Phanerochaete chrysosporium Multienzyme Catabolic System for in

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Phanerochaete chrysosporium Multi-Enzyme Catabolic System for In Vivo Modification of Synthetic Lignin to Succinic Acid Chang-Young Hong, Sun-Hwa Ryu, Hanseob Jeong, Sung-Suk Lee, Myungkil Kim, and In-Gyu Choi ACS Chem. Biol., Just Accepted Manuscript • Publication Date (Web): 02 May 2017 Downloaded from http://pubs.acs.org on May 7, 2017

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ACS Chemical Biology

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Title

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Phanerochaete chrysosporium Multi-Enzyme Catabolic System for In Vivo

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Modification of Synthetic Lignin to Succinic Acid

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Authors

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Chang-Young Hong,† Sun-Hwa Ryu,† Hanseob Jeong,†Sung-Suk Lee,† Myungkil

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Kim,*,† and In-Gyu Choi*,‡,§,║

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Affiliations

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†

Division of Wood Chemistry & Microbiology, Department of Forest Products, National Institute of Forest Science, Seoul, Republic of Korea

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‡

Department of Forest Sciences, Seoul National University, Seoul, Republic of Korea

§

Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, Republic of Korea

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║

Green Eco Engineering, Institutes of Green Bio Science and Technology, Seoul National University, Pyeongchang, Republic of Korea

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Corresponding Authors:

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Myungkil Kim

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Division of Wood Chemistry & Microbiology, Department of Forest Products, National

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Institute of Forest Science, Seoul, Republic of Korea

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In-Gyu Choi

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Department of Forest Sciences, College of Agriculture and Life Sciences, Seoul

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National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of Korea

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E-mail: [email protected] 1

ACS Paragon Plus Environment


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ABSTRACT: Whole cells of the basidiomycete fungus Phanerochaete chrysosporium

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(ATCC 20696) were applied to induce the biomodification of lignin in an invivo system.

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Our results indicated that P. chrysosporium has a catabolic system that induces

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characteristic biomodifications of synthetic lignin through a series of redox reactions,

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leading not only to the degradation of lignin but also to its polymerization. The reducing

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agents ascorbic acid and α-tocopherol were used to stabilize the free radicals generated

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from the ligninolytic process. The application of P. chrysosporium in combination with

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reducing agents produced aromatic compounds and succinic acid as well as degraded

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lignin polymers. P. chrysosporium selectively catalyzed the conversion of lignin to

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succinic acid, which has an economic value. A transcriptomic analysis of P.

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chrysosporium suggested that the bond cleavage of synthetic lignin was caused by

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numerous enzymes, including extracellular enzymes such as lignin peroxidase and

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manganese peroxidase, and that the aromatic compounds released were metabolized in

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both the short-cut and classical tricarboxylic acid cycles of P. chrysosporium. In

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conclusion, P. chrysosporium is suitable as a biocatalyst for lignin degradation to

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produce a value-added product.

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INTRODUCTION

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Lignin, an aromatic macromolecule that is highly abundant in nature, has been

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recognized as a candidate source for replacing petroleum-derived chemicals.(1) Lignin

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modification is essential in terms of economic analyses of lignocellulosic

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biorefineries;(2) however, owing to the high cost of such technologies, industrial-scale

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lignin utilization has been mainly limited to combustion to produce heat and power.

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Recently, the concept of lignin modification using ligninolytic enzyme systems has

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emerged. Catabolic systems of bacteria such as Bacillus subtilis and Amycolatopsis spp.

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have been discovered to degrade lignin.(3-5) Some research groups have studied

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integrated biological funnel systems with the objective of degrading lignin.(6,7) This

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concept suggests that bacteria expressing oxidative enzymes utilize aromatic compounds

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as a carbon source. These powerful oxidative enzymes depolymerize lignin and then the

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dioxygenase system in bacteria catalyzes the cleavage of aromatic rings, leading to the

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production of intermediary metabolites such as polyhydroxyalkanoates that can be

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converted to renewable chemicals and materials such as hydrocarbons and hydroxy acid

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monomers.(7,8) However, questions remain regarding which ligninolytic enzymes are

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secreted from microbes, how they act on natural lignin, and which aromatic compounds

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they can produce from lignin.

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The degradation of lignin by bacteria was recently shown to have a low efficiency

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compared with that by white rot basidiomycetes.(9) Specifically, bacterial lignin

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removal efficiency of Rhodococci, Streptomyces, and Pseudomonas was much less than

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that of white rot fungi such as Phanerochaete chrysosporium. Furthermore, white rot

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fungi were reported to be capable of demethylating lignin, which facilitates lignin

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depolymerization.(10) White rot basidiomycetes contains three systems for lignolysis: 3



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peroxidase-based, laccase-based, and Fenton reaction-based, and is thus versatile and

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highly efficient at lignin removal.(11) In addition, biocatalysis by basidiomycetes can

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induce the conversion from lignin to value-added products under a mild condition,

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which is not normally possible using the chemical processes.(3) Therefore, lignin

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modification by biocatalysts can be a powerful tool in industrial biotechnology for

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producing intermediates that can be used to produce fuels and valuable chemicals.(12)

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Accordingly, white rot basidiomycetes have been recognized to be suitable for lignin

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modification through their ligninolytic enzyme system with high redox potential.(13,14)

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Their use is of particular interest in green chemistry and biorefineries(13,15) because of

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their ecological sustainability and potential economic advantages.(16)

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However, it has been difficult to implement an efficient biomodification process

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using basidiomycetes owing to the lack of genetic tools for these organisms and the

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challenge of controlling their enzyme systems to produce value-added products. Whole

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cells of basidiomycetes have complex enzyme systems including extracellular and

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intracellular enzymes,(13,17) and it is necessary to understand the mechanisms by

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which they can modify lignin and devise a method for controlling their enzyme and

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metabolic systems. In this study, we investigated the biomodification mechanism of

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synthetic lignin by Phanerochaete chrysosporium, which is a well-studied model

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basidiomycete. In addition, a transcriptomic analysis was performed to investigate the

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complex enzyme systems of P. chrysosporium related to the biomodification of

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dehydrogenative polymer (DHP; a synthetic lignin).

94 95 96

RESULTS AND DISCUSSION Degradation and Polymerization of Synthetic Lignin by P. chrysosporium. To 4


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examine the structural changes of DHP induced by incubation with P. chrysosporium,

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the molecular weight (MW) and the contents of phenolic OH groups and nitrobenzene

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oxidation (NBO) products were analyzed. Table 1A shows the MW change of DHP

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during the incubation period. Compared to the weight-average MW of DHP, the MW of

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the control was similar to that of DHP during the incubation period. The MW of DHP

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treated with P. chrysosporium (ATCC 20696) showed a tendency to decrease by

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incubation day 10. The measured MWs on incubation days 5 and 10 were 3,106 and

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3,240, respectively. Thereafter, the MW increased between days 15 and 25, and reached

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its peak values at days 15 (3,897 Da), and 25 (3,930 Da).

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The phenolic OH content of DHP was 4.6%, which was similar to that of other

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lignins such as kraft lignin (4.5%), soda lignin (4.4%), and lignosulfonate (2.0%).(18)

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The phenolic OH content of the control was 6.6% during the incubation, a value that

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was slightly higher than that of DHP. The phenolic OH content of DHP treated by

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fungus was increased by 10.5% on incubation day 10 (Figure 1). The phenolic OH

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contents of DHP modified by fungus maintained a higher value than that of the control.

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The amount of NBO products of DHP present during the fungal treatment declined

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compared with that of the control except on incubation day 20 (Table 2A).

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These results showed that the incubation of DHP with P. chrysosporium led to the

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cleavage of ether bonds; thus, DHP was degraded to yield a high phenolic OH group

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content during the initial incubation days. However, during the latter part of the

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incubation period, polymerization occurred with an increase in MW despite the decrease

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in ether bonding in DHP. The latter result implied that DHP was condensed by other

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types of linkages, rather than ether bonds such as α-O-4 and β-O-4, under the enzyme-

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controlled condition, and was predicted to assume a rigid form.(19) With respect to the 5



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structure of lignin, the phenolic OH groups are the most reactive sites.(20) Because DHP

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modified by fungus could be very unstable with a high phenolic OH content, its

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structure could be modified easily by the enzymes of P. chrysosporium. This explains

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why the observed structural changes of DHP occurred during the incubation period.

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In the present study, P. chrysosporium simultaneously induced the degradation and

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polymerization of DHP. The ligninolytic enzyme system of white rot fungus was

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previously reported to catalyze polymerization as well as degradation via radical

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reactions.(13,21) Consequently, P. chrysosporium catalyzed both the bond cleavage and

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polymerization of synthetic lignin. The unstable intermediate compounds derived from

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synthetic lignin seemed to be polymerized under oxidative conditions. Therefore, it was

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necessary to devise a method for the depolymerization of lignin to produce

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economically valuable compounds under the ligninolytic treatment by whole cells of P.

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chrysosporium.

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DHP Depolymerization by incubation with added Reducing Agents. On the basis

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of the above results, we decided to add the reducing agents ascorbic acid and α-

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tocopherol to stabilize the compounds generated during the degradation of lignin by P.

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chrysosporium. Ascorbic acid is one of the most extensively studied hydrophilic

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antioxidants and can directly scavenge O2, O2−, and OH•.(22) Furthermore, it was

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previously reported to inhibit the further oxidation of phenolic products in an enzymatic

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reaction.(23) α-Tocopherol is a well-known lipophilic reducing agent that can scavenge

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and quench reactive oxygen species and peroxyl radicals.(22,24) Furthermore, ascorbic

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acid regenerates tocopherol from tocopheroxyl radicals, which increases its

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effectiveness as an antioxidant.(25) As both water-soluble and water-insoluble lignins 6


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were formed during the modification process, in this study, for the depolymerization of

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DHP (lipophilic and hydrophilic) and the production of DHP-derived chemicals

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(hydrophilic), two reducing agents were used.

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The results of gel permeation chromatography analysis showed that the MW of DHP

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was decreased slightly when these two reducing agents were included in the culture

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medium during incubation with P. chrysosporium (Table 1B). The MWs of the control

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on incubation days 10 and 20 were 3,132 and 3,034 Da, respectively. On incubation day

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25, the MW of DHP treated by P. chrysosporium was 2,919 Da. The phenolic OH

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content of DHP progressively declined during the incubation period, while the amount

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of NBO products after fungal treatment was generally lower than that of the control

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(Figure 1, Table 2B). These results indicated that reducing agents played a significant

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role in stabilizing the DHP-containing high phenolic OH group by cleavage of the ether

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bonds. In particular, α-tocopherol as a lipophilic reducing agent had a stabilizing effect

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on DHP, because preliminary experiments in which ascorbic acid was added without α-

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tocopherol affected only the degradation of lignin oligomers derived from DHP in the

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culture medium. Accordingly, the MW of DHP incubated with P. chrysosporium in the

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presence of reducing agents showed no significant fluctuation throughout the incubation

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period. When both α-tocopherol and ascorbic acid were added as reducing agents, P.

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chrysosporium could degrade DHP.

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Previous studies on the biomodification of synthetic lignin by white rot fungi

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demonstrated the crucial role of ligninolytic enzymes in synthetic lignin degradation

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along with the production of volatile organic acid from lignin.(26,27) However, the

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potential findings from measurement of changes in the MW of DHPs or detection of

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volatile compounds released from

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C-labeled synthetic lignin are limited.(27,28) 7



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Alternatively, our results suggested that reducing agents were necessary for the

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depolymerization of synthetic lignin because otherwise, enzymatic degradation and

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polymerization occurred simultaneously during incubation with whole cells of

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basidiomycetes. In addition, we also examined the changes in bonds in lignin effected

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by P. chrysosporium. Therefore, the findings described above represent a

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methodological advancement towards biocatalytic lignin modification by applying the

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catalytic system of P. chrysosporium.

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Lignin-Derived Compounds Production by Incubation with added Reducing

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Agents. To achieve a better understanding of the characteristic biomodification

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mechanism of synthetic lignin by P. chrysosporium, the MWs and components of

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degraded products dissolved in culture medium were also analyzed. The MW of lignin

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oligomers progressively decreased during the incubation period (Table 1D) compared

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with non- addition of reducing agent (Table 1C), which showed a slight fluctuation of

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molecular weight. Ascorbic acid, which is a hydrophilic reducing agent, had a positive

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effect on the degradation of lignin oligomers derived from DHP.

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The results from analyzing the degradation products of DHP by gas

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chromatography-mass spectrometry (GC-MS) showed that various products were

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present, including aromatic and acid compounds. On incubation day 10, succinic acid,

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2,6-dimethoxy-1,4-benzodiol, and syringic acid were detected (Figure 2). Because α-

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tocopherol stabilized DHP, the catalytic enzyme system of P. chrysosporium

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preferentially induced the formation of various degradation intermediates from DHP,

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rather than polymerization. Syringic acid released from DHP was oxidized to 2,6-

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dimethoxy-1,4-benzodiol. Thereafter, succinic acid was formed through the metabolic 8


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pathway of P. chrysosporium. Because succinic acid is one of the primary metabolites of

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the fungus, it was considered that the metabolic pathway of the fungus was involved in

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production of succinic acid from synthetic lignin. In our previous study, we found that P.

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chrysosporium degraded syringic acid to 4-hydroxybenzoic acid, hydroquinone, and

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succinic acid, as well as degrading hydroquinone to succinic acid.(29) Accordingly,

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aromatic compounds hydroxylated at the para position such as 2,6-dimethoxy-1,4-

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benzodiol were assumed to be cleaved by homogentisate-1,2-dioxygenase and then

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degraded to succinic acid by primary metabolism of P. chrysosporium. Our

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transcriptomic results described subsequently also supported this hypothesis.

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During the incubation with DHP, P. chrysosporium secreted approximately 0.1 mg/L

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of succinic acid as the primary metabolite (Figure 3), and also secreted a maximum of

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1.46 mg/L of succinic acid in the presence of reducing agents. In comparison, P.

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chrysosporium exposed to DHP secreted 0.75–18.51 mg/L of succinic acid between

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incubation days 1 and 25 (Figure 3). This yield of succinic acid is low compared with

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that reported by other studies on the production of acid compounds from lignin by

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biological treatment.(6,30) However, our results showed that basidiomycetes could be

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used as a biocatalyst for lignin modification. In particular, P. chrysosporium represents a

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powerful tool for the utilization of the aromatic biomass, because acid compounds

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produced

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polyhydroxyalkanoate, which is a useful bioplastic. The derivation of acid compounds

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from lignin is a promising research area and to date studies on the catabolism of lignin

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using bacteria to produce aromatic and acid compounds have been carried out

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intensively.(6,8,30) Succinic acid, which was the final degradation product of lignin in

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this study, is an economically valuable chemical that can also be produced by the

from

lignin

can

be

converted

into

myriad

9


products,

including


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biochemical transformation of sugars via biorefinery methods.(31) Succinic acid has

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been utilized as a surfactant/detergent, food additive, and health-promoting agent, and it

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is recognized to have the potential for industrial use.(32) Therefore, the formation of

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succinic acid from DHP by incubation with P. chrysosporium in this study is noteworthy.

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Accordingly, for investigating the secretome of P. chrysosporium related to the

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production of succinic acid from lignin, we performed an analysis of the differential

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expression of enzyme-coding genes related to lignin degradation by transcriptomic

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profiling.

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Functional Analysis of Enzymes Related to the Lignin Degradation of P. chrysosporium

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Extracellular enzymes related to bond cleavage of lignin in P. chrysosporium.

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Extracellular lignin peroxidase and manganese peroxidase have been isolated from P.

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chrysosporium, the best studied white rot basidiomycetes. These representative enzymes

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play key roles in lignin degradation.(13) However, it was difficult to explain, by

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examining extracellular enzymes alone, how succinic acid was produced from lignin.

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Therefore, a transcriptomic analysis was carried out to investigate the differential

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expression of enzyme-coding genes when P. chrysosporium degraded synthetic lignin

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under the presence of reducing agents.

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Figure 4 shows the expression levels of extracellular enzymes related to lignin

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degradation in the presence of reducing agents. Each enzyme-coding unigene was

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identified to have a high identity with a corresponding annotated enzyme in the NCBI

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database as determined by BLAST searches. Numerous extracellular enzymes, such as

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lignin peroxidase, manganese peroxidase, copper radical oxidase, multicopper oxidase 10


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(MCO), cellobiose dehydrogenase, and glyoxal oxidase activities, were up-regulated at

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the transcript level as synthetic lignin was added. These enzymes were already well

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known to play important roles in lignin degradation through interacting with one another.

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It is important to recognize that the expression levels of specific enzyme-coding

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unigenes increased, including pclip1 and pcmco1. The fragments per kilobase of exon

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per million fragments mapped (FPKM) level of pclip1 reached 22.9 with a fold-change

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value of 7.9 and that of pcmco1 reached 19.4 (Figure 4). As mentioned above,

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extracellular lignin peroxidase plays a key role in the bond cleavage of lignin.(13,14)

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MCO secreted from P. chrysosporium has been reported to modulate the Fenton reaction

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through Fe2+ oxidation by ferroxidase activity.(33,34)

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Analysis of the conserved domains of each gene identified pclip1 as encoding a

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protein that contained a heme-binding site with two axial histidine residues, termed

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proximal (Histidine-67) and distal (Histidine-204) histidines, corresponding to

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Histidine-47 and Histidine-176 in LiP-H8, as well as a substrate-binding site with an R_

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_ FH motif.(14) However, phylogenetic analysis of 7 lignin peroxidase genes showed

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that pclip1 was distant from other genes. In addition, the pcmco1-encoded protein was

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identified to have a differently-shaped conserved domain compared with that of other

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MCOs. The general conserved MCO domain has three cupredoxin domains composed

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of several copper binding motifs.(35) The conserved domain of Pcmco1 was identified

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to have three cupredoxin domains but the second cupredoxin domain did not contain

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interface motifs. Therefore, it was assumed that the different structure of the Pcmco1

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domain was associated with its different expression, compared with that of other genes

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Therefore, it would likely be valuable to conduct further studies on the characteristics of

264

these genes. We are investigating the differences in gene sequence and function of 11



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pclip1 and pcmco1 of P. chrysosporium.

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Consequently, P. chrysosporium attacks lignin polymers utilizing these complex

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extracellular enzymes. Under the experimental conditions used in the current study,

268

reducing agents facilitated predominantly bond cleavage of lignin by stabilizing radicals

269

despite the enhanced activities of extracellular lignin-degrading enzymes. Thus,

270

synthetic lignin was degraded by the extracellular enzymes system of P. chrysosporium

271

in the presence of reducing agents.

272 273

Intracellular enzymes related to aromatic catabolism in P. chrysosporium.

274

Transcriptomic analysis using next-generation sequencing (NGS) technology has the

275

advantage of being able to simultaneously analyze the functions of extracellular and

276

intracellular enzymes because it provides multidimensional examinations of cellular

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transcriptomes.(36) In this study, the differentially expressed gene (DEG) results

278

demonstrated the involvement of various intracellular enzymes in lignin degradation.

279

Cytochrome P450 monooxygenase (CYP450), 1,4-benzoquinone reductase (QR), and

280

aryl alcohol dehydrogenase (AAD) were expressed (Figure 5A).

281

The expression of CYP450 was increased after the addition of DHP on incubation

282

day 5 (Figure 5A). Intracellularly, the monooxygenation reaction by CYP450 is well

283

known to play an important role in the biodegradation of lignin and aromatic

284

compounds.(37) CYP450 is implicated in the oxidative elimination of hydrophobic

285

substances.(38)

286

AAD and QR were also overexpressed (Figure 5A). AAD can convert aromatic

287

aldehydes into their corresponding alcohols, and QR plays a role in the conversion of

288

quinone to the corresponding hydroquinone needed to produce an additional Fenton 12


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reagent.(39,40) The expression of AAD was highest in the fungal sample treated with

290

DHP and reducing agents on incubation day 25, and the expression of QR was also high

291

in the samples exposed to DHP (Figure 5A). The enhancement of AAD and QR

292

activities was coincident with which the occurrence of lignin degradation, strongly

293

suggesting their involvement in lignin degradation. Finally, the overexpression of genes

294

related to dioxygenases was observed (Figure 5A). Homogentisate 1,2-dioxygenase

295

(HDO), a type of dioxygenase, is responsible for the ring cleavage step in the

296

degradation pathway and was previously reported to be up-regulated by ethylbenzene

297

treatment.(41) In this study, the FPKM levels of the dioxygenase annotated as HDO

298

reached 33.8 on day 5 and 25.6 on day 25 (Figure 5A). Other types of dioxygenases

299

except for homogentisate 1,2-dioxygenase were rarely up-regulated in fungal samples

300

exposed to DHP. Accordingly, because hydroquinone was detected as an intermediate in

301

aromatic degradation,(29) homogentisate 1,2-dioxygenase was assumed to be involved

302

in ring cleavage. Although we could not detect C6-type acid compounds derived from

303

aromatics, it was supposed that ring-opened intermediates stimulated the metabolic

304

pathway, resulting in the production of succinic acid.

305

Consequently, the degradation of synthetic lignin involved the intracellular enzymes

306

of P. chrysosporium as well as its extracellular enzymes in the presence of reducing

307

agents. Aromatic fragments released from lignin by extracellular enzymes were oxidized

308

by intracellular enzymes such as CYP450, AAD, QR, and HDO.

309 310

Intracellular Enzymes Related to the Production of Succinic Acid in P.

311

chrysosporium. The above-mentioned results confirmed that lignin degradation was

312

mediated by extracellular and intracellular enzymes. The production of succinic acid 13



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from DHP was observed as described previously. We carried out a Kyoto Encyclopedia

314

of Genes and Genomes (KEGG) pathway analysis based on the DEG results to

315

investigate enzymes related to the production of succinic acid from DHP, and to

316

understand the metabolic processes of P. chrysosporium at the transcriptional level.

317

The KEGG pathway analysis results were indicative of the involvement of the

318

tricarboxylic acid (TCA) metabolic pathways in producing succinic acid. Figure 5B

319

presents the FPKM levels of enzymes of the TCA cycle and glyoxylate cycle.

320

Basidiomycetes have been reported to have a unique metabolic system termed the

321

“short-cut TCA/glyoxylate bicycle system”.(42,43) Citrate synthase, isocitrate

322

dehydrogenase (ICD), and 2-oxoglutarate dehydrogenase, all of which function in the

323

TCA cycle, were reportedly up-regulated when P. chrysosporium was exposed to

324

vanillin. This pathway converts isocitrate to succinate-producing intermediates such as

325

3-carboxy-1-hydroxypropyl-thiamine diphosphate and succinyl coenzyme A.(43)

326

Alternatively, isocitrate can be converted to succinate and glyoxylate directly in the

327

short-cut TCA cycle.(44) Notably, the glyoxylate cycle exists in basidiomycetes (43) as

328

well as in plants and certain other microorganisms.(45)

329

We found that 7 enzymes in the TCA cycle and 3 in the glyoxylate cycle were up-

330

regulated in the presence of reducing agents, especially on incubation day 25 (Figure

331

5B). The activities of isocitrate lyase, citrate synthase, and aconitase were sharply

332

enhanced on day 25 in the presence of reducing agents. Additionally, 2-oxoglutarate

333

dehydrogenase and succinyltransferase were up-regulated in the fungus exposed to DHP

334

(Figure 5B). On the whole, the addition of the synthetic lignin DHP drastically enhanced

335

the activities of enzymes functioning in the TCA and glyoxylate cycles. Particularly, the

336

up-regulation of three enzymes in the short-cut TCA cycle is indicative of an 14


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337

enhancement of succinate production. Thus, it was assumed that aromatic compounds

338

derived from synthetic lignin were degraded by dioxygenases, resulting in the formation

339

of acid compounds that are important intermediates for entering fungal metabolic

340

pathways. These intermediates were metabolized by various enzymes functioning in the

341

TCA cycles of P. chrysosporium. This assumption was supported by the up-regulation of

342

enzymes functioning in the TCA cycle and the short-cut TCA cycle. The glyoxylate

343

cycle has been reported to exist alongside the short-cut TCA cycle in basidiomycetes.(43)

344

Consequently, the short-cut TCA cycle was the major metabolic pathway in P.

345

chrysosporium that produced succinic acid from DHP (Figure 6).

346 347

CONCLUSION

348

P. chrysosporium simultaneously induced the degradation and polymerization of

349

synthetic lignin. The addition of reducing agents induced the degradation of DHP as

350

well as the production of aromatic compounds (syringic acid and hydroquinone) and

351

succinic acid under ligninolytic treatment by P. chrysosporium. We established the

352

biomodification mechanism of synthetic lignin by P. chrysosporium. The unique

353

catabolic system of P. chrysosporium induced a one-step conversion from synthetic

354

lignin to succinic acid. A transcriptomic analysis of P. chrysosporium provided

355

information about enzymes related to the lignin degradation and aromatic catabolic

356

pathways in the presence of reducing agents. Consequently, the extracellular catalytic

357

system of P. chrysosporium attacked synthetic lignin, resulting in the production of

358

aromatic compounds derived from lignin molecules. Thereafter, aromatic compounds

359

were metabolized in the short-cut TCA cycle of P. chrysosporium and were finally

360

converted to succinic acid. Our results showed that P. chrysosporium has promise as a 15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

361

tool for lignin valorization in a lignocellulosic biorefinery.

362 363

MATERIALS AND METHODS

364

Synthesis of Monolignols and Synthetic Lignin. Coniferyl alcohol and sinapyl

365

alcohol were synthesized from ferulic acid and sinapic acid (Sigma-Aldrich),

366

respectively, using the methods of Quideau and Ralph with slight modifications.(46) The

367

yields of coniferyl alcohol and sinapyl alcohol obtained from ferulic acid and sinapic

368

acid were more than 70% and 55%, respectively. The successful synthesis of coniferyl

369

alcohol and sinapyl alcohol were confirmed by GC-MS analysis.

370

DHP was used as a synthetic lignin. For the synthesis of DHP, horseradish

371

peroxidase (199 U/mg, MW = 44,000 Da) was purchased from Sigma-Aldrich, and three

372

solutions were prepared to synthesize DHP as follows: Solution 1, potassium phosphate

373

buffer (0.025 N, pH 6); Solution 2, mixture of dioxane (10 mL) and buffer solution (40

374

mL) containing 1.43 mmol of monolignols (molar feed ratio of sinapyl alcohol to

375

coniferyl alcohol, 6:4); and Solution 3, hydrogen peroxide (0.140 mL; 28 wt.%) in

376

buffer (50 mL). Horseradish peroxidase (5,000 U) was added to Solution 1 (100 mL),

377

and then Solution 2 and Solution 3 were added drop-wise to Solution 1 for 5 h at room

378

temperature with stirring at 400 rpm (end-wise polymerization). The mixture was then

379

stirred for an additional 5 h. The precipitates were separated from the buffer solution by

380

centrifugation (12,000 rpm, 15 min) and then lyophilized to obtain DHP.(47)

381 382

Microorganism and Culture Preparation. P. chrysosporium (ATCC 20696) was

383

grown on 3.9% potato dextrose agar medium, incubated at 28 °C for 7 days, and then

384

stored at 4 °C. After 7 days, the mycelium had fully grown. Mycelia covering the agar 16


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385

medium were separated from the medium and homogenized in 20 mL of distilled water

386

at 5,000 rpm for 3 min. Finally, the dry weight of the fungal suspension was calculated

387

on a drying machine at 105 °C to ensure the addition of an equal amount of fungal

388

biomass in each flask.

389

Shallow stationary culture (SSC) medium was used as a nitrogen-limited medium.

390

SSC medium was proposed as a culture medium for the activation of specific enzymes,

391

such as ligninolytic enzymes. To prepare SSC medium, the 6 major components

392

(glucose, ammonium tartrate, KH2PO4, MgSO4, CaCl2, and thiamine HCl) of the

393

medium were dissolved in 990 mL of distilled water. Then, 10 mL of the solution

394

containing various mineral components were added after filtration through a 0.2-µm

395

syringe membrane filter and then the medium was autoclaved at 121 °C for 15 min.(48)

396

The fungus (dried weight: 0.04 g) was inoculated into 200 mL of SSC medium in a 500-

397

mL Erlenmeyer flask, which was sealed with a silicone rubber stopper. After the fungal

398

culture was incubated at 28 °C for 4 days, 200 mg of synthetic lignin was spiked into the

399

medium, followed by incubation under the static condition. To induce the

400

depolymerization reaction of synthetic lignin during the procedure of biomodification by

401

white rot fungus,(23) 5 mM of ascorbic acid (Sigma-Aldrich, ≥99%) and 1 mM of α-

402

tocopherol (Sigma-Aldrich, 96%) were added at incubation times 3, 7, 12, 17, and 22

403

days after preincubation.

404 405

Analytical Methods for Modified Lignin and Lignin-Derived Compounds. We

406

prepared the three types of samples as follows: 1) synthetic lignin in medium, 2) fungi in

407

medium, and 3) synthetic lignin treated by the fungus in medium. Each sample was

17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

408

centrifuged at 12,000 rpm for 15 min at 4 °C to separate the medium and precipitates

409

containing the fungi and synthetic lignin. After centrifugation, 50 mL of dioxane was

410

added to the precipitates containing mycelia to facilitate the extraction of DHP adsorbed

411

onto the mycelia. DHP in dioxane was lyophilized. To identify the structural changes to

412

the precipitated DHP, the MW and the contents of phenolic hydroxyl groups and NBO

413

products were analyzed (see Supporting Information).

414 415

Analysis of Degradation Products of DHP. After centrifugation, the supernatant

416

was extracted with 100 mL of ethyl acetate in triplicate, evaporated, and then dissolved

417

in 10 mL of ethyl acetate. To analyze the characteristics of the liquid fraction, gel

418

permeation chromatography was carried out as described in the Supporting Information

419

and GC-MS was performed using a HP7890A GC instrument (Agilent) with a HP5975A

420

mass selective detector (Agilent) and a DB-5 capillary column (30 m × 0.25 mm ID ×

421

0.25 µm coating thickness; Agilent). The initial oven temperature of the GC was 50 °C

422

for 5 min, after which the temperature was increased at a rate of 3 °C/min up to 300 °C

423

and maintained there for 10 min. The temperatures of the injector and detector were

424

220 °C and 300 °C, respectively, and the carrier gas was helium at a flow rate of 1

425

mL/min. Peak identification was based on comparison of the mass spectra with the

426

NIST (National Institute of Standards and Technology) library.

427 428

Transcriptomic Analysis of P. chrysospporium Exposed to DHP. To identify enzymes

429

of P. chrysosporium that are involved in lignin degradation, a transcriptomic analysis

430

was carried out using a HiSeq™ 2500 platform (Illumina). To analyze DEGs between

431

fungal samples, fungal samples were divided into 6 groups depending on the addition of 18


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432

reducing agents and synthetic lignin as follows: P. chrysosporium on 5 and 25 days (5 and

433

25 PCH), P. chrysosporium treated with reducing agents on 5 and 25 days (5 and 25

434

PCH+RA), and P. chrysosporium treated with DHP and reducing agents on incubation 5 and

435

25 days (5 and 25 PCH+DHP+RA).

436

For the transcriptomic analysis, total RNA was isolated. After total RNA extraction,

437

the NGS library was prepared for the transcriptomic analysis using a kit purchased from

438

BIOO Scientific. Sequencing was carried out using the HiSeq™ 2500 platform and the

439

resulting transcriptomic data were filtered by removing adapter sequences, empty reads,

440

low-quality reads, and reads with more than 20% Q < 20 bases. RNA-Seq data obtained

441

for the mycelium of P. chrysosporium were mapped onto a reference dataset through the

442

re-sequencing method. FPKM levels were determined after a normalization process. To

443

identify the DEGs among the six samples, the edgeR package was used based on the

444

FPKM level (see Supporting Information). KEGG metabolic pathway analysis was

445

performed by aligning genes with sequences from the NCBI non-redundant database

446

and automatically assigning gene functions to corresponding KEGG terms (see

447

Supporting Information). All experiments related to transcriptomic analysis were

448

performed in the National Instrumentation Center for Environmental Management

449

(NICEM) at Seoul National University, Korea.

450 451

Supporting Information

452

The Supporting Information is available free of charge via the Internet

453 454

Gel permeation chromatography (GPC), content of phenolic hydroxyl (OH) groups,

455

nitrobenzene oxidation (NBO), total RNA extraction and NGS library preparation, 19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

456

analysis of Differently Expressed Genes (DEGs), Kyoto Encyclopedia of Genes and

457

Genomes (KEGG) annotation (PDF)

458 459

Acknowledgments

460

This research was supported by the Research Program of the National Institute of Forest

461

Science (NIFoS), Seoul, Republic oc Korea.

20


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C-labelled lignin (14C-DHP) during solid-state fermentation of

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41. Gunsch, C. K., Cheng, Q., Kinney, K. A., Szaniszlo, P. J., and Whitman, C. P. (2005) Identification of a homogentisate-1, 2-dioxygenase gene in the fungus Exophiala lecanii-corni: analysis and implications, Appl. Microbiol. Biotechnol. 68, 405–411. 42. Munir, E., Yoon, J.-J., Tokimatsu, T., Hattori, T., and Shimada, M. (2001) New role for glyoxylate cycle enzymes in wood-rotting basidiomycetes in relation to biosynthesis of oxalic acid, J. Wood Sci. 47, 368–373. 43. Shimizu, M., Yuda, N., Nakamura, T., Tanaka, H., and Wariishi, H. (2005) Metabolic regulation at the tricarboxylic acid and glyoxylate cycles of the lignin‐degrading basidiomycete Phanerochaete chrysosporium against exogenous addition of vanillin, Proteomics, 5, 3919–3931. 44. Munir, E., Yoon, J.J., Tokimatsu, T., Hattori, T., and Shimada, M. (2001) A physiological role for oxalic acid biosynthesis in the wood-rotting basidiomycete Fomitopsis palustris, Proc. Nat. Acad. Sci. U S A. 98, 11126–11130. 45. Cozzone, A. J. (1998) Regulation of acetate metabolism by protein phosphorylation in enteric bacteria, Annu. Rev. Microbiol. 52, 127–164. 46. Quideau, S., and Ralph, J. (1992) Facile large-scale synthesis of coniferyl, sinapyl, and p-coumaryl alcohol, J. Agric. Food. Chem. 40, 1108–1110. 47. Moon, S.-J., Kwon, M., Choi, D., Won, K., Kim, Y. H., Choi, I.-G., and Choi, J. W. (2012) In vitro analysis of the monolignol coupling mechanism using dehydrogenative polymerization in the presence of peroxidases and controlled feeding ratios of coniferyl and sinapyl alcohol, Phytochem. 82, 15–21. 48. Hong, C. Y., Park, S. Y., Kim, S. H., Lee, S. Y., Choi, W. S., and Choi, I. G. (2016) Degradation and polymerization of monolignols by Abortiporus biennis, and induction of its degradation with a reducing agent, J. Microbiol. 54, 675–685. 26


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1

Table 1. Molecular weight change of dehydrogenative polymer (DHP)(A & B) and oligomers derived from DHP(C & D) after treated by P.

2

chrysosporium with / without addition of reducing agents (Control: DHP in medium, PCH+ DHP: DHP treated by P. chrysosporium)

(A)Non addition of reducing agents

Control

0d

5d

10d

15d

20d

25d

1d

5d

10d

15d

20d

25d

Mw

a

3686

3527

3676

3701

3457

3563

3077

-

3132

-

3034

-

Mn

b

1832

2038

2393

2426

1978

1988

1128

-

1201

-

1134

-

Mw/Mn

DHP PCH+DHP

(B)Addition of reducing agents

2.01

1.73

1.54

1.53

1.75

1.79

2.72

-

2.61

-

2.68

-

Mw

a

3686

3106

3240

3897

3580

3930

2987

3290

3507

3445

3300

2919

Mn

b

1832

1603

1656

1627

1717

1877

1187

1333

1285

1489

1391

1198

2.01

1.94

1.96

2.40

2.08

2.09

2.51

2.47

2.73

2.31

2.37

2.44

Mw/Mn

(C)Non addition of reducing agents 0d

5d

10d

15d

20d

25d

1d

5d

10d

15d

20d

25d

1021

-

1009

-

-

848

-

852

-

920

-

846

-

793

-

-

490

-

495

-

476

-

Mw/Mn

1.21

-

1.27

-

-

1.73

-

1.72

-

1.93

-

Mw a

941

978

1028

966

949

1276

1061

1015

1049

930

854

Mn b

565

631

646

643

643

741

682

609

577

541

512

1.66

1.55

1.59

1.50

1.48

1.72

1.56

1.67

1.82

1.72

1.67

Mw a

Oligomers derived from DHP

Control

PCH+DHP

(D) Addition of reducing agents

Mn

b

Mw/Mn

*DHP: Mw: 3686, Mn: 1832, Polydispersity: 2.01

3

*DHP: Mw: 3421, Mn: 1476, Polydispersity: 2.32

4 5

a

weight-average molecular weight (Daltons), b number-average molecular weight (Daltons), - : Not detection 27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

6 7

Table 2. Nitrobenzene oxidation products (µmol/g sample) of dehydrogenative polymer (DHP) treated by P. chrysosporium with / without addition of reducing agents (Control: DHP in medium, PCH+DHP: DHP treated by P. chrysosporium) (A)Non addition of reducing agents Control

(B)Addition of reducing agents

PCH+DHP 823.8±50.2

Control

PCH+DHP

1570.1±99.4

985.4±37.1

0d

1022.3±140.3

5d

872.7±29.5

585.7±59.6

-

1076.4±177.7

10d

1274.2±345.1

314.8±57.3

1669.8±265.2

812.1±4.3

15d

840.8±22.4

799.2±191.3

-

254.7±168.4

20d

1111.3±98.8

1409.3±810.2

1123.8±212.4

376.8±2.2

25d

957.9±56.3

719.3±7.6

-

404.8±16.2

*DHP: Sum of G & S units: 1301.1±138.5 µmol/g sample

8

*DHP: Sum of G & S units : 1772.6±.467.2 µmol/g sample

9 10

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* - : Not detection

11 12 13

28


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Figure catption Figure 1. Phenolic hydroxyl group content of dehydrogenative polymer (DHP) treated by P. chrysosporium with / without addition of reducing agents (Control: DHP in medium, PCH+DHP: DHP treated by P. chrysosporium, Control+R.A.: DHP with reducing agents in medium, PCH+DHP+R.A.: DHP treated by P. chrysosporium with reducing agents) *Phenolic OH content of DHP: 4.78±0.17%

Figure 2. Total ion chromatograms of control (A) and sample treated by P. chrysosporium on incubation day 10 (B) and 20 (C)

Figure 3. Amount of succinic acid by P. chrysosporium in 200 ml of culture medium (A), and concentration of succinic acid from DHP by fungus with reducing agent (B) (PCH: P. chrysosporium, PCH+DHP: P. chrysosporium exposed to DHP, PCH+DHP+R.A: P. chrysosporium exposed to DHP with reducing agents)

Figure 4. Heat map of genes of extracellular enzymes of P. chrysosporium depending on addition of DHP on incubation time 5 and 25 days

Figure 5. Heat map of genes of intracellular enzymes of P. chrysosporium related to aromaitcs oxidation (A) and TCA cycle (B) depending on addition of DHP on incubation time 5 and 25 days Figure. 6. Biomodification mechanism of DHP by P. chrysosporium (ATCC 20696) with addition of ascorbic acid and α-tocopherol

29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. Phenolic hydroxyl group content of dehydrogenative polymer (DHP) treated by P. chrysosporium with / without addition of reducing agents (Control: DHP in medium, PCH+DHP: DHP treated by P. chrysosporium, Control+R.A.: DHP with reducing agents in medium, PCH+DHP+R.A.: DHP treated by P. chrysosporium with reducing agents) *Phenolic OH content of DHP: 4.78±0.17% *DHP : dehydrogenative polymer *R.A. : reducing agents

30


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(A)

(B)

(C)

Figure 2. Total ion chromatograms of control (A) and sample treated by P. chrysosporium on incubation day 10 (B) and 20 (C)

31



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3. Concentration (mg/L) of succinic acid by P. chrysosporium (A), and concentration of succinic acid from DHP by fungus with reducing agent (B) (PCH: P. chrysosporium, PCH+R.A.: P. chrysosporium adding the reducing agents, PCH+DHP+R.A: P. chrysosporium exposed to DHP with reducing agents)

32


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Heat map of genes of extracellular enzymes of P. chrysosporium depending on addition of DHP on incubation time 5 and 25 days *LiP: lignin peroxidase, *MnP: manganese peroxidase, *CRO: copper radical oxidase, *GLOX: glyoxal oxidase, * MCO: multicopper oxidase, * CDH: cellobiose dehydrogenase

33



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(A)

(B)

Figure 5. Heat map of genes of intracellular enzymes of P. chrysosporium related to aromaitcs oxidation (A) and TCA cycle (B) depending on addition of DHP on incubation time 5 and 25 days * CYP450: Cytochrome P450 monooxygenase, *AAD: aryl alcohol dehydrogenase, * QR: 1,4benzoquinone reductase, * HDO: homogentisate-1,2- dioxygenase

34


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. Biomodification mechanism of DHP by P. chrysosporium (ATCC 20696) with addition of ascorbic acid and α-tocopherol *LiP: lignin peroxidase, * MCO: multicopper oxidase, * CYP450: Cytochrome P450 monooxygenase, *AAD: aryl alcohol dehydrogenase, * QR: 1,4-benzoquinone reductase, * HDO: homogentisate-1,2- dioxygenase, * ICL : isocitrate lyase, * AC : aconitase, * ICD : isocitrate dehydrogenase, *OGD : oxoglutarate dehydrogenase, *ST : succinyltransgerase, *LG : ligase, *SD : succinate dehydrogenase, *CS : citrate synthase

35



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40x20mm (600 x 600 DPI)


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Phanerochaete chrysosporium Multienzyme Catabolic System for in

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