Genome Editing in Filamentous Fungus Penicillium Verruculosum Using the CRISPR/Cas9 System

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.

Influence of Mono- and Oligosaccharides on the cbh1 Gene Transcripion in the Penicillium verruculosum Filamentous Fungus

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).

Cloning, Isolation, and Properties of a New Recombinant Tannase from the Aspergillus niger Fungus

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 verruculosum This work was supported by the Ministry of Science and Higher Education of the Russian Federation.

Comparative Study of the Convertibility of Agricultural Residues and Other Cellulose-Containing Materials in Hydrolysis with Penicillium verruculosum Cellulase Complex

Non-edible cellulose-containing biomass is a promising and abundant feedstock for simple sugar production. This study presents the results of different cellulose-containing materials (CCM) hydrolysis experiments with P. verruculosum enzyme complexes in laboratory conditions. Among the non-pretreated substrates, only a few had a relatively high convertibility—soy bean husks (31%) and sugar beat pulp (20%)—while wheat straw, oat husks, sunflower peals, and corn stalks had a low convertibility of 3% to 12%. This indicates that a major part of CCM needs pretreatment. Steam-exploded (with Ca(OH)2) soy bean and oat husks (76% and 58%), fine ball-milled aspen wood and nitric acid-pretreated aspen wood (62% and 78%), and steam-exploded (with sulfuric acid) corn stalks (55%) had a high convertibility. Woody biomass pretreated with pulp and paper mills also had a high convertibility (56–78%)—e.g., never dried kraft hardwood and softwood pulp (both bleached and unbleached). These results demonstrate that effective cellulose-containing material processing into simple sugars is possible. Simple sugars derived from CCM using P. verruculosum preparation are a promising feedstock for the microbiological production of biofuels (bioethanol and biobutanol), aminoacids, and organic acids (e.g., lactic acid for polylactic acid production).