Publications by authors named "Naofumi Kamimura"

43 Publications

Lignin valorization through efficient microbial production of β-ketoadipate from industrial black liquor.

Bioresour Technol 2021 Oct 2;337:125489. Epub 2021 Jul 2.

Bio-based Solution Division, Kantechs Co. Ltd., Bunkyo, Tokyo 112-0004, Japan.

Vanillin and vanillate are the major lignin-derived aromatic compounds produced through the alkaline oxidation of softwood lignin. Because the production of higher-value added chemicals from these compounds is essential for lignin valorization, the microbial production of β-ketoadipate, a promising raw material for the synthesis of novel nylons, from lignin was considered. Pseudomonas putida KT2440 was engineered to convert vanillin and vanillate to β-ketoadipate. By examining the culture conditions with an initial culture volume of 1 L, the engineered strain completely converted 25 g of vanillin and 25 g of vanillate and produced approximately 23 g of β-ketoadipate from each of them with a yield of 93% or higher. Furthermore, this strain showed the ability to efficiently produce β-ketoadipate from softwood lignin extracts in black liquor, a byproduct of pulp production. These results suggest that the production of β-ketoadipate from industrial black liquor is highly feasible for substantial lignin valorization.
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http://dx.doi.org/10.1016/j.biortech.2021.125489DOI Listing
October 2021

Caffeoylquinic acids: chemistry, biosynthesis, occurrence, analytical challenges, and bioactivity.

Plant J 2021 Sep 23;107(5):1299-1319. Epub 2021 Jul 23.

Department of Chemistry, Oregon State University, Corvallis, OR, USA.

Caffeoylquinic acids (CQAs) are specialized plant metabolites we encounter in our daily life. Humans consume CQAs in mg-to-gram quantities through dietary consumption of plant products. CQAs are considered beneficial for human health, mainly due to their anti-inflammatory and antioxidant properties. Recently, new biosynthetic pathways via a peroxidase-type p-coumaric acid 3-hydroxylase enzyme were discovered. More recently, a new GDSL lipase-like enzyme able to transform monoCQAs into diCQA was identified in Ipomoea batatas. CQAs were recently linked to memory improvement; they seem to be strong indirect antioxidants via Nrf2 activation. However, there is a prevalent confusion in the designation and nomenclature of different CQA isomers. Such inconsistencies are critical and complicate bioactivity assessment since different isomers differ in bioactivity and potency. A detailed explanation regarding the origin of such confusion is provided, and a recommendation to unify nomenclature is suggested. Furthermore, for studies on CQA bioactivity, plant-based laboratory animal diets contain CQAs, which makes it difficult to include proper control groups for comparison. Therefore, a synthetic diet free of CQAs is advised to avoid interferences since some CQAs may produce bioactivity even at nanomolar levels. Biotransformation of CQAs by gut microbiota, the discovery of new enzymatic biosynthetic and metabolic pathways, dietary assessment, and assessment of biological properties with potential for drug development are areas of active, ongoing research. This review is focused on the chemistry, biosynthesis, occurrence, analytical challenges, and bioactivity recently reported for mono-, di-, tri-, and tetraCQAs.
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http://dx.doi.org/10.1111/tpj.15390DOI Listing
September 2021

Characterization of aromatic acid/proton symporters in Pseudomonas putida KT2440 toward efficient microbial conversion of lignin-related aromatics.

Metab Eng 2021 03 4;64:167-179. Epub 2021 Feb 4.

Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan. Electronic address:

Pseudomonas putida KT2440 (hereafter KT2440) is a well-studied platform bacterium for the production of industrially valuable chemicals from heterogeneous mixtures of aromatic compounds obtained from lignin depolymerization. KT2440 can grow on lignin-related monomers, such as ferulate (FA), 4-coumarate (4CA), vanillate (VA), 4-hydroxybenzoate (4HBA), and protocatechuate (PCA). Genes associated with their catabolism are known, but knowledge about the uptake systems remains limited. In this work, we studied the KT2440 transporters of lignin-related monomers and their substrate selectivity. Based on the inhibition by protonophores, we focused on five genes encoding aromatic acid/H symporter family transporters categorized into major facilitator superfamily that uses the proton motive force. The mutants of PP_1376 (pcaK) and PP_3349 (hcnK) exhibited significantly reduced growth on PCA/4HBA and FA/4CA, respectively, while no change was observed on VA for any of the five gene mutants. At pH 9.0, the conversion of these compounds by hcnK mutant (FA/4CA) and vanK mutant (VA) was dramatically reduced, revealing that these transporters are crucial for the uptake of the anionic substrates at high pH. Uptake assays using C-labeled substrates in Escherichia coli and biosensor-based assays confirmed that PcaK, HcnK, and VanK have ability to take up PCA, FA/4CA, and VA/PCA, respectively. Additionally, analyses of the predicted protein structures suggest that the size and hydropathic properties of the substrate-binding sites of these transporters determine their substrate preferences. Overall, this study reveals that at physiological pH, PcaK and HcnK have a major role in the uptake of PCA/4HBA and FA/4CA, respectively, and VanK is a VA/PCA transporter. This information can contribute to the engineering of strains for the efficient conversion of lignin-related monomers to value-added chemicals.
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http://dx.doi.org/10.1016/j.ymben.2021.01.013DOI Listing
March 2021

Roles of two glutathione S-transferases in the final step of the β-aryl ether cleavage pathway in Sphingobium sp. strain SYK-6.

Sci Rep 2020 11 26;10(1):20614. Epub 2020 Nov 26.

Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata, 940-2188, Japan.

Sphingobium sp. strain SYK-6 is an alphaproteobacterial degrader of lignin-derived aromatic compounds, which can degrade all the stereoisomers of β-aryl ether-type compounds. SYK-6 cells convert four stereoisomers of guaiacylglycerol-β-guaiacyl ether (GGE) into two enantiomers of α-(2-methoxyphenoxy)-β-hydroxypropiovanillone (MPHPV) through GGE α-carbon atom oxidation by stereoselective Cα-dehydrogenases encoded by ligD, ligL, and ligN. The ether linkages of the resulting MPHPV enantiomers are cleaved by stereoselective glutathione (GSH) S-transferases (GSTs) encoded by ligF, ligE, and ligP, generating (βR/βS)-α-glutathionyl-β-hydroxypropiovanillone (GS-HPV) and guaiacol. To date, it has been shown that the gene products of ligG and SLG_04120 (ligQ), both encoding GST, catalyze GSH removal from (βR/βS)-GS-HPV, forming achiral β-hydroxypropiovanillone. In this study, we verified the enzyme properties of LigG and LigQ and elucidated their roles in β-aryl ether catabolism. Purified LigG showed an approximately 300-fold higher specific activity for (βR)-GS-HPV than that for (βS)-GS-HPV, whereas purified LigQ showed an approximately six-fold higher specific activity for (βS)-GS-HPV than that for (βR)-GS-HPV. Analyses of mutants of ligG, ligQ, and both genes revealed that SYK-6 converted (βR)-GS-HPV using both LigG and LigQ, whereas only LigQ was involved in converting (βS)-GS-HPV. Furthermore, the disruption of both ligG and ligQ was observed to lead to the loss of the capability of SYK-6 to convert MPHPV. This suggests that GSH removal from GS-HPV catalyzed by LigG and LigQ, is essential for cellular GSH recycling during β-aryl ether catabolism.
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http://dx.doi.org/10.1038/s41598-020-77462-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7691349PMC
November 2020

Author Correction: Iron acquisition system of Sphingobium sp. strain SYK-6, a degrader of lignin-derived aromatic compounds.

Sci Rep 2020 Oct 2;10(1):16731. Epub 2020 Oct 2.

Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata, 940-2188, Japan.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-73843-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530733PMC
October 2020

The Syringate -Demethylase Gene of sp. Strain SYK-6 Is Regulated by DesX, while Other Vanillate and Syringate Catabolism Genes Are Regulated by DesR.

Appl Environ Microbiol 2020 10 28;86(22). Epub 2020 Oct 28.

Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan

Syringate and vanillate are the major metabolites of lignin biodegradation. In sp. strain SYK-6, syringate is O demethylated to gallate by consecutive reactions catalyzed by DesA and LigM, and vanillate is O demethylated to protocatechuate by a reaction catalyzed by LigM. The gallate ring is cleaved by DesB, and protocatechuate is catabolized via the protocatechuate 4,5-cleavage pathway. The transcriptions of , , and are induced by syringate and vanillate, while those of and are negatively regulated by the MarR-type transcriptional regulator DesR, which is not involved in regulation. Here, we clarified the regulatory system for transcription by analyzing the IclR-type transcriptional regulator , located downstream of Quantitative reverse transcription (RT)-PCR analyses of a mutant indicated that the transcription of was negatively regulated by DesX. In contrast, DesX was not involved in the regulation of and The ferulate catabolism genes (), under the control of a MarR-type transcriptional regulator, FerC, are located upstream of RT-PCR analyses suggested that the -SLG_25010- gene cluster consists of the operon and the SLG_25010- operon. Promoter assays revealed that a syringate- and vanillate-inducible promoter is located upstream of SLG_25010. Purified DesX bound to this promoter region, which overlaps an 18-bp inverted-repeat sequence that appears to be essential for the DNA binding of DesX. Syringate and vanillate inhibited the DNA binding of DesX, indicating that the compounds are effector molecules of DesX. Syringate is a major degradation product in the microbial and chemical degradation of syringyl lignin. Along with other low-molecular-weight aromatic compounds, syringate is produced by chemical lignin depolymerization. Converting this mixture into value-added chemicals using bacterial metabolism (i.e., biological funneling) is a promising option for lignin valorization. To construct an efficient microbial lignin conversion system, it is necessary to identify and characterize the genes involved in the uptake and catabolism of lignin-derived aromatic compounds and to elucidate their transcriptional regulation. In this study, we found that the transcription of , encoding syringate -demethylase in SYK-6, is regulated by an IclR-type transcriptional regulator, DesX. The findings of this study, combined with our previous results on (encoding a MarR transcriptional regulator that controls the transcription of and ), provide an overall picture of the transcriptional-regulatory systems for syringate and vanillate catabolism in SYK-6.
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http://dx.doi.org/10.1128/AEM.01712-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642091PMC
October 2020

Iron acquisition system of Sphingobium sp. strain SYK-6, a degrader of lignin-derived aromatic compounds.

Sci Rep 2020 07 22;10(1):12177. Epub 2020 Jul 22.

Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata, 940-2188, Japan.

Iron, an essential element for all organisms, acts as a cofactor of enzymes in bacterial degradation of recalcitrant aromatic compounds. The bacterial family, Sphingomonadaceae comprises various degraders of recalcitrant aromatic compounds; however, little is known about their iron acquisition system. Here, we investigated the iron acquisition system in a model bacterium capable of degrading lignin-derived aromatics, Sphingobium sp. strain SYK-6. Analyses of SYK-6 mutants revealed that FiuA (SLG_34550), a TonB-dependent receptor (TBDR), was the major outer membrane iron transporter. Three other TBDRs encoded by SLG_04340, SLG_04380, and SLG_10860 also participated in iron uptake, and tonB2 (SLG_34540), one of the six tonB comprising the Ton complex which enables TBDR-mediated transport was critical for iron uptake. The ferrous iron transporter FeoB (SLG_36840) played an important role in iron uptake across the inner membrane. The promoter activities of most of the iron uptake genes were induced under iron-limited conditions, and their regulation is controlled by SLG_29410 encoding the ferric uptake regulator, Fur. Although feoB, among all the iron uptake genes identified is highly conserved in Sphingomonad strains, the outer membrane transporters seem to be diversified. Elucidation of the iron acquisition system promises better understanding of the bacterial degradation mechanisms of aromatic compounds.
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http://dx.doi.org/10.1038/s41598-020-68984-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7376174PMC
July 2020

Identification of enzymatic genes with the potential to reduce biomass recalcitrance through lignin manipulation in .

Biotechnol Biofuels 2020 29;13:97. Epub 2020 May 29.

Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588 Japan.

Background: During the chemical and biochemical decomposition of lignocellulosic biomasses, lignin is highly recalcitrant. Genetic transformation of plants to qualitatively and/or quantitatively modify lignin may reduce these recalcitrant properties. Efficient discovery of genes to achieve lignin manipulation is thus required.

Results: To screen for new genes to reduce lignin recalcitrance, we heterologously expressed 50 enzymatic genes under the control of a cinnamate 4-hydroxylase () gene promoter, derived from a hybrid aspen, which is preferentially active in tissues with lignified cell walls in plants. These genes encode enzymes that act on metabolites in shikimate, general phenylpropanoid, flavonoid, or monolignol biosynthetic pathways. Among these genes, 30, 18, and 2 originated from plants, bacteria, and fungi, respectively. In our first screening step, 296 independent transgenic plants (T generation) harboring single or multiple transgenes were generated from pools of seven strains used for conventional floral-dip transformation. Wiesner and Mäule staining patterns in the stems of the resultant plants revealed seven and nine plants with apparent abnormalities in the two respective staining analyses. According to genomic PCR and subsequent direct sequencing, each of these 16 plants possessed a gene encoding either coniferaldehyde dehydrogenase (), feruloyl-CoA 6'-hydroxylase (), hydroxycinnamoyl-CoA hydratase/lyase (), or ferulate 5-hydroxylase (), with one transgenic plant carrying both and . The effects of these genes on lignin manipulation were confirmed in individually re-created T transgenic plants. While no difference in lignin content was detected in the transgenic lines compared with the wild type, lignin monomeric composition was changed in the transgenic lines. The observed compositional change in the transgenic plants carrying , , and led to improved sugar release from cell walls after alkaline pretreatment.

Conclusions: Simple colorimetric characterization of stem lignin is useful for simultaneous screening of many genes with the potential to reduce lignin recalcitrance. In addition to , the positive control, we identified three enzyme-coding genes that can function as genetic tools for lignin manipulation. Two of these genes ( and ) accelerate sugar release from transgenic lignocelluloses.
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http://dx.doi.org/10.1186/s13068-020-01736-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7260809PMC
May 2020

Development of the production of 2-pyrone-4,6-dicarboxylic acid from lignin extracts, which are industrially formed as by-products, as raw materials.

J Biosci Bioeng 2020 Jul 29;130(1):71-75. Epub 2020 Mar 29.

College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa 252-0880, Japan.

Lignosulfonate is a by-product of the cooking process by sulfite pulping for paper manufacturing. The treatment of wood chips by various salts of sulfurous acid solubilizes lignin to produce a cellulose-rich wood pulp. Developing a technique for the conversion of lignosulfonate by-product to high value materials has an important industrial utility. Sphingobium sp. strain SYK-6, which was isolated from pulping wastewater, is one of the best enzymatically or genetically characterized bacteria for degrading lignin-derived aromatics. We have previously established a system for the production of 2-pyrone-4,6-dicarboxylic acid (PDC), a novel platform chemical that can produce a variety of bio-based polymers, by introducing of ligA, ligB, and ligC genes from SYK-6 into a mutant strain of Pseudomonas putida PpY1100. In this study, extracts from lignosulfonates, which were desulphonated and depolymerized by alkaline oxidation, were evaluated as substrates for microbiological conversion to PDC by the transgenic bacteria.
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http://dx.doi.org/10.1016/j.jbiosc.2020.02.002DOI Listing
July 2020

Erratum: Publisher Correction: A TonB-dependent receptor constitutes the outer membrane transport system for a lignin-derived aromatic compound.

Commun Biol 2019;2:476. Epub 2019 Dec 17.

1Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata Japan.

[This corrects the article DOI: 10.1038/s42003-019-0676-z.].
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http://dx.doi.org/10.1038/s42003-019-0724-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6917759PMC
December 2019

A TonB-dependent receptor constitutes the outer membrane transport system for a lignin-derived aromatic compound.

Commun Biol 2019 22;2:432. Epub 2019 Nov 22.

1Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata Japan.

TonB-dependent receptors (TBDRs) mediate substrate-specific transport across the outer membrane, utilizing energy derived from the proton motive force transmitted from the TonB-ExbB-ExbD complex located in the inner membrane (TonB system). Although a number of TonB systems involved in the uptake of siderophores, vitamin B12 and saccharides have been identified, their involvement in the uptake and catabolism of aromatic compounds was previously unknown. Here, we show that the outer membrane transport of a biphenyl compound derived from lignin is mediated by the TonB system in a Gram-negative bacterium capable of degrading lignin-derived aromatic compounds, sp. strain SYK-6. Furthermore, we found that overexpression of the corresponding TBDR gene enhanced the uptake of this biphenyl compound, contributing to the improved rate of platform chemical production. Our results will provide an important basis for establishing engineered strains optimized for use in lignin valorisation.
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http://dx.doi.org/10.1038/s42003-019-0676-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6874591PMC
May 2020

Regulation of vanillate and syringate catabolism by a MarR-type transcriptional regulator DesR in Sphingobium sp. SYK-6.

Sci Rep 2019 12 2;9(1):18036. Epub 2019 Dec 2.

Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata, 940-2188, Japan.

Vanillate and syringate are major intermediate metabolites generated during the microbial degradation of lignin. In Sphingobium sp. SYK-6, vanillate is O demethylated to protocatechuate by LigM; protocatechuate is then catabolized via the protocatechuate 4,5-cleavage pathway. Syringate is O demethylated to gallate by consecutive reactions catalyzed by DesA and LigM, and then gallate is subjected to ring cleavage by DesB. Here, we investigated the transcriptional regulation of desA, ligM, and desB involved in vanillate and syringate catabolism. Quantitative reverse transcription-PCR analyses indicated that the transcription of these genes was induced 5.8-37-fold in the presence of vanillate and syringate. A MarR-type transcriptional regulator, SLG_12870 (desR), was identified as the gene whose product bound to the desB promoter region. Analysis of a desR mutant indicated that the transcription of desB, ligM, and desR is negatively regulated by DesR. Purified DesR bound to the upstream regions of desB, ligM, and desR, and the inverted repeat sequences similar to each other in these regions were suggested to be essential for DNA binding of DesR. Vanillate and syringate inhibited DNA binding of DesR, indicating that these compounds are effector molecules of DesR. The transcription of desA was found to be regulated by an as-yet unidentified regulator.
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http://dx.doi.org/10.1038/s41598-019-54490-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6888825PMC
December 2019

Discovery of novel enzyme genes involved in the conversion of an arylglycerol-β-aryl ether metabolite and their use in generating a metabolic pathway for lignin valorization.

Metab Eng 2019 09 4;55:258-267. Epub 2019 Aug 4.

Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan. Electronic address:

Microbial conversions known as "biological funneling" have attracted attention for their ability to upgrade heterogeneous mixtures of low-molecular-weight aromatic compounds obtained by chemical lignin depolymerization. β-hydroxypropiovanillone (HPV) and its analogs can be obtained by chemoselective catalytic oxidation of lignin using 2,3-dichloro-5,6-dicyano-1,4-benzoquinone/tert-butyl nitrite/O, followed by cleavage of arylglycerol-β-aryl ether with zinc. Sphingobium sp. strain SYK-6 can degrade HPV generated by the catabolism of arylglycerol-β-aryl ether through 2-pyrone-4,6-dicarboxylate (PDC), a promising platform chemical. Therefore, production of PDC from HPV can be achieved using the HPV catabolic pathway. However, the pathway and genes involved in the catabolism of vanilloyl acetic acid (VAA) generated during HPV catabolism have not been investigated. In the present study, we isolated SLG_24960 (vceA), which encodes an enzyme that converts VAA into a coenzyme A (CoA) derivative of vanillate (vanilloyl-CoA) from SYK-6, by shotgun cloning. The analysis of a vceA mutant indicated that this gene is not required for VAA conversion in vivo, but it encodes a major enzyme catalyzing CoA-dependent VAA conversion in vitro. We also identified SLG_12450 (vceB), whose product can convert vanilloyl-CoA to vanillate. Enzyme genes besides vceA and vceB, which are necessary for the conversions of HPV to VAA and of vanillate to PDC, were introduced and expressed in Pseudomonas putida. The resulting engineered strain completely converted 1  mM HPV into PDC after 24  h. Our results suggest that the enzyme genes that are not required for the catabolic pathway in microorganisms but can be used for the conversion of target substrates are buried in microbial genomes. These genes are, thus, useful for designing metabolic pathways to produce value-added metabolites.
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http://dx.doi.org/10.1016/j.ymben.2019.08.002DOI Listing
September 2019

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

J Ind Microbiol Biotechnol 2019 Aug 27;46(8):1071-1080. Epub 2019 May 27.

Department of Bioengineering, Nagaoka University of Technology, Kamitomioka, Nagaoka, Niigata, 940-2188, Japan.

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.
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http://dx.doi.org/10.1007/s10295-019-02190-6DOI Listing
August 2019

Advances in microbial lignin degradation and its applications.

Curr Opin Biotechnol 2019 04 11;56:179-186. Epub 2018 Dec 11.

Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan. Electronic address:

Lignocellulosic biomass represents a crucial resource for achieving sustainable development by replacing petroleum-based production systems. Lignin, a major component of plant cell walls, has significant potential as a bioresource; however, it is an obstacle in lignocellulosic biomass utilization due to its recalcitrance. Consequently, decomposition or removal of lignin is a crucial step to utilize cell wall components. In nature, lignin may be degraded via two stages: depolymerization and the mineralization of the resulting heterogeneous low-molecular-weight aromatic species. Microbial enzymes responsible for the former could be attractive tools for lignin decomposition during biomass pretreatment, and enzymes involved in the latter are useful for lignin valorization through the production of value-added chemicals. Moreover, specific microbial enzymes could reduce the recalcitrance of lignocellulosic biomass via plant cell wall bioengineering. This review focuses on microbial enzymes that are responsible for lignin degradation and on their applications to biological lignocellulosics pretreatment and biotechnological lignin engineering.
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http://dx.doi.org/10.1016/j.copbio.2018.11.011DOI Listing
April 2019

DdvK, a Novel Major Facilitator Superfamily Transporter Essential for 5,5'-Dehydrodivanillate Uptake by Sphingobium sp. Strain SYK-6.

Appl Environ Microbiol 2018 10 1;84(20). Epub 2018 Oct 1.

Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan

The microbial conversion of lignin-derived aromatics is a promising strategy for the industrial utilization of this large biomass resource. However, efficient application requires an elucidation of the relevant transport and catabolic pathways. In sp. strain SYK-6, most of the enzyme genes involved in 5,5'-dehydrodivanillate (DDVA) catabolism have been characterized, but the transporter has not yet been identified. Here, we identified SLG_07710 () and SLG_07780 (), genes encoding a putative major facilitator superfamily (MFS) transporter and MarR-type transcriptional regulator, respectively. A mutant of SYK-6 completely lost the capacity to grow on and convert DDVA. DdvR repressed the expression of the DDVA -demethylase oxygenase component gene (), while DDVA acted as the gene inducer. A DDVA uptake assay was developed by employing this DdvR-controlled transcriptional regulatory system. A UT26S transformant expressing acquired DDVA uptake capacity, indicating that encodes the DDVA transporter. DdvK, probably requiring the proton motive force, was suggested to be a novel MFS transporter on the basis of the amino acid sequence similarity. Subsequently, we evaluated the effects of overexpression on the production of the DDVA metabolite 2-pyrone-4,6-dicarboxylate (PDC), a building block of functional polymers. A SYK-6 mutant of the PDC hydrolase gene () cultured in DDVA accumulated PDC via 5-carboxyvanillate and grew by utilizing 4-carboxy-2-hydroxypenta-2,4-dienoate. The introduction of a -expression plasmid into a mutant increased the growth rate in DDVA and the amounts of DDVA converted and PDC produced after 48 h by 1.35- and 1.34-fold, respectively. These results indicate that enhanced transporter gene expression can improve metabolite production from lignin derivatives. The bioengineering of bacteria to selectively transport and metabolize natural substrates into specific metabolites is a valuable strategy for industrial-scale chemical production. The uptake of many substrates into cells requires specific transport systems, and so the identification and characterization of transporter genes are essential for industrial applications. A number of bacterial major facilitator superfamily transporters of aromatic acids have been identified and characterized, but many transporters of lignin-derived aromatic acids remain unidentified. The efficient conversion of lignin, an abundant but unutilized aromatic biomass resource, to value-added metabolites using microbial catabolism requires the characterization of transporters for lignin-derived aromatics. In this study, we identified the transporter gene responsible for the uptake of 5,5'-dehydrodivanillate, a lignin-derived biphenyl compound, in sp. strain SYK-6. In addition to characterizing its function, we applied this transporter gene to the production of a value-added metabolite from 5,5'-dehydrodivanillate.
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http://dx.doi.org/10.1128/AEM.01314-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6182893PMC
October 2018

Identification of the protocatechuate transporter gene in Sphingobium sp. strain SYK-6 and effects of overexpression on production of a value-added metabolite.

Appl Microbiol Biotechnol 2018 Jun 19;102(11):4807-4816. Epub 2018 Apr 19.

Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata, 940-2188, Japan.

Sphingobium sp. strain SYK-6 expresses the best characterized catabolic systems for lignin-derived aromatic compounds. However, the uptake systems for these aromatics remain unknown. In this study, we identified and characterized the protocatechuate (PCA) transporter gene SLG_12880 (pcaK) in SYK-6. Sequence comparisons revealed that PcaK belongs to the aromatic acid/H symporter family of major facilitator superfamily transporters. Further, pcaK was highly conserved among Sphingomonadales possessing catabolic genes for vanillate and PCA. The growth of an SYK-6 pcaK mutant was significantly delayed on PCA medium and PCA uptake rate was only 8% of wild type. In addition, vanillate uptake rate was 78% of wild type, although the pcaK mutant grew as well as the wild type on vanillate. When pcaK was expressed in Sphingobium japonicum UT26S, the transformant acquired the capacity to uptake both PCA and vanillate. These results indicate that pcaK is responsible for the major proportion of PCA uptake and a minor fraction of vanillate uptake in SYK-6. The productivity of 2-pyrone-4,6-dicarboxylate (PDC), a building block of functional polymers, was evaluated using a PDC hydrolase SYK-6 mutant harboring a pcaK plasmid. The mutant exhibited 1.27-fold greater PCA conversion and 1.24-fold greater PDC production compared to the control strain, suggesting that enhanced expression of transporter genes for lignin-derived aromatics can be used to increase the production of value-added metabolites.
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http://dx.doi.org/10.1007/s00253-018-8988-3DOI Listing
June 2018

Two novel decarboxylase genes play a key role in the stereospecific catabolism of dehydrodiconiferyl alcohol in Sphingobium sp. strain SYK-6.

Environ Microbiol 2018 05 26;20(5):1739-1750. Epub 2018 Mar 26.

Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan.

Sphingobium sp. strain SYK-6 is able to use a phenylcoumaran-type biaryl, dehydrodiconiferyl alcohol (DCA), as a sole source of carbon and energy. In SYK-6 cells, the alcohol group of the B-ring side chain of DCA was first oxidized to the carboxyl group, and then the alcohol group of the A-ring side chain was oxidized to generate 5-(2-carboxyvinyl)-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-2,3-dihydrobenzofuran-3-carboxylate (DCA-CC). We identified phcF, phcG and phcH, which conferred the ability to convert DCA-CC into 3-(4-hydroxy-3-(4-hydroxy-3-methoxystyryl)-5-methoxyphenyl)acrylate (DCA-S) in a host strain. These genes exhibited no significant sequence similarity with known enzyme genes, whereas phcF and phcG, which contain a DUF3237 domain of unknown function, showed 32% amino acid sequence identity with each other. The DCA-CC conversion activities were markedly decreased by disruption of phcF and phcG, indicating that phcF and phcG play dominant roles in the conversion of DCA-CC. Purified PhcF and PhcG catalysed the decarboxylation of the A-ring side chain of DCA-CC, producing DCA-S, and showed enantiospecificity towards (+)- and (-)-DCA-CC respectively. PhcF and PhcG formed homotrimers, and their K for DCA-CC were determined to be 84 μM and 103 μM, and V were 307 μmol⋅min ⋅mg and 137 μmol⋅min ⋅mg respectively. In conclusion, PhcF and PhcG are enantiospecific decarboxylases involved in phenylcoumaran catabolism.
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http://dx.doi.org/10.1111/1462-2920.14099DOI Listing
May 2018

Bacterial Catabolism of β-Hydroxypropiovanillone and β-Hydroxypropiosyringone Produced in the Reductive Cleavage of Arylglycerol-β-Aryl Ether in Lignin.

Appl Environ Microbiol 2018 04 19;84(7). Epub 2018 Mar 19.

Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan

sp. strain SYK-6 converts four stereoisomers of arylglycerol-β-guaiacyl ether into achiral β-hydroxypropiovanillone (HPV) via three stereospecific reaction steps. Here, we determined the HPV catabolic pathway and characterized the HPV catabolic genes involved in the first two steps of the pathway. In SYK-6 cells, HPV was oxidized to vanilloyl acetic acid (VAA) via vanilloyl acetaldehyde (VAL). The resulting VAA was further converted into vanillate through the activation of VAA by coenzyme A. A syringyl-type HPV analog, β-hydroxypropiosyringone (HPS), was also catabolized via the same pathway. SLG_12830 (), which belongs to the glucose-methanol-choline oxidoreductase family, was isolated as the HPV-converting enzyme gene. An mutant completely lost the ability to convert HPV and HPS, indicating that is essential for the conversion of both the substrates. HpvZ produced in oxidized both HPV and HPS and other 3-phenyl-1-propanol derivatives. HpvZ localized to both the cytoplasm and membrane of SYK-6 and used ubiquinone derivatives as electron acceptors. Thirteen gene products of the 23 aldehyde dehydrogenase (ALDH) genes in SYK-6 were able to oxidize VAL into VAA. Mutant analyses suggested that multiple ALDH genes, including SLG_20400, contribute to the conversion of VAL. We examined whether the genes encoding feruloyl-CoA synthetase () and feruloyl-CoA hydratase/lyase ( and ) are involved in the conversion of VAA. Only FerA exhibited activity toward VAA; however, disruption of did not affect VAA conversion. These results indicate that another enzyme system is involved in VAA conversion. Cleavage of the β-aryl ether linkage is the most essential process in lignin biodegradation. Although the bacterial β-aryl ether cleavage pathway and catabolic genes have been well documented, there have been no reports regarding the catabolism of HPV or HPS, the products of cleavage of β-aryl ether compounds. HPV and HPS have also been found to be obtained from lignin by chemoselective catalytic oxidation by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone/-butyl nitrite/O, followed by cleavage of the β-aryl ether with zinc. Therefore, value-added chemicals are expected to be produced from these compounds. In this study, we determined the SYK-6 catabolic pathways for HPV and HPS and identified the catabolic genes involved in the first two steps of the pathways. Since SYK-6 catabolizes HPV through 2-pyrone-4,6-dicarboxylate, which is a building block for functional polymers, characterization of HPV catabolism is important not only for understanding the bacterial lignin catabolic system but also for lignin utilization.
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http://dx.doi.org/10.1128/AEM.02670-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5861824PMC
April 2018

Bacterial catabolism of lignin-derived aromatics: New findings in a recent decade: Update on bacterial lignin catabolism.

Environ Microbiol Rep 2017 Dec;9(6):679-705

Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan.

Lignin is the most abundant phenolic polymer; thus, its decomposition by microorganisms is fundamental to carbon cycling on earth. Lignin breakdown is initiated by depolymerization catalysed by extracellular oxidoreductases secreted by white-rot basidiomycetous fungi. On the other hand, bacteria play a predominant role in the mineralization of lignin-derived heterogeneous low-molecular-weight aromatic compounds. The outline of bacterial catabolic pathways for lignin-derived bi- and monoaryls are typically composed of the following sequential steps: (i) funnelling of a wide variety of lignin-derived aromatics into vanillate and syringate, (ii) O demethylation of vanillate and syringate to form catecholic derivatives and (iii) aromatic ring-cleavage of the catecholic derivatives to produce tricarboxylic acid cycle intermediates. Knowledge regarding bacterial catabolic systems for lignin-derived aromatic compounds is not only important for understanding the terrestrial carbon cycle but also valuable for promoting the shift to a low-carbon economy via biological lignin valorisation. This review summarizes recent progress in bacterial catabolic systems for lignin-derived aromatic compounds, including newly identified catabolic pathways and genes for decomposition of lignin-derived biaryls, transcriptional regulation and substrate uptake systems. Recent omics approaches on catabolism of lignin-derived aromatic compounds are also described.
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http://dx.doi.org/10.1111/1758-2229.12597DOI Listing
December 2017

Phthalates impact human health: Epidemiological evidences and plausible mechanism of action.

J Hazard Mater 2017 Oct 19;340:360-383. Epub 2017 Jun 19.

Enzyme Technology Laboratory, School of Biosciences, University of Calicut, Kerala 673 635, India.

Disregarding the rising alarm on the hazardous nature of various phthalates and their metabolites, ruthless usage of phthalates as plasticizer in plastics and as additives in innumerable consumer products continues due low their cost, attractive properties, and lack of suitable alternatives. Globally, in silico computational, in vitro mechanistic, in vivo preclinical and limited clinical or epidemiological human studies showed that over a dozen phthalates and their metabolites ingested passively by man from the general environment, foods, drinks, breathing air, and routine household products cause various dysfunctions. Thus, this review addresses the health hazards posed by phthalates on children and adolescents, epigenetic modulation, reproductive toxicity in women and men; insulin resistance and type II diabetes; overweight and obesity, skeletal anomalies, allergy and asthma, cancer, etc., coupled with the description of major phthalates and their general uses, phthalate exposure routes, biomonitoring and risk assessment, special account on endocrine disruption; and finally, a plausible molecular cross-talk with a unique mechanism of action. This clinically focused comprehensive review on the hazards of phthalates would benefit the general population, academia, scientists, clinicians, environmentalists, and law or policy makers to decide upon whether usage of phthalates to be continued swiftly without sufficient deceleration or regulated by law or to be phased out from earth forever.
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http://dx.doi.org/10.1016/j.jhazmat.2017.06.036DOI Listing
October 2017

The crystal structure of a new O-demethylase from Sphingobium sp. strain SYK-6.

FEBS J 2017 06 11;284(12):1855-1867. Epub 2017 May 11.

Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), Tsukuba, Ibaraki, Japan.

In the cell, tetrahydrofolate (H folate) derivatives with a C1 unit are utilized in various ways, such as for the synthesis of amino acids and nucleic acids. While H folate derivatives with the C1 unit are typically produced in the glycine cleavage system, Sphingobium sp. strain SYK-6, which can utilize lignin-derived aromatic compounds as a sole source of carbon and energy, lacks this pathway, probably due to its unique nutrient requirements. In this bacterium, H folate-dependent O-demethylases in catabolic pathways for lignin-derived aromatic compounds seem to be involved in the C1 metabolism. LigM is one of the O-demethylases and catalyzes a C1-unit transfer from vanillate (VNL) to H folate. As the primary structure of LigM shows a similarity to T-protein in the glycine cleavage system, we hypothesized that LigM has evolved from T-protein, acquiring its unique biochemical and biological functions. To prove this hypothesis, structure-based understanding of its catalytic reaction is essential. Here, we determined the crystal structure of LigM in apo form and in complex with substrates and H folate. These crystal structures showed that the overall structure of LigM is similar to T-protein, but LigM has a few distinct characteristics, particularly in the active site. Structure-based mutational analysis revealed that His60 and Tyr247, which are not conserved in T-protein, are essential to the catalytic activity of LigM and their interactions with the oxygen atom in the methoxy group of VNL seem to facilitate a methyl moiety (C1-unit) transfer to H folate. Taken together, our structural data suggest that LigM has evolved divergently from T-protein.

Databases: All atomic coordinates of the crystal structures determined in this study have been deposited to PDB. LigM: 5X1I, LigM-VNL complex: 5X1J, LigM-3-O-methylgallate complex: 5X1K, LigM-H folate complex: 5X1IL, LigM-H folate-protocatechuate (PCA) complex (P2 2 2): 5X1M, LigM-H folate-PCA complex (P3 21): 5X1N.
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http://dx.doi.org/10.1111/febs.14085DOI Listing
June 2017

A bacterial aromatic aldehyde dehydrogenase critical for the efficient catabolism of syringaldehyde.

Sci Rep 2017 03 15;7:44422. Epub 2017 Mar 15.

Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan.

Vanillin and syringaldehyde obtained from lignin are essential intermediates for the production of basic chemicals using microbial cell factories. However, in contrast to vanillin, the microbial conversion of syringaldehyde is poorly understood. Here, we identified an aromatic aldehyde dehydrogenase (ALDH) gene responsible for syringaldehyde catabolism from 20 putative ALDH genes of Sphingobium sp. strain SYK-6. All these genes were expressed in Escherichia coli, and nine gene products, including previously characterized BzaA, BzaB, and vanillin dehydrogenase (LigV), exhibited oxidation activities for syringaldehyde to produce syringate. Among these genes, SLG_28320 (desV) and ligV were most highly and constitutively transcribed in the SYK-6 cells. Disruption of desV in SYK-6 resulted in a significant reduction in growth on syringaldehyde and in syringaldehyde oxidation activity. Furthermore, a desV ligV double mutant almost completely lost its ability to grow on syringaldehyde. Purified DesV showed similar k/K values for syringaldehyde (2100 s·mM) and vanillin (1700 s·mM), whereas LigV substantially preferred vanillin (8800 s·mM) over syringaldehyde (1.4 s·mM). These results clearly demonstrate that desV plays a major role in syringaldehyde catabolism. Phylogenetic analyses showed that DesV-like ALDHs formed a distinct phylogenetic cluster separated from the vanillin dehydrogenase cluster.
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http://dx.doi.org/10.1038/srep44422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5353671PMC
March 2017

Identification of natural rubber degradation gene in Rhizobacter gummiphilus NS21.

Biosci Biotechnol Biochem 2017 Mar 23;81(3):614-620. Epub 2017 Jan 23.

a Department of Bioengineering , Nagaoka University of Technology , Nagaoka , Japan.

A Gram-negative rubber-degrading bacterium, Rhizobacter gummiphilus NS21 grew and produced aldehyde metabolites on a deproteinized natural rubber (DPNR)-overlay agar medium forming a clearing zone. A transposon-insertion mutant, which had lost the ability to degrade DPNR, was isolated to identify the rubber degradation genes. Sequencing analysis indicated that the transposon was inserted into a putative oxygenase gene, latA. The deduced amino acid sequence of latA has 36% identity with that of roxA, which encodes a rubber oxygenase of Xanthomonas sp. strain 35Y. Phylogenetic analysis revealed that LatA constitutes a distinct group from RoxA. Heterologous expression in a Methylibium host and deletion analysis of latA indicated that the latA product is responsible for the depolymerization of DPNR. The quantitative reverse transcription-PCR analysis indicated that the transcription of latA is induced during the growth on DPNR. These results strongly suggest that latA is directly involved in the degradation of rubber in NS21.
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http://dx.doi.org/10.1080/09168451.2016.1263147DOI Listing
March 2017

Overcoming a hemihedral twinning problem in tetrahydrofolate-dependent O-demethylase crystals by the microseeding method.

Acta Crystallogr F Struct Biol Commun 2016 12 30;72(Pt 12):897-902. Epub 2016 Nov 30.

Department of Accelerator Science, School of High Energy Accelerator Science, SOKENDAI (The Graduate University for Advanced Studies), 1-1 Oho, Tsukuba, Ibaraki 305-0031, Japan.

A tetrahydrofolate-dependent O-demethylase, LigM, from Sphingobium sp. SYK-6 was crystallized by the hanging-drop vapour-diffusion method. However, the obtained P321 or P321 crystals, which diffracted to 2.5-3.3 Å resolution, were hemihedrally twinned. To overcome the twinning problem, microseeding using P321/P321 crystals as microseeds was performed with optimization of the reservoir conditions. As a result, another crystal form was obtained. The newly obtained crystal diffracted to 2.5-3.0 Å resolution and belonged to space group P222, with unit-cell parameters a = 102.0, b = 117.3, c = 128.1 Å. The P222 crystals diffracted to better than 2.0 Å resolution after optimizing the cryoconditions. Phasing using the single anomalous diffraction method was successful at 3.0 Å resolution with a Pt-derivative crystal. This experience suggested that microseeding is an effective method to overcome the twinning problem, even when twinned crystals are utilized as microseeds.
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http://dx.doi.org/10.1107/S2053230X16018665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5137467PMC
December 2016

Achromobacter denitrificans SP1 efficiently utilizes 16 phthalate diesters and their downstream products through protocatechuate 3,4-cleavage pathway.

Ecotoxicol Environ Saf 2016 Dec 10;134P1:172-178. Epub 2016 Sep 10.

Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940 2188, Japan.

This study provides physical and analytical evidences for the efficient utilization of most of the commercially available phthalate diesters by Achromobacter denitrificans SP1, coupled with the demonstration of a plausible degradation pathway. We tested 17 phthalate diesters [viz., ditridecyl phthalate, diisodecyl phthalate (DIDP), di(2-ethylhexyl)phthalate (DEHP), di-n-octyl phthalate (DOP), bis(2-ethylhexyl)isophthalate (BEIP), dihexyl phthalate (DHP), dibutyl phthalate (DBP), dicyclohexyl phthalate (DCHP), diphenyl phthalate (DPP), benzyl butyl phthalate (BBP), diamyl phthalate (DAmP), diisobutyl phthalate, dipropyl phthalate, dially phthalate (DAlP), diethyl phthalate, diethyl terephthalate and dimethyl phthalate (DMP)], and their major degradation products for the degradation efficiency of A. denitrificans SP1 in Wx medium. It efficiently utilized 16 phthalate diesters (except DAlP), and showed general preference toward phthalate diesters with longer side chains (utilized ~10mM in 48h) than those with shorter side chains and with cyclic structures (utilized ~5mM in 48h) accompanied by a sharp decline of pH to ~5 from initial 7. In a detailed study, about 37mM (~15g/L) DEHP was utilized in 48h. Moreover, A. denitrificans SP1 produced reddish-pink pigment when DIDP, DEHP, DOP, DHP, DBP, DIBP, BBP, DAmP, DCHP, DPP or DMP was supplied in the medium. From the available evidences, it seems that its putative phthalate diester degradation pathway contains the following check points: phthalate diesters, phthalate monoesters, phthalate (4,5-dioxygenase), protocatechuate (3,4-dioxygenase), and TCA cycle. Nonspecificity toward utilization of phthalate diesters, especially with greater specificity to phthalate diesters having longer side chain, and the characteristic sticky reddish-pink (or colorless) cell clump formation in the presence of phthalate diesters makes A. denitrificans SP1 a very attractive candidate to be employed as an efficient biofactory in waste water treatment processes.
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http://dx.doi.org/10.1016/j.ecoenv.2016.08.028DOI Listing
December 2016

Expression and functional analyses of a putative phenylcoumaran benzylic ether reductase in Arabidopsis thaliana.

Plant Cell Rep 2016 Mar 25;35(3):513-26. Epub 2015 Nov 25.

Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo, 184-8588, Japan.

Key Message: A candidate gene for phenylcoumaran benzylic ether reductase in Arabidopsis thaliana encodes a peptide with predicted functional activity and plays a crucial role in secondary metabolism. Phenylcoumaran benzylic ether reductase (PCBER) is thought to be an enzyme crucial in the biosynthesis of 8-5'-linked neolignans. Genes of the enzyme have been isolated and characterized in several plant species. In this study, we cloned cDNA and the 5'-untranslated region of one PCBER candidate gene (At4g39230, designated AtPCBER1) from Arabidopsis thaliana. At the amino acid level, AtPCBER1 shows high sequence identity (64-71 %) with PCBERs identified from other plant species. Expression analyses of AtPCBER1 by reverse transcriptase-polymerase chain reaction and histochemical analysis of transgenic plants harboring the 5'-untranslated region of AtPCBER1 linked with gus coding sequence indicate that expression is induced by wounding and is expressed in most tissues, including flower, stem, leaf, and root. Catalytic analysis of recombinant AtPCBER1 with neolignan and lignans in the presence of NADPH suggests that the protein can reduce not only the 8-5'-linked neolignan, dehydrodiconiferyl alcohol, but also 8-8' linked lignans, pinoresinol, and lariciresinol, with lower activities. To investigate further, we performed metabolomic analyses of transgenic plants in which the target gene was up- or down-regulated. Our results indicate no significant effects of AtPCBER1 gene regulation on plant growth and development; however, levels of some secondary metabolites, including lignans, flavonoids, and glucosinolates, differ between wild-type and transgenic plants. Taken together, our findings indicate that AtPCBER1 encodes a polypeptide with PCBER activity and has a critical role in the biosynthesis of secondary metabolites in A. thaliana.
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http://dx.doi.org/10.1007/s00299-015-1899-1DOI Listing
March 2016

Methanogenic degradation of lignin-derived monoaromatic compounds by microbial enrichments from rice paddy field soil.

Sci Rep 2015 Sep 24;5:14295. Epub 2015 Sep 24.

Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo, Hokkaido 062-8517, Japan.

Anaerobic degradation of lignin-derived aromatics is an important metabolism for carbon and nutrient cycles in soil environments. Although there are some studies on degradation of lignin-derived aromatics by nitrate- and sulfate-reducing bacteria, knowledge on their degradation under methanogenic conditions are quite limited. In this study, methanogenic microbial communities were enriched from rice paddy field soil with lignin-derived methoxylated monoaromatics (vanillate and syringate) and their degradation intermediates (protocatechuate, catechol, and gallate) as the sole carbon and energy sources. Archaeal community analysis disclosed that both aceticlastic (Methanosarcina sp.) and hydrogenotrophic (Methanoculleus sp. and Methanocella sp.) methanogens dominated in all of the enrichments. Bacterial community analysis revealed the dominance of acetogenic bacteria (Sporomusa spp.) only in the enrichments on the methoxylated aromatics, suggesting that Sporomusa spp. initially convert vanillate and syringate into protocatechuate and gallate, respectively, with acetogenesis via O-demethylation. As the putative ring-cleavage microbes, bacteria within the phylum Firmicutes were dominantly detected from all of the enrichments, while the dominant phylotypes were not identical between enrichments on vanillate/protocatechuate/catechol (family Peptococcaceae bacteria) and on syringate/gallate (family Ruminococcaceae bacteria). This study demonstrates the importance of cooperation among acetogens, ring-cleaving fermenters/syntrophs and aceticlastic/hydrogenotrophic methanogens for degradation of lignin-derived aromatics under methanogenic conditions.
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http://dx.doi.org/10.1038/srep14295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4585845PMC
September 2015

Membrane-associated glucose-methanol-choline oxidoreductase family enzymes PhcC and PhcD are essential for enantioselective catabolism of dehydrodiconiferyl alcohol.

Appl Environ Microbiol 2015 Dec 11;81(23):8022-36. Epub 2015 Sep 11.

Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan

Sphingobium sp. strain SYK-6 is able to degrade various lignin-derived biaryls, including a phenylcoumaran-type compound, dehydrodiconiferyl alcohol (DCA). In SYK-6 cells, the alcohol group of the B-ring side chain of DCA is initially oxidized to the carboxyl group to generate 3-(2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-7-methoxy-2,3-dihydrobenzofuran-5-yl) acrylic acid (DCA-C). Next, the alcohol group of the A-ring side chain of DCA-C is oxidized to the carboxyl group, and then the resulting metabolite is catabolized through vanillin and 5-formylferulate. In this study, the genes involved in the conversion of DCA-C were identified and characterized. The DCA-C oxidation activities in SYK-6 were enhanced in the presence of flavin adenine dinucleotide and an artificial electron acceptor and were induced ca. 1.6-fold when the cells were grown with DCA. Based on these observations, SLG_09480 (phcC) and SLG_09500 (phcD), encoding glucose-methanol-choline oxidoreductase family proteins, were presumed to encode DCA-C oxidases. Analyses of phcC and phcD mutants indicated that PhcC and PhcD are essential for the conversion of (+)-DCA-C and (-)-DCA-C, respectively. When phcC and phcD were expressed in SYK-6 and Escherichia coli, the gene products were mainly observed in their membrane fractions. The membrane fractions of E. coli that expressed phcC and phcD catalyzed the specific conversion of DCA-C into the corresponding carboxyl derivatives. In the oxidation of DCA-C, PhcC and PhcD effectively utilized ubiquinone derivatives as electron acceptors. Furthermore, the transcription of a putative cytochrome c gene was significantly induced in SYK-6 grown with DCA. The DCA-C oxidation catalyzed by membrane-associated PhcC and PhcD appears to be coupled to the respiratory chain.
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http://dx.doi.org/10.1128/AEM.02391-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4651077PMC
December 2015

Draft Genome Sequence of Comamonas sp. Strain E6 (NBRC 107749), a Degrader of Phthalate Isomers through the Protocatechuate 4,5-Cleavage Pathway.

Genome Announc 2015 Jun 18;3(3). Epub 2015 Jun 18.

Department of Bioengineering, Nagaoka University of Technology, Kamitomioka, Nagaoka, Niigata, Japan

Comamonas sp. strain E6 can degrade o-phthalate, terephthalate, and isophthalate via the protocatechuate 4,5-cleavage pathway. Here, we report the draft genome sequence of E6 in order to provide insights into its mechanisms in o-phthalate catabolism and its potential use for biotechnological applications.
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http://dx.doi.org/10.1128/genomeA.00643-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4472898PMC
June 2015
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