Lignin peroxidase expression in the white rot fungus Phanerochaete chrysosporium:
The most intensively studied white rot fungus, Phanerochaete chrysosporium, secretes an array of peroxidases which are able to extensively degrade lignin, the most aboundant aromatic polymer. Two major families of hydrogen peroxide (H2O2)-requiring extracellular heme-peroxidases designated lignin peroxidase (LIP) and manganese-dependent peroxidase (MNP) are secreted. The non-specific nature and exceptional oxidation potential of the lignin peroxidases has attracted considerable interest in organopollutants degradation and fiber bleaching. The regulation of the gene families encoding extracellular peroxidases is poorly understood, but it’s clear that oxygen stress is a key factor. LIP expression requires nutrient or carbon starvation, signaled by a rise in intracellular cAMP concentrations and concomitant pulses of pure oxygen gas in the headspace. The effect of such elevated oxygen concentrations on LIP expression can be reproduced by depleting Mn2+ ions from cultures, in the presence of atmospheric air. Development of a putative oxidative stress is detected in LIP-producing cultures of P. chrysosporium, either oxygenated or Mn2+-deficient. In oxygenated cultures, increased expression of MnSOD was the major response of the antioxidant system. In contrast, in Mn2+-deficient cultures, negligible activity of MnSOD was detected.
Addition of a OH. scavenger, dimethylsulfoxide, to the cultures completely abolished LIP transcription (both mRNA and heme-protein were undetectable), indicating that these ROS, coupled to high levels of cAMP, are indeed involved in the induction of LIP expression. Since the difference between both types of LIP-producing cultures consist in activation or lack of MnSOD activity, it is possible that ROS production in general and OH. generation in particular occurs by different mechanisms. The long-term objective of this research is to study the role of ROS in the regulation or induction of LIP expression in the white-rot fungus P. chrysosporium as well as the sources, ways and mechanisms of their generation.
The construction of conditional MnSOD(-) mutants will enable us to ascertain the role or influence of MnSOD, Mn2+ ions as well as other antioxidant enzymes in the production of the relevant ROS necessary for LIP induction. The results of this research are expected to advance the understanding of the pathways, sources and nature of ROS as second messengers in LIP expression.
Work program:
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Effect of reactive oxygen expression (ROS) on lignin peroxidase expression in the white rot fungus P. chrysososporium.
It was found that hydroxyl radical is the principal ROS for enhancement of lignin peroxidase expression. The sources of this radical in different lignin peroxidase producing cultures are under investigation.
- Role of manganese superoxide dismutase in lignin peroxidase expression.
Mutants lacking manganese superoxide dismutase were prepared by RNA silencing. Those mutants will be characterized and they will be used for understanding the role of manganese ions and manganese superoxide dismutase in lignin peroxidase expression.
- Signal transduction pathway and lignin peroxidase expression.
The relationship between protein kinase C and lignin peroxidase will be investigated.
- Improvement of lignin peroxidase production by P. chrysosoporium.
Optimization of lignin peroxidase production by changing different growth parameters and medium composition will be performed.
Homologous expression of lignin peroxidase in yeasts:
Potential applicability of LIP depends on the ability to produce high quantities of the enzyme by efficient growth and purification technologies.
Lignin peroxidase production by submerged fermentation of P. chrysosporium is hampered by several factors, such as expression under nutrient limitation together with oxidative stress, and in the sensitivity of this basidiomycete fungus to high shear forces in a fermenter. Moreover, the purification stage of the enzyme from the growth liquid medium of P. chrysosporium is tedious and expensive.
Homologous expression of LIP in yeasts can be an alternative and more efficient process for LIP production, since less incubation time is needed for the organism growth and only LIP-H2 could be expressed, avoiding the purification step from other isoenzymes.
The present study addresses the production of lignin peroxidase H2 in the yeast Pichia pastoris.
Work program
- Cloning of the encoding region of LIP-H2 into the expression vector pPIC9 from the expression system of P. pastoris (invitrogene).
- Transformation to P. pastoris.
- Induction of LIP enhanced expression, collection and purification of the secreted enzyme
- Structural and activity comparison between the enzyme expressed by P. pastoris and the enzyme produced by P. chrysosporium
Large scale lignin peroxidase production:
- Development and scale up for production of lignin peroxidase by different white rot fungi.
- Scale up of downstream process.
- Optimization of spore production at large quantities
Development of formulations containing lignin peroxidase for skin whitening:
- Test of compatibility of the different formula components with the enzyme.
- Determination of optimal melanin oxidation by lignin peroxidase formulation