RU-2861689-C2 - RECOMBINANT LACCASE FROM PARAMYROTHECIUM RORIDUM VKM F-3565 POLYMERISING PHENYLPROPANOIDS IN NEUTRAL-ALKALINE MEDIUM, AND METHOD FOR PRODUCING SAME

RU2861689C2RU 2861689 C2RU2861689 C2RU 2861689C2RU-2861689-C2

Abstract

FIELD: biotechnology; genetic engineering. SUBSTANCE: there is provided a method for producing recombinant laccase from Paramyrothecium roridum VKM F-3565. The method involves inserting into the genomic DNA of Komagataella phaffii GS115 an expression cassette based on the vector pPIC9K, having a size of 10917 bp, shown in Fig. 1 and carrying the AOX1 promoter, the Saccharomyces cerevisiae a-factor, a spliced gene for alkalophilic laccase from Paramyrothecium roridum VKM F-3565, the nucleotide sequence of which is represented by SEQ ID NO: 1, the AOX1 gene terminator, the HIS4 marker gene, the kanamycin resistance gene KanR, the AOX1 3′-fragment, ori, the ampicillin resistance gene AmpR, the AmpR promoter, culturing the transformed Komagataella phaffii GS115 cells, followed by expression of the alkalophilic laccase into the culture medium. EFFECT: efficient production of recombinant laccase from Paramyrothecium roridum VKM F-3565. 1 cl, 4 dwg, 3 tbl, 13 ex

Inventors

Renfeld Zhanna Vladimirovna
KOLOMYTSEVA MARINA PAVLOVNA
Gorina Sofya Yuryevna
Chernykh Aleksei Mikhailovich

Dates

Publication Date: 20260507
Application Date: 20240703

Claims (1)

A method for producing recombinant laccase from Paramyrothecium roridum VKM F-3565, which involves inserting into the genomic DNA of Komagataella phaffii GS115 an expression cassette based on the pPIC9K vector, having a size of 10917 bp, shown in Fig. 1 and carrying the AOX1 promoter, the α-factor of the yeast Saccharomyces cerevisiae, the spliced gene of the alkaliphilic laccase from Paramyrothecium roridum VKM F-3565, the nucleotide sequence of which is represented by SEQ ID NO: 1, the AOX1 gene terminator, the HIS4 marker gene, the KanR kanamycin resistance gene, the AOX1 3'-fragment, ori, the AmpR ampicillin resistance gene, the AmpR promoter, and culturing the transformed Komagataella phaffii cells GS115 followed by expression of alkaliphilic laccase in the culture medium.

Description

Field of technology to which the invention relates The invention relates to the field of biotechnology and genetic engineering and concerns the production of recombinant laccase of the ascomycete Paramyrothecium roridum VKM F-3565, which oxidizes phenylpropanoids in a neutral-alkaline medium with the formation of oligomeric products, and a method for producing it using a transformant of the yeast Komagataella phaffii (Pichia pastoris), containing in the chromosomal DNA an expression cassette, which includes a gene sequence encoding the target enzyme. The recombinant alkaliphilic laccase of P. roridum VKM F-3565 can be used in the cellular biosynthesis of pharmacologically valuable lignan-like oligomeric compounds or the production of other compounds difficult to access for chemical synthesis, as well as in the textile, pulp and paper and food industries, for the transformation and biodegradation of xenobiotics, for the creation of biosensors and biofuel cells for implantable devices without the additional costs associated with the use of mediators. State of the art Laccase (EC 1.10.3.2, "blue" oxidase) is an industrially valuable enzyme capable of performing one-electron oxidation of a wide range of phenolic, non-aromatic, and inorganic compounds with concomitant four-electron reduction of molecular oxygen to water [1-3]. The main natural producers of laccases are fungi, particularly basidiomycetes, due to their natural function of decomposing plant products lignin and cellulose. Laccases have also been found in plants, bacteria, archaea, lichens, insects, sponges, mollusks, nematodes, and even humans [4]. Fungal and bacterial laccases are actively used in various biotechnologies: destruction of xenobiotics, in the production of synthetic detergents, delignification and bleaching of dyes in textile production waste, in cosmetics (hair dye, whitening toothpaste), in the food industry (removal of oxygen in food products to increase shelf life), in organic synthesis, etc. [4, 5]. Possessing a high redox potential and, consequently, broad substrate specificity without the use of mediators, fungal laccases offer significant advantages over bacterial laccases and laccase-like enzymes. However, most typical fungal laccases are functionally active at acidic pH, significantly complicating their use in technological processes occurring in neutral-alkaline environments. With the advent of new technologies implemented in neutral-alkaline environments (cellular platforms for the biosynthesis of pharmacologically and industrially valuable compounds, biosensors, biofuel cells for implantable devices, 3D nanodevices for biomedical purposes, synthesis of C-N heteropolymer dyes, etc.), there is a growing interest in the search for and study of biotechnologically valuable atypical fungal laccases active in neutral-alkaline environments [3, 6]. However, alkaliphilic fungal laccases are the least represented in nature [1, 7]. Polymeric derivatives of phenylpropanoids (lignans, stilbenes, flavonoids, etc.) are pharmacologically valuable compounds, exerting a wide range of effects on living organisms: cytostatic, carcinolytic, antibacterial, fungicidal, antiparasitic, antioxidant, stimulating, adaptogenic, etc. [3, 6]. The ability of typical fungal laccases to polymerize a wide range of phenolic compounds is limited by their low efficiency in neutral-alkaline conditions, which are necessary for cellular technologies of biosynthesis of pharmacologically valuable compounds [8]. Currently, laccases that are most active in neutral-alkaline environments have been discovered in several fungi. Thus, laccase from the basidiomycete Rhizoctonia praticola showed maximum activity with syringaldazine at pH 6.0-7.0 [9], laccase from the basidiomycete Coprinopsis cinerea showed maximum activity with guaiacol at pH 6.5 [10], and laccase from the basidiomycete Moniliophthora perniciosa FA553 was most active with 2,6-dimethoxyphenol at pH 6.5 and with syringaldazine at pH 7.5 [11]. Of the listed basidiomycete alkaliphilic laccases, there is a report of a recombinant laccase from Coprinopsis cinerea expressed in Pichia pastoris GS115 using the pPIC9K plasmid and with a yield of 1.750-3.138 U/l [12]. However, this enzyme had a pH optimum for the reaction with syringaldazine at pH 6.0, but retained 50-60% of activity at pH 7.0-8.0 [12]. Also known is a recombinant laccase Lcc9 from Coprinus cinereus, expressed in Aspergillus niger MA70.15 with a yield of 150 U/l and 3138±62 U/l (under optimized conditions), most active with syringaldazine at pH 6.0 [12, 13]. The same laccase was able to decolorize M2GE, KD8B, KM8B, K7R, 6B, and IC dyes at pH 7.5. Lcc9 laccase was also expressed in P. pastoris cells using the pPIC9K-PIE5K expression vector with an unknown yield and was most active with guaiacol and indigo blue at pH 7.0–8.5 [14]. There is also a report of recombinant Lcc1 laccase from Coprinus cinereus expressed in Aspergillus oryzae fungal cells with