Display of both N- and C-terminal target fusion proteins on the Aspergillus oryzae cell surface using a chitin-binding module

A novel cell surface display system in Aspergillus oryzae was established by using a chitin-binding module (CBM) from Saccharomyces cerevisiae as an anchor protein. CBM was fused to the N or C terminus of green fluorescent protein (GFP) and the fusion proteins (GFP-CBM and CBM-GFP) were expressed using A. oryzae as a host. Western blotting and fluorescence microscopy analysis showed that both GFP-CBM and CBM-GFP were successfully expressed on the cell surface. In addition, cell surface display of triacylglycerol lipase from A. oryzae (tglA), while retaining its activity, was also successfully demonstrated using CBM as an anchor protein. The activity of tglA was significantly higher when tglA was fused to the C terminus than N terminus of CBM. Together, these results show that CBM used as a first anchor protein enables the fusion of both the N and/or C terminus of a target protein

Direct IBE fermentation from mandarin orange wastes by combination of Clostridium cellulovorans and Clostridium beijerinckii

Abstract For a resolution of reducing carbon dioxide emission and increasing food production to respond to the growth of global population, production of biofuels from non-edible biomass is urgently required. Abundant orange wastes, such as peel and strained lees, are produced as by-product of orange juice, which is available non-edible biomass. However, d-limonene included in citrus fruits often inhibits yeast growth and makes the ethanol fermentation difficult. This study demonstrated that isopropanol-butanol-ethanol fermentation ability of Clostridium beijerinckii and cellulosic biomass degrading ability of C. cellulovorans were cultivated under several concentrations of limonene. As a result, C. cellulovorans was able to grow even in the medium containing 0.05% limonene (v/v) and degraded 85% of total sugar from mandarin peel and strained lees without any pretreatments. More interestingly, C. beijerinckii produced 0.046 g butanol per 1 g of dried strained lees in the culture supernatant together with C. cellulovorans

Biomethane production from sugar beet pulp under cocultivation with Clostridium cellulovorans and methanogens

Abstract This study was demonstrated with a coculture fermentation system using sugar beet pulp (SBP) as a carbon source combining the cellulose-degrading bacterium Clostridium cellulovorans with microbial flora of methane production (MFMP) for the direct conversion of cellulosic biomass to methane (CH4). The MFMP was taken from a commercial methane fermentation plant and extremely complicated. Therefore, the MFMP was analyzed by a next-generation sequencing system and the microbiome was identified and classified based on several computer programs. As a result, Methanosarcina mazei (1.34% of total counts) and the other methanogens were found in the MFMP. Interestingly, the simultaneous utilization of hydrogen (H2) and carbon dioxide (CO2) for methanogenesis was observed in the coculture with Consortium of C. cellulovorans with the MFMP (CCeM) including M. mazei. Furthermore, the CCeM degraded 87.3% of SBP without any pretreatment and produced 34.0 L of CH4 per 1 kg of dry weight of SBP. Thus, a gas metabolic shift in the fermentation pattern of C. cellulovorans was observed in the CCeM coculture. These results indicated that degradation of agricultural wastes was able to be carried out simultaneously with CH4 production by C. cellulovorans and the MFMP

Comparison of Different Carbon Sources on Biomethane Production with <i>Clostridium cellulovorans</i> and Methanogens

Methane (CH4) has attracted attention as not only one of the hydrogen carriers in terms of energy density, but also synthetic natural gas. In nature, the decomposition of organic compounds is performed with bacterial ecosystems that can produce CH4. Clostridium cellulovorans as a decomposer was cultivated with pig manure (PM) as an unused biomass in this study. As a result of high-performance liquid chromatography (HPLC) analysis, while formate and lactate were decreased in the C. cellulovorans medium containing 0.5% PM, acetate and butyrate were increased in it. Accordingly, in order to compare with the effect of carbon sources for methane production, the cocultivation of C. cellulovorans and the methanogenesis of Methanosarcina mazei or microbial flora of methane production (MFMP) was carried out in the C. cellulovorans medium. As a result, only the cocultivation with C. cellulovorans and MFMP showed methane production in 0.5% acetate medium. Moreover, in comparison with a carbon source in either 1% acetate or 1% methanol medium, MFMP was only cultivated after being precultivated with 0.5% glucose medium for 12 h. The results revealed that MFMP with a 1% methanol medium produced methane approximately eight times higher than with 1% acetate medium. After cultivation with 1% acetate or 1% methanol, next-generation sequencing (NGS) analysis of MFMP was carried out. Interestingly, Methanofollis (0.211%), belonging to methanogens through the CO2 reduction pathway, was dominant in the 1% acetate medium for 72 h cultivation, while Methanosarcina siciliae (1.178%), M. barkeri (0.571%), and Methanofollis (0.490%) were major species in 1% methanol medium for 72 h cultivation. Since Methanosarcina spp. belong to acetoclasts (acetoclastic pathway), methanol could promote the growth of Methanosarcina spp., rather than acetate. Therefore, it seems that Methanosarcina spp. may play a key methanogenesis role in MFMP. Thus, these results will provide important information for low-cost biomethane production

Enzymatic Characterization of Unused Biomass Degradation Using the Clostridium cellulovorans Cellulosome

The cellulolytic system of Clostridium cellulovorans mainly consisting of a cellulosome that synergistically collaborates with non-complexed enzymes was investigated using cellulosic biomass. The cellulosomes were isolated from the culture supernatants with shredded paper, rice straw and sugarcane bagasse using crystalline cellulose. Enzyme solutions, including the cellulosome fractions, were analyzed by SDS-PAGE and Western blot using an anti-CbpA antibody. As a result, C. cellulovorans was able to completely degrade shredded paper for 9 days and to be continuously cultivated by the addition of new culture medium containing shredded paper, indicating, through TLC analysis, that its degradative products were glucose and cellobiose. Regarding the rice straw and sugarcane bagasse, while the degradative activity of rice straw was most active using the cellulosome in the culture supernatant of rice straw medium, that of sugarcane bagasse was most active using the cellulosome from the supernatant of cellobiose medium. Based on these results, no alcohols were found when C. acetobutylicum was cultivated in the absence of C. cellulovorans as it cannot degrade the cellulose. While 1.5 mM of ethanol was produced with C. cellulovorans cultivation, both n-butanol (1.67 mM) and ethanol (1.89 mM) were detected with the cocultivation of C. cellulovorans and C. acetobutylicum. Regarding the enzymatic activity evaluation against rice straw and sugarcane bagasse, the rice straw cellulosome fraction was the most active when compared against rice straw. Furthermore, since we attempted to choose reaction conditions more efficiently for the degradation of sugarcane bagasse, a wet jet milling device together with L-cysteine as a reducing agent was used. As a result, we found that the degradation activity was almost twice as high with 10 mM L-cysteine compared with without it. These results will provide new insights for biomass utilization

Novel Carbohydrate-Binding Module of β-1,3-Xylanase from a Marine Bacterium, Alcaligenes sp. Strain XY-234

A β-1,3-xylanase gene (txyA) from a marine bacterium, Alcaligenes sp. strain XY-234, has been cloned and sequenced. txyA consists of a 1,410-bp open reading frame that encodes 469 amino acid residues with a calculated molecular mass of 52,256 Da. The domain structure of the β-1,3-xylanase (TxyA) consists of a signal peptide of 22 amino acid residues, followed by a catalytic domain which belongs to family 26 of the glycosyl hydrolases, a linker region with one array of DGG and six repeats of DNGG, and a novel carbohydrate-binding module (CBM) at the C terminus. The recombinant TxyA hydrolyzed β-1,3-xylan but not other polysaccharides such as β-1,4-xylan, carboxymethylcellulose, curdlan, glucomannan, or β-1,4-mannan. TxyA was capable of binding specifically to β-1,3-xylan. The analysis using truncated TxyA lacking either the N- or C-terminal region indicated that the region encoding the CBM was located between residues 376 and 469. Binding studies on the CBM revealed that the K(d) and the maximum amount of protein bound to β-1,3-xylan were 4.2 μM and 18.2 μmol/g of β-1,3-xylan, respectively. Furthermore, comparison of the enzymatic properties between proteins with and without the CBM strongly indicated that the CBM of TxyA plays an important role in the hydrolysis of β-1,3-xylan

Perturbation by Antimicrobial Bacteria of the Epidermal Bacterial Flora of Rainbow Trout in Flow-Through Aquaculture

The bacterial flora of the epidermal mucus of fish is closely associated with the host&rsquo;s health and susceptibility to pathogenic infections. In this study, we analyzed the epidermal mucus bacteria of rainbow trout (Oncorhynchus mykiss) reared in flow-through aquaculture under environmental perturbations. Over ~2 years, the bacteria present in the skin mucus and water were analyzed based on the 16S rDNA sequences. The composition of the mucus bacterial community showed significant monthly fluctuations, with frequent changes in the dominant bacterial species. Analysis of the beta- and alpha-diversity of the mucus bacterial flora showed the fluctuations of the composition of the flora were caused by the genera Pseudomonas, Yersinia, and Flavobacterium, and some species of Pseudomonas and Yersinia in the mucus were identified as antimicrobial bacteria. Examination of the antimicrobial bacteria in the lab aquarium showed that the natural presence of antimicrobial bacteria in the mucus and water, or the purposeful addition of them to the rearing water, caused a transition in the mucus bacteria community composition. These results demonstrate that specific antimicrobial bacteria in the water or in epidermal mucus comprise one of the causes of changes in fish epidermal mucus microflora