The Catabolic System of Acetovanillone and Acetosyringone in Sphingobium sp. Strain SYK-6 Useful for Upgrading Aromatic Compounds Obtained through Chemical Lignin Depolymerization.

Acetovanillone is a major aromatic monomer produced in oxidative/base-catalyzed lignin depolymerization. However, the production of chemical products from acetovanillone has not been explored due to the lack of information on the microbial acetovanillone catabolic system. Here, the acvABCDEF genes were identified as specifically induced genes during the growth of Sphingobium sp. strain SYK-6 cells with acetovanillone and these genes were essential for SYK-6 growth on acetovanillone and acetosyringone (a syringyl-type acetophenone derivative). AcvAB and AcvF produced in Escherichia coli phosphorylated acetovanillone/acetosyringone and dephosphorylated the phosphorylated acetovanillone/acetosyringone, respectively. AcvCDE produced in Sphingobium japonicum UT26S carboxylated the reaction products generated from acetovanillone/acetosyringone by AcvAB and AcvF into vanilloyl acetic acid/3-(4-hydroxy-3,5-dimethoxyphenyl)-3-oxopropanoic acid. To demonstrate the feasibility of producing cis,cis-muconic acid from acetovanillone, a metabolic modification on a mutant of Pseudomonas sp. strain NGC7 that accumulates cis,cis-muconic acid from catechol was performed. The resulting strain expressing vceA and vceB required for converting vanilloyl acetic acid to vanillic acid and aroY encoding protocatechuic acid decarboxylase in addition to acvABCDEF successfully converted 1.2 mM acetovanillone to approximately equimolar cis,cis-muconic acid. Our results are expected to help improve the yield and purity of value-added chemical production from lignin through biological funneling. IMPORTANCE In the alkaline oxidation of lignin, aromatic aldehydes (vanillin, syringaldehyde, and p-hydroxybenzaldehyde), aromatic acids (vanillic acid, syringic acid, and p-hydroxybenzoic acid), and acetophenone-related compounds (acetovanillone, acetosyringone, and 4'-hydroxyacetophenone) are produced as major aromatic monomers. Also, base-catalyzed depolymerization of guaiacyl lignin resulted in vanillin, vanillic acid, guaiacol, and acetovanillone as primary aromatic monomers. To date, microbial catabolic systems of vanillin, vanillic acid, and guaiacol have been well characterized, and the production of value-added chemicals from them has also been explored. However, due to the lack of information on the microbial acetovanillone and acetosyringone catabolic system, chemical production from acetovanillone and acetosyringone has not been achieved. This study elucidated the acetovanillone/acetosyringone catabolic system and demonstrates the potential of using these genes for the production of value-added chemicals from these compounds.

Isolation of a novel platform bacterium for lignin valorization and its application in glucose-free cis,cis-muconate production.

Microbial production of cis,cis-muconate (ccMA) from phenolic compounds obtained by chemical depolymerization of lignin is a promising approach to valorize lignin. Because microbial production requires a large amount of carbon and energy source, it is desirable to establish a ccMA-producing strain that utilizes lignin-derived phenols instead of general sources like glucose. We isolated Pseudomonas sp. strain NGC7 that grows well on various phenolic compounds derived from p-hydroxyphenyl, guaiacyl, and syringyl units of lignin. An NGC7 mutant of protocatechuate (PCA) 3,4-dioxygenase and ccMA cycloisomerase genes (NGC703) lost the ability to grow on vanillate and p-hydroxybenzoate but grew normally on syringate. Introduction of a plasmid carrying genes encoding PCA decarboxylase, flavin prenyltransferase, vanillate O-demethylase, and catechol 1,2-dioxygenase into NGC703 enabled production of 3.2 g/L ccMA from vanillate with a yield of 75% while growing on syringate. This strain also produced ccMA from birch lignin-derived phenols. All these results indicate the utility of NGC7 in glucose-free ccMA production.

Heterologous expression of Trametes versicolor laccase in Saccharomyces cerevisiae.

Laccase is used in various industrial fields, and it has been the subject of numerous studies. Trametes versicolor laccase has one of the highest redox potentials among the various forms of this enzyme. In this study, we optimized the expression of laccase in Saccharomyces cerevisiae. Optimizing the culture conditions resulted in an improvement in the expression level, and approximately 45 U/L of laccase was functionally secreted in the culture. The recombinant laccase was found to be a heavily hypermannosylated glycoprotein, and the molecular weight of the carbohydrate chain was approximately 60 kDa. These hypermannosylated glycans lowered the substrate affinity, but the optimum pH and thermo-stability were not changed by these hypermannosylated glycans. This functional expression system described here will aid in molecular evolutionary studies conducted to generate new variants of laccase.

Accumulation of proanthocyanidins and/or lignin deposition in buff-pigmented soybean seed coats may lead to frequent defective cracking.

MAIN CONCLUSION: Defective cracking frequently occurs in buff-pigmented soybean seed coats, where proanthocyanidins accumulate and lignin is deposited, suggesting that proanthocyanidins and/or lignin may change physical properties and lead to defective cracking. In the seed production of many yellow soybean (Glycine max) cultivars, very low percentages of self-pigmented seeds are commonly found. This phenomenon is derived from a recessive mutation of the I gene inhibiting seed coat pigmentation. In Japan, most of these self-pigmented seeds are buff-colored, and frequently show multiple defective cracks in the seed coat. However, it is not known why cracking occurs specifically in buff seed coats. In this study, quantitative analysis was performed between yellow and buff soybean seed coats. Compared with yellow soybeans, in which defective cracking rarely occurs, contents of proanthocyanidins (PAs) and lignin were significantly higher in buff seed coats. Histochemical data of PAs and lignin in the seed coats strongly supported this result. Measurements of the physical properties of seed coats using a texture analyzer showed that a hardness value was significantly decreased in the buff seed coats. These results suggest that PA accumulation and/or lignin deposition may affect the physical properties of buff seed coats and lead to the defective cracking. This work contributes to understanding of the mechanism of defective cracking, which decreases the seed quality of soybean and related legumes.

Expression of a fungal laccase fused with a bacterial cellulose-binding module improves the enzymatic saccharification efficiency of lignocellulose biomass in transgenic Arabidopsis thaliana.

Delignification is effective for improving the saccharification efficiency of lignocellulosic biomass materials. We previously identified that the expression of a fungal laccase (Lac) fused with a bacterial cellulose-binding module domain (CBD) improved the enzymatic saccharification efficiency of rice plants. In this work, to evaluate the ability of the Lac-CBD fused chimeric enzyme to improve saccharification efficiency in a dicot plant, we introduced the chimeric gene into a dicot model plant, Arabidopsis thaliana. Transgenic plants expressing the Lac-CBD chimeric gene showed normal morphology and growth, and showed a significant increase of enzymatic saccharification efficiency compared to control plants. The transgenic plants with the largest improvement of enzymatic saccharification efficiency also showed an increase of crystalline cellulose in their cell wall fractions. These results indicated that expression of the Lac-CBD chimeric protein in dicotyledonous plants improved the enzymatic saccharification of plant biomass by increasing the crystallinity of cellulose in the cell wall.

Enzymatic activity of cell-free extracts from Burkholderia oxyphila OX-01 bio-converts (+)-catechin and (-)-epicatechin to (+)-taxifolin.

This study characterized the enzymatic ability of a cell-free extract from an acidophilic (+)-catechin degrader Burkholderia oxyphila (OX-01). The crude OX-01 extracts were able to transform (+)-catechin and (-)-epicatechin into (+)-taxifolin via a leucocyanidin intermediate in a two-step oxidation. Enzymatic oxidation at the C-4 position was carried out anaerobically using H2O as an oxygen donor. The C-4 oxidation occurred only in the presence of the 2R-catechin stereoisomer, with the C-3 stereoisomer not affecting the reaction. These results suggest that the OX-01 may have evolved to target both (+)-catechin and (-)-epicatechin, which are major structural units in plants.

A novel approach to recycle bacterial culture waste for fermentation reuse via a microbial fuel cell-membrane bioreactor system.

Biochemical production processes require water and nutrient resources for culture media preparation, but aqueous waste is generated after the target products are extracted. In this study, culture waste (including cells) produced from a lab-scale fermenter was fed into a microbial fuel cell-membrane bioreactor (MFC-MBR) system. Electrical energy was generated via the interaction between the microbial consortia and the solid electrode in the MFC. The treated wastewater was reclaimed in this process which was reused as a solvent and a nutrient source in subsequent fermentation. Polarization testing showed that the MFC produced a maximum current density of 37.53 A m(-3) with a maximum power density of 5.49 W m(-3). The MFC was able to generate 0.04 kWh of energy per cubic meter of culture waste treated. The lab-scale fermenters containing pure cultures of an engineered Pseudomonas spp. were used to generate 2-pyrone-4,6-dicarboxylic acid (PDC), a high value platform chemical. When the MFC-MBR-treated wastewater was used for the fermenter culture medium, a specific bacterial growth rate of 1.00 ± 0.05 h(-1) was obtained with a PDC production rate of 708.11 ± 64.70 mg PDC L(-1) h(-1). Comparable values for controls using pure water were 0.95 ± 0.06 h(-1) and 621.01 ± 22.09 mg PDC L(-1) h(-1) (P > 0.05), respectively. The results provide insight on a new approach for more sustainable bio-material production while at the same time generating energy, and suggest that the treated wastewater can be used as a solvent and a nutrient source for the fermentation production of high value platform chemicals.

Enhanced production of reducing sugars from transgenic rice expressing exo-glucanase under the control of a senescence-inducible promoter.

We generated transgenic rice plants that express EXG1 exo-glucanase under the control of a senescence-inducible promoter. When a GUS coding sequence was connected to a promoter region of STAY GREEN (SGR) gene of rice and introduced into rice, GUS activity was specifically observed along with senescence. When an EXG1 cDNA was connected to the SGR promoter and introduced into rice, higher cellulase activities were detected after senescence. The EXG1 transgenic plants showed enhanced enzymatic saccharification efficiencies after senescence, but no significant difference of saccharification efficiencies was observed before senescence. The saccharification efficiencies were correlated with the cellulase activities in the transgenic plants. The EXG1 transgenic plants showed neither morphological abnormality nor sterility, both of which were observed when EXG1 was constitutively overexpressed. These results indicate that expression of cell wall degrading enzymes such as cellulase by a senescence-inducible promoter is one of the ways to enhance the saccharification ability of cellulosic biomass without affecting plant growth for efficient production of biofuels.

Direct catalytic oxidation of rice husk lignin with hydroxide nanorod-modified copper foam and muconate production by engineered Pseudomonas sp. NGC7.

For the direct alkaline oxidation of rice husk lignin, we developed a copper foam-based heterogeneous catalyst that offers advantages in its recovery after the reaction mixture. The depolymerized products were utilized for muconate production by an engineered Pseudomonas sp. NGC7-based strain. A hydroxide nanorod-modified copper foam was prepared by the surface oxidation of copper foam, followed by alkaline oxidation of rice husk lignin over the catalyst. The catalyst was easily separated from the cellulosic residues in the reaction mixture, and the residues were then recovered by filtration. The resulting lignin stream was composed of a variety of aromatic monomers containing p-hydroxyphenyl, guaiacyl, and syringyl compounds. The catabolic activity of Pseudomonas sp. NGC7 was demonstrated to be more suitable for muconate production from a mixture comprising 4-hydroxybenzoate (a typical p-hydroxyphenyl compound), vanillate (a guaiacyl compound), and syringate (a syringyl compound), owing to its natural ability to grow on syringate. Thus, it was applied to produce muconate from a rice husk lignin stream prepared through hydroxide nanorod-modified copper foam-catalyzed alkaline oxidation by conferring the genes responsible for converting the acetophenone derivatives to their corresponding aromatic acids and protocatechuate decarboxylase to an NGC7-based strain deficient in protocatechuate 3,4-dioxygenase and muconate cycloisomerase. As a result, the engineered NGC7-based muconate-producing strain produced muconate selectively from the rice husk lignin stream at 93.7 mol% yield.

Influence of biochar addition on methane metabolism during thermophilic phase of composting.

CH(4) is known to be generated during the most active phase of composting, even in well-managed composting piles. In this manuscript, we studied the influence of biochar on the CH(4) metabolism during composting of cattle manure and local organic wastes. We evaluated the presence of methanogens and methanotrophs in the composting piles quantified by the level of mcrA encoding methyl coenzyme M reductase alpha subunit and pmoA encoding particulate methane monooxygenase. A decrease of methanogens (mcrA) and an increase of methanotrophs (pmoA) were measured in the composting mixture containing biochar during the most active phase of composting. During the thermophilic phase, the mcrA/pmoA ratios obtained in the composting piles with biochar were twofold lower than in the pile without biochar.

Successful selective production of vanillic acid from depolymerized sulfite lignin and its application to poly(ethylene vanillate) synthesis.

Towards lignin upgrading, vanillic acid (VA), a lignin-derived guaiacyl compound, was produced from sulfite lignin for successfully synthesizing poly(ethylene vanillate), an aromatic polymer. The engineered Sphingobium sp. SYK-6-based strain in which the genes responsible for VA/3-O-methyl gallic acid O-demethylase and syringic acid O-demethylase were disrupted was able to produce vanillic acid (VA) from the mixture consisting of acetovanillone, vanillin, VA, and other low-molecular-weight aromatics obtained by Cu(OH)2-catalyzed alkaline depolymerization of sulfite lignin and membrane fractionation. From the bio-based VA, methyl-4-(2-hydroxyethoxy)-3-methoxybenzoate was synthesized via methylesterification, hydroxyethylation, and distillation, and then it was subjected to polymerization catalyzed by titanium tetraisopropoxide. The molecular weight of the obtained poly(ethylene vanillate) was evaluated to be Mw = 13,000 (Mw/Mn = 1.99) and its melting point was 261 °C. The present work proved that poly(ethylene vanillate) is able to be synthesized using VA produced from lignin for the first time.

Pseudomonas sp. NGC7 as a microbial chassis for glucose-free muconate production from a variety of lignin-derived aromatics and its application to the production from sugar cane bagasse alkaline extract.

cis,cis-Muconate (ccMA) is a promising platform for use in synthesizing various polymers. A glucose-free ccMA production using Pseudomonas sp. NGC7 from hardwood lignin-derived aromatic compounds was previously reported. In that system, syringyl nucleus compounds were essential for growth. Here, it is shown that NGC7 is available for glucose-free ccMA production even from a mixture of lignin-derived aromatics that does not contain syringyl nucleus compounds. By introducing a gene set for the protocatechuate (PCA)-shunt consisting of PCA 3,4-dioxygenase and PCA decarboxylase into an NGC7-derived strain deficient in PCA 3,4-dioxygenase and ccMA cycloisomerase, it was succeeded in constructing a ccMA-producing strain that grows on a lignin-derived aromatics mixture containing no syringyl nucleus compounds. Finally, it is demonstrated that the engineered strain produced ccMA from sugar cane bagasse alkaline extract in 18.7 mol%. NGC7 is thus shown to be a promising microbial chassis for biochemicals production from lignin-derived aromatics.

Methoxyl groups of lignin are essential carbon donors in C1 metabolism of Sphingobium sp. SYK-6.

Sphingobium sp. SYK-6 can utilize lignin biphenyl compounds, such as 2,2'-dihydroxy-3,3'-dimethoxy-5,5'-dicarboxybiphenyl (DDVA). In the metabolism of DDVA, this microorganism exploits two O -demethylation systems with different substrate specificities, namely, a DDVA-specific oxygenative O -demethylation system and a vanillate-specific (VA-specific) tetrahydrofolate-dependent (THF-dependent) methyltransferase system. We examined the way in which these two systems interact in the metabolism of lignin in Sphingobium sp. SYK-6. Our results indicate that THF accepts methyl groups derived not only from the THF-dependent O -demethylation of VA but also from the oxygenative O -demethylation of DDVA. Thus, the methoxyl groups of lignin model compounds are an essential source of carbon in Sphingobium sp. SYK-6. ((c) 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).

Influence of biochar addition on the humic substances of composting manures.

Application of biochar (10% v/v) to a manure composting matrix was investigated to evaluate its effect on the chemical composition of humic substances during the composting process. The characteristics of the humic acid (HA) and fulvic acid (FA) fractions were analyzed in compost mixtures originating from two different manures (poultry manure (PM) and cow manure (CM)). The C contents of HA and FA from the manure compost/biochar blends (PM+B and CM+B) were higher than those from PM and CM, with an enhanced recalcitrant fraction, as determined by thermogravimetric analysis. Spectroscopic analysis showed that enrichment of aromatic-C and carboxylic-C occurred in the FA fractions of PM+B and CM+B to a greater extent than in PM and CM. Biochar addition into the composting mixture improved the final compost quality, especially for the light humified fraction (FA).

Beta-ketoadipic acid and muconolactone production from a lignin-related aromatic compound through the protocatechuate 3,4-metabolic pathway.

In this work, the effects of PcaJ (beta-ketoadipate:succinyl-coenzyme A transferase)- and PcaD (beta-ketoadipate enol-lactone hydrolase)-inactivation on protocatechuic acid metabolism in Pseudomonas putida KT2440 were evaluated. Beta-ketoadipic acid was produced from protocatechuic acid by the inactivation of PcaJ as expected; however, a portion of the produced beta-ketoadipic acid was converted to levulinic acid through a purification step consisting of extraction from the culture and recrystallization. On the other hand, muconolactone was purified from the culture of the PcaD-inactivated mutant of KT2440, although beta-ketoadipate enol-lactone was supposed to be produced because it is the substrate of PcaD. Under aerobic conditions, it has been reported that lignin-related aromatics are metabolized through PCA 2,3- or 3,4- or 4,5-ring cleavage pathways, and muconolactone is an intermediate observed in the metabolism of catechol, not protocatechuic acid. Our results will provide a prospective route to produce muconolactone with a high yield through the protocatechuate-3,4-metabolic pathway.

Functional coupling between vanillate-O-demethylase and formaldehyde detoxification pathway.

Pseudomonas putida vanillate-O-demethylase consisting of VanA and VanB was expressed in Escherichia coli strain K-12. Recombinant E. coli strain K-12 cells expressing VanAB efficiently converted vanillate into protocatechuate with glucose consumption. Mutant lacking either pgi or zwf showed higher or lower converting activity than the parental strain, respectively. Formaldehyde, which is the by-product of the demethylation, was converted into formate in the cellular reaction. Formate accumulation was blocked by gene disruption of the E. coli frmA that coded glutathione-dependent formaldehyde dehydrogenase.

Water-insoluble material from apple pomace makes changes in intracellular NAD⁺/NADH ratio and pyrophosphate content and stimulates fermentative production of hydrogen.

Apple pomace is one of the major agricultural residues in Aomori prefecture, Japan, and it would be useful to develop effective applications for it. As apple pomace contains easily fermentable sugars such as glucose, fructose and sucrose, it can be used as a feedstock for the fermentation of fuels and chemicals. We previously isolated a new hydrogen-producing bacterium, Clostridium beijerinckii HU-1, which could produce H2 at a production rate of 14.5 mmol of H2/L/h in a fed-batch culture at 37 °C, pH 6.0. In this work we found that the HU-1 strain produces H2 at an approximately 20% greater rate when the fermentation medium contains the water-insoluble material from apple pomace. The water-insoluble material from apple pomace caused a metabolic shift that stimulated H2 production. HU-1 showed a decrease of lactate production, which consumes NADH, accompanied by an increase of the intracellular pyrophosphate content, which is an inhibitor of lactate dehydrogenase. The intracellular NAD(+)/NADH ratios of HU-1 during H2 fermentation were maintained in a more reductive state than those observed without the addition of the water insoluble material. To correct the abnormal intracellular redox balance, caused by the repression of lactate production, H2 production with NADH oxidation must be stimulated.

Enhancement of protocatechuate decarboxylase activity for the effective production of muconate from lignin-related aromatic compounds.

The decarboxylation reaction of protocatechuate has been described as a bottleneck and a rate-limiting step in cis,cis-muconate (ccMA) bioproduction from renewable feedstocks such as sugar. Because sugars are already in high demand in the development of many bio-based products, our work focuses on improving protocatechuate decarboxylase (Pdc) activity and ccMA production in particular, from lignin-related aromatic compounds. We previously had transformed an Escherichia coli strain using aroY, which had been used as a protocatechuate decarboxylase encoding gene from Klebsiella pneumoniae subsp. pneumoniae A170-40, and inserted other required genes from Pseudomonas putida KT2440, to allow the production of ccMA from vanillin. This recombinant strain produced ccMA from vanillin, however the Pdc reaction step remained a bottleneck during incubation. In the current study, we identify a way to increase protocatechuate decarboxylase activity in E. coli through enzyme production involving both aroY and kpdB; the latter which encodes for the B subunit of 4-hydroxybenzoate decarboxylase. This permits expression of Pdc activity at a level approximately 14-fold greater than the strain with aroY only. The expression level of AroY increased, apparently as a function of the co-expression of AroY and KpdB. Our results also imply that ccMA may inhibit vanillate demethylation, a reaction step that is rate limiting for efficient ccMA production from lignin-related aromatic compounds, so even though ccMA production may be enhanced, other challenges to overcome vanilate demethylation inhibition still remain.

Glycosylation of tyrosinase is a determinant of melanin production in cultured melanoma cells.

The majority of malignant melanoma cell types are able to produce melanin and the degree of melanin synthesis in various types of cultured cell line differs. In this study, we evaluated three types of cultured cell line, MNT­1, HM3KO and G­361, with differing melanin production levels. The level was greatest in the MNT­1 cells, lower in the HM3KO cells and lowest in the G­361 cells. In addition, a positive correlation between melanin production and tyrosinase activity was observed. The molecular masses of tyrosinases from HM3KO and G­361 cells were marginally lower than those from MNT­1 cells. Glycosylation inhibitor treatment on MNT­1 cells caused decreases in the molecular mass of tyrosinase, its activity and melanin production. An immunoprecipitation assay using anti­tyrosinase indicated that the immature glycosylated tyrosinases were associated with a type of chaperone, Hsp70. The interaction between tyrosinase and Hsp70 was also detected in HM3KO and G­361 cells. The results indicated that the immature glycosylation of tyrosinase has a critical effect on the melanin-producing ability of melanoma cells.

Application of fungal laccase fused with cellulose-binding domain to develop low-lignin rice plants.

We observed a reduction of lignin content linked to the expression of fungal laccase in rice plants. The lignin content of L-4, which showed the highest LAC activity among transgenic lines produced, was lower than that of the control line. However, this change was not reflected to the saccharification efficiency.