Food Science and Biotechnology, Kyungpook National University, Daegu 41566, Korea.

Marine algae are considered to be an abundant sources of bioactive compounds with cosmeceutical potential. Recently, a great deal of interest has focused on the health-promoting effects of marine bioactive compounds. Carbohydrates are the major and abundant constituent of marine algae and have been utilized in cosmetic formulations, as moisturizing and thickening agents for example. In addition, marine carbohydrates have been suggested as promising bioactive biomaterials for their various properties beneficial to skin, including antioxidant, anti-melanogenic and skin anti-aging properties. Therefore, marine algae carbohydrates have potential skin health benefits for value-added cosmeceutical applications. The present review focuses on the various biological capacities and potential skin health benefits of bioactive marine carbohydrates.
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http://dx.doi.org/10.3390/md16110459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6266229PMC
November 2018

Novel Two-Step Process Utilizing a Single Enzyme for the Production of High-Titer 3,6-Anhydro-l-galactose from Agarose Derived from Red Macroalgae.

J Agric Food Chem 2018 Nov 7;66(46):12249-12256. Epub 2018 Nov 7.

Department of Biotechnology, Graduate School , Korea University , Seoul 02841 , South Korea.

3,6-Anhydro-l-galactose (l-AHG), a major component of agarose derived from red macroalgae, has excellent potential for industrial applications based on its physiological activities such as skin whitening, moisturizing, anticariogenicity, and anti-inflammation. However, l-AHG is not yet commercially available due to the complexity, inefficiency, and high cost of the current processes for producing l-AHG. Currently, l-AHG production depends on a multistep process requiring several enzymes. Here, we designed and tested a novel two-step process for obtaining high-titer l-AHG by using a single enzyme. First, to depolymerize agarose preferentially into agarobiose (AB) at a high titer, the agarose prehydrolysis using phosphoric acid as a catalyst was optimized at a 30.7% (w/v) agarose loading, which is the highest agarose or agar loading reported so far. Then AB produced by the prehydrolysis was hydrolyzed into l-AHG and d-galactose (d-Gal) by using a recently discovered enzyme, Bgl1B. We suggest that this simple and efficient process could be a feasible solution for the commercialization and mass production of l-AHG.
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http://dx.doi.org/10.1021/acs.jafc.8b04144DOI Listing
November 2018

Biosynthesis of a Functional Human Milk Oligosaccharide, 2'-Fucosyllactose, and l-Fucose Using Engineered Saccharomyces cerevisiae.

ACS Synth Biol 2018 11 24;7(11):2529-2536. Epub 2018 Oct 24.

Carl R. Woose Institute for Genomic Biology , University of Illinois at Urbana-Champaign , Urbana , Illinois 61801 , United States.

2'-fucosyllactose (2-FL), one of the most abundant human milk oligosaccharides (HMOs), has received much attention due to its health-promoting activities, such as stimulating the growth of beneficial gut microorganisms, inhibiting pathogen infection, and enhancing the host immune system. Consequently, large quantities of 2-FL are on demand for food applications as well as in-depth investigation of its biological properties. Biosynthesis of 2-FL has been attempted primarily in Escherichia coli, which might not be the best option to produce food and cosmetic ingredients due to the presence of endotoxins on the cell surface. In this study, an alternative route to produce 2-FL via a de novo pathway using a food-grade microorganism,  Saccharomyces cerevisiae, has been devised. Specifically, heterologous genes, which are necessary to achieve the production of 2-FL from a mixture of glucose and lactose, were introduced into S. cerevisiae. When the lactose transporter (Lac12), de novo GDP-l-fucose pathway (consisting of GDP-d-mannose-4,6-dehydratase (Gmd) and GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase (WcaG)), and α1,2-fucosyltransferase (FucT2) were introduced, the resulting engineered strain (D452L-gwf) produced 0.51 g/L of 2-FL from a batch fermentation. In addition, 0.41 g/L of l-fucose was produced when α-l-fucosidase was additionally expressed in the 2-FL producing strain (D452L-gwf). To our knowledge, this is the first report of 2-FL and l-fucose production in engineered S. cerevisiae via the de novo pathway. This study provides the possibility of producing HMOs by a food-grade microorganism S. cerevisiae and paves the way for more HMO production in the future.
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http://dx.doi.org/10.1021/acssynbio.8b00134DOI Listing
November 2018

Multi-omic characterization of laboratory-evolved Saccharomyces cerevisiae HJ7-14 with high ability of algae-based ethanol production.

Appl Microbiol Biotechnol 2018 Oct 18;102(20):8989-9002. Epub 2018 Aug 18.

Department of Bio and Fermentation Convergence Technology, and BK21 PLUS Program, Kookmin University, Seoul, 02707, South Korea.

In this study, an evolved Saccharomyces cerevisiae HJ7-14 with high ability of algae-based ethanol production was characterized by multi-omic approaches. Genome sequencing of the HJ7-14 revealed a point mutation in the GAL83 gene (G703A) involved in the catabolite repression as well as the galactose metabolism. Cultural and transcriptional analyses of a S. cerevisiae mutant with chromosomal GAL83(G703A) indicated that the catabolite repression onto the galactose metabolism was considerably relieved in all cell growth stages. Untargeted metabolomic approach revealed that metabolic phenotypes between the control D452-2 and HJ7-14 strains were clearly discriminated in time-dependent manner. Especially in early growth stage at 6 h, the HJ7-14 showed dramatic and coordinated alteration in central carbon and amino acid metabolisms. Through metabolomic re-organization, fold changes in fatty acid metabolism and metabolites related to stress response system were also found upon glucose depletion and active galactose utilization. Multi-omic characterization using genome sequencing, transcription, and metabolome profiling clearly unveiled that the GAL83 gene mutation partially relieved glucose-dependent catabolite repression and allowed the evolved HJ7-14 to efficiently convert algal sugars to ethanol. Our finding could be applicable for engineering of S. cerevisiae able to covert red algal biomass to other biofuels and biochemicals.
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http://dx.doi.org/10.1007/s00253-018-9306-9DOI Listing
October 2018

Production of a human milk oligosaccharide 2'-fucosyllactose by metabolically engineered Saccharomyces cerevisiae.

Microb Cell Fact 2018 Jun 27;17(1):101. Epub 2018 Jun 27.

Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA.

Background: 2'-Fucosyllactose (2-FL), one of the most abundant oligosaccharides in human milk, has potential applications in foods due to its health benefits such as the selective promotion of bifidobacterial growth and the inhibition of pathogenic microbial binding to the human gut. Owing to the limited amounts of 2-FL in human milk, alternative microbial production of 2-FL is considered promising. To date, microbial production of 2-FL has been studied mostly in Escherichia coli. In this study, 2-FL was produced alternatively by using a yeast Saccharomyces cerevisiae, which may have advantages over E. coli.

Results: Fucose and lactose were used as the substrates for the salvage pathway which was constructed with fkp coding for a bifunctional enzyme exhibiting L-fucokinase and guanosine 5'-diphosphate-L-fucose phosphorylase activities, fucT2 coding for α-1,2-fucosyltransferase, and LAC12 coding for lactose permease. Production of 2-FL by the resulting engineered yeast was verified by mass spectrometry. 2-FL titers of 92 and 503 mg/L were achieved from 48-h batch fermentation and 120-h fed-batch fermentation fed with ethanol as a carbon source, respectively.

Conclusions: This is the first report on 2-FL production by using yeast S. cerevisiae. These results suggest that S. cerevisiae can be considered as a host engineered for producing 2-FL via the salvage pathway.
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http://dx.doi.org/10.1186/s12934-018-0947-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6020385PMC
June 2018