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.

A new nag1 gene encoding N-acetyl-D-glucosaminidase from Streptomyces kursanovii was cloned. The soluble form of N-acetyl-D-glucosaminidase was obtained after improving its expression in E. coli ArcticExpressTM (DE3) cells. The homogeneous form of the enzyme was isolated by affinity chromatography, and its bacteriolytic ability against cells of the Gram-positive bacterium Micrococcus lysodeikticus was studied. It was demonstrated that the new enzyme's bacteriolytic capacity surpasses that of chicken lysozyme.

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 interest in peroxidases of the basidiomycete secreted enzyme complex is due to their wide substrate specificity and the ability of these enzymes to participate in the biodegradation of such difficult to degrade biopolymers as lignin. However, due to the difficulty of isolating these enzymes from native sources, their study is difficult. In this work, expression vectors were created that carried the sequence encoding the T. hirsuta LE-BIN072 versatile peroxidase VP2, which was transformed into the genome of the P. canescens strain. Screening of transformants showed the presence of peroxidase activity up to 1 U/mL. Fragments of the target protein in the culture liquids of the selected transformants were identified by mass spectrometric analysis. A new strain, P. canescens pVP2D-6, a producer of the recombinant versatile peroxidase VP2 of T. hirsuta LE-BIN072, was obtained for the first time, and the ability of the enzyme complex secreted by it to modify alkaline lignin was shown.

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.

PurposeTo adapt CRISPR/Cas9 genome editing method for use in filamentous fungus Penicillium verruculosum, which is industrial producer of carbohydrases. ResultsFor the first time the CRISPR/Cas9 method was adapted for genome editing in the filamentous fungi Penicillium verrucullosum. Using the nitrate reductase gene (niaD) as a selective marker with the CRISPR/Cas9 system we performed double knockout of niaD and cellobiohydrolase 1 (cbh1) genes. The efficiency of double editing was 50%. At the same time, it was unexpected that the specific cellobiohydrolase activity rised after knockout of cbh1 gene due to the increase CBH2 expression. ConclusionWe developed effective method for genome editing in P. verruculosum, which can be used to improve qualities of industrial strains.

A method has been developed for the analysis of the induction of the cbh1 gene transcription in the filamentous fungus Penicillium verruculosum after treatment with mono- and oligosaccharides. The method allows obtaining mRNA, which encodes cellobiohydrolase-1 (CBH1) in an amount sufficient for RT-PCR. Citrate, a compound that does not cause carbon catabolic repression, was used as a carbon source. It was shown for the first time that xylose, gentiobiose, and mainly cellobiose induce the expression of the cbh1 gene in P. verruculosum, while sophorose and a mixture of xylooligosaccharides with a degree of polymerization from 3 to 5 are most likely to serve as precursors of inducers. Penicillium verruculosum, qPCR, RT-PCR, monosaccharides, oligosaccharides, inducer The work was supported by the Russian Foundation for Basic Research (RFBR) (grant no.18-29-07070).

The cas9 gene of the Streptococcus pyogenes bacterium, which encodes Cas9 nuclease of the class 2 CRISPR/Cas system, is expressed in the filamentous fungus Penicillium verruculosum under the control of the constitutive autologous promoter of the glyceraldehyde-3-phosphate dehydrogenase gpdA gene, which is cloned and sequenced for the first time in the present work. The functionality of the gpdA promoter is confirmed by the expression of the heterologous β-glucosidase gene of Aspergillus niger in P. verruculosum . The relative copy number and expression level of the cas9 gene in recombinant strains are determined by real-time PCR. In order to detect and determine the localization of Cas9 nuclease in P. verruculosum fungal cells, the chimeric Cas9 nuclease with the fluorescent eGFP protein added to the C-terminus was obtained. The fluorescence microscopy of the growing mycelium confirms the intracellular expression of the chimeric construct in the cells of recombinant strains.

A novel Aspergillus nidulans lac№4 (argB–) strain, a producer of recombinant laccase A from basidiomycete Trametes hirsuta 072, was obtained. This strain was used for the biocatalytic transformation of progesterone (PG). The major biotransformation products are 11α-hydroxy-PG, 11α-acetoxy-PG and 6β, 11α-dihydroxy-PG. Macronet MN-200 sorbent was used to study the adsorption kinetics of PG, and the main biotransformation products from the transformation medium. The solid-phase extraction of steroids without preliminary removal of the mycelium was shown to be efficient.

pCXSN-Ch140mb, almost complete attenuation of the GFP fluorescence was observed. At the same time, qPCR analysis confirmed that there were no changes in GFP expression in all samples, indicating that the amount GFP was only regulated post-translationally. Thus, we have created the system which allows us to perform the selective degradation of plant proteins via the UPS. Supported by RFBR #15-04-09365.

A system for the production of mutant recombinant human alpha-fetoprotein (rhAFP0) lacking the glycosylation site has been engineered in the yeast Pichia pastoris. A strain of the methylotrophic yeast Pichia pastoris GS115/pPICZαA/rhAFP0, which produces unglycosylated rhAFP0 and secretes it to the culture medium, has been constructed. Optimization and scale-up of the fermentation technology have resulted in an increase in the rhAFP0 yield to 20 mg/L. A scheme of isolation and purification of biologically active rhAFP0 has been developed. The synthesized protein has the antitumor activity, which is analogous to the activity of natural human embryonic alpha-fetoprotein.

The gene xylE encoding endo-1,4-b-xylanase from the 10th family of gly cosyl hydrolases produced by the mycelial fungus Penicillium canescens has been expressed under the control of the strong promoter of the bgaS gene encod- ing b-galactosidase from P. canescens. As a result, a strain-producer of endoxylanase XylE was developed. The recombinant enzyme was isolated and purified to homogeneity with specific activity of 50 U/mg. The physicochemical and biochemical properties of the endoxylanase were studied. The maximal enzymatic activity was observ ed at pH 6.0 and 70°C. Endoxylanase XylE was shown to be a highly thermostable enzyme with half-inactivation period t1/2 of 7 h at 60°C. The kinetic parameters were 0.52 mg/ml (Km) and 75 µmol/min per mg ( Vmax) using birch xylan as the substrate. Crystals of endoxylonase XylE were obtained, and the 3D structure was solved at 1.47 A resolution. The 3D structure of an endo-1,4- b-xylanase from the 10th family containing carbohydrate and unique cyclic structure located at the C-terminus of the polypeptide chain was obtained for the first time.

Ген xylE эндо 1,4-ƒ-ксиланазы десятого семейства гликозил-гидролаз мицелиального гриба Penicillium canescens экспрессирован под контролем сильного промотора гена bgaS, кодирующего ƒ-галактозидазу P. canescens и получен штамм - продуцент эндоксиланазы XylE. Фермент выделен в гомогенном состоянии (специфическая активность 50 ед/мг) и исследованы его физико-химические и биохимические свойства. Максимальная ферментативная активность наблюдалась при рН 6,0 и 70. Эндоксиланаза XylE является вы- сокостабильным ферментом, период полуинактивации фермента ƒ1/2 при 60 равен 7 ч. Кинетические пара- метры, определенные по березовому ксилану, равнялись 0,52 мг/мл (Km) и 75 мкМоль/мин/мг (Vmax). Полу- чены кристаллы эндоксиланазы XylE и решена трехмерная структура с разрешением 1,47 Е. Впервые опре- делена кристаллическая структура эндо 1,4-ƒ-ксиланазы 10 семейства, содержащая углеводы и уникальную циклическую структуру на С-конце.

The gene xylE encoding endo-1,4-β-xylanase from the 10th family of glycosyl hydrolases produced by the mycelial fungus Penicillium canescens has been expressed under the control of the strong promoter of the bgaS gene encoding β-galactosidase from P. canescens. As a result, a strain-producer of endoxylanase XylE was developed. The recombinant enzyme was isolated and purified to homogeneity with specific activity of 50 U/mg. The physicochemical and biochemical properties of the endoxylanase were studied. The maximal enzymatic activity was observed at pH 6.0 and 70°C. Endoxylanase XylE was shown to be a highly thermostable enzyme with half-inactivation period τ1/2 of 7 h at 60°C. The kinetic parameters were 0.52 mg/ml (K m) and 75 μmol/min per mg (V max) using birch xylan as the substrate. Crystals of endoxylonase XylE were obtained, and the 3D structure was solved at 1.47 Å resolution. The 3D structure of an endo-1,4-β-xylanase from the 10th family containing carbohydrate and unique cyclic structure located at the C-terminus of the polypeptide chain was obtained for the first time.

Penicillium canescens strain F178 is a natural producer of β-galactosidase and endo-1,4-β-xylanase. Transcription of bgaS and xylA genes coding these proteins is subject to carbon catabolite repression. A system of direct selection of P. canescens regulatory mutants has been developed. Two mutant strains from the obtained collection have been studied in detail. Both mutations have been shown to be complemented by the creA gene coding global regulator of carbon catabolite repression in filamentous fungi. Also, creA − alleles contain frameshift mutations in the CreA C-domain. It has been found that the xylA gene is derepressed in mutants at the transcription level in the presence of D-glucose. The transcription of the creA gene in mutants is also derepressed proving the effect of autoregulation for this gene.