Expression of recombinant proteins is important for studying their biological function. Most often, the expression system of the E. coli is used for the primary description of protein properties. However, under overexpression conditions, the rate of aggregation of target proteins often exceeds the rate of proper folding, resulting in the formation of insoluble inclusion bodies. Inclusion bodies are a clear disadvantage of the E. coli expression system because they interfere with the release of target recombinant proteins. One solution to the existing problem is the use of chaperone-like proteins in vitro to refold the target protein. In this work, the recombinant protein MF3 was taken as an example of a chaperone-like protein, which increased the yield of soluble plant chitinase by 92% compared to the yield of this protein using the standard refolding procedure.

The Penicillium verruculosum filamentous fungus is a highly active producer of cellulolytic complex enzymes, cellobiohydrolases, endoglucanases and β-glucosidases. Using the CRISPR/Cas9 genome editing system, previously adapted to P. verruculosum, a strain with a knockout of the xlnR gene encoding XlnR, one of the main transcription factors of filamentous fungi, has been obtained. The transcription level of cellulolytic genes was determined by quantitative PCR for the P. verruculosum B1-221-151 strain and the new P. verruculosum ΔxlnR strain. The XlnR protein was shown to activate transcription of the cbh1 , egl2 , and bgl1 genes encoding cellobiohydrolase 1, endoglucanase 2, and β-glucosidase, respectively, in the presence of xylose and xylooligosaccharides in the growth medium. It was found that other factors are also involved in the activation of transcription of these genes by cellobiose, cellotriose, sophorose, and gentiobiose, which has a complex effect on the biosynthesis of the cellulolytic complex of enzymes produced by the P. verruculosum fungus.

Methods of the pretreatment of reeds (grinding, hydrothermal treatment, treatment with acid or alkali solutions, organosolv, deep eutectic solvents) and their effect on the subsequent enzymatic hydrolysis by cellulase and hemicellulase complexes have been studied. Substrates with the highest reactivity were obtained by exposing the reeds to a weakly alkaline deep eutectic solvent (DES) and an alkali solution. The depth of enzymatic hydrolysis of these pretreated substrates was 63 and 59

The reactivity during enzymatic hydrolysis of eight industrially produced samples of pulps and semichemical pulps by enzyme preparations of glycosyl hydrolases B151 and F10 produced by a strain of the ascomycete fungus Penicillium verruculosum has been determined. It is shown for the first time that among fibrous pulps available on the market of pulp and paper industry in Russia, the highest level of yield of glucose from the initial wood during biocatalysis using cellulases and hemicellulases is characteristic of semichemical pulps obtained after green liquor cooking of hardwood. A high degree of enzymatic conversion of softwood bleached kraft pulp has been established, which in combination with the possibility of obtaining modified polysaccharide materials from non-hydrolysable residue makes this cellulosic substrate the most promising for the development of biological processes at pulp and paper industries. It is shown that drying of pulp negatively affects the efficiency of cellulose hydrolysis, while mechanical refining improves the performance of the enzymatic saccharification process.

Exhaustive enzymatic hydrolysis is performed for semi-bleached sulfate hardwood cellulose (a semi-finished pulp and paper product) at ultra-high concentrations of it in a reaction mixture (up to 300 g/L per dry compound). Russian commercial enzyme preparations are used for hydrolysis. The best seems to be Agroxil Plus, which has high cellulase and endoxylanase activities. A total of 290 g/L of sugars (including 210 g/L of glucose and 30 g/L of xylose) is obtained using Agroxil Plus (20 mg protein/1 g substrate) in combination with an auxiliary β-glucosidase enzyme preparation (2 mg protein/1 g substrate) at an initial semi-bleached cellulose concentration of 300 g/L. The dosage of Agroxil Plus can be halved (10 mg of protein/1 g of substrate with a total concentration of semi-bleached cellulose of 300 g/L) with a high yield of hydrolysis product (270 g/L of sugars, including 200 g/L of glucose and 30 g/L of xylose), due to the fractional addition of a substrate.

Микроскопические грибы широко используются для промышленного производства технических ферментов; важным условием для этого является создание высокоактивных штаммов – продуцентов ферментов. Показана возможность использования для получения таких штаммов двух подходов: индуцированного мутагенеза и методов генетической инженерии. Разработаны схемы индуцированного мутагенеза, обеспечивающие получение высокоактивных продуцентов технических ферментов. В результате гамма-мутагенеза получены мутантные штаммы грибов рода Trichoderma – продуценты целлюлаз, ксиланаз и пектиназы, с различным компонентным составом карбогидразного комплекса, позволяющие получать в культуральной жидкости (КЖ) до 35–40 г/л внеклеточного белка. С помощью комбинированной обработки УФ и гамма-облучением получен стабильный мутантный штамм Aspergillus awamori с увеличенной продукцией глюкоамилазы. Создана система экспрессии Penicillium verruculosum В1-537 (ΔniaD), позволяющая трансформировать реципиентный штамм экспрессионными конструкциями, содержащими целевые гетерологичные или гомологичные гены, функционально связанные с промотором и терминатором сильного индуцибельного промотора гена мажорного секреторного белка целлобиогидролазы I (cbh1) P. verruculosum. Технология создания и отбора активных рекомбинантных штаммов проста и надежна, состав питательной среды и условий культивирования рекомбинантных штаммов – продуцентов различных целевых ферментов стандартизирован. Затраты времени на получение продуцента целевого фермента составляют от 3 до 6 мес. Реципиентный штамм P. verruculosum характеризуется высокой секреторной способностью (до 60 г/л внеклеточного белка в КЖ), а рекомбинантные штаммы-продуценты – высокой продуктивностью целевых ферментов; полученные с их помощью ферментные препараты (ФП) содержат 30‒70 % целевых рекомбинантных ферментов (в некоторых случаях до 80 %) от общего пула белка. Созданы рекомбинантные штаммы – продуценты целлюлаз, β-глюканаз, ксиланаз, фитазы, кислой протеазы, пектин-лиазы, β-глюкозидазы, экзо- и эндоинулиназ. Мутантные и рекомбинантные штаммы – продуценты различных ферментов – используются для промышленного производства на заводе ООО «Агрофермент» различных технических ФП для пищевой промышленности и кормопроизводства. Microscopic fungi are widely used for the industrial production of technical enzymes; an important condition for this is the creation of highly active strains – producers of enzymes. The possibility of using such approaches to obtain highly active strains as induced mutagenesis and genetic engineering methods is shown. Schemes of induced mutagenesis have been developed that ensure the production of highly active producers of technical enzymes. As a result of gamma-mutagenesis, mutant fungal strains of Trichoderma genus were obtained – producers of cellulases, xylanases and pectinase, with different component composition of the carbohydrase complex. The methods used made it possible to obtain up to 35–40 g/l of extracellular protein in the cultural liquid (CL). Using UV and gamma rays combined treatment, a stable mutant Aspergillus awamori strain with increased glucoamylase production was obtained. An expression system for Penicillium verruculosum B1-537 (ΔniaD) has been developed, which makes it possible to transform a recipient strain with expression constructs containing target heterologous or homologous genes functionally linked to the promoter and terminator of the strong inducible promoter of the P. verruculosum major secretory protein cellobiohydrolase I (cbh1) gene. The technology for creating and selection of active recombinant strains is simple and reliable, the composition of the nutrient medium and cultivating conditions for recombinant strains producing various target enzymes are standardized. The time spent on obtaining the target enzyme producer is from 3 to 6 months. The recipient P. verruculosum strain is characterized by a high secretory capacity (up to 60 g/l of extracellular protein in CL), and the recombinant strains are characterized by a high productivity of target enzymes. Enzyme preparations (EP) derived from the recombinant strains contain 30–70 % of the target recombinant enzymes in the total protein pool (in some cases, up to 80 %). Recombinant strains producing cellulases, β-glucanases, xylanases, phytase, acid protease, pectin-lyase, β-glucosidase, exo- and endo-inulinases have been developed. Mutant and recombinant strains – producers of various enzymes for the food industry and feed production are successfully used for industrial production of various technical EPs at the plant LLC «Agroferment».

To increase the frequency of homologous recombination (HR) during the transformation of the industrial strain Penicillium verruculosum 221-151 (VKM F-3972D), the ku70 gene encoding the Ku70, which binds at the sites of double-stranded DNA breaks and is involved in the repair process by the non-homologous end joint (NHEJ), was knocked out by the CRISPR/CAS9 method. Presumably, the new host strain, P. verruculosum ΔniaDΔku70, should have had an increased frequency of homologous recombination during transformation in comparison with the host strain P. verruculosum ΔniaD due to the integrative insertion of the expression cassette only by the HR mechanism. The pep1 gene encoding homologous aspartate protease was chosen as a marker. However, it was shown that the knockout of the ku70 gene led to a dramatic decrease in the frequency of co-transformation in the P. verruculosum ΔniaDΔku70 strain compared to the P. verruculosum ΔniaD strain at the same load of exogenous DNA (3 μg). The number of copies of the pep1 gene in recombinant strains of the P. verruculosum Pep1 (with a native Ku70) series ranged from 3 to 28 copies, which indicated the predominance of the non-homologous recombination mechanism.

The possibility of exhaustive enzymatic hydrolysis of semi-bleached sulfate hardwood pulp, a semi-finished product of pulp and paper production, at its super high concentrations in the reaction mixture (up to 300 g/l) is shown. For hydrolysis, Russian commercial enzyme preparations were used, the best of them was Agroxyl Plus, which has a high activity of cellulases and endoxylanase. With the help of Agroxyl Plus (at its dosage of 20 mg protein/g substrate) in the presence of an auxiliary enzyme preparation of β-glucosidase (2 mg protein/g substrate) at an initial concentration of semi-bleached cellulose of 300 g/l, 290 g/l sugars (210 g/l glucose, 30 g/l xylose) were obtained. Due to fed-batch enzymatic hydrolysis, it was possible to halve the dosage of Agroxyl Plus (10 mg protein/g of substrate at a total concentration of semi-bleached cellulose of 300 g/l) while maintaining a high yield of hydrolysis products – 270 g/l sugars (200 g/l glucose, 30 g/l xylose).

Cloning and expression of the full-length endo- processive-type xyloglucanase from the Trichoderma reesei (TrXeg74A) fungus, as well as its catalytic domain TrXeg74A-CD, in the Penicillium verruculosum B1-537 recipient strain have been carried out. P. verruculosum is a highly effective producer of cellulases. The levels of protein secretion after culturing the obtained recombinant strains in a laboratory fermenter were 35.4 and 31.4 g/L, respectively. TrXeg74A accounted for at least 30% of the total protein, while TrXeg74A-CD was expressed to a much lesser extent. Both forms of the recombinant enzyme were isolated in purified state and their properties were studied. TrXeg74A and TrXeg74A-CD were characterized by a similar degree of processivity when exposed to tamarind xyloglucan and the same Michaelis constant (0.35-0.38 g/L), close to that for the native enzyme (0.30 g/L), while the catalytic constant for TrXeg74A-CD was 1.5 times higher than the corresponding parameter for full-length xyloglucanase. The obtained new recombinant P. verruculosum strains can be useful in the development of composite enzyme preparations for efficient hydrolysis of renewable lignocellulosic raw materials.

New recombinant strains of Penicillium verruculosum with a high level of expression of homologous endo-β-1,4-glucanase II and heterologous phytase A of A. niger, as well as heterologous endo-1,4-β-xylanase E of P. canescens and phytase A of A. niger, are created. This allows us to obtain highly active feed enzyme preparations (EPs) capable of simultaneously significantly reducing the viscosity of nonstarch polysaccharides (NPSs), as well as increasing the bioavailability of phosphorus and minerals of grain-based feed and, thus, increasing the digestibility of nutrients of agricultural animal and poultry feed. Specific EP activities for specific substrates (xylan, β-glucan, and phytate) are studied and the qualitative and quantitative component composition of new EPs is determined. A recipient strain δnia D, into which heterologous endo-1,4-β-xylanase E of P. canescens is successfully cloned, is obtained based on a new enzyme preparation containing homologous endo-β-1,4-glucanase II and heterologous phytase A of A. niger. In this way, a promising producer of three target enzymes is obtained simultaneously.

The tacA gene, encoding the TacA repressor protein, was cloned by “walking the uncloned DNA” method from the genomic DNA of the fungus Penicillium verruculosum B1-221-151. Knockout of the tacA and niaD genes by the CRIS-PR/CAS9 led to the production of a new host strain P. verruculosum ΔniaDΔtacA, characterized by a higher rate of extracellular protein biosynthesis. Analysis of the transcription and expression of the cbhI gene in the original P. verruculosum B1-221-151 strain and in the P. verruculosum ΔniaDΔtacA strain showed a sharp increase in the level of cbhI gene transcription 2 h after the start of induction with cellobiose, cellotriose, gentiobiose, and a mixture of di- and trisaccharides in comparison with the transcription of the cbhI gene in the original strain. The speci c activity of cellobiohydrolase I, the main enzyme of the cellulolytic complex of the fungus P. verruculosum, by 96 h of fermentation of the ΔtacA strain increased 3 times compared to the original strain.

The review describes the history of the development of research on carbohydrasеs conducted at the Department of Chemical Enzymology from the mid-1970s to the present. The results concerning the study of the mechanism and kinetics of the processes of enzymatic conversion of cellulose and renewable plant raw materials under the action of a multi-enzyme cellulases complexes; the role of individual components of these complexes - basic (endoglucanases and cellobiohydrolases) and auxiliary enzymes (polysaccharide monooxygenase, β-glucosidase, xylanase) and their synergistic interaction. The features of using reactors of various designs for bioconversion of plant raw materials are described: periodic type, continuous column type, reactor for hydrolysis in a constant electric field, reactor with intensive mixing by ferromagnetic particles in magnetic field. The possibilities of increasing the reactivity of plant raw materials using various pretreatment methods, as well as the influence of the structural and physico-chemical properties of cellulose on the efficiency of its enzymatic conversion are discussed. Data on the creation of highly active strains of microscopic fungi-producers of cellulases and other carbohydrases using methods of induced mutagenesis - Trichoderma (Hypocrea), Penicillium (Talaromyces), Aspergillus, Chrysosporium (Myceliophtora) spp., as well as data on the composition of the enzyme complexes produced by them and the properties of the enzymes forming them are presented. It describes the creation of expression systems based on P. canescens and P. verruculosum and the production of recombinant producer strains with their help, which made it possible to obtain enzyme preparations (EP) that ensure high efficiency of bioconversion processes of plant raw materials, as well as to create producers of a wide range of carbohydrases for practical use in various fields of industry and agriculture. A number of industrially important EP obtained using the P. verruculosum expression system are currently being produced at the Agroferment LLC plant.

As a result of cloning of the inuA, inu1, aglC, and fopA genes encoding endoinulinase (endoINU), exoinulinase (exoINU), α-galactosidase C (AGLС) and sucrase (SUC), respectively, into the recipient strain Penicillium verruculosum B1-537 (ΔniaD), recombinant producer strains were obtained that are capable of producing target recombinant enzymes with a high yield (32‒50

The thermostability of industrially important enzymes (exoinulinase, phytase, xylanase, endoglucanase) was increased through the rational design of α-helices.

Based on the recipient strain of Penicillium verruculosum B1-537 (ΔniaD) using the promoter of the cellobiohydrolase I gene, a producer of homologous lytic polysaccharide monooxygenase (PMO) and heterologous β-glucosidase Aspergillus niger (BG), auxiliary enzymes for the “basic” cellulase complex consisting of endoglucanases and cellobiohydrolase, was obtained. The enzyme preparation PMO–BG was obtained, containing 34% PMO and 43% BG, which was used as a source of auxiliary enzymes that increase the effectiveness of basic cellulases P. verruculosum by 20–100% in the bioconversion of various types of cellulose-containing raw materials: microcrystalline cellulose, pretreated with alkali common reed and semi-bleached sulfate hardwood pulp of Arkhangelsk Pulp and Paper Mill JSC.

A new recombinant Aspergillus niger tannase (tannin acyl hydrolase) produced by the Penicillium verruculosum fungus has been studied. A strain with a high level of extracellular tannase (TAN2) secretion (80% of the total extracellular protein) was obtained by cloning the tan2 gene (PDB Acc. No: MT828303) into the recipient strain. The tannase enzyme preparation degraded tannins in black tea extracts. TAN2 was isolated in homogeneous form using chromatographic methods; the enzyme had a high activity against gallotannin (53 U/mg) and less activity against propyl gallate (4.7 U/mg). Homogeneous TAN2 showed temperature and pH optima of 45 °C and 3.5, respectively. At a temperature of 50 °C, TAN2 retained above 80% activity for 3 h; at 60 °C, it retained about 75% of its activity for 90 min; at 70 °C, the enzyme was completely inactivated within 10 min. Tannase was characterized by a high tolerance to NaCl, the activity against gallotannin exceeded 50% of the initial value in solutions with a salt concentration of up to 5 M. The tannase activity was stimulated by Ca2+, Mg2+, Zn2+, Mn2+, Cu2+, Cd2+, Pb2+ by 3--64% and inhibited by 4-65% in the presence of Co2+, Fe3+ and Fe2+ ions.tannase, tannins, Aspergillus niger, Penicillium verruculosumThis work was supported by the Ministry of Science and Higher Education of the Russian Federation.