Bioenergy and Biorefinery: Feedstock,Biotechnological Conversion,and Products

Biorefinery has been suggested to provide relevant substitutes to a number of fossil products. Feedstocks and conversion technologies have, however, been the bottleneck to the realization of this concept. Herein, feedstocks and bioconversion technologies under biorefinery have been reviewed. Over the last decade, research has shown possibilities of generating tens of new products but only few industrial implementations. This is partly associated with low production yields and poor cost‐competitiveness. This review addresses the technical barriers associated with the conversion of emerging feedstocks into chemicals and bioenergy platforms and summarizes the developed biotechnological approaches including advances in metabolic engineering. This summary further suggests possible future advances that would expand the portfolio of biorefinery and speed up the realization of biofuels and biochemicals.     

Protein–protein interactions and selection: yeast-based approaches that exploit guanine nucleotide-binding protein signaling

For elucidating protein–protein interactions, many methodologies have been developed during the past two decades. For investigation of interactions inside cells under physiological conditions, yeast is an attractive organism with which to quickly screen for hopeful candidates using versatile genetic technologies, and various types of approaches are now available.Among them, a variety of unique systems using the guanine nucleotide-binding protein (G-protein) signaling pathway in yeast have been established to investigate the interactions of proteins for biological study and pharmaceutical research. G-proteins involved in various cellular processes are mainly divided into two groups: small monomeric G-proteins,and heterotrimeric G-proteins. In this minireview, we summarize the basic principles and applications of yeast-based screening systems, using these two types of G-protein, which are typically used for elucidating biological protein interactions but are differentiated from traditional yeast two-hybrid systems.     

Adaptive mutation related to cellulose producibility in <Emphasis Type="Italic">Komagataeibacter medellinensis</Emphasis> (<Emphasis Type="Italic">Gluconacetobacter xylinus</Emphasis>) NBRC 3288

Gluconacetobacter xylinus (formerly Acetobacter xylinum and presently Komagataeibacter medellinensis) is known to produce cellulose as a stable pellicle. However, it is also well known to lose this ability very easily. We investigated the on and off mechanisms of cellulose producibility in two independent cellulose-producing strains, R1 and R2. Both these strains were isolated through a repetitive static culture of a non-cellulose-producing K. medellinensis NBRC 3288 parental strain. Two cellulose synthase operons, types I and II, of this strain are truncated by the frameshift mutation in the bcsBI gene and transposon insertion in the bcsCII gene, respectively. The draft genome sequencing of R1 and R2 strains revealed that in both strains the bcsBI gene was restored by deletion of a nucleotide in its C-rich region. This result suggests that the mutations in the bcsBI gene are responsible for the on and off mechanism of cellulose producibility. When we looked at the genomic DNA sequences of other Komagataeibacter species, several non-cellulose-producing strains were found to contain similar defects in the type I and/or type II cellulose synthase operons. Furthermore, the phylogenetic relationship among cellulose synthase genes conserved in other bacterial species was analyzed. We observed that the cellulose genes in the Komagataeibacter shared sequence similarities with the γ-proteobacterial species but not with the α-proteobacteria and that the type I and type II operons could be diverged from a same ancestor in Komagataeibacter.     

Regulation of 5-aminolevulinic acid-dependent protoporphyrin IX accumulations in human histiocytic lymphoma U937 cells

The aim of the present work is to clarify the mechanism(s) that regulates the accumulation of protoporphyrin IX (PpIX) in human histiocytic lymphoma cell line U937 incubated with 5-aminolevulinic acid (ALA). Biosynthesis and accumulation of PpIX in the cells was determined after incubation with 0.1-5 mM ALA using a flow cytometric technique. The synthesized endogenous PpIX was found to localize predominantly in the mitochondrial region of the cells. The ALA-enhanced PpIX synthesis was suppressed by the presence of either beta-alanine, a competitive inhibitor of beta-transporters on cell membranes, or carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, an uncoupler of mitochondrial oxidative phosphorylation. In contrast, cellular accumulation of PpIX was enhanced by the presence of either deferoxamine (an iron chelater), MnCl2 (a ferrochelatase inhibitor), or Sn-mesoporphyrin (heme oxygenase inhibitor). These results suggest that ALA-enhanced accumulation of PpIX in U937 cells was regulated by cellular uptake and conversion of ALA to PpIX and by degradation of Heme.     

Influence of length on cytotoxicity of multi-walled carbon nanotubes against human acute monocytic leukemia cell line THP-1 in vitro and subcutaneous tissue of rats in vivo

Carbon nanotubes (CNTs) are single- or multi-cylindrical graphene structures that possess diameters of a few nanometers, while the length can be up to a few micrometers. These could have unusual toxicological properties, in that they share intermediate morphological characteristics of both fibers and nanoparticles. To date, no detailed study has been carried out to determine the effect of length on CNT cytotoxicity. In this paper, we investigated the activation of the human acute monocytic leukemia cell line THP-1 in vitro and the response in subcutaneous tissue in vivo to CNTs of different lengths. We used 220 nm and 825 nm-long CNT samples for testing, referred to as "220-CNTs" and "825-CNTs", respectively. 220-CNTs and 825-CNTs induced human monocytes in vitro, although the activity was significantly lower than that of microbial lipopeptide and lipopolysaccharide, and no activity appeared following variation in the length of CNTs. On the other hand, the degree of inflammatory response in subcutaneous tissue in rats around the 220-CNTs was slight in comparison with that around the 825-CNTs. These results indicated that the degree of inflammation around 825-CNTs was stronger than that around 220-CNTs since macrophages could envelop 220-CNTs more readily than 825-CNTs. However, no severe inflammatory response such as necrosis, degeneration or neutrophil infiltration in vivo was observed around both CNTs examined throughout the experimental period.     

Pnlip encoding pancreatic lipase is possible candidate for obesity QTL in the OLETF rat

The Otsuka Long-Evans Tokushima Fatty (OLETF) rat exhibits polygenic obesity, and one of quantitative trait loci (QTLs) responsible for a susceptibility to obesity in the OLETF, Nidd6/of, has been mapped to the approximately 10-cM genomic region between D1Rat166 and D1Rat90 on chromosome 1 in (OLETF x normal) F2 intercross. In this study, we have attempted to identify the causal gene for the Nidd6/of QTL. A Nidd6/of congenic strain, constructed by introgressing the OLETF allele on the mapped Nidd6/of region in the normal F344 rat strain, confirmed the existence of the Nidd6/of as obesity QTL. The Nidd6/of region was refined to a approximately 2.3-cM genomic region between D1Rat225 and D1Rat90, using informative recombinants selected from (Nidd6/of congenic x F344) F1 x Nidd6/of congenic backcross progenies. Among 46 genes located within the approximately 2.3-cM region, pancreatic lipase gene, Pnlip, was regarded as the most prominent and physiologically relevant positional candidate for the Nidd6/of QTL. We found that Pnlip possesses an OLETF allele-specific increase of mRNA levels in the pancreas, and that the OLETF allele is longer in variable number of tandem repeat (VNTR) within the 5'-flanking region than normal alleles. We further showed that the Nidd6/of QTL completely cosegregates with Pnlip VNTR in the informative recombinants from (Nidd6/of congenic x F344) F1 x Nidd6/of congenic backcross progenies. These results suggest that Pnlip is possible candidate for the Nidd6/of QTL.     

Kinetics and mechanism of a reaction catalyzed by PST-01 protease from Pseudomonas aeruginosa PST-01

The initial rates of carboxybenzoyl-alanyl-l-leucyl-amide (Z-L-Ala-L-Leu-NH(2)) synthesis from carboxybenzoyl-L-alanine (Z-L-Ala) and L-leucineamide (L-Leu-NH(2)) and Z-L-Ala-L-Leu-NH(2) hydrolysis in a homogeneous dimethyl sulfoxide-aqueous buffer solution [1:1 (v/v)] system catalyzed by PST-01 protease from Pseudomonas aeruginosa were measured under a wide range of Z-L-Ala, L-Leu-NH(2) and Z-L-Ala-L-Leu-NH(2) concentrations. The initial rates of the synthetic reaction, in which Z-L-Ala-L-Leu-NH(2) was produced from Z-L-Ala and L-Leu-NH(2), were inhibited by the substrates. Furthermore, the initial rates of the synthetic reaction were not inhibited by the product Z-L-Ala-L-Leu-NH(2), and those of the hydrolytic reaction were inhibited by Z-L-Ala and L-Leu-NH(2). All the initial rate data of the synthetic and hydrolytic reactions were well correlated with the rate equation derived based on the proposed reaction scheme.     

Phylogenetic analysis of manganese-oxidizing fungi isolated from manganese-rich aquatic environments in Hokkaido, Japan

Three strains of Mn-oxidizing fungi were isolated from manganese-rich aquatic environments: sediment in a stream (Komanoyu) in Mori-machi and inflow to an artificial wetland in Kaminokuni-cho, Hokkaido, Japan. The characteristics of each strain were then established. Genetic analysis based on the ribosomal RNA (rRNA) gene was performed to clarify their classification. The sequences of the 18S rRNA and internal transcribed spacer (ITS1)-5.8S rRNA-ITS2 genes showed that all three strains are Ascomycetes. Based on its morphology, it seems probable that the KY-1 strain from Mori-machi belongs to the genus Phoma or Ampelomyces. The phylogenetic analysis indicates that this strain belongs to Phoma rather than Ampelomyces. Morphological identification of WL-1 and WL-2 strains from Kaminokuni-cho was impossible because of the lack of a sexual stage and specific organs. Phylogenetic analysis of the sequence in the ITS1-5.8S rRNA-ITS2 gene suggests that the WL-1 strain corresponds to Paraconyothyrium sporulosum and that WL-2 also belongs to the genus Paraconiothyrium. Because the ability to oxidize Mn has not been evaluated for most species of Phoma or Paraconiothyrium (Coniothyrium), further study is needed to confirm the status of these three strains.     

Protein-encapsulated bio-nanocapsules production with ER membrane localization sequences

Bio-nanocapsules (BNCs) are hollow nanoparticles composed of the L protein of hepatitis B virus (HBV) surface antigen (HBsAg), which can specifically introduce genes and drugs into various kinds of target cells. Although the classic electroporation method has typically been used to introduce highly charged molecules such as DNA, it is rarely adopted for proteins due to its very low efficiency. In this study, a novel approach to the preparation of BNC was established whereby a target protein was pre-encapsulated during the course of nanoparticle formation. Briefly, because of the process of BNC formation in a budding manner on the endoplasmic reticulum (ER) membrane, the association of target proteins to the ER membrane with lipidation sequences (ER membrane localization sequences) could directly generate protein-encapsulating BNC in collaboration with co-expression of the L proteins. Since the membrane-localized proteins are automatically enveloped into BNCs during the budding event, this method can be protect the proteins and BNCs from damage caused by electroporation and obviate the need for laborious consideration to study the optimal conditions for protein encapsulation. This approach would be a useful method for encapsulating therapeutic candidate proteins into BNCs.