Search

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.

The phylum Actinobacteria includes important human pathogens like Mycobacterium tuberculosis and Corynebacterium diphtheriae and renowned producers of secondary metabolites of commercial interest, yet only a small part of its diversity is represented by sequenced genomes. Here, we present 824 actinobacterial isolate genomes in the context of a phylum-wide analysis of 6,700 genomes including public isolates and metagenome-assembled genomes (MAGs). We estimate that only 30%–50% of projected actinobacterial phylogenetic diversity possesses genomic representation via isolates and MAGs. A comparison of gene functions reveals novel determinants of host-microbe interaction as well as environment-specific adaptations such as potential antimicrobial peptides. We identify plasmids and prophages across isolates and uncover extensive prophage diversity structured mainly by host taxonomy. Analysis of >80,000 biosynthetic gene clusters reveals that horizontal gene transfer and gene loss shape secondary metabolite repertoire across taxa. Our observations illustrate the essential role of and need for high-quality isolate genome sequences. The work (proposal DOI[s]: https://doi.org/10.46936/10.25585/60001024; https://doi.org/10.46936/10.25585/60000886; https://doi.org/10.46936/10. 25585/60001401; https://doi.org/10.46936/10.25585/60001087; https:// doi.org/10.46936/10.25585/60001079; https://doi.org/10.46936/10.25585/ 60001044) conducted by the US Department of Energy Joint Genome Institute (https://ror.org/04xm1d337), a DOE Office of Science User Facility, is supported by the Office of Science of the US Department of Energy operated under contract no. DE-AC02-05CH11231. Design and synthesis of peptide constructs were supported by the Biosystems Design program (DOE Office of Science contract DE-SC0018260) and the US Department of Energy Joint Genome Institute (DOE Office of Science contract DEAC02-05CH1123), respectively. Characterization of the peptide was supported by the Secured Biosystem Design project entitled ‘‘Rapid Design and Engineering of Smart and Secure Microbiological Systems’’ (DOE Office of Science contract DE-AC02-05CH1123). We thank the following researchers for their support of this study by providing free use of their public genome data: Kristen De-Angelis, Grace Pold, Mallory Choudoir, Camila Carlos-Shanley, Paul Carini, H. Corby C. Kistler, James Elkins, Javier A. Izquierdo, Dimitris Hatzinikolaou, Daniel Schachtman, Paul R. Jensen, Aindrila Mukhopadhyay, John Vogel, Carolin Frank, Paul M. D’Agostino, Ann M. Hirsch, Satoshi Yuzawa, Regina Lamendella, Bernhard Fuchs, Dale Pelletier, Laila P. Partida-Martinez, Cameron Currie, Seth De-Bolt, Jeff Dangl, David Mead, Susannah Tringe, David A. Baltrus, Seung Bum Kim, Linda Kinkel, Kelly Wrighton, William Mohn, Ludmila Christoserdova, Sarah Lebeis, Janet Janssen, Sandra Baena Garzon, and Nicholas Coleman. Special thanks to Marie Louise Ballon at the JGI Communications and Outreach office for all her help with the graphical abstract and many other details.

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.

Various health beneficial outcomes associated with red seaweeds, especially their polysaccharides, have been claimed, but the molecular pathway of how red seaweed polysaccharides are degraded and utilized by cooperative actions of human gut bacteria has not been elucidated. Here, we investigated the enzymatic and metabolic cooperation between two human gut symbionts, Bacteroides plebeius and Bifidobacterium longum ssp. infantis, with regard to the degradation of agarose, the main carbohydrate of red seaweed. More specifically, B. plebeius initially decomposed agarose into agarotriose by the actions of the enzymes belonging to glycoside hydrolase (GH) families 16 and 117 (i.e., BpGH16A and BpGH117) located in the polysaccharide utilization locus, a specific gene cluster for red seaweed carbohydrates. Then, B. infantis extracted energy from agarotriose by the actions of two agarolytic β-galactosidases (i.e., Bga42A and Bga2A) and produced neoagarobiose. B. plebeius ultimately acted on neoagarobiose by BpGH117, resulting in the production of 3,6-anhydro-l-galactose, a monomeric sugar possessing anti-inflammatory activity. Our discovery of the cooperative actions of the two human gut symbionts on agarose degradation and the identification of the related enzyme genes and metabolic intermediates generated during the metabolic processes provide a molecular basis for agarose degradation by gut bacteria.

α-Neoagarobiose (NAB)/neoagarooligosaccharide (NAO) hydrolase plays an important role as an exo-acting 3,6-anhydro-α-(1,3)-L-galactosidase in agarose utilization. Agarose is an abundant polysaccharide found in red seaweeds, comprising 3,6-anhydro-L-galactose (AHG) and D-galactose residues. Unlike agarose degradation, which has been reported in marine microbes, recent metagenomic analysis of Bacteroides plebeius, a human gut bacterium, revealed the presence of genes encoding enzymes involved in agarose degradation, including α-NAB/NAO hydrolase. Among the agarolytic enzymes, BpGH117 has been partially characterized. Here, we characterized the exo-acting α-NAB/NAO hydrolase BpGH117, originating from B. plebeius. The optimal temperature and pH for His-tagged BpGH117 activity were 35 °C and 9.0, respectively, indicative of its unique origin. His-tagged BpGH117 was thermostable up to 35 °C, and the enzyme activity was maintained at 80% of the initial activity at a pre-incubation temperature of 40 °C for 120 min. Km and Vmax values for NAB were 30.22 mM and 54.84 U/mg, respectively, and kcat/Km was 2.65 s−1 mM−1. These results suggest that His-tagged BpGH117 can be used for producing bioactive products such as AHG and agarotriose from agarose efficiently.

Recently, the utilization of renewable biomass instead of fossil fuels for producing fuels and chemicals is receiving much attention due to the global climate change. Among renewable biomass, marine algae are gaining importance as third generation biomass feedstocks owing to their advantages over lignocellulose. Particularly, red macroalgae have higher carbohydrate contents and simpler carbohydrate compositions than other marine algae. In red macroalgal carbphydrates, 3,6-anhydro-L-galactose (AHG) is the main sugar composing agarose along with D-galactose. However, AHG is not a common sugar and is chemically unstable. Thus, not only AHG but also red macroalgal biomass itself cannot be efficiently converted or utilized. Here, we biologically upgraded AHG to a new platform chemical, its sugar alcohol form, 3,6-anhydro-l-galactitol (AHGol), an anhydrohexitol. To accomplish this, we devised an integrated process encompassing a chemical hydrolysis process for producing agarobiose (AB) from agarose and a biological process for converting AB to AHGol using metabolically engineered Saccharomyces cerevisiae to efficiently produce AHGol from agarose with high titers and yields. AHGol was also converted to an intermediate chemical for plastics, isosorbide. To our knowledge, this is the first demonstration of upgrading a red macroalgal biomass component to a platform chemical via a new biological route, by using an engineered microorganism.

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

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.

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.

Vibrio 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 VejGH50A, VejGH50B, VejGH50C, and VejGH50D and the α-neoagarooligosaccharide (NAOS) hydrolase VejGH117, 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, VejGH50A, VejGH50B, and VejGH50C 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 VejGH50D and the α-NAOS hydrolase VejGH117. Second, via the newly found alternative agarolytic pathway, NeoDP4 was depolymerized into AHG and agarotriose (AgaDP3) by VejGH117, and AgaDP3 then was completely depolymerized into monomeric sugars by sequential reactions of the agarolytic β-galactosidases (ABG) VejABG and VejGH117. 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.IMPORTANCE 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, Vibrio 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.

Background Understanding the global metabolic network, significantly perturbed upon promiscuous activities of foreign enzymes and different carbon sources, is crucial for systematic optimization of metabolic engineering of yeast Saccharomyces cerevisiae. Here, we studied the effects of promiscuous activities of overexpressed enzymes encoded by foreign genes on rerouting of metabolic fluxes of an engineered yeast capable of assimilating sugars from renewable biomass by profiling intracellular and extracellular metabolites. Results Unbiased metabolite profiling of the engineered S. cerevisiae strain EJ4 revealed promiscuous enzymatic activities of xylose reductase and xylitol dehydrogenase on galactose and galactitol, respectively, resulting in accumulation of galactitol and tagatose during galactose fermentation. Moreover, during glucose fermentation, a trisaccharide consisting of glucose accumulated outside of the cells probably owing to the promiscuous and transglycosylation activity of β-glucosidase expressed for hydrolyzing cellobiose. Meanwhile, higher accumulation of fatty acids and secondary metabolites was observed during xylose and cellobiose fermentations, respectively. Conclusions The heterologous enzymes functionally expressed in S. cerevisiae showed promiscuous activities that led to unintended metabolic rerouting in strain EJ4. Such metabolic rerouting could result in a low yield and productivity of a final product due to the formation of unexpected metabolites. Furthermore, the global metabolic network can be significantly regulated by carbon sources, thus yielding different patterns of metabolite production. This metabolomic study can provide useful information for yeast strain improvement and systematic optimization of yeast metabolism to manufacture bio-based products.

Marine red macroalgae have received much attention as sustainable resources for producing bio-based products. Therefore, understanding the metabolic pathways of carbohydrates from red macroalgae, in fermentative microorganisms, is crucial for efficient bioconversion of the carbohydrates into bio-based products. Recently, the novel catabolic pathway of 3,6-anhydro-l-galactose (AHG), the main component of red macroalgae, was discovered in a marine bacterium, Vibrio sp. strain EJY3. However, the global metabolic network in response to AHG remains unclear. Here, the intracellular metabolites of EJY3 grown on AHG, glucose, or galactose were comparatively profiled using gas chromatography/time-of-flight mass spectrometry. The global metabolite profiling results revealed that the metabolic profile for AHG significantly differed from those for other common sugars. Specifically, the metabolic intermediate of the AHG pathway, 3,6-anhydrogalactonate, was detected during growth only in the presence of AHG; thus, the recently discovered key steps in AHG catabolism was found not to occur in the catabolism of other common sugars. Moreover, the levels of metabolic intermediates related to glycerolipid metabolism and valine biosynthesis were higher with AHG than those with other sugars. These comprehensive metabolomic analytical results for AHG in this marine bacterium can be used as the basis for having fermentative microbial strains to engineered to efficiently utilize AHG from macroalgal biomass.

3,6-Anhydro- l -galactose (AHG) is a rare sugar found in red macroalgae. The key metabolic steps in AHG catabolism involve its oxidation into 3,6-anhydrogalactonate (AHGA), followed by cycloisomerization of AHGA into 2-keto-3-deoxy-galactonate. These steps were recently discovered in a marine bacterium Vibrio sp. strain EJY3. In this study, we characterized NAD(P)+-dependent AHG dehydrogenase (AHGD) involved in the first step of AHG catabolism. AHGD displayed high substrate specificity for AHG, but showed no catalytic activity toward other aldehyde sugars, including D-form of AHG, glucose, and galactose. This high substrate specificity of AHGD may be associated with the unique chemical structure of its substrate AHG. Unlike other common aldehyde sugars, AHG mainly exists in its hydrated form under aqueous conditions. Growth of EJY3 in presence of AHG, agar, and Gelidium amansii but glucose, as the sole carbon source resulted in a significant increase in the AHGD activity of cell-free EJY3 lysates. Amino acid sequence analysis revealed that AHGD is highly homologous to other aldehyde dehydrogenases from agar-degrading bacteria, suggestive of its key role in agar-related metabolism in marine bacteria utilizing red macroalgae. Therefore, AHGD may serve as an important enzyme involved in the bioconversion of red macroalgal biomass to value-added chemicals.

Agar is a major cell wall carbohydrate of red macroalgae (Rhodophyta). Sugars derived from agar, such as agarooligosaccharides (AOSs), neoagarooligosaccharides (NAOSs), neoagarobiose (NAB), and 3,6-anhydro-L-galactose (L-AHG), possess various physiological activities. These agar-derived sugars can be produced by hydrolysis using chemicals or agarolytic enzymes. Despite the industrial potential of agar-derived sugars, their application has been hampered mainly due to the absence of efficient processes for the liquefaction and saccharification of agar. In this review, we have focused on strategies for producing high value-added sugars from agarose via chemical or enzymatic liquefaction and enzymatic saccharification. The liquefaction of agarose is a key step for preventing gelling and increasing the solubility of agarose in water by prehydrolyzing agarose into AOSs or NAOSs. For the industrial use of agar-derived sugars, AOS, NAOS, NAB, and L-AHG can be used as functional biomaterials owing to their physiological activities such as antiinflammation, skin whitening, and moisturizing. Recently, it was reported that AHG could be considered as a new anticariogenic sugar to replace xylitol. This review provides a comprehensive overview of processes for the hydrolysis of agar or agarose to produce high value-added sugars and the industrial application of these sugars.

Vibrio 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 VejGH50A, VejGH50B, VejGH50C, and VejGH50D and the α-neoagarooligosaccharide (NAOS) hydrolase VejGH117, 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, VejGH50A, VejGH50B, and VejGH50C 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 VejGH50D and the α-NAOS hydrolase VejGH117. Second, via the newly found alternative agarolytic pathway, NeoDP4 was depolymerized into AHG and agarotriose (AgaDP3) by VejGH117, and AgaDP3 then was completely depolymerized into monomeric sugars by sequential reactions of the agarolytic β-galactosidases (ABG) VejABG and VejGH117. 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. IMPORTANCE 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, Vibrio 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.

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.

Numerous health benefits of diets containing red seaweeds or agar-derived sugar mixtures produced by enzymatic or acid hydrolysis of agar have been reported. However, among various agar-derived sugars, the key components that confer health-beneficial effects, such as prebiotic and anti-colon cancer activities, remain unclear. Here, we prepared various agar-derived sugars by multiple enzymatic reactions using an endo-type and an exo-type of β-agarase and a neoagarobiose hydrolase and tested their in vitro prebiotic and anti-colon cancer activities. Among various agar-derived sugars, agarotriose exhibited prebiotic activity that was verified based on the fermentability of agarotriose by probiotic bifidobacteria. Furthermore, we demonstrated the anti-colon cancer activity of 3,6-anhydro-l-galactose, which significantly inhibited the proliferation of human colon cancer cells and induced their apoptosis. Our results provide crucial information regarding the key compounds derived from red seaweeds that confer beneficial health effects, including prebiotic and anti-colon cancer activities, to the host.

Highly porous and monodispersed TiO2 spheres (SPs) with diameters in the range of 100–200 nm, notably smaller than the conventional SPs reported thus far, were selectively controlled, and found to be an outstanding building block for the mesoporous TiO2 layer in dye-sensitized solar cells (DSCs). The prepared TiO2 films employing these SPs are highly transparent, and the fabricated DSCs exhibit significantly enhanced photovoltaic performances. Particularly, the 100 nm-sized TiO2 SP (SP100) provides a photovoltaic conversion efficiency (PCE) of 10.66% with a JSC of 17.98 mA/cm2, VOC of 808 mV, and FF of 0.734, which is higher than the PCE of DSCs with the typical 20 nm-sized TiO2 nanoparticle (NP20) or conventional 250 nm-sized SP (SP250). Transient photoelectron spectroscopic analyses revealed that both the electron diffusion coefficient and electron lifetime of the TiO2 layer increase as the size of the SPs decreases. The tiny TiO2 SPs prepared in this work offer distinct advantages in electron transport as well as dye-soaking and electrolyte diffusion.

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.

In thin film solid-state heterojunction solar cells (HSCs), titanium-dioxide (TiO2) electrodes need to be optimized to have large specific surface area, controllable pore sizes, and superior light scattering properties. In this study, we synthesize hierarchical nanoporous TiO2 beads with sub-micron diameters by a template-free, fast, and low-temperature synthetic scheme to satisfy the aforementioned requirements for HSCs. These nanoporous TiO2 beads are composed of numerous TiO2 nano crystallites that provide mesopores, and the inter-particle distances of size-controlled TiO2 beads can provide additional controllable macropores. We report the first successful application of TiO2 bead films (SP250, SP450) with controllable hierarchical nanostructure to be sensitized with Sb2S3 for all-solid-state heterojunction solar cells (Sb-HSCs). The Sb-HSCs made using the controlled TiO2 beads as photoanodes exhibit a superior light-to electricity conversion efficiency of 4.8%, yielding more than 15% enhancement in comparison with that (3.6%) of commercial TiO2 nanoparticle (NP40) electrodes. The well-tailored photoanode with high surface area, fewer grain boundaries, multi-scale pore structure, and enhanced optical scattering results in much better infiltration of hole-conducting materials, decreased recombination with increased electron lifetime, and enhanced light scattering, which result in the enhanced photovoltaic properties.

Highly ordered, vertically aligned, one side-opened, and regularly porous anatase TiO2 nanotube arrays (TNAs) have been facilely grown by anodizing Ti foil in mixed viscous solvents of ethylene glycol (EG) and glycerol. By changing the volume ratio of two solvents, we have controlled the structural properties of TNAs such as tube diameters, wall thicknesses, and tube lengths. Our prepared TNAs have been found to have enhanced (004) planes, which are reactive in catalysis reactions. We have demonstrated that TNAs grown in 2:1 (v/v) EG and glycerol have the lowest band-gap energy and the largest mean crystallite diameter. TNAs grown on Ti foil have been directly employed for photocatalytic materials and the working electrode of photovoltaic dye-sensitized solar cells. Among our prepared samples, TNAs grown in 2:1 (v/v) EG and glycerol have shown the best photocatalytic activity for the degradation of methylene blue and the highest photovoltaic conversion efficiency of 4.08 %.

To fabricate flexible dye-sensitized solar cells (DSCs) utilizing full solar spectrum, the double-layered TiO 2 films, selectively loading two different dyes in discrete layers, were formed on a plastic substrate by transferring the high-temperature-processed N719/TiO 2 over an organic dye (TA-St-CA)-sensitized TiO 2 film by a typical compression process at room temperature. It was found that interface control between two TiO 2 layers is crucial for the efficient transport of photo-injected electrons from the N719/TiO 2 to the TA-St-CA/TiO 2 layer. Electron impedance spectra (EIS) and transient photoelectron spectroscopic analyses exhibited that introduction of a thin interfacial TiO 2 layer between the two TiO 2 layers remarkably decreased the resistance at the interface, while increasing the electron diffusion constant ( D e ) by ∼10 times. As a result, the photovoltaic conversion efficiency ( η ) of the flexible tandem DSC was 6.64%, whereas that of the flexible cell derived from the single TA-St-CA/TiO 2 layer was only 2.98%. Another organic dye (HC-acid), absorbing a short wavelength region of solar spectrum, was also applied to fabricate flexible tandem DSC. The η of the cell derived from the HC-acid/TiO 2 layer was only 2.91%, but that of the tandem cell obtained by transferring the high-temperature-processed N719/TiO 2 was 6.86%.

Transparent TiO 2 films in various thicknesses were prepared by sol‐gel and MOCVD method, respectively, and their photocatalytic activities in decomposing gaseous 2‐propanol were evaluated. The surfaces and grain structures of the prepared films were characterized by FESEM, XRD, and AFM. It was found that the photocatalytic activities of TiO 2 films were greatly dependent on the film thickness and surface roughness: The photocatalytic activity increases with the increase of film thickness, while it decreases with the increase of surface roughness. We have proposed that these phenomena originate from the transfer of photogenerated electron and hole pairs from the bulk to the surface of TiO 2 film. Several experimental evidences supporting this mechanism have also been provided. INTRODUCTION TiO 2 photocatalysts in thin film form have promising industrial applications for the elimination of organic pollutants in aqueous solution or in gas phase, and they are also well‐known as self‐cleaning and super‐hydrophilic smart materials working under an irradiation of light.

The main carbohydrate of red macroalgae is agarose, a heterogeneous polysaccharide composed of D-galactose and 3,6-anhydro-L-galactose. When saccharifying agarose by enzymes, the unique physical properties of agarose, namely the sol-gel transition and the near-insolubility of agarose in water, limit the accessibility of agarose to the enzymes. Due to the lower accessibility of agarose to enzymes in the gel state than to the sol state, it is important to prevent the sol-gel transition by performing the enzymatic liquefaction of agarose at a temperature higher than the sol-gel transition temperature of agarose. In this study, a thermostable endo-type β-agarase, Aga16B, originating from Saccharophagus degradans 2-40T, was characterized and introduced in the liquefaction process. Aga16B was thermostable up to 50 °C and depolymerized agarose mainly into neoagarooligosaccharides with degrees of polymerization 4 and 6. Aga16B was applied to enzymatic liquefaction of agarose at 45 °C, which was above the sol-gel transition temperature of 1 % (w/v) agarose (∼35 °C) when cooling agarose. This is the first systematic demonstration of enzymatic liquefaction of agarose, enabled by determining the sol-gel temperature of agarose under specific conditions and by characterizing the thermostability of an endo-type β-agarase.

Red macroalgae are currently considered as renewable resources owing to their high carbohydrate and low lignin and hemicellulose contents. However, utilization of red macroalgae has been limited owing to the lack of established methods for pretreatment and an effective saccharification system. Furthermore, marine red macroalgae consist of the non-favorable mixed sugars for industrial microorganisms. In this review, we suggest strategies for converting red macroalgae to bio-based products, focusing on the pretreatment and saccharification of red macroalgae to produce fermentable sugars and the microbial fermentation of these sugars by industrial microorganisms. In particular, some recent breakthroughs for the efficient utilization of red macroalgae include the discovery of key enzymes for the complete monomerization of red macroalgal carbohydrate and the catabolic pathway of 3,6-anhydro-l-galactose, the most abundant sugar in red macroalgae. This review provides a comprehensive perspective for the efficient utilization of red macroalgae as sustainable resources to produce bio-based products.