Eun Ju Yun

The novel catabolic pathway of 3,6‐anhydro‐L‐galactose, the main component of red macroalgae, in a marine bacterium

The catabolic fate of the major monomeric sugar of red macroalgae, 3,6-anhydro-L-galactose (AHG), is completely unknown in any organisms. AHG is not catabolized by ordinary fermentative microorganisms, and it hampers the utilization of red macroalgae as renewable biomass for biofuel and chemical production. In this study, metabolite and transcriptomic analyses of Vibrio sp., a marine bacterium capable of catabolizing AHG as a sole carbon source, revealed two key metabolic intermediates of AHG, 3,6-anhydrogalactonate (AHGA) and 2-keto-3-deoxy-galactonate; the corresponding genes were verified in vitro enzymatic reactions using their recombinant proteins. Oxidation by an NADP(+) -dependent AHG dehydrogenase and isomerization by an AHGA cycloisomerase are the two key AHG metabolic processes. This newly discovered metabolic route was verified in vivo by demonstrating the growth of Escherichia coli harbouring the genes of these two enzymes on AHG as a sole carbon source. Also, the introduction of only these two enzymes into an ethanologenic E. coli strain increased the ethanol production in E. coli by fermenting both AHG and galactose in an agarose hydrolysate. These findings provide not only insights for the evolutionary adaptation of a central metabolic pathway to utilize uncommon substrates in microbes, but also a metabolic design principle for bioconversion of red macroalgal biomass into biofuels or industrial chemicals.

PHO13 Deletion-Induced Transcriptional Activation Prevents Sedoheptulose Accumulation during Xylose Metabolism in Engineered Saccharomyces cerevisiae

The deletion of PHO13 (pho13Δ) in Saccharomyces cerevisiae, encoding a phosphatase enzyme of unknown specificity, results in the transcriptional activation of genes related to the pentose phosphate pathway (PPP) such as TAL1 encoding transaldolase. It has been also reported that the pho13Δ mutant of S. cerevisiae expressing a heterologous xylose pathway can metabolize xylose efficiently compared to its parental strain. However, the interaction between the pho13Δ-induced transcriptional changes and the phenotypes of xylose fermentation was not understood. Thus we investigated the global metabolic changes in response to pho13Δ when cells were exponentially growing on xylose. Among the 134 intracellular metabolites that we identified, the 98% reduction of sedoheptulose was found to be the most significant change in the pho13Δ mutant as compared to its parental strain. Because sedoheptulose-7-phosphate (S7P), a substrate of transaldolase, reduced significantly in the pho13Δ mutant as well, we hypothesized that limited transaldolase activity in the parental strain might cause dephosphorylation of S7P, leading to carbon loss and inefficient xylose metabolism. Mutants overexpressing TAL1 at different degrees were constructed, and their TAL1 expression levels and xylose consumption rates were positively correlated. Moreover, as TAL1 expression levels increased, intracellular sedoheptulose concentration dropped significantly. Therefore, we concluded that TAL1 upregulation, preventing the accumulation of sedoheptulose, is the most critical mechanism for the improved xylose metabolism by the pho13Δ mutant of engineered S. cerevisiae.

Red macroalgae as a sustainable resource for bio-based products

Red macroalgae are being actively investigated as a renewable biomass source because of their advantageous characteristics such as abundant carbohydrate contents, low lignin contents, and the absence of conflicts with food production. With recent technological advances, the efficient utilization of red macroalgae for biofuel and chemical production is now possible.

Pretreatment and saccharification of red macroalgae to produce fermentable sugars

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.

Food metabolomics: from farm to human

Metabolomics, one of the latest components in the suite of systems biology, has been used to understand the metabolism and physiology of living systems, including microorganisms, plants, animals and humans. Food metabolomics can be defined as the application of metabolomics in food systems, including food resources, food processing and diet for humans. The study of food metabolomics has increased gradually in the recent years, because food systems are directly related to nutrition and human health. This review describes the recent trends and applications of metabolomics to food systems, from farm to human, including food resource production, industrial food processing and food intake by humans.

A novel agarolytic β-galactosidase acts on agarooligosaccharides for complete hydrolysis of agarose into monomers.

ABSTRACT Marine red macroalgae have emerged to be renewable biomass for the production of chemicals and biofuels, because carbohydrates that form the major component of red macroalgae can be hydrolyzed into fermentable sugars. The main carbohydrate in red algae is agarose, and it is composed of d-galactose and 3,6-anhydro-l-galactose (AHG), which are alternately bonded by β1-4 and α1-3 linkages. In this study, a novel β-galactosidase that can act on agarooligosaccharides (AOSs) to release galactose was discovered in a marine bacterium (Vibrio sp. strain EJY3); the enzyme is annotated as Vibrio sp. EJY3 agarolytic β-galactosidase (VejABG). Unlike the lacZ-encoded β-galactosidase from Escherichia coli, VejABG does not hydrolyze common substrates like lactose and can act only on the galactose moiety at the nonreducing end of AOS. The optimum pH and temperature of VejABG on an agarotriose substrate were 7 and 35°C, respectively. Its catalytic efficiency with agarotriose was also similar to that with agaropentaose or agaroheptaose. Since agarotriose lingers as the unreacted residual oligomer in the currently available saccharification system using β-agarases and acid prehydrolysis, the agarotriose-hydrolyzing capability of this novel β-galactosidase offers an enormous advantage in the saccharification of agarose or agar in red macroalgae for its use as a biomass feedstock for fermentable sugar production.

High temperature and low acid pretreatment and agarase treatment of agarose for the production of sugar and ethanol from red seaweed biomass

To obtain fermentable sugar from agarose, pretreatment of agarose by using acetic acid was conducted for short durations (10-30 min) at low acid concentrations (1-5% (w/v)) and high temperatures (110-130 °C). On testing the pretreated agarose by using an endo-β-agarase I (DagA), an exo-β-agarase II (Aga50D), and neoagarobiose hydrolase (NABH), we observed that the addition of the endo-type agarase did not increase the sugar yield. Use of the crude enzyme of Vibrio sp. EJY3 in combination with Aga50D and NABH including acetic acid pretreatment resulted in a 1.3-fold increase in the final reducing sugar yield (62.8% of theoretical maximum based on galactose and 3,6-anhydrogalactose in the initial agarose), compared to those obtained using Aga50D and NABH only after acetic acid pretreatment. The simultaneous saccharification and fermentation of pretreated agarose yielded ethanol of 37.1% theoretical maximum yield from galactose contained in the pretreated agarose.

Genome Sequence of Vibrio sp. Strain EJY3, an Agarolytic Marine Bacterium Metabolizing 3,6-Anhydro-l-Galactose as a Sole Carbon Source

The metabolic fate of 3,6-anhydro-L-galactose (L-AHG) is unknown in the global marine carbon cycle. Vibrio sp. strain EJY3 is an agarolytic marine bacterium that can utilize L-AHG as a sole carbon source. To elucidate the metabolic pathways of L-AHG, we have sequenced the complete genome of Vibrio sp. strain EJY3.

Optimal production of 4-deoxy- l -erythro-5-hexoseulose uronic acid from alginate for brown macro algae saccharification by combining endo- and exo-type alginate lyases

Abstract Algae are considered as third-generation biomass, and alginate is the main component of brown macroalgae. Alginate can be enzymatically depolymerized by alginate lyases into uronate monomers, such as mannuronic acid and guluronic acid, which are further nonenzymatically converted to 4-deoxy-l-erythro-5-hexoseulose uronic acid (DEH). We have optimized an enzymatic saccharification process using two recombinant alginate lyases, endo-type Alg7D and exo-type Alg17C, for the efficient production of DEH from alginate. When comparing the sequential and simultaneous additions of Alg7D and Alg17C, it was found that the final yield of DEH was significantly higher when the enzymes were added sequentially. The progress of saccharification reactions and production of DEH were verified by thin layer chromatography and gas chromatography–mass spectrometry, respectively. Our results showed that the two recombinant enzymes could be exploited for the efficient production of DEH that is the key substrate for producing biofuels from brown macro algal biomass.

Linalool is a PPARα ligand that reduces plasma TG levels and rewires the hepatic transcriptome and plasma metabolome

We investigated the hypotriglyceridemic mechanism of action of linalool, an aromatic monoterpene present in teas and fragrant herbs. Reporter gene and time-resolved fluorescence resonance energy transfer assays demonstrated that linalool is a direct ligand of PPARα. Linalool stimulation reduced cellular lipid accumulation regulating PPARα-responsive genes and significantly induced FA oxidation, and its effects were markedly attenuated by silencing PPARα expression. In mice, the oral administration of linalool for 3 weeks reduced plasma TG concentrations in Western-diet-fed C57BL/6J mice (31%, P < 0.05) and human apo E2 mice (50%, P < 0.05) and regulated hepatic PPARα target genes. However, no such effects were seen in PPARα-deficient mice. Transcriptome profiling revealed that linalool stimulation rewired global gene expression in lipid-loaded hepatocytes and that the effects of 1 mM linalool were comparable to those of 0.1 mM fenofibrate. Metabolomic analysis of the mouse plasma revealed that the global metabolite profiles were significantly distinguishable between linalool-fed mice and controls. Notably, the concentrations of saturated FAs were significantly reduced in linalool-fed mice. These findings suggest that the appropriate intake of a natural aromatic compound could exert beneficial metabolic effects by regulating a cellular nutrient sensor.