Evolved Fusarium oxysporum laccase expressed in Saccharomyces cerevisiae

Abstract

Fusarium oxysporum laccase was functionally expressed in Saccharomyces cerevisiae and engineered towards higher expression levels and higher reactivity towards 2,6-dimethoxyphenol, that could be used as a mediator for lignin modification. A combination of classical culture optimization and protein engineering led to around 30 times higher activity in the culture supernatant. The winner mutant exhibited three times lower Km, four times higher kcat and ten times higher catalytic efficiency than the parental enzyme. The strategy for laccase engineering was composed of a combination of random methods with a rational approach based on QM/MM MD studies of the enzyme complex with 2,6-dimethoxyphenol. Laccase mediator system with 2,6-dimethoxyphenol caused fulvic acids release from biosolubilized coal. Laccases are oxidoreductases that catalyze the 4-electron reduction of O 2 to water with simultaneous oxidation of organic substrates. Laccases are able to catalyze oxidation of a substrate of interest directly or indirectly by the formation of a radical, which then may take part in a non-enzymatic event that effects in the oxidation of a substrate of interest. Laccases oxidize phenolic substrates but are also able to oxidize non-phenolic or bigger substrates by Laccase Mediator System (LMS), where a small phenolic compound acts as a mediator 1. Laccases contain 4 copper atoms buried in their 3D structure, which are located in two separate Cu centers. T1 copper ion is a mononuclear center, whereas T2 copper ion and two T3 copper ions are positioned in T2/T3 copper center. The substrate is oxidized closed to T1 copper ion and then the electrons are transferred to the tri-nuclear center where O 2 is reduced to water. The coppers are coordinated by nearby located residues: the T1 copper ion by one Cys and two His residues, the T2 copper by two His residues and a solvent molecule and T3 copper to three His residues 2,3. Biosolubilization of brown coal is a clean coal technology that aims at the conversion of the lignite to its cleaner form or to change its structure to gain new features 4,5. Such solubilized material could be used as a source of value-added products 4. The liquid form of coal-microorganisms, such as Fusarium oxysporum reported before as an excellent coal solubilizer 6 , uses their metabolites, alkaline substances, biosurfactants and enzymes to turn the solid polymer to black liquid. Laccases, from bacteria and fungi, for example from species Pleurotus 7 or Streptomyces 8 , are known to take part in the degradation of lignin and lignite. These two polymers are similar in structure, thus, the mechanism of their degradation is expected to be the same. Lignin comprises only 10-20% of phenolic subunits. In theory, laccase could act on those subunits present on the lignin surface and modify the polymer. However, the possibility of lignin subunits entering the laccase active site is very limited. Moreover, it is not known to what extent lignin could be modified in this way 9. According to literature data, the treatme...

Elaboration of a Phytoremediation Strategy for Successful and Sustainable Rehabilitation of Disturbed and Degraded Land

“…To date, LAC remains the only candidate enzyme identified and functionally characterized from a coal-degrading fungus. Thus, heterologous expression of a Fusarium oxysporum LAC in the yeast Pichia pastoris was shown to depolymerize and liquefy solubilized brown coal with the release of humic and fulvic acids [58,59].…”

Bright Side of Fusarium oxysporum: Secondary Metabolites Bioactivities and Industrial Relevance in Biotechnology and Nanotechnology

“…Laccases are belonging to oxidoreductases that catalyze the O 2 reduction to H 2 O with simultaneous organic substrates oxidation. They oxidize phenolic substrates but are also able to oxidize bigger or non-phenolic substrates by LMS (laccase-mediator system), where a small phenolic compound acts as a mediator [164]. Additionally, they have been reported as potential lignin-degrading enzymes and as solubilizing agents [165,166].…”

“…Kwiatos et al, expressed F. oxysporum Gr2 laccase in Saccharomyces cerevisiae and engineered it for getting higher effect against 2,6-dimethoxyphenol and higher expression levels. The resulted laccase had a promising potential for different industrial uses such as solubilization of brown coal, which is a clean coal technology, aiming at converting lignite to its cleaner form [164]. In 2018, Kwiatos et al, reported that F. oxysporum LOCK-1134 isolated from brown coal, efficiently bio-solubilized lignite, producing liquefied products that had over 99% less Hg and 50% less sulfur than the crude coal.…”

“…It showed maximum bioethanol yield and production of 0.84 g/g and 2.47 g/L, respectively, indicating its importance as a bio-agent for single-pot local bio-refinery [179]. Moreover, F. oxysporum BN converted imidazolium-based ionic liquid (IL)-pretreated rice straw to bioethanol via CBP with 64.2% of the theoretical yield of 0.125 g ethanol/g rice straw [164]. It secreted a novel IL-tolerant cellulase that can direct the conversion of ILpretreated lignocellulose residue to ethanol, which had a significant potential to bring a breakthrough in commercial ethanol production by the reduction of the overall cost [180].…”

Coal-Degrading Bacteria Display Characteristics Typical of Plant Growth Promoting Rhizobacteria

“…Coal mining not only visibly disrupts the aesthetics of a landscape but also disrupts all of the soil components including soil horizons, structure, microorganisms and nutrient cycles that are crucial to sustaining a healthy ecosystem [9,10]. While a number of fungi show potential as rehabilitation biocatalysts and degrade coal into a soil-like humic-rich material [11][12][13][14][15][16][17][18], the only candidate enzymes identified and functionally characterised from coal-degrading fungi so far are laccase and esterase. Heterologous expression of Fusarium oxysporum laccase in Pichia pastoris was shown to depolymerise and liquefy solubilised brown coal with the release of humic and fulvic acids [18].…”