**2.3 Methods for increasing the efficiency of bioconversion of plant materials under the action of an enzyme complex produced by** *P. verruculosum*

An auxotrophic mutant B1-537 (ΔniaD) was obtained for genetic engineering manipulations based on the highly productive strain *P. verruculosum* B151. It has a defective nitrate reductase gene, which makes it possible to use it as a basis for obtaining recombinant producers of homologous and heterologous proteins by selecting transformants on the medium with nitrate [55–57, 64].

The expression of the gene of the target enzyme can be carried out under the control of various "promoter-terminator" systems selected by the researcher. This may be a proprietary system inherent in the target protein gene, or a promoter and terminator of another gene. To express various genes in the strain B1-537 (ΔniaD), a promoter and a transcription terminator of the inducible CBH I gene *P. verruculosum* (*cbh1*) [55–57, 65, 66], which is a major component of the cellulase complex of the fungus *P. verruculosum*, were used. The CBH I expression system makes it possible to obtain enzyme preparations with a high content of the target protein. For example, on the basis of strain B1-537 (ΔniaD) using the promoter of the c*bh1* gene, a recombinant strain F10 was created—a superproducer of highly active BGL *A. niger*. The enzyme preparations based on this strain contain up to 80% of the heterologous enzyme from the total secreted protein [56, 62, 63]. The strain F10 contained a large number of copies of the aspergillus BGL gene under the *cbh 1* promoter, whereas the expression of *P. verruculosum*'s own enzymes (and primarily CBH I) was largely suppressed. However, in many other cases, the use of the *cbh1* gene promoter made it possible to obtain recombinant *P. verruculosum* strains capable of secreting a largely preserved cellulase complex of this fungus together with homologously or heterologously expressed additional enzymes of various specificity to enhance the action of cellulases during hydrolysis of various types of cellulose-containing raw materials (or for use in other biotechnological processes), examples are given in **Table 4**.

One of the approaches to obtain recombinant strains is so-called "fusion construct", consisting, for example, of sequentially connected genes encoding CBH I, EG II *P. verruculosum*, and BGL *A. niger*, expressed under the promoter and terminator cbh1 [65]. The use of such a construct made it possible to obtain an enzyme preparation enriched with the three most important components for cellulose hydrolysis (**Table 4**). The use of this preparation together with the initial preparation P*. verruculosum* B151 led to an increase in the yield of sugars during the hydrolysis of pretreated pine and aspen wood by up to 70% [65].

Another example, as described in the previous chapter, is the composite preparation of *P. verruculosum*, which was obtained on the basis of strains B151 and F10 and which, during hydrolysis of various types of lignocellulose raw materials, provided a sugar yield comparable to that demonstrated by modern commercial enzyme preparations based on a new generation of mutant strains of *T. reesei* (Accelerase 1500 *Cellulases from Mycelial fungi* Penicillium verruculosum *as a Real Alternative to* Trichoderma*… DOI: http://dx.doi.org/10.5772/intechopen.111851*


*\*The content of enzymes in % of the total protein content in the preparation obtained using the corresponding recombinant strain of P. verruculosum is given. In parentheses the content of the expressed enzymes in the control preparation P. verruculosum B151 is indicated.*

#### **Table 4.**

*Using the promoter and terminator of the cbh1 gene to produce recombinant strains and enzyme preparations of Piptochaetium verruculosum with homologously or heterologously expressed enzymes.*

and DUET, Cellic CTec1 and CTec2) [62]. In most cases, the composite preparation of *P. verruculosum* was superior to the indicated preparations of *T. reesei* in terms of effectiveness.

To obtain the strains with a moderate level of expression of target recombinant enzymes alternative expression systems can be used. This type includes, for example, a constitutive expression system based on the histone gene promoter hist4.1 [64]. Its use makes it possible to increase the production of the target protein without significantly changing the composition of the main secreted enzyme complex. Using an expression system based on a histone promoter, the B1\_PrHist enzyme preparation was obtained with heterologous *A. niger* BGL, the content of which was about 13% of the total secreted protein. This preparation provided a higher yield of glucose (by 10–21%) during the hydrolysis of MCC and crushed aspen wood compared to the control preparation *P. verruculosum* B151. Together with the heterologous *A. niger* BGL under the control of a histone promoter, the expression of *P. verruculosum*'s own CBH I was increased, as a result, the enzyme preparation obtained on the basis of the recombinant strain demonstrated a 29–100% gain in sugar yield compared to the control preparation [57].

Thus, both of the genetic engineering approaches discussed above, in which the target protein is expressed either under the control of a strong inducible (*cbh1*) promoter or under the control of a weaker constitutive (*hist4.1*) promoter, can significantly increase the hydrolytic ability of secreted cellulase complexes. The choice of one or another approach largely depends on the specific application of the final

enzyme preparation in a particular biotechnological process. In the case of enzymatic hydrolysis of cellulose, it depends on the type of plant material and the method of its pretreatment.

Cellulases are an example of enzymes that are extremely demanded in practice and are used in Europe, the USA, and a number of other countries to implement the ideas of bioeconomics, implying the replacement of non-renewable fossil raw materials and chemical processes of its processing with alternative biotechnological processes with integrated use of renewable raw materials and various wastes. In particular, with the use of cellulases in a number of countries (USA, Italy, Brazil), the concept of biofactory (biorefinery) has been practically implemented, consisting in the realization of large-scale processes of enzymatic conversion of renewable lignocellulose plant biomass (agricultural waste) into sugars, followed by the production of alcohols (second-generation biofuels) from them [67]. Other biotechnological processes based on the use of lignocellulose raw materials have been tested and are close to practical implementation in order to obtain solvents, organic and amino acids, biologically active substances, monomers for the synthesis of various polymers, etc. In addition, cellulases are traditionally used in the textile industry, pulp and paper industry, and various branches of the food industry, such as brewing, alcohol, and bakery, as well as feed additives in poultry and animal husbandry [68].
