Caffeic acid production by simultaneous saccharification and fermentation of kraft pulp using recombinant Escherichia coli

Caffeic acid (3,4-dihydroxycinnamic acid) serves as a building block for thermoplastics and a precursor for biologically active compounds and was recently produced from glucose by microbial fermentation. To produce caffeic acid from inedible cellulose, separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) reactions were compared using kraft pulp as lignocellulosic feedstock. Here, a tyrosine-overproducing Escherichia coli strain was metabolically engineered to produce caffeic acid from glucose by introducing the genes encoding a 4-hydroxyphenyllactate 3-hydroxylase (hpaBC) from Pseudomonas aeruginosa and tyrosine ammonia lyase (fevV) from Streptomyces sp. WK-5344. Using the resulting recombinant strain, the maximum yield of caffeic acid in SSF (233 mg/L) far exceeded that by SHF (37.9 mg/L). In the SSF with low cellulase loads (≤2.5 filter paper unit/g glucan), caffeic acid production was markedly increased, while almost no glucose accumulation was detected, indicating that the E. coli cells experienced glucose limitation in this culture condition. Caffeic acid yield was also negatively correlated with the glucose concentration in the fermentation medium. In SHF, the formation of by-product acetate and the accumulation of potential fermentation inhibitors increased significantly with kraft pulp hydrolysate than filter paper hydrolysate. The combination of these inhibitors had synergistic effects on caffeic acid fermentation at low concentrations. With lower loads of cellulase in SSF, less potential fermentation inhibitors (furfural, 5-hydroxymethyfurfural, and 4-hydroxylbenzoic acid) accumulated in the medium. These observations suggest that glucose limitation in SSF is crucial for improving caffeic acid yield, owing to reduced by-product formation and fermentation inhibitor accumulation.

     

Occurrence and reproduction of tropical fishes in ocean warming hotspots of Japanese temperate reefs

Reproduction of tropical species beyond their geographic range associated with ocean warming is regarded as the key indicator of a range shift. However, the lack of historical breeding records poses challenges for detecting distinct range shifts of tropical fishes. To obtain baseline data of the current status of the occurrence and breeding activity of tropical pomacentrid and apogonid fishes in ocean warming hotspots of temperate reefs (Kochi and Wakayama, 33°N) of Japan, we conducted a two-year underwater visual survey and synthesized those data with recently published information. By combining data from the present as well as past studies, the results confirmed the occurrence of 52 pomacentrid and 34 apogonid species, whereas breeding activity was confirmed for 19 and 16 species, respectively. Species richness and abundance of recruitment periphery and breeding active species were high at the warmer site adjacent to the Kuroshio Current. Most observed species were found beyond their known geographic range. Some species showing active breeding were widespread tropical fishes (e.g., Amphiprion clarkii, Pomacentrus coelestis and Apogon notatus) and probably have established breeding populations irrespective of recent global warming. The winter sea water temperature around the study sites will continue to rise, increasing by >2 °C by the end of the century; therefore, our results are highly relevant and represent the first step to elucidate the potential range extension of tropical fishes into temperate reefs with climate change.     

Nakijiquinones G-I, new sesquiterpenoid quinones from marine sponge

Three new sesquiterpenoid quinones, nakijiquinones G-I (1-3), containing a different amino group derived from amino acids have been isolated from Okinawan marine sponges of the family Spongiidae, and the structures and relative stereochemistry of 1-3 were elucidated on the basis of the spectral data. Nakijiquinones G-I (1-3) showed modest cytotoxicity and inhibitory activity against HER2 kinase, while nakijiquinone H (2) exhibited antimicrobial activity.     

Recruitment of Tom1L1/Srcasm to endosomes and the midbody by Tsg101

Tom1 (target of Myb 1) and its related proteins (Tom1L1/Srcasm and Tom1L2) constitute a protein family, which share an N-terminal VHS (Vps27, Hrs and STAM) domain and a following GAT (GGA and Tom1) domain. Tom1L1 has potential binding sequences for Tsg101, which is one of key regulators of the multivesicular body (MVB) formation. To obtain a clue to the role of Tom1L1 in the MVB formation, we have characterized the Tom1L1-Tsg101 interaction. We have found that not only the PTAP sequence in the GAT domain but also the PSAP sequence in the C-terminal region of Tom1L1 is responsible for its interaction with the UEV domain of Tsg101 and competes with the HIV-1 Gag protein for the Tsg101 interaction. Furthermore, we show that, by means of Tsg101, Tom1L1 associates with the midbody during cytokinesis as well as endosomes. Taken into account the topological equivalency among the events of the MVB formation, viral egress from the cell, and cytokinesis, the data obtained here suggest that Tom1L1 is implicated in these three distinct cellular processes.     

Novel strategy using an adsorbent-column chromatography for effective ethanol production from sugarcane or sugar beet molasses

Molasses-based distilleries generate large volumes of a highly polluted and dark brown-colored wastewater. The present work describes the way in which an adsorbent-column chromatography can effectively remove the colorant and produce biomass ethanol from sugarcane or sugar beet molasses. It was found that the color and chemical oxygen demand of the resulting wastewater was respectively reduced by approximately 87% and 28% as compared with conventional molasses fermentation. Gas chromatography showed that the decolorized molasses maintained good ethanol productivity almost equal to that of the original molasses. Furthermore, it was revealed that the colorant concentrations of about 5 mg ml⁻¹ in the medium were the most favorable for ethanolic fermentation. In summary, we have concluded that this method is the most effective when the adsorbent chromatography is performed just before molasses fermentation and that the decolorized molasses is an ideal substrate for fuel ethanol production.     

SGT1-HSP90 complex is required for CENP-A deposition at centromeres

The centromere plays an essential role in accurate chromosome segregation, and defects in its function lead to aneuploidy and thus cancer. The centromere-specific histone H3 variant CENP-A is proposed to be the epigenetic mark of the centromere, as active centromeres require CENP-A–containing nucleosomes to direct the recruitment of multiple kinetochore proteins. CENP-A K124 ubiquitylation, mediated by CUL4A-RBX1-COPS8 E3 ligase activity, is required for CENP-A deposition at the centromere. However, the mechanism that controls the E3 ligase activity of the CUL4A-RBX1-COPS8 complex remains obscure. We have discovered that the SGT1-HSP90 complex is required for recognition of CENP-A by COPS8. Thus, the SGT1-HSP90 complex contributes to the E3 ligase activity of the CUL4A complex that is necessary for CENP-A ubiquitylation and CENP-A deposition at the centromere.     

Evaluation of the bitterness of green tea catechins by a cell-based assay with the human bitter taste receptor hTAS2R39

Catechins have a broad range of physiological functions and act as the main taste ingredient of green tea. Although catechins show a strong bitterness, the bitter taste receptor for catechins has not been fully understood. The objective of this study was to identify the receptor for the major green tea catechins such as (−)-epicatechin (EC), (−)-epicatechin gallate (ECg), (−)-epigallocatechin (EGC), and (−)-epigallocatechin gallate (EGCg). By the cell-based assay using cultured cells expressing human bitter taste receptor, a clear response of hTAS2R39-expressing cells was observed to 300 μM of either ECg or EGCg, which elicit a strong bitterness in humans. The response of hTAS2R39-expressing cells to ECg was the strongest among the tested catechins, followed by EGCg. Because the cellular response to EC and EGC is much weaker than those of ECg and EGCg, galloyl groups was strongly supposed to be involved in the bitter intensity. This finding is similar to the observations of taste intensity obtained from a human sensory study. Our results suggest the participation of hTAS2R39 in the detection of catechins in humans, indicating the possibility that bitterness of tea catechins can be evaluated by using cells expressing hTAS2R39.     

Basophil-derived mouse mast cell protease 11 induces microvascular leakage and tissue edema in a mast cell-independent manner

Mouse mast cell protease 11 (mMCP-11) is the most recently identified member of the mouse mast cell tryptase family. This tryptase is preferentially produced by basophils in contrast to other members that are expressed by mast cells but not basophils. Although blood-circulating basophils have long been considered as minor and redundant relatives of tissue-resident mast cells, recent studies illustrated that basophils and mast cells play distinct roles in vivo. To explore the in vivo role of basophil-derived mMCP-11, here we prepared recombinant mMCP-11 and its protease-dead mutant. Subcutaneous injection of the wild-type mMCP-11 but not the mutant induced edematous skin swelling with increased microvascular permeability in a dose-dependent manner. No apparent infiltration of proinflammatory cells including neutrophils and eosinophils was detected in the skin lesions. The cutaneous swelling was abolished by the pretreatment of mice with indomethacin, a cyclooxygenase inhibitor, suggesting the major contribution of prostaglandins to the microvascular leakage. Of note, the cutaneous swelling was elicited even in mast cell-deficient mice, indicating that mast cells are dispensable for the mMCP-11-induced cutaneous swelling. Thus, basophil-derived mMCP-11 can induce microvascular leakage via prostaglandins in a mast cell-independent manner, and may contribute to the development of basophil-mediated inflammatory responses.