Source: MICHIGAN STATE UNIV submitted to
ENZYMOLOGY AND MOLECULAR BIOLOGY OF LIGNIN-MODIFYING ENZYMES OF WHITE-ROT FUNGI
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0153318
Grant No.
(N/A)
Project No.
MICL01629
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Apr 1, 2008
Project End Date
Dec 31, 2011
Grant Year
(N/A)
Project Director
Reddy, C. A.
Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824
Performing Department
MICROBIOLOGY AND MOLECULAR GENETICS
Non Technical Summary
Plant biomass is the most abundant and renewable repository of photosynthetic energy in the biosphere. Cellulose and hemicellulose, which are, respectively, polymers of glucose and pentose sugars are literally inexhaustible renewable sources of bioenergy which can be transformed into biofuels such as ethanol, animal feeds, and value added organic chemicals. Another major polymer in biomass, which is roughly equivalent to or slightly higher in quantity than hemicellulose, is lignin. It is a highly complex, heterogeneous, three-dimensional aromatic polymer that is recalcitrant to biodegradation. Because of its close physical and chemical association with cellulose and hemicellulose, lignin is the single most important limiting factor in lowering the efficiency of conversion of biomass to biofuels and other value added products. Hence there in increasingly intensive research worldwide to use microbes to delignify lignocellulosic materials and increase the efficiency of transformation of biomass into biofuels and other useful products. In the foreseeable future, biomass is potentially a very important source of energy that can give the United States energy independence, by decreasing our dependency on imported fossil fuels, stimulate economic growth, and give environmental benefits. Delignification of lignocellulosic feeds (straw, silage, grasses, etc.) is also known to contribute enhanced digestibility of 30% or more to ruminant animals, which are an important source of milk and meat to people worldwide. It has become apparent because of research in our lab and several other labs that ligninolytic enzyme systems of microbes gratuitously degrade a variety of chroaromatic pollutants and thus contribute to enhancement of environmental quality and for detoxification of toxic wastes that pose a serious health hazard to humans and animals. Last but not least, research on lignin degradation has important applications to increase the efficiency of operation of pulp and paper industries in North America. For nearly three decades or so, the research in our laboratory has been designed to obtain a better understanding of the ecology, physiology, enzymology, and molecular biology of the ligninolytic systems of white-rot fungi, which are perhaps the most efficient lignin degraders in the microbial world. Previous studies have shown that three families of lignin-modifying enzymes (LME) designated lignin peroxidases, manganese peroxidases, and laccases play an important role in the initial depolymerization and degradation of lignin. In continuation of our past research with these enzymes, the proposed study is primarily geared towards molecular understanding of the expression of key genes encoding some of the LMEs and to understand the regulation of expression of these genes so that eventually, we will be use this knowledge for selective and efficient delignification of lignocellulosic biomass.
Animal Health Component
30%
Research Effort Categories
Basic
60%
Applied
30%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1234020100020%
1234020104030%
1234020110230%
1334020100010%
1334020110210%
Goals / Objectives
An important focus of the research for the next few years would be on understanding the regulation of expression of key lignin-modifying enzymes in two models of white rot fungi. One of the organism we selected is P. chrysosporium, one of the most extensively studied white-rot fungus, which produces copious amounts of LIP and MNP enzymes, but marginal levels of laccase. Multiple isoforms of LIP and MNP enzymes are produced and a separate gene encodes each. For example, our lab has shown that this fungus produces over 10 LIP isozymes and several MNP isozymes and that each isozyme appears to be encoded by a separate gene. Ganoderma is an important white rot fungus with extensive worldwide distribution and it produces some of the highest levels of laccase recorded to date, but negligible levels of LIP and MNP. This organism produces multiple isoforms of laccase but whether or not all these isozymes are encoded by a separate gene is not known. However, studies with other white rot fungi such as Trametes sp. showed that a separate gene encodes individual isozymes. Very recently, we have isolated several laccase clones of this organism and two of the clones have been cloned and characterized. Several other clones are yet to be sequenced and characterized. These genomic clones and the corresponding cDNAs would be good tools for analyzing the selective expression of different laccase genes. To get a better understanding of the regulation of expression of LIPs, MNPs, and laccases, the two organisms would be grown under varying environmental conditions such as C, N, and different types of wood substrates and the nature and extent of expression would be characterized. Preliminary evidence suggests that different subsets of LIP genes are expressed under varying concentrations of C and N in the medium. This proposal represents one of the first detailed studies on the differential expression of different LME genes in two model organisms as influenced by a variety of environmental parameters of significance to the ecological niche of these ligninolytic white rot fungi. The study will provide information about the structural similaritiesand differences between different LME genes and the presence of regulatory signal sequences that may be unique to the ecological niches occupied by these two basidiomycetes. We also propose conducting X ray crystallography studies of laccase in Ganoderma lucidum and the data obtained will contribute to a fundamental biochemical understanding of laccase structure and function. Given below are the specific objectives. SPECIFIC OBJECTIVES Regulation of Expression of the Lignin Peroxidase and Manganese Peroxidase genes of Phanerochaete chrysosporium Characterization and Regulation of Expression of the Laccase genes of Ganoderma lucidum X ray crystallography of two major laccase isozymes from Ganoderma lucidum
Project Methods
An important focus of the research for the next few years would be on understanding the regulation of expression of key lignin modifying enzymes in two models of white rot fungi. Phanerochaete chrysosporium, perhaps the most extensively studied white rot fungus, produces copious amounts of LIP and MNP enzymes, but only marginal levels of laccase. Multiple isoforms of LIP and MNP enzymes are produced and a separate gene encodes each. Ganoderma lucidum is an important white rot fungus with extensive worldwide distribution and it produces some of the highest levels of laccase recorded to date, but almost no LIP and marginal levels of MNP. This organism produces multiple isoforms of laccase but whether or not a separate gene encodes these isozymes is not clearly known. However, studies with other white rot fungi such as Trametes sp. showed that separate gene encodes each individual isozyme. Very recently, we have isolated several laccase clones of this organism and two of the clones have been sequenced. Several other clones are yet to be sequenced and characterized. These genomic clones and the corresponding cDNAs would be good tools for analyzing the selective expression of different laccase genes. To get a better understanding of the regulation of expression of LIPs, MNPs, and laccases, each organism would be grown under varying environmental conditions such as C, N, and different types of wood substrates and the nature and extent of expression would be characterized. Preliminary evidence suggests that different subsets of LIP genes are expressed under varying concentrations of C and N in the defined medium. We will be studying differential expression of various LMEs as influenced by a varied environmental parameters of significance to the ecological niche of these ligninolytic white rot fungi. The study will provide information about the structural similarities and differences between different LME genes and the presence of regulatory signal sequences that may be unique to the ecological niches occupied by these two basidiomycetes. we propose to study in detail the effect of C, N, and Mn on the regulation of lip and mnp gene expression in wood grown cultures of P. chrysosporium. The pattern of lip gene expression in wood-grown white rot fungi can be precisely understood only if these fungi are grown directly on different species of wood rather than in defined medium. We will also be testing the hypothesis that the expression of lip genes in the white rot fungi is substantially different when they are grown on wood as primary substrate as compared to the expression of lip genes reported in defined medium cultures. We will also be testing the regulation of expression of LME when the given organism is grown with individual wood substrate and a combination of woods such as soft wood plus hard wood. We also propose conducting X ray crystallography studies of laccase in Ganoderma lucidum and the data obtained will contribute to a fundamental biochemical understanding of laccase function-structure in this organism relative to other fungi.

Progress 04/01/08 to 12/31/11

Outputs
OUTPUTS: Lignin modifying enzymes (LME) of white-rot basidiomycete fungi are the major players in nature in the degradation of lignin, the second most abundant organic polymer in the biosphere, and thus play an important role in global carbon cycling, in improving the efficiency of conversion of lignocelluloses to value added products. LMEs include three families of enzymes designated lignin peroxidases, manganese peroxidases, and laccases, which are relatively non-specific and degrade a broad spectrum of highly toxic environmental pollutants such as PCBs, dioxins, chlorophenols and a number of other compounds. Thus, white-rot fungi potentially can play a key role in alleviating environmental pollution and protecting health of humans and animals. As explained in my annual report for 2009, we have reoriented research in my laboratory due to loss of key research personnel as well as due to the growing worldwide interest in increasing food production compatible with sustainable agriculture, to meet the needs of the rapidly expanding human population worldwide. We have been successful in developing a polymicrobial formulation for substantially increasing the growth of a broad spectrum of crops. This product, SumaGrow, has received much press report because of its ability to increase productivity of a variety of vegetables, food crops, and fodder crops. MSU has applied for a patent and has licensed a biotech company (BioSoil Enhancers Inc., Hattiesburg, Mississippi) for commercial production of this product. An international conference has been scheduled for Jan18-19, 2012 in Biloxi, MS to exchange data from field trials done with Suma Grow on a variety of crops, in a variety of soil types, and in diverse geographical regions of the USA. During the first two years of the reporting period we published four papers and one review in this area of xenobiotic degradation and co-directed the Ph. D. research of Dr. Marco-Urrea from the University of Barcelona in Spain. In collaboration with a scientist from Egypt, we published a peer-reviewed paper on decolorization of the dye Victoria Blue. I presented a six-week course on "Fungal Degradation of Xenobiotics" at the University of Barcelona and presented two public seminars to a larger audience from multiple departments. I also participated as a main speaker in two international symposia. A peer-reviewed proceedings paper has been published describing the polymicrobial formulation enhancing crop productivity. I mentored an undergraduate research student who demonstrated the ability of Trichoderma to facilitate profuse root nodulation in garden beans, substantially improving nitrogen economy of the plant and increasing yields. He won the prestigious Gilmore Award as well as Gerhardt Award in 2008 and 2009, respectively. He presented his results at the MSU undergraduate research forum. He is now doing Ph. D. in a large public university. I also mentored Shraddha, who did research on Bacterial Diversity in root nodules of green beans. I also hosted a visiting scientist from India for two years who played a key role in developing the polymicrobial formulation mentioned above. PARTICIPANTS: C. A. Reddy (PI), Visiting Scientist Lalithakumari Janarthanam from the Biology faculty at the University of Guyana; Ernest Marco-Urrea [mentee and collaborator from the Department of Chemical Engineering at the University of Barcelona in Spain]; Ola M. Gomaa [mentee and visiting scientist from the National Center for Radiation Research and Technology, Nasr City, Cairo, Egypt]; Shraddha Narayanan, Masters graduate student at Michigan State University; Mark Charboneau, research undergraduate student at Michigan State University; and R. S. Saravanan(Ph. D.), postdoctoral researcher. TARGET AUDIENCES: Target audiences include governmental agencies such as the U. S. Department of Agriculture, U. S. Environmental Protection Agency, U.S. Department of Energy, industrries involved in production of environmental pollutants and interested in bioremediation to meet the regulatory guidelines imposed by State or Federal Government agencies; Industries involved in palnt biotechnology worldwide; Farmers in the USA and abroad interested in sustainable agriculture and increasing crop productivity; Turf grass industry, deer hunting clubs/community which have showed a great interest; Beef production industry; and farmers involved in production of fodders. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The white-rot fungus Trametes versicolor degraded trichloroethylene (TCE), a highly oxidized chloroethene, and one of the priority pollutant chemicals on the EPA list. Using [13C]-TCE as the substrate, [2,2,2]-trichloroethanol and carbon dioxide were the main products of degradation. Laccase (nor LiP or MnP), does not appear to play a role in TCE degradation, but cytochrome P-450 appears to be involved as evidenced by marked inhibition of TCE degradation in the presence of 1- aminobenzotriazole, a known inhibitor of cytochrome P-450. The results indicate that the TCE degradation pathway in T. versicolor is similar to that previously reported in mammals and is mechanistically quite different from bacterial TCE degradation. Further studies showed that mixtures of TCE and polychloroethene were also degraded by T. versicolor. Synthetic textile dyes are among the most dangerous chemical pollutants released in industrial wastewater streams and a number of these dyes are readily broken degraded by the white rot fungus Phanerochaete chrysosporium. Victoria Blue B (VB),one of the more important polluting dyes, was degraded by the fungus over a relatively broad concentration range. Sodium azide and aminotriazole inhibited endogenous catalase and cytochrome P-450 oxygenase activities and there was 100% and 70% reduction in VB decolorization, respectively. Adding benzoate to trap hydrogen peroxide-derived hydroxyl radicals resulted in 50% inhibition in VB decolorization. Collectively, these data suggest that laccase, and/or oxygenase/oxidase plays a role in VB decolorization by P. chrysosporium. Considering the current human population of over 7 billion and the projected need to raise world food production by 110% in the next 50 years, there is an urgent need to develop stable, efficacious, and eco-friendly microbial formulations designed to enhance productivity of a broad spectrum of crops consistent with the principles of sustainable agriculture. The desired beneficial effects of such a formulation are: enhancement of nitrogen fixation, direct or indirect inhibition of plant pathogens, solubilization and mobilization of insoluble phosphates, induce plant resistance to pathogens and production of plant growth-promoting factors. We constructed a polymicrobial formulation containing selected bacteria and fungi, using humate (12%) as a carrier. This product, designated SumaGrow, showed substantial increase in productivity of a broad spectrum of crops including: vegetables such as garden beans, tomatoes, peas, okra, eggplant, and squash; major food crops such corn, soybean, rice, peanuts, and sorghum; fodder crops such as alfalfa; and biofuel grasses such as switch grass. In general, SumaGrow-treated plants appeared healthier and showed early flowering and fruiting with good root nodulation (in the case of legumes). Yields obtained in field trials were consistent with those from the greenhouse experiments according to a company licensed by MSU to produce the product. SumaGrow's forage boost product has been chosen as a "2011 Best of What's New" grand award winner in the green technology category by Popular Science magazine.

Publications

  • 1. Marco-Urrea, E. and Reddy, C. A. 2012. Degradation of chloro-organic pollutants by white rot fungi. In S. N. Singh (ed.) Microbial Degradation of Xenobiotics. Springer-Verlag, Berlin


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: The results on our remarkably effective polymicrobial growth formulation for boosting crop productivity have been presented at the International Congress on Conservation Agriculture in New Delhi, 2009. Our results were also featured on the opening page of Michigan State University web page for one month and also in the local news paper, State News. Our results also received wide publicity nationwide by a feature article in Popular Science magazine. One of the pictures from our results were picked as one of the ten best scientific pictures of the year by the Fobes magazine. News items of our results were also published in a number of trade magazines related to agriculture. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: We intend to stay on course with the research described in this report.

Impacts
As explained in the 'project modifications' section of 2009 progress report, the research in my lab was modified substantially to still stress on biomass but focus more on crop productivity. This was necessary because of the availability of substantial research funding and more importantly to address the growing problem of food shortage for people around the world.The rationale and the results are described below.Considering the current population of 6.5 billion humans on this planet and the projected need to raise world food production by 110% in the next 50 years to meet the growing food needs of the fast rising population, there is urgent need for research aimed at developing cost-effective and eco-friendly polymicrobial formulations to obtain increased crop yields with minimal input of chemical nitrogen fertilizers and pesticides, conserve soil health, and preserve environmental quality. Our aim is to develop stable, efficacious, and eco-friendly microbial formulations containing naturally occurring diverse phylogenetic groups of microbes with complementary functions designed to enhance productivity of a broad spectrum of crops. The formulation is designed to provide the observed beneficial effects through enhancement of nitrogen fixation, direct or indirect inhibition of plant pathogens, solubilization and mobilization of insoluble phosphates, and production of plant hormonal substances. We constructed a formulation (F2),now designated SumaGrow, using humate (12%) as a carrier. Sumagrow showed substantial increase in productivity of a broad spectrum of crops including Zea mays (corn), Sorghum bicolor (sorghum), Glycine max (soybean), Phaseolus vulgaris (Garden bean), Pisum sativum (pea), Phaseolus sp. (wonder bush bean), Arachis hypogea (peanut), Oryza sativa (rice), Lycopersicon esculentum (tomato), Solanum melongena (eggplant), Hibiscus esculentus (okra), and Cucurbita maxima (squash). For example, when compared to controls, corn production increased by 65%, eggplant 41%; wonder bush beans 40%, tomato 88%, soybeans 96%, pea purple hull 50%, okra 50% and rice yield by 40%. In general, SumaGrow-treated plants appeared healthier and showed early flowering and fruiting with good root nodulation (in the caselegumes). Yields obtained in field trials were consistent with those from the greenhouse experiments. For example, suash, tomato, bell peper, and corn yields increased 61%, 75%, 27.5%, and 40%. We conclude that the polymicrobial growth formulation described here causes remarkable enhancement of a broad spectrum of vegetable, leguminous and cereal crops, and grasses. These results enabled us to file a 'Disclosure of Invention' and submission of a formal patent application.This research has also attracted wide industry attention. More importantly, Michigan State University has granted an exclusive license for the large scale manufacture and distribution of our product to farmers nationwide. The product is also being sold to a number of golf courts because of the desirable qualities of our product.

Publications

  • Reddy, C. A. and Lalithakumari, J. 2009. Poly microbial formulation for enhanced productivity of a broad spectrum of crops. Wold. Congr. Conserv. Agric. Symposium: Lead Papers, p 94-101, Indian Council of Agricultural Research, New Delhi.


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: Lignin peroxidases, manganese-dependent peroxidases,and laccases are three families of lignin modifying oxidative enzymes, produced by white rot basidiomycete fungi, that play a key role in lignin degradation and in the breakdown of many toxic environmental polutants. Trametes versicolor and Phanerochate chrysosporium are two of the best studied white rot fungi worldwide. In this report we present our results on the mechanisms of degradation of trichloroethylene (TCE) by T. versicolor and decolorization of dye pollutants by P. chrysosporium. Using [13C]-TCE as the substrate, we showed that T. versicolor degraded TCE and produced 2,2,2-trichloroethanol and carbon dioxide as the main products of degradation, based on the results obtained . For a range of concentrations of TCE between 2 and 20 mg per liter, 53% of the theoretical maximum chloride expected from complete degradation of TCE was observed. Laccase was shown to be induced by TCE, but did not appear to play a role in TCE degradation. Cytochrome P-450 appears to be involved in TCE degradation,as evidenced by marked inhibition of degradation of TCE in the presence of 1- aminobenzotriazole, a known inhibitor of cytochrome P-450. Our results indicated that trichloroacetaldehyde is a key intermediate in TCE degradation suggesting that the pathway for TCE degradation in T. versicolor is similar to that of mammals but is much different from bacterial TCE degradation. Parallel studies, in collaboration with Ola Gomma from Egypt, focused on degradation of selected synthetic textile dyes that are among the more toxic chemical pollutants released in industrial wastewater streams. Recognizing the adverse environmental impact of these dyes, the ability of P. chrysosporium to decolorize these textile dyes was investigated. P. chrysosporium decolorized 6 of the 14 structurally diverse dyes with varying efficiency (14% to 52%), but there was no discernable pattern of decolorization even among dyes of the same chemical class, suggesting that attack on the dyes is relatively non-specific. Victoria Blue B (VB) was chosen for further analysis because the ability of the fungus to decolorize VB was nearly independent over a relatively broad concentration range. Blocking lignin peroxidase (LiP) and manganese peroxidase (MnP) production by the fungus did not substantially affect VB decolorization. Inhibition of laccase production by adding various inhibitors to shaken cultures reduced VB decolorization significantly suggesting a role for laccase in VB decolorization. When sodium azide and aminotriazole were used to inhibit endogenous catalase and cytochrome P-450 oxygenase activities, there was 100% and 70% reduction in VB decolorization, respectively. Adding benzoate to trap hydrogen peroxide-derived hydroxyl radicals resulted in 50% decolorization of VB. Boiling the extracellular fluid (ECF) for 30 min resulted in approximately 50% reduction in VB decolorization. Collectively, these data suggest that laccase, and/or oxygenase/oxidase and a heat-stable non-enzymatic factor, but not lignin peroxidase or manganese peroxidase, play a role in VB decolorization by P. chrysosporium. PARTICIPANTS: TCE work is a collaboration between C. A. Reddy from Michigan State University and Ernest Marco-Urrea and other colleagues from the Department of Chemical Engineering at the University of Barcelona in Spain. The research on dye decolorization is a collaboration between C. A. Reddy and J. E. Linz from Michigan State University and Ola M. Gomaa from the National Center for Radiation Research and Technology in Cairo, Egypt. TARGET AUDIENCES: The target audience include Government agencies such as Environmental Protection Agency (EPA), USDA, and DOE and industries involved in generating the pollutants and interested in bioremediation to meet the regulatory guidelines imposed by agencies such as EPA. PROJECT MODIFICATIONS: Necessitated by loss of key research personnel in the laboratory, growing interest in sustanable agriculture and emphasis on increasing crop productivity (by non-chemical means)using synergistic microbial mixtures, we have actively been Pursuing development of polymicrobial formulations that contain multiple functional groups including those that: fix nitrogen, solubilize phosphate and make it more available to the plant, inhibit plant pathogenic microbes (i.e. act as biopesticides), induce systemic resistance in the plant against pathogens, and produce phytohormone - like growth promoters. The initial results have been very promising and these results would be presented in detail in next year's progress report.

Impacts
One of the very important unanticipated beneficial offshoots of research on lignin biodegradation by white rot basidiomycete fungi was that these organisms produce degradative enzymes that are relatively non-specific and gratuitously catalyze free radical mediated degradation of a very broad spectrum of toxic aromatic environmental polutants which have similarity to some substructures in the lignin polymer. In our curiosity to examine if the degradative ability of white rot fungi extended to halogenated aliphatic compunds, we made the discovery that important and toxic chloroaliphatic compunds (exemplified by trichloroethylene and listed as priority poillutants by U. S. Environmental Protection Agency) are also degraded by white rot basidiomycetes. A continuation of these studies has contributed to an expansion of our basic knowledge in terms of understanding how different biological systems degrade trichloroethylene (TCE) and plychloroethylene (PCE). We not only demonstrated for the first time that TCE and PCE are degraded by white rot fungi but also that these organisms use novel biodegradation pathways different from those of bacteria but somewhat similar to mammalian systems. Furthermore, our studies reported here showed that P. chrysosporium has the ability to decolorize an array of structurally diverse textile dyes. The initial decolorization of Victoria Blue B was 41% and this increased to 83% by altering the media components to block other enzymatic pathways and by adding CuSO4 to enhance laccase activity. The fact that this fungus degrades the dye Victoria Blue B without prior adaptation is an advantage in practical bioremediation applications. Additional biochemical data suggest that oxygenases/oxidases are involved in VB decolorization by P. chrysosporium. More detailed studies on the mechanisms of degradation of these dyes would add to our knowledge on the biochemical and physiological factors influencing dye degradation by P. chrysosporium.

Publications

  • Gomaa, O. M., LinzJ. E., and ReddyC. A. (2008). Decolorization of Victoria blue by the white rot fungus, Phanerochaete chrysosporium. World Journal of Microbiology and Biotechnology. 24:2349-2356.


Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: Lignin-degrading basidiomycete fungi (commonly referred to as white-rot fungi) have assumed much prominence in recent years as important microbial agents for degrading a broad variety of toxic environmental pollutants. These fungi produce three families of lignin-modifying oxidative enzymes, designated lignin peroxidase, manganese-dependent peroxidase, and laccase, as major components of their ligninolytic system. The lignin-degrading enzyme system of these fungi is relatively non-specific and fortuitously degrades a wide range of toxic aromatic environmental pollutants. We have been investigating the role of white-rot fungi in degrading polychlorinated ethylenes, trichloroethylene (TCE) and perchloroethylene (also called tetrachloroethylene), which have been widely used in industrial cleaning solutions and are on the list of priority environmental pollutants published by EPA. Most studies to date have focused on degradation of TCE and PCE by individual bacteria or consortia but studies have been few on the degradation of PCE and TCE by white-rot fungi. We demonstrate for the first time that the white-rot fungus (WRF) P. chrysosporium mineralizes TCE. We further showed, also for the first time, that Trametes versicolor, another important WRF that is widely distributed around the world, degrades PCE by a novel metabolic pathway involving cytochrome P-450 and the formation of trichloroacetic acid (TCA) as an intermediate, that has not previously been shown in bacteria or fungi. We also showed unequivocally that T. versicolor mineralizes TCE and that cytochrome P-450 appears to be involved in the degradation of TCE as well. TCE degradation ability is now extended to two more important widely distributed white-rot fungi, Ganoderma lucidum and Irpex lacteus. To the best of our knowledge this is the first report on the ability of G. lucidum to degrade a xenobiotic environmental pollutant. TCE and PCE are often found together in polluted environments but little is known about the ability of microorganisms to simultaneously degrade TCE and PCE. Our studies showed that T. versicolor does degrade mixtures of TCE and PCE. The extent of PCE and TCE degradation when added as mixture was, respectively, 34% and 48%. Thus, T. versicolor degrades mixtures of TCE and PCE almost as well as its ability to degrade individually added TCE or PCE. The results suggest potential promise of T. versicolor for bioremediation of TCE and PCE in the environment. Results have been communicated at two international meetings and journal paper has been published. The results of the study have been disseminated by presentation of papers at two international scientific meetings and through two peer-reviewed publications. PARTICIPANTS: C. A. Reddy, Principal Investigator Ernest Marco-Urrea, Collaborator from Department of Chemical Engineering at the University of Barcelona, Spain. TARGET AUDIENCES: The target audience are industry and government agencies interested in bioremediation of contaminated sites. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Our laboratory is primarily interested in studying and obtaining a better understanding of the enzymology and molecular biology of lignin-modifying enzymes of white-rot fungi. The results of this research, besides making a number of fundamental contributions to basic science, has important practical applications. These include: more efficient production of cellulosic ethanol, an important bioenergy goal that has attained much prominence now; more efficient production of pulp and paper with minimal generation or complete elimination of toxic waste streams; enzymatic pretreatments of lignocellulosic feed materials to make these more efficient sources of energy for ruminant animals, which are the prime source milk and meat to people worldwide; and make significant advances in our ability to degrade toxic environmental pollutants (i.e. efficient bioremediation). White-rot fungi, which are the basic experimental tools used in our lab, have received much attention as bioremediation agents and the list of pollutants degraded by these remarkable organisms is getting to be quite long. Until recently, practically all the bioremediation work using white-rot fungi has been done with done with a wide range of aromatic environmental pollutants. To the best of our knowledge, our lab was the first to initiate studies on understanding the degradation of aliphatic halogenated compounds by white-rot fungi. We chose the study of degradation of trichloroethylene (TCE) and polychloroethylene PCE), two of the more important pollutants in their class. We showed that both TCE and PCE are degraded by several white-rot fungi as individual compounds or as a mixture of the two compounds. Thus, the results open up an interesting new area for detailed studies on the physiology, biochemistry, and molecular biology of chloroaliphatics (exemplified by TCE and PCE) using white-rot fungi (which are ubiquitous in nature) as model organisms. It is significant that the studies lead to the novel finding that the mechanism of degradation of TCE is quite different from known pathways for degradation of TCE or PCE by any of the bacteria or other fungi and is much more similar to the pathway used in mammalian systems; hence, expanded studies in this area may be applicable to the understanding of analogous systems in humans. Simultaneous degradation of TCE and PCE by a pure culture of Trametes versicolor is of particular interest because not much is known about this in other microbes. These results indicate that use T. versicolor may be a viable approach for practical bioremediation of TCE and PCE contaminated sites.

Publications

  • Marco-urrea, E., Gabarrell, X., Caminal, G., Vicent, T., and Reddy, C. A. (2008). Aerobic degradation by white-rot fungi of trichloroethylene (TCE) and mixtures of TCE and perchloroethylene. Journal of Chemical Technology and Biotechnology 83: 1190-1196.
  • Marco-urrea, E., Parella, T., Gabarrell, X., Caminal, G., Vicent, T., and Reddy, C. A. (2008). Mechanistics of trichloroethylene mineralization by the white-rot fungus Trametes versicolor. Chemosphere 70:404-410.


Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: Lignin is a highly complex aromatic polymer and accounts for 20-30% of the dry weight of lignocellulosic plant biomass. It is the second most abundant organic polymer in the biosphere after cellulose. Lignin occurs in close physical and chemical association with cellulose and hemicellulose in plant biomass and limits the efficient and economic conversion of lignocellulosic plant mass to biofuels and other useful products. Studies in our laboratory (as well several other laboratories) have shown that white rot basidiomycete fungi are the most efficient degraders of lignin in nature. The lignin-degrading enzyme systems of these fungi are relatively non-specific and fortuitously catalyze the degradation of a wide range of toxic environmental pollutants. Recently we reported for the first time the ability of the white rot fungus, Trametes versicolor, to degrade polychlorinated ethylene (PCE) aerobically using a novel pathway that has not previously been shown in prokaryotes or lower eukaryotes. Inhibitor studies suggested that cytochrome P-450 system is involved in the mineralization of PCE by T. versicolor. We now describe the results of our studies on degradation of trichloroethylene by T. versicolor. Trichloroethylene (TCE) is a highly oxidized toxic halogenated aliphatic organic compound that has been widely used once as a solvent in industrial cleaning solutions. TCE is one of the most common organic pollutants found in soils and groundwater around the world. It is a suspected carcinogen. The anaerobic degradation of TCE by bacteria via reductive dechlorination has been studied extensively. TCE is transformed to relatively non-toxic ethene as a final product. Some bacteria, however, transform TCE to cis-dichloroethylene or vinyl chloride (VC), which are toxic and VC is a known carcinogen. Very little is known about the aerobic degradation of TCE by bacteria. The results of this study unequivocally show that TCE is mineralized by T versicolor. Using [13C]-TCE as the substrate, T. versicolor was shown to degrade trichloroethylene (TCE) and produce 2,2,2-trichloroethanol and carbon dioxide as the main products of degradation. For a range of concentrations of TCE between 2 and 20 mg per liter, 53% of the theoretical maximum chloride expected from complete degradation of TCE was observed. The lignin-modifying enzyme laccase of T. versicolor, a blue copper oxidase that catalyzes one electron oxidation of organic substrate coupled to 4-electron reduction of molecular oxygen to water, was shown to be induced by TCE but did not appear to play a role in TCE degradation. Cytochrome P-450 appears to be involved in TCE degradation, as evidenced by marked inhibition of degradation of TCE in the presence of 1-aminobenzotriazole, a known inhibitor of cytochrome P-450. Our results suggested that chloral (trichloroacetaldehyde) was an intermediate of the TCE degradation pathway. The results indicate that the TCE degradation pathway in T. versicolor appears to be similar to that previously reported in mammals and is mechanistically quite different from bacterial TCE degradation. PARTICIPANTS: C. A. Reddy and Ernest Marco-Urrea, a collaborating scientist team from the Department of Chemical Engineering at the Universitat Autonoma Barcelona, Barcelona, Spain. TARGET AUDIENCES: The target audience are industry and government agencies involved in environmental clean-up. PROJECT MODIFICATIONS: A revised project has been submitted from Jan 2008 to Dec. 2012.

Impacts
The long range goals of our lab are to understand the key enzyme systems involved in lignin degradation by white rot fungi, which are known to be the most efficient lignin degraders in nature. The knowledge gained from such studies would help in the more efficient conversion of lignocellulosic biomass, the most abundant renewable organic resource on earth, into biofuels, feedstock chemicals, and feeds. Such a knowledge would also contribute to considerable conservation of energy and minimize pollutant waste streams by the the pulp and paper industry. In view of the sky rocketing costs of imported fossil fuels, there is intense interest now to use plant biomass as a source of ethanol and to make the process efficient by effective removal of lignin from the plant biomass. In view of these potential benefits, our lab has extensively studied the biochemistry, physiology and molecular biology of the three key families of lignin-modifying enzymes: lignin peroxidases, manganese peroxidases and laccases. An important and unanticipated benefit of the basic research was the recognition that the three lignin modifying enzymes are relatively non-specific and they gratuitously degrade a wide range of toxic halogenated aromatic pollutants that persist in the environment and constitute a serious hazard to human and animal health. Classic examples of such compounds degraded by ligninolytic enzymes of white rot fungi include polycholrinated biphenyls (PCBs), dioxins, various chlorophenols including pentachlorophenol, and wide range of close to 150 compounds. Practically all the emphasis in previous years was on aromatic pollutants as thse have some structural similarites to the substructures in lignin. However, our curiosity to see if the ability of white rot fungi to degrade toxic environmental pollutants extended to aliphatic (straight chain) chloroorganic compounds, led us to make the discovery that trichloroethylene was mineralized by the white rot fungus Phanerochaete chrysosporium. However, we did not determine the metabolic intermediates in the TCE degradation pathway. A continuation of these studies have now led to the finding that TCE degradation by white rot fungi is very different from those used by bacteria contributing to a more fundamental biochemical understanding of the pathway. Furthermore, these studies have helped us to report for the first time that polychlorinated ethylene (PCE) is also efficiently degraded by white rot fungi and that the degradation mechanism used is more similar to that seen in mammalian systems than in microbes. Thus, it is obvious that applied studies opened important leads to initiate fundamental studies and vice versa. The novel findings generated in the last 18 months have greatly contributed in opening and partially understanding a new area of research on degradation of choloroaliphatics by white rot fungi.

Publications

  • One paper has been accepted for publication but it has a Jan 2008 date.


Progress 01/01/06 to 12/31/06

Outputs
Lignin is a highly complex, three dimensional, amorphous aromatic polymer consisting of phenylpropanoid monomer units and is the second most abundant organic polymer in the biosphere (after cellulose). White rot fungi are the most active degraders of lignin in nature. The lignin-degrading enzyme system (LDS) of these fungi consists of enzymes that are relatively non-specific. Efficient degradation of a variety of chloroaromatic xenobiotic compounds by white rot fungi is well documented and is believed to be due to fortuitous side reactions catalyzed by LDS enzymes of these fungi. In spite of the vast amount of literature on the degradation of chloroaromatics by the white rot fungi, relatively little is known about the degradation of chlorinated aliphatic compounds, such as perchloroethylene and trichloroethylene, by these organisms. Perchloroethylene (PCE) is one of the most important groundwater pollutants around the world. It is a suspected carcinogen and is believed to be recalcitrant to microbial degradation. We report here, for the first time, aerobic degradation of PCE by the ubiquitous white rot fungus, Trametes versicolor, to less hazardous products. Aerobic degradation rate of PCE was 0.20 and 0.28 nmol per hour per mg dry weight of fungal biomass. Our results showed that three major families of lignin-modifying enzymes, lignin peroxidases, manganese peroxidases and laccases, which play a major role in fungal lignin degradation as well as in the degradation of a number of chroaromatic pollutants, are not involved in PCE degradation. It is noteworthy that vinyl chloride and dichloroethene, frequently seen as products of reductive dehalogenation by bacteria under anaerobic conditions, were not observed as products of PCE degradation by T. versicolor. Instead, using [2-C13]-PCE as the substrate, T. versicolor was shown to degrade PCE to trichloroacetyl chloride (TCAC), which was rapidly hydrolyzed in water (abiotically) to trichloroacetic acid (TCA). This evidence was further corroborated by demonstrating the expected stoichiometry between micromoles of PCE degraded, chloride released, and TCA generated. Our studies using 1-aminobenzotriazole (ABT), an inhibitor of cytochrome P-450, suggested that a cytochrome P-450 type oxygenase system may be involved in PCE degradation by T. versicolor. These results are of particular interest because TCA production from PCE has not been reported to date in bacteria or fungi. The results of this study not only suggest the potential of T. versicolor for bioremediation of the important chloroaliphatic pollutant, PCE, at contaminated sites, but also demonstrate a novel pathway for the microbial degradation of PCE, that has been demonstrated only in mammalian systems so far. Studies are currently in progress to determine the rate and extent of trichloroethylene degradation and the probable mechanism of its degradation by T. versicolor.

Impacts
Laccases are important enzymes involved in lignin degradation with potential applications for bioremediation; conversion of plant biomass to feeds, fuels and chemicals; and in biopulping and biobleaching. Thus, continued research on fungal laccases should have important potential benefits in realizing the industrial potential of these enzymes. We expect that the isolation of laccase genes, as described here, should help us in eventual over expression of laccases and in the efficient biotehnological applications of these enzymes. It was of interest, however, that laccases, which are involved in the degradation of a number of chloroaromatic pollutants, do not appear to be involved in perchloroethylene (PCE)degradation. Instead, the white rot fungus Trametes versicolor appears to use P-450 type oxygenase(s) in PCE degradation, using a novel degradation pathway that has not previously been reported in microbial systems. Our studies further expand the spectrum of environmental pollutants degrded by white rot fungi.

Publications

  • Marco-Urrea, E., Xavier, G. Montserra, T, Caminal, G. Vicent, T, and Reddy. C. A. 2006. Novel aerobic perchloroethylene degradation by the white-rot fungus Trametes versicolor. Environ. Sci. Technol. 40:7796-7802
  • Kumar, K. and Reddy, C. A. 2006. Isolation and analysis of laccase genes of the white rot fungus Ganoderma lucidum. Abstr. Annu. Mtg. Am. Soc. Microbiol.


Progress 01/01/05 to 12/31/05

Outputs
Laccases are one of the three major classes of lignin-modifying enzymes (LME) and are widely distributed among wood-degrading basidiomycetes from habitats such as forest soils, agricultural soils, and tropical mangrove ecosystems. These key oxidative enzymes are found in all the three domains of life: Eukaryota, Prokarya, and Archaea. Biochemically, laccases are blue copper oxidases that catalyze 1-electron oxidation of organic substrates coupled to 4-electron reduction of molecular oxygen to water. We have recently shown that Ganoderma lucidum (strain no.103561), a major white rot fungus with world wide distribution, produces laccase with some of the highest activity reported for any laccases to date. Our results showed that this organism produces at least 11 different isoforms of laccase based on variation in molecular weight and/or isoelectric point. Optimization of culture parameters for laccase activity followed by partial purification lead to 17,000 micro katals per mg of protein which is much higher relative to other fungal laccases. We also report in this study isolation of 2 laccase genomic clones of G. lucidum using inverse PCR and forward/reverse primers corresponding to the sequences of the conserved copper binding region in the N-terminal domain of one of the laccases of this organism. The coding sequence of lac1 gene is interrupted by 6 introns ranging in size from 37-55 nt and encodes a mature protein consisting of 456 aa (Mr: 50,160), preceded by a putative 37-aa signal sequence. This predicted molecular mass is in agreement with the range of those previously reported by us for the laccases of G. lucidum. The deduced aa sequence of LAC1 showed relatively high degree of homology with laccases of other basidiomycetes. It showed 96% homology to full-length LAC4 protein and 47-53% similarity to unpublished partial laccase sequences of other G. lucidum strains. Among the other basidiomycete laccases, LAC1 showed the highest similarity of 53 - 55% to Trametes versicolor LAC3 and LAC4. The consensus copper-binding domains found in other basidiomycete laccases are conserved in the LAC1 protein of G.lucidum. Eight putative N-glycosylation sites as well as consensus eukaryotic promoter sequence and polyadenylation signal sequences are also found. Coding sequence of lac4 is interrupted by 7 introns (similar in size to those of lac1), encodes a mature protein of 525aa (Mr: 57,750), and has 98% nt homology to lac1, but was otherwise identical. Eight other laccase clones, distinct from lac1 and lac4 have recently been isolated. Our results show the existence of a laccase multi-gene family in G. lucidum in agreement with our earlier results showing multiple isoforms of laccase in this organism.

Impacts
Laccases are important enzymes involved in lignin degradation with potential applications for bioremediation; conversion of plant biomass to feeds, fuels and chemicals; and in biopulping and biobleaching. Thus, continued research on fungal laccases should have important potential benefits in realizing the industrial potential of these enzymes. We expect that the isolation of laccase genes, as described here, should help us in eventual over expression of laccases and in the efficient biotehnological applications of these enzymes.

Publications

  • Pointing,S. B.,Pellingg, A. L., Smith, G. J. D.,Hyde, K. D., Reddy, C. A. 2005. Screening of basidiomycetes and xylareous fungi for lignin peroxidase and laccase gene-specific sequences. Mycological Research 109: 115-124


Progress 01/01/04 to 12/31/04

Outputs
Laccases are one of the three major classes of lignin modifying enzymes (LME) in basidiomycete fungi. In comparison to the relatively large body of literature on the LMEs of wood-degrading basidiomycetes, little is known about the LMEs of soil basidiomycetes and marine basidiomycetes. Our studies showed that laccases are the predominant LMEs among a large number of soil basidiomycetes investigated. Lignin peroxidases and manganese peroxidase activities were either not found or were expressed in very low amounts. Yet, a substantial number of these fungi contained DNA sequences with homology to known lignin and manganese peroxidase sequences of wood decay fungi. The results with a large number of marine basidiomycetes isolated from tropical environments were quite similar. Furthermore, detailed studies with several individual isolates showed the presence of diversity of isoforms of laccases with substantial differences in molecular weights and electrophoretic mobilities. Marked ability to degrade a number of toxice environmental pollutants is a characteristic shared by the new isolates with the well studies white-rot basidiomycetes. In contrast to this, investigation of the wood decay enzymes in tropical Xylariaceae showed that a number of taxa were capable of cellulose and xylan degradation but few produced enzymes involved in lignin breakdown. Interestingly, however, a number of these fungi showed lignin solubilization similar to those shown by white-rot basidiomycetes suggesting the probable presence of ligninolytic systems, albeit different from systems known to date based on research with white-rot basidiomycetes. Furthermore, in continuation of studies on laccases of Ganoderma lucidum, which produces very high levels of laccase, and is of considerable industrial and medicinal interest, we investigated methods to obtain laccase gene sequences using inverse PCR procedure. Preliminary results indicate a multigene family of laccases in this organism, in conformation with our earlier biochemical data. Detailed studies are under way to obtain a better understanding of the molecular architecture of of this laase gene family.

Impacts
Laccases are important enzymes involved in lignin degradation with potential applications for bioremediation; conversion of plant biomass to feeds, fuels and chemicals; and in biopulping and biobleaching. Thus, continued research on fungal laccases should have important potential benefits in realizing the industrial potential of these enzymes.

Publications

  • Bucher, V.V.C., Pointing,S.B.,Hyde,K.D.,Reddy, C.A. 2004. Production of wood decay enzymes, loss of mass and lignin solubilization in wood by diverse freshwater fungi. Microbial Ecology 48:329-335.
  • Bucher, V.V.C., Hyde,K.D.,Pointing,S.B.,Reddy, C.A. 2004. Production of wood decay enzymes, loss of mass and lignin solubilization in wood by diverse marine fungi. Fungal Diversity 15:1-14.


Progress 01/01/03 to 12/31/03

Outputs
Laccases are an important family of blue multi-copper oxidases that catalyze oxidation of various organic and inorganic substrates coupled to the four-electron reduction of oxygen to water. Laccases are so widely distributed that they are found in all the three domains of life: Eukarya, Prokarya, and Archaea. Ganoderma lucidum, a lignin degrading basidiomycete fungus distributed worldwide, is the model organism we have been using for studies on laccases in our laboratory. This organism is known to be one of the highest laccase producers studied so far and it produces multiple molecular forms of laccase. One area of focus has been to clone and characterize laccase gene sequences from G. lucidum.This is an important first step in producing major isoforms of laccase from this organism in sufficient quantity to conduct detailed structure-function studies, which are necessary for the eventual realization of the biotechnological potential of these enzymes. A lambda based genomic library of G.lucidum was constructed in Escherichia coli to isolate the laccase genes. Specific primers, homologous to the conserved 200 bp copper-binding region of the basidiomycete laccase sequences, to isolate laccase gene fragments which were then used as probes to isolate the laccase genes from the library. During the screening of the potential positive clones, PCR studies showed additional fragments besides the 3 usual fragments around 200 bp. This leads us to believe that the additional PCR fragments result from potential primer annealing sites on genomic DNA of E.coli in addition to the annealing sites on G.lucidum laccase gene sequences. To analyze this we isolated chromosomal DNA from four different strains of E.coli and G.lucidum and performed PCR reactions using specific primers homologous to the conserved copper-binding region of basidiomycete laccase sequences previously isolated from our lab. Furthermore, using 200 bp laccase gene probe, southern hybridizations were performed on the PCR reaction products and several E.coli genomic fragments were found to strongly hybridize suggesting the presence of laccase gene sequences in E. coli. Four different strains of E. coli gave different PCR fragment patterns suggestive of heterogeneity among the strains. A search of the protein database of E. coli has revealed the presence of at least 3 laccase-like proteins in E.coli: PcoA, CueO (previously called YackA), and Suf1. A multiple sequence alignment of the E. coli proteins with three basidiomycete laccases reveals that 1 cysteine and 10 histidines residues involved in the coordination of 4 copper atoms are conserved in PcoA and YackA, whereas only 3 histidines are conserved in SuF1. Further multiple alignments of laccase DNA sequences and laccase activity assays with cell extracts of E.coli strains, using standard laccases substrates, should give a better understanding of the novel presence of laccase gene sequences in E. coli and the potential possibility for cloning and over expression of G. lucidum laccase genes in E. coli.

Impacts
Laccases are important enzymes involved in lignin degradation with potential applications for bioremediation; conversion of plant biomass to feeds, fuels and chemicals; and in biopulping and biobleaching. Thus, continued research on fungal laccases should have important potential benefits in realizing the industrial potential of these enzymes.

Publications

  • No publications reported this period


Progress 01/01/02 to 12/31/02

Outputs
Laccases along with lignin peroxidases and manganese-dependent peroxidases constitute three major classes of enzymes produced by white rot fungi, that are believed to be the most important organisms involved in degrading lignin, the second most abundant renewable organic polymer on earth after cellulose. Among the three classes of lignin modifying enzymes, laccases are very distributed in wood-degrading fungi. Even the extensively studied Phanerochaete chrysosporium, that is widely reported to lack laccase, was shown by us to be positive for laccase production. Physiological, biochemical, and molecular biological studies on laccases of white rot fungi continue to be active areas of interest in our laboratory. Biochemically, laccases belong to a family of blue multicopper oxidases that couple four electron reduction of molecular oxygen to water. One of the model organisms of interest to us for studying laccase is Ganoderma lucidum, which is one of the most important and widely distributed white-rot fungi in North America and is associated with the degradation of a variety of hard woods. G lucidum produces multiple isoforms of laccase, which vary substantially in different media. Regulation of expression of different isoforms of laccase is regulated differently when the organism is grown in the presence of different wood species and by cultural factors such as veratryl alcohol. Substantial research efforts towards optimization of laccase production has resulted in laccase levels of over 160 to 200 mkatal/L, a level higher than that reported for most (if not all) other fungi. Time course studies showed peak production occurs on day 12 followed by a secondary minor peak around day 15. Substrate specificity studies showed that laccase gives maximum activity with ABTS (2,2'-azino-bis [3-ethylbenzthiazoline-6-sulfonic acid]) as the substrate and sixteen-fold less activity was observed with the next best substrate, 2-6 dimethoxy-phenol. Activity was much lower with several other substrates tested. Laccase production was negligible when copper was deleted from the medium, analogous to previous observations with another basidiomycete fungus Irpex lacteus. Addition of copper to HN medium minus copper on the eighth day of incubation resulted in almost complete restoration of activity. Preliminary Northern blot studies indicate that regulation of laccase production by copper occurs at the transcriptional level. Substantial efforts at enzyme purification have shown that G. lucidum grown in HN medium produces 11 different isoforms of laccase. The pI of the isoforms range from 4.7-3.3. Scale-up studies for isolating the four major isoforms in sufficient quantity for crystallographic studies are in progress.

Impacts
There has been growing interest in laccases because of their importance in: lignin degradation (a key event in global carbon cycling), increasing the efficiency of conversion of lignocellulosic plant biomass to fuels (such as methane and ethanol) and chemicals, degrading toxic environmental pollutants, decolorization and detoxification of pulp waste effluents, and in biobleaching and biopulping. Additionally, laccases have other applications as in construction of biosensors, and enzyme immunoassays. Laccases are also of interest because they share with the terminal oxidase of aerobic respiration the ability to reduce molecular oxygen directly to water. Thus, our research contributions on laccase will have significant future impact in realizing the industrial potential of these enzymes.

Publications

  • Mathew,Z., Zalinski,D. and Reddy, C. A.2002. Characterization of laccases from Ganoderma lucidum. Abstr. Annu. Mtg. AM. Soc. Microbiol. #Q44, p 386.
  • Reddy, C. A. 2002. Overview of fungal lignin degradation. IMC7-Book of Abstracts, #198, p 63.


Progress 01/01/01 to 12/31/01

Outputs
Our long term objective is to obtain a better understanding of the physiology, molecular biology, and applications of the lignin degrading enzyme systems of White rot fungi (WRF), which are the most important group of wood-rot fungi involved in mineralizing lignin. These organisms have also attracted worldwide attention because their ligninolytic enzymes are relatively non-specific and are believed to be important in degrading a broad range of environmental pollutants. The white-rot fungus Phanerochaete chrysosporium, which grows rapidly and mineralizes lignin more extensively than most other white-rot fungi, has also been shown to degrade important environmental pollutants such as dioxins, polychlorinated biphenyls (PCBs), chlorophenols, chlorinated pesticides, and polycyclic aromatic hydrocarbons (PAHs). Trichloroethylene (TCE), a volatile halocarbon compound, is a common industrial degreasing solvent as well as a precursor for industrial chemical synthesis. Large scale production and usage of TCE combined with improper waste disposal practices and landfill leaching, have led to the contamination of soils, air, and aquifers with this chemical. TCE is carcinogenic and causes hematological, cardiac, and neurological problems. TCE is one of the top ten most commonly detected chemicals at the hazardous waste sites. Our studies showed for the first time that P. chrysosporium degrades TCE. Lignin peroxidases (LiPs) and manganese dependent peroxidases (MnPs), which were shown to be important in degrading lignin and a number of pollutants are not involved in TCE degradation. TCE degradation by P. chrysosporium (Fig.1) in ME, high N, and low N media was 46%, 14 %, and 6%, respectively, after three weeks of incubation. Time course of TCE degradation in ME medium showed that, after a slight lag in the first two days, there was a steep increase in the rate of degradation, coincident with the increase in the fungal biomass up to 6 days. The percent degradation of TCE decreased with the increase in TCE concentrations beyond 50 ppm, although the net degradation of TCE was still greater at the higher concentrations. However, the effect on mycelial dry weight was relatively insignificant upto 100 ppm. Most of the mineralization (27.4%) occurred during the first week of incubation. Cultures that were reoxygenated after the first week showed higher level of mineralization (38.5%) than control cultures suggesting that cultures may be oxygen-limited after the first week of incubation. Ability of P. chrysosporium to tolerate relatively high concentrations of TCE and its ability to degrade TCE under nutrient-rich as well as under nutrient-limited conditions indicate the desirability of this fungus for TCE bioremediation in the environment.

Impacts
There is much interest in the utilization of white-rot fungi in general and P. chrysosporium in particular for bioremediation of environmentally contaminated sites. This study shows that P. chrysosporium has the ability to mineralize trichloroethylene, a priority pollutant listed by the U.S.-EPA. Furthermore, P. chrysosporium which has previously been shown to degrade a broad array of aromatic environmental pollutants, also degrades TCE, an important aliphatic chemical pollutant. This adds to the versatility of pollutant degradation by this organism.

Publications

  • LaMontagne,M.L., Michel,F.C.Jr., Reddy,C.A. 2001. Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost for community analysis. J. Microbiol. Methods (in press)
  • Pointing,S.B.and C. A. Reddy.2001. Applications of lignocellulose degrading fungi. In: Pointing, S.B. and Hyde, K.D.(eds),Bio-Exploitation of Filamentous Fungi. Fungal Diversity Press,The University of Hong Kong, Hong Kong.


Progress 01/01/00 to 12/31/00

Outputs
White rot fungi (WRF) are the most important group of wood-rot fungi in the biosphere in mineralizing lignin, a major structural constituent of wood. Our long term objective is to obtain a better understanding of the physiology, molecular biology, and applications of the lignin degrading enzyme systems of WRF. Laccases (p-diphenol oxidases), a major class of ligninolytic enzymes in WRF, are copper-containing oxidases that catalyze the one-electron oxidation of organic substrates to the four-electron reduction of dioxygen to water. In comparison to the relatively large body of literature on the LMEs of wood-rot basidiomycetes, little is known about the LMEs of soil basidiomycetes, which are reported to play a key role in lignocellulose degradation in the soil ecosystem. In our efforts to know more about the distribution and diversity of LME in soil basidiomycetes, we isolated over 70 strains of soil basidiomycetes from the Long Term Ecological Research (LTER) plots at the Kellogg Biological Station of Michigan State University. Our studies showed that laccase is the predominant LME produced by a large majority of the isolates. Of these, Irpex lacteus, produced more laccase than most of the isolates in defined media as well as in wood-grown cultures. Of a number of media tested, maximal levels of laccase activity (70 mkatals/l) were obtained in a high nitrogen-high carbon (HNHC) medium that contained veratryl alcohol (VA). Presence of both VA and 2,5-xylidine in HNHC medium gave an additional three-fold increase in laccase production. Laccase levels in cultures grown with oak wood powder as the sole source of carbon/energy/nitrogen were lower than those observed in HNHC medium but were much higher than those seen in cultures grown with pine, poplar, or spruce wood. SDS-PAGE of the concentrated extracellular fluid (ECF) from HNHC cultures showed two broad bands with Mr of 50 kDa and 90 kDa while that from oak-grown cultures gave two bands of 50 and 60 kDa. Isoelectric focusing (IEF) of ECF from HNHC cultures gave two distinct groups of 14 laccase isozymes (four in the pI range of 3.5 to 3.9 and ten in the pI range of 4.5 to 6.3). Oak-grown cultures showed four laccase isozymes (pI range of 3.5-3.9) while spruce and poplar cultures showed 4-7 isozymes both in the low and high pI range. PCR amplification, using total DNA of AX1 as the template and primers corresponding to the conserved copper-binding region of basidiomycete laccases, gave a 199 bp fragment, the deduced aa sequence of which had a high degree of similarity to the corresponding fragments in other basidiomycete laccases. Five major laccase isozymes were purified to homogeneity from cultures grown in HNHC medium: four had pI values of 5.8, 5.5, 5.2 and 4.8 and an Mr of 90 kDa while the fifth had a pI of 3.9 and an Mr of 50 kDa. Further studies will focus on large-scale purification of the major isozymes and study their kinetic and structural properties.

Impacts
Major Lignin degrading enzymes exemplified by laccases, lignin peroxidases, and manganese peroxidases are believed to be important in the ability of white rot fungi to degrade a broad variety of toxic chloroaromatic environmental pollutants such s polychlorinated biphenyls, dioxins, chlorophenols, and munition wastes. Hence, a better understanding of these enzymes will not only be of great significance in understanding the nature of lignin degradation by these fungi, but also in the use of these organisms for biopulping, manufacture of fuels and chemicals from woody materials, and in cleaning-up the environment.

Publications

  • Yadav, S. J., C. A. Reddy, J. F. Quensen, J. M. Tiedje. 2000. Degradation of polychlorinated biphenyl mixtures in soil using Phanerochaete chrysosporium in nutrient rich, non-ligninolytic conditions. U. S. Patent No. 6,107,079 dt. 8/22/2000
  • Yadav, S. J., C. Bethea, and C. A. Reddy. 2000. Mineralization of trichloroethylene(TCE) by the white rot fungus Phanerochaete chrysosporium. Bull. Environ. Contam. Toxicol. 65: 28-34.


Progress 01/01/99 to 12/31/99

Outputs
Basidiomycete fungi are believed to be the most active of the microbial groups in nature in degrading the plant polymer lignin, the second most abundant renewable organic polymer in the biosphere. Laccases along with lignin peroxidases (LIPs) and manganese peroxidases (MNPs)are the three major classes of extracellular lignin-modifying enzymes (LMEs). In this study, we characterized the LMEs of Ganoderma lucidum, one of the most important and widely distributed white-rot fungi involved in the white-rot decay of a broad variety of hard woods in North America. Our results show that this organism produces laccase as the dominant LME and the levels of laccase were much higher than those reported for most other white-rot fungi described to date. At least five isoforms of laccase (PIs 3.0 to 5.1) are produced. There is a marked synergy in laccase production when the organism is grown on pine and poplar woods compared to production with either one alone. It produces MnP when grown on poplar but not when grown on pine. In other studies, we have also described for the first time the LMEs produced by a marine basidiomycete isolate, Flavodon flavus. This organism produces relatively high levels of laccase and MNP activities but low levels of LIP. LMEs were produced both in the presence and absence of sea water. Multiple molecular forms were produced for each class of LME.

Impacts
This is the first detailed characterization of LMEs in Ganoderma lucidum and a marine isolate of Flavodon flavus. LMEs have important biotechnological potential not only in biopulping and biobleaching but also in the efficient bioconversion of wood materials for the production of feeds, fuels, and petrochemical feed stock and in bioremediation of toxic chloroaromatic environmental pollutants such as dioxins and PCBs.

Publications

  • D'Souza, T. M., Merritt, C. S. and Reddy, C. A. 1999. Lignin-modifying enzymes of the white rot basidiomycete Ganoderma lucidum. Appl. Environ. Microbiol. 65:5307-5313.
  • Raghukumar, C., D'Souza, T. M., Thorn, R. G. and Reddy, C. A. 1999. Lignin-modifying enzymes of Flavodon flavus, a basidiomycete isolated from a coastal marine environment. Appl. Environ. Microbiol. 65: 2103-2111.


Progress 01/01/98 to 12/31/98

Outputs
Soil basidiomycetes are considered to be the major players in lignocellulose degradation in soils. Studying the lignin modifying enzymes (LME) of these organisms is an important focus of research in my laboratory. A majority of the soil basidiomycetes produce laccase as the major LME. Irpex lacteus (#AX1), a soil basidiomycete, is known to produce particularly high levels of laccase, a copper-containing polyphenol oxidase that is believed to be important in lignin degradation. In this study, we investigated the effect of substitution of the divalent metals nickel, cobalt or manganese for copper on laccase production by I. lacteus. In HCHN medium containing 168 mM glucose, 24 mM ammonium tartrate and 7 ppm Cu++, this organism produces up to 70 mkatals/l of laccase in the extracellular fluid (ECF) and at least fourteen different isoforms. No laccase activity was demonstrable in HCHN cultures without Cu++. Cultures grown in Cu++-free HCHN medium with added Ni++, Co++ or Mn++ produced active laccase, albeit at low levels. In the presence of Ni++, laccase activity was much lower (23 mkatals/l) than that observed in HCHN medium with Cu++. Even lower levels of laccase activity were observed in HCHN cultures grown with Co++ and Mn++. Addition of 2,5-xylidine, a known inducer of fungal laccases, stimulated laccase activity 4-fold in HCHN cultures containing Cu++ but caused only a marginal increase in laccase activity in cultures grown with Ni++, Co++ or Mn++. SDS-PAGE analysis of the ECF from HCHN cultures grown with Cu++ showed two laccase activity bands corresponding to Mrs of 50, 000 and 90, 000. Cultures grown with Ni++, Co++ or Mn++ also showed two laccase activity bands but the molecular masses were slightly different. Laccase isozyme profiles in the ECF of cultures grown with Cu++, Ni++ or Mn++ were comparable whereas cultures grown with Co++ lacked four isoforms of laccase with pI values in the range, 3.9-3.5. To the best of our knowledge this is the first demonstration of fungal laccase production in the absence of added Cu++ and that substitution of Ni++, Mn++, or Co++ for Cu++ results in active laccase production.In parallel studies we are focusing on the purification and characterization of laccase from I. lacteus and to study the patterns of paccase production when the organism is grown on different types of wood.

Impacts
(N/A)

Publications

  • 1.THOMPSON, D. N., B. R. HAMES, H. E. GRETHLEIN, C. A. REDDY. 1998. In vitro degradation of insoluble lignin in aqueous media by the lignin peroxidase and manganese peroxidase. Appl. Biochem. Biotechnol. 70-72: 967-981.
  • 2.THOMPSON, D. N., B. R. HAMES, H. E. GRETHLEIN, C. A. REDDY. 1998. 1998. In vitro degradation of natural insoluble lignin in aqueous media by the extracellular peroxidases of Phanerochaete chrysosporium. Biotehnol. Bioeng. 57: 704-717.
  • 3. REDDY, C. A., T. M. D'SOUZA. 1998. Application of PCR in studying lignocellulose degradation by basidiomycetes, p 206-243. In P. D. Bridge, D. K. Arora, C. A. Reddy, and R. P. Elander (eds.), Applications fo PCR in Mycology, CAB International, Wallingford, Oxon, UK.


Progress 01/01/97 to 12/31/97

Outputs
Ganoderma lucidum is one of the most important and widely distributed ligninolytic basidiomycete fungi that causes white-rot decay of wood in North America. Little is known about the production of the key ligninolytic enzymes lignin peroxidase (LIP), manganese peroxidase (MNP), and laccase by this organism. Our studies showed that in defined media it produces laccase activity at levels higher than those produced by most other white-rot fungi, but does not produce LIP or MNP. Laccase production is also seen in cultures grown with pine or poplar wood as the sole C and N source. However,cultures grown with both pine and poplar produced 5- to 10-fold higher levels of laccase than those seen in poplar or pine alone cultures. Addition of syringic acid to pine cultures also results in laccase levels comparable to those seen in pine plus poplar cultures. Isoelectric focusing studies showed that G. lucidum produces at least five isoforms of laccase. Biotechnological potential of G. lucidum is enhanced considerably because of the many industrial applications of laccase and the fact that it produces high levels of this enzyme. In related studies, we published a new selective procedure for the isolation of soil basidiomycetes, which are important in lignocellulose breakdown in soils. This should stimulate a flurry of scientific studies on this much neglected group of organisms.

Impacts
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Publications

  • D'SOUZA,T.M., K.BOOMINATHAN, C.A.REDDY. 1996. Isolation of laccase gene-specific sequences from white rot and brown rot fungi by PCR. Appl.Environ.Microbiol. 62:3739-3744.
  • THORN,R.G., C.A.REDDY, D.HARRIS, E.PAUL. 1996. Isolation of saprophytic basidiomycete from soil. Appl.Environ.Microbiol. 62:4288-4292.
  • RAGHUKUMAR,C., D.CHANDRAMOHAN, F.C.MICHEL,JR., C.A.REDDY. 1996. Degradation of lignin and decolorization of bleach plant effluent (BPE) by marine fungi. Biotechnol. Lett. 18: 105-106.


Progress 01/01/96 to 12/30/96

Outputs
Lignin is a major component of woody plants & the 2nd most abundant renewable polymer in the biosphere. White-rot fungi, which are known to mineralize lignin efficiently, 3 groups of lignin modifying enzymes (LMF) designated lignin peroxidases (LIPs), manganese peroxidases (MNPs), and laccases (Lac). Very little is known about the LMF of a 2nd major group of wood-degrading fungi called brown-rot fungi. The possibility of rapidly isolating & characterizing laccase gene specific sequences from different wood-degrading fungi using PCR methodology was investigated. Degenerate primers corresponding to the consensus sequences of the copper-binding regions in the N-terminal domains of known basidiomycete laccases were used to isolate laccase gene-specific sequences from strains representing 9 genera of wood-rot fungi. All, except 3, gave the expected PCR product of 200 bp. Computer searches identified the sequence of the PCR products analyzed as a laccase gene sequence suggesting the specificity of the primers. PCR products of the white-rot fungi G. lucidum, P. brevispora, & T. versicolor, showed (64-97%) nucleotide (nt) sequence similarity; the similarity in deduced amino acid (aa) sequences was 68-100%. The PCR products of L. edodes and L. tigrinus, on the other hand, showed relatively low nt and aa similarities. Demonstration of laccase activity in G. trabeum and several other brown-rot fungi was of particular interest because these organisms were not previously shown to produce laccases.

Impacts
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Publications


    Progress 01/01/95 to 12/30/95

    Outputs
    Three classes of enzymes designated lignin peroxidases (LIPs), manganese-dependent peroxidases (MNPs), & laccases play an important role in fungal degradation of lignin. Phanerochaete chrysosporium, a lignin-degrading white-rot basidiomycete, has been extensively studied as a model for fungal lignin degradation. This organism produces LIPs & MNPs but has been widely reported as negative for laccase production. Most earlier investigations employed media which contained glucose as the energy source. Our recent studies showed the presence of extracellular laccase in P. chrysosporium, using 2,2'-azino-bis-(3-ethylbenzathiazoline-6-sulfonic acid) (ABTS) as the substrate. Laccase was produced in cultures grown in either low nitrogen (2.4mM) or high nitrogen (24mM) media containing cellulose, but not glucose, as the carbon source. Laccase activity was seen with 6-hydroxy dopamine or syringaldazine as the assay substrates. Activity was seen in cultures grown at 25C or at 37C. Oxygenation of cultures improved production of laccase while unoxygenated cultures showed little or no laccase activity. SDS-PAGE of concentrated extracellular culture fluid followed by activity staining showed the presence of a laccase band with an estimated Mr of 46,500.

    Impacts
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    Publications

    • YADAV, J., WALLACE, R., REDDY, C. 1995. Degradation of mono- & dichlorobenzenes & simultaneous degradation of chloro- & methyl-substituted benzenes by the white-rot fungus P. chrysosporium. Appl. Env. Micro. 61:667-680.
    • DASS, B.C., DOSORETZ, C., REDDY, C.A., GRETHLEIN, H.E. 1995. Electrophoretic characterization of the proteases in white-rot fungus Phanerochaete chrysosporium. Arch. Microbiol. 163:254-258.
    • YADAV, J., ET AL. 1995. Degradation of commercial PCB mixtures (Aroclors 1242, 1254, & 1260) by the white-rot fungus P. chrysosporium through congener-specific analysis. Appl. Env. Micro. 61:2560-2565.
    • MICHEL, F., REDDY, C., FORNEY, L. 1995. Microbial degradation & humification of the lawn care pesticide 2,4-D during the composting of year trimmings. Appl. Env. Micro. 61:2566-2571.
    • REDDY, C.A. 1995. The potential for white-rot fungi in the treatment of pollutants. Curr. Opinion Biotechnol. 6:320-328.
    • SRINIVASAN, C., D'SOUZA, T., BOOMINATHAN, K., REDDY, C.A. 1995. Demonstration of laccase activity in the white-rot fungus P. chrysosporium. Appl. Environ. Microbiol. in-press.
    • RAGHUKUMAR, C., CHANDRAMOHAN, D., MICHEL, F.C., REDDY, C.A. 1995. Degradation of lignin and decolorization of paper mill bleach plant effluent (BPE) by marine fungi. Biotechnol. Lett. 18:105-106.


    Progress 01/01/94 to 12/30/94

    Outputs
    There is a wealth of information on the main lignin modifying enzymes (lignin peroxidases, manganese peroxidases, and laccases) in white-rot basidiomycetes. However, very little is known about the occurrence of lignin modifying enzymes in marine fungi. Our results show that a majority of the marine fungi produce laccase, but none produced lignin peroxidase (LIP); only 3 of the 17 fungi produced manganese peroxidase (MNP). #White-rot and brown-rot fungi play a major role in wood degradation in nature. Studies on the interspecific interactions among the common species of white-rot and brown-rot fungi when they colonize wood showed that most of the brown-rot fungi deadlocked with some or all of the white-rot fungi tested. However, some of the brown-rot fungi were capable of invading and occupying domains within white-rot fungal communities in decaying wood. Further studies showed that interspecific mycelial interactions among brown-rot fungi also result in either deadlock or replacement of one fungus by the other. #More recent studies showed that the fungus, Phanerochaete chrysosporium, mineralizes both monochlorobenzenes and dichlorobenzenes. LIPs and MNPs do not appear to be involved in chlorobenzene degradation by this organism.

    Impacts
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    Publications


      Progress 01/01/93 to 12/30/93

      Outputs
      Lignin peroxidases (LIPs) & manganese-dependent peroxidases (MNPs) are 2 families of extracellular heme proteins that play an import. role in lignn degradation by wood-rotting fungi. Genetic regulation of LIP & MNP product. was investigated using a number of previously characterized regulatory mutants of Phanerochaete chrysosporium. Results of crosses between various mutants indicated that at least three independently assorting regulatory loci (der, glm, & per) are involved in controlling LIP & MNP production. The der mutation (i.e. nitrogen deregulated phenotype) appears to be epistatic to glm (lack of LIP, MNP, & glucose-1 oxidase) while the per mutation (lack of LIP & MNP only) is epistatic to der. Heterokaryon analyses indicated that both the wild type (glm+ der+) & der are dominant to glm. FPLC profiles & quantita. data on LIPs & MNPs suggest that the der gene product is a transacting factor. Based on this & other published data, a heirarchial model for the genetic regulation of LIP & MNP production in P. chrysosporium has been proposed. In a parallel study, electrophoretic karyotyping of 2 of the most widely studied strains of P. chrysosporium BKMF-1767 & ME-446 showed that genomic DNA of BKMF-1767 is resolvable into 10 chromosomes ranging in size from 1.8 to 5.0 Mb (total genome size = 29 Mb) while the genomic DNA of strain me-446 was resolvable into 11 chromos. (total genome size = 32 Mb). Lignin peroxidase genes have been localized to 5 chromos. in BKMF-1767 & to 4 chromos. in ME-446.

      Impacts
      (N/A)

      Publications

      • D'SOUZA, T.M., DASS, S.B., RASOOLY, A., REDDY, C. A. 1993. Electrophoretic karyotyping of the lignin-degrading basidiomycete Phanerochaete chrysosporium. Mol. Microbiol. 8:803-807.
      • REDDY, C. A. 1993. An overview of the recent advances on the physiology and molecular biology of lignin peroxidases of Phanerochaete chrysosporium. J. Biotechnol. 30:91-107.
      • YADAV, J. S., REDDY, C. A. 1993. Degradation of Benzene, Toluene, Ethylbenzene, and xylenes (BTEX) by the lignin-degrading basidiomycete Phanerochaete chrysosporium. Appl. Environ. Microbiol. 59:756-762.
      • YADAV, J. S., REDDY, C. A. 1993. Mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D) and mixtures of 2,4-D and 2,,5-Trichlorophenoxyacetic acid by Phanerochaete chrysosporium. Appl. Environ. Microbiol. 59:2904-2908.
      • BOOMINATHAN, K., D'SOUZA, T. M., NAIDU, P. S., DOSORETZ, C., REDDY, C. A. 1993. Temporal expression of the major lignin peroxidase genes of Phanerochaete chrysosporium. Appl. Environ. Microbiol. 59:3946-3950.


      Progress 01/01/92 to 12/30/92

      Outputs
      Phanerochaete chrysosporium produces two families of extracellular peroxidases designated lignin peroxidases (LIPs) and manganese-dependent peroxidases (MNPS) that are two of the key components of the lignin degrading enzyme system of this organism. Production of LIPS and MNPs is tightly regulated by N, C, and Mn(II) concentrations in the medium. Our studies using a N-deregulated mutant showed that LIP and MNP production is independently regulated by N, C, and Mn(II). Further studies showed that cAMP, a key regulator of metabolism both in prokaryotic and eukaryotic systems, plays an important role in the regulation of expression of LIP and MNP gene families of P. chrysosporium. A sharp rise in intracellular cAMP concentration was observed just prior to the appearance of LIPs and MNPs. A positive correlation was observed between intracellular concentration of cAMP and the level of expression of LIP and MNP gene families. The results further showed that expression of LIP and MNP gene families is differentially regulated by cAMP in that LIP gene expression was more sensitive to the decrease in cAMP concentration than was MNP gene expression. Northern blot analyses indicated that cAMP affects expression of LIP and MNP gene families at the level of transcription. We then investigated the temporal patterns of production of LIP and MNP isozymes and their corresponding gene transcripts.

      Impacts
      (N/A)

      Publications

      • BOOMINATHAN, K., and REDDY, C.A. 1992. cAMP-mediated differential regulation of lignin peroxidase and manganese-dependent peroxidase production in the white-rot basidiomycete Phanerochaete chrysosporium. Proc. Natl. Acad. Sci. USA 89:5586-.
      • RANDALL, T.A., and REDDY, C.A. 1992. The nature of extra-chromosomal maintenance of transforming plasmids in the filamentous basidiomycete Phanerochaete chrysosporium. Curr. Genet. 21:255-260.
      • YADAV, J.S., and REDDY, C.A. 1992. Non-involvement of lignin peroxidases and manganese peroxidases in 2,4,5-trichlorophenoxyacetic acid degradation by Phanerochaete chrysosporium. Biotechnol. Lett. 14:1089-1092.
      • MICHEL, F.C. JR., GRULKE, E.A., and REDDY, C.A. 1992. A kinetic model for the fungal pellet lifecycle. AIChE J. 38:1449-1460.
      • MICHEL, F.C. JR., GRULKE, E.A., and REDDY, C.A. 1992. Determination of the respiration kinetics for mycelial pellets of Phanerochaete chrysosporium. Appl. Environ. Microbiol. 58:1740-1745.


      Progress 01/01/91 to 12/30/91

      Outputs
      Phanerochaete chrysosporium is a lignin-degrading white rot fungus that producestwo families of extracellular, glycosylated, heme protein, designated lignin peroxidases (LIPs) and manganese peroxidases (MNPs), which are produced in response to nutrient starvation during secondary metabolism. LIPs play a key role in the initial depolymerization and degradation of lignin by this organism. At least six LIP isozymes designated H1, H2, H6, H7, H8, and H10 are seen in lignin-degrading cultures of P. chrysosporium. We have recently isolated and sequenced LIP genes encoding the major LIP isozymes H2, H8, and H10. Our results show that each LIP gene encodes a mature protein that contains 344 aa and is preceded by a signal peptide that contains 27 to 28 aa. All the LIP protein sequences contain 1-2 potential N-glycosylation sites and a number of O-glycosylation sites. The coding region of each LIP gene is interrupted by 8-9 small introns from 50-63 bp. A comparison of the nucleotide homologies of the LIP genes as well as the aa homologies of the proteins encoded by these genes showed that the H(subscript 2)-encoding LIP gene constitutes a distinct subclass within the LIP gene family of P. chrysosporium. Recently, we have isolated and sequenced the LIP gene (VLG1) of another important white rot fungus Trametes versicolor. VLG1 encodes a mature LIP protein that is 341 aa long and is preceded by a 25 aa-long signal peptide. The LIP protein encoded by VLG1 has 55-60% homology to the LIP proteins of P. chrysosporium.

      Impacts
      (N/A)

      Publications

      • ZHANG, Y.Z., REDDY, C.A., and RASSOLY, A. 1991. Cloning of several lignin peroxidase (LIP)-encoding genes: sequence analysis of the LIP6 gene from the white-rot basidiomycete, Phanerochaete chrysosporium. Gene 97:191-198.
      • BLACK, A.K., and REDDY, C.A. 1991. Cloning and characterization of a lignin peroxidase gene from the white-rot fungus, Trametes versicolor. Biochem. Biophys. Res. Commun. 179:428-435.
      • RANDALL, T., REDDY, C.A., and BOOMINATHAN, K. 1991. A novel extrachromosomally maintained transformation vector for the lignin-degrading basidiomycete Phanerochaete chrysosporium. J. Bacteriol. 173:776-782.
      • RANDALL, T., and REDDY, C.A. 1991. An improved transformation vector for the lignin-degrading white-rot basidiomycete Phanerochaete chrysosporium. 103:125-130.