Development of a powerful synthetic hybrid promoter to improve the cellulase system of Trichoderma reesei for efficient saccharification of corncob residues

Background The filamentous fungus Trichoderma reesei is a widely used workhorse for cellulase production in industry due to its prominent secretion capacity of extracellular cellulolytic enzymes. However, some key components are not always sufficient in this cellulase cocktail, making the conversion of cellulose-based biomass costly on the industrial scale. Development of strong and efficient promoters would enable cellulase cocktail to be optimized for bioconversion of biomass. Results In this study, a synthetic hybrid promoter was constructed and applied to optimize the cellulolytic system of T. reesei for efficient saccharification towards corncob residues. Firstly, a series of 5’ truncated promoters in different lengths were established based on the strong constitutive promoter Pcdna1. The strongest promoter amongst them was Pcdna1-3 (− 640 to − 1 bp upstream of the translation initiation codon ATG), exhibiting a 1.4-fold higher activity than that of the native cdna1 promoter. Meanwhile, the activation region (− 821 to − 622 bp upstream of the translation initiation codon ATG and devoid of the Cre1-binding sites) of the strong inducible promoter Pcbh1 was cloned and identified to be an amplifier in initiating gene expression. Finally, this activation region was fused to the strongest promoter Pcdna1-3, generating the novel synthetic hybrid promoter Pcc. This engineered promoter Pcc drove strong gene expression by displaying 1.6- and 1.8-fold stronger fluorescence intensity than Pcbh1 and Pcdna1 under the inducible condition using egfp as the reporter gene, respectively. Furthermore, Pcc was applied to overexpress the Aspergillus niger β-glucosidase BGLA coding gene bglA and the native endoglucanase EG2 coding gene eg2, achieving 43.5-fold BGL activity and 1.2-fold EG activity increase, respectively. Ultimately, to overcome the defects of the native cellulase system in T. reesei, the bglA and eg2 were co-overexpressed under the control of Pcc promoter. The bglA-eg2 double expression strain QPEB70 exhibited a 178% increase in total cellulase activity, whose cellulase system displayed 2.3- and 2.4-fold higher saccharification efficiency towards acid-pretreated and delignified corncob residues than the parental strain, respectively. Conclusions The synthetic hybrid promoter Pcc was generated and employed to improve the cellulase system of T. reesei by expressing specific components. Therefore, construction of synthetic hybrid promoters would allow particular cellulase genes to be expressed at desired levels, which is a viable strategy to optimize the cellulolytic enzyme system for efficient biomass bioconversion.

Preparation of nanocellulose by hydrolysis with ionic liquids and two-step hydrolysis with ionic liquids and enzymes

The aim of this study was to compare parameters of nanocellulose obtained by two different procedures: hydrolysis with ionic liquids (1-allyl-3-methylimidazolium chloride and 1-ethyl-3-methylimidazolium acetate) and hydrolysis with ionic liquids in combination with hydrolysis using a cellulolytic enzyme from Trichoderma reesei. Avicel cellulose was treated with two ionic liquids: 1-allyl-3-methylimidazolium chloride (AmimCl) and 1-ethyl 3-methylimidazolium acetate (EmimOAc). In the two-step hydrolysis cellulose after treatment with ionic liquids was additionally hydrolyzed with a solution of enzymes. In order to characterize the obtained material, the following analyses were used: infrared spectroscopy, X-ray diffraction and dynamic light scattering. The results indicated that cellulose obtained by two-step nanocellulose production methods (first hydrolysis with ionic liquids and then with enzymes) showed similar parameters (particle size, XRD patterns and degree of crystallinity) as the material after the one-step process, i.e. hydrolysis with ionic liquids.

Cellulose-containing waste recycling using fungi

Accumulation of plant waste is a serious environmental problem. Mushrooms with high cellulolytic activity can process it into valuable products that will be useful in solving various industries and agriculture problems. The enzymes of the cellulolytic complex include 1,4-β-D-glucan-4-glucanohydrolase, exo-1,4-β-glucosidase, cellobiohydrolase, β-glucosidase. 1,4-β-D-glucan-4-glucanohydrolases destroy β-1,4-glycosidic bonds within the chain of cellulose and lichenin polysaccharides. Exoglucanases destroy β-1,3- and β-1,4-glycosidic bonds at the end of the molecule. Cellobiohydrolases cleave β-1,4-glycosidic bonds to form cellobiose and glucose. β-glucosidase complete the process of destruction. Fungi with high cellulolytic activity include both representatives of the Ascomycota and Basidiomycota divisions. Ascomycete Chaetomium globosum produces endoglucanases of two families and 8 cellobiohydrolases. Myceliophthora thermophila also produces endoglucanases and cellobiohydrolases, the most abundant of which is Mt Cel7A. The fungus is a promising producer of thermostable enzymes. Trichoderma reesei has a long history of safe use as a source of highly active cellulolytic enzymes and other valuable metabolites. LPMOs of the cellulolytic fungus Thielavia terrestris are considered auxiliary enzymes, but can negatively affect the main enzymes of the complex. Irpex lacteus also produces LPMO and a complete cellulolytic enzyme complex. The cellulolytic activity of fungi and their ability to grow on cheap substrates can be used to bioconvert plant waste into valuable products. One of the ways to utilize them is to convert into compound feed with a high protein content through the use of starter cultures. The use of mushrooms will increase the content of protein and simple carbohydrates, enrich the feed with fats. Another method is to obtain cellulases, which are widely used in many industries. Thanks to the production of biodiesel and bioethanol from cellulose-containing raw materials it is possible to solve the problem of lack of fuel by replacing energy carriers from non-renewable energy sources with their environmentally friendly counterparts. They are less toxic than diesel and gasoline and are also made from renewable resources.

Defining the Frontiers of Synergism between Cellulolytic Enzymes for Improved Hydrolysis of Lignocellulosic Feedstocks

Lignocellulose has economic potential as a bio-resource for the production of value-added products (VAPs) and biofuels. The commercialization of biofuels and VAPs requires efficient enzyme cocktail activities that can lower their costs. However, the basis of the synergism between enzymes that compose cellulolytic enzyme cocktails for depolymerizing lignocellulose is not understood. This review aims to address the degree of synergism (DS) thresholds between the cellulolytic enzymes and how this can be used in the formulation of effective cellulolytic enzyme cocktails. DS is a powerful tool that distinguishes between enzymes’ synergism and anti-synergism during the hydrolysis of biomass. It has been established that cellulases, or cellulases and lytic polysaccharide monooxygenases (LPMOs), always synergize during cellulose hydrolysis. However, recent evidence suggests that this is not always the case, as synergism depends on the specific mechanism of action of each enzyme in the combination. Additionally, expansins, nonenzymatic proteins responsible for loosening cell wall fibers, seem to also synergize with cellulases during biomass depolymerization. This review highlighted the following four key factors linked to DS: (1) a DS threshold at which the enzymes synergize and produce a higher product yield than their theoretical sum, (2) a DS threshold at which the enzymes display synergism, but not a higher product yield, (3) a DS threshold at which enzymes do not synergize, and (4) a DS threshold that displays anti-synergy. This review deconvolutes the DS concept for cellulolytic enzymes, to postulate an experimental design approach for achieving higher synergism and cellulose conversion yields.

Isolation and characterization of soil actinobacteria as cellulolytic enzyme producer from Aceh Besar, Indonesia

Fitri L, Bessania MA, Septi N, Suhartono S. 2021. Isolation and characterization of soil actinobacteria as cellulolytic enzyme producer from Aceh Besar, Indonesia. Biodiversitas 22: 5169-5180. Cellulolytic actinobacteria are cellulase-producing bacteria capable of degrading cellulose. This study aimed to isolate, characterize, evaluate the cellulolytic ability, and to determine physiological characterization of soil cellulolytic actinobacteria isolated from the Ujung Pancu area, Aceh Besar. Isolation of actinobacteria from soil samples was performed using serial dilution method on Yeast Malt Agar (YMA) medium. Morphological characterization was carried out by growing isolates on YMA, Oatmeal Agar (OA), and Yeast Starch Agar (YSA) media. Cellulolytic ability was determined by calculating the cellulolytic index (IS) on 1% carboxymethyl cellulose (CMC) medium after adding 0.1% congo red solution. Physiological characterization of cellulolytic actinobacteria tested in this study was salinity, pH, and carbon source in liquid Yeast Malt (liquid YM), and the growth was measured at a wavelength of 581nm. The results showed that a total of nine isolates of actinobacteria were isolated, which belonged to the genus Streptomyces. Cellulolytic test results showed that eight isolates had the ability to degrade cellulose. Isolates AUP-04, AUP-03, and AUP-01 had the highest cellulolytic index value. Physiological characterization results revealed that three isolates had different tolerances for salinity levels, pH, and types of carbon sources. AUP-03 isolate grew well at 10% salinity with an OD value of 0.88, isolate AUP-01 grew at 5% salinity with an OD value of 0.49, whereas isolate AUP-04 grew well on media that did not contain salinity. All three isolates grew well at pH 6 with OD values of 0.93, 1.12, and 1.27. AUP-03 and AUP-01 isolates grew well on media containing dextrose as carbon source with OD values of 0.154 and 0.17, respectively, while isolate AUP-04 grew well on glucose-containing media with an OD value of 0.22.

Evaluating the effects of cellulolytic enzymes and Lactobacillus bulgaricus on mycotoxins production and the quality of maize silage

Fungal spoilage and mycotoxin contamination are two of the greatest hazards of silage. The present work was carried out to evaluate the impact of Lactobacillus bulgaricus and cellulolytic enzymes on the maize silage (MS) quality. Fungal analysis of different MS samples showed different mycotoxigenic fungi. The highest frequency (62.8%) was associated with Fusarium spp. Four species with different relative densities were found: F. graminearum (71.1%), F. culmorum (15.2%), F. proliferatum (11.2%), and F. oxysporum (2.50 %). High-performance liquid chromatography analysis showed the presence of trichothecene, nivalenol, zearalenone, and fumonisins mycotoxins in MS inoculated by F. graminearum. The inhibition % of trichothecene, nivalenol, and zearalenone synthesis was 50.2%, 47.5%, and 23.5%, respectively, in MS inoculated by Lactobacillus bulgaricus after a 30 d incubation period. Trichoderma harzianum succeeded in producing cellulolytic enzymes, i.e., carboxymethyl cellulase, manganase peroxidase, and laccase, with a maximum production of 350 µg/mL, 5.47 µg/mL, and 16.0 µg/mL, respectively, after 21 d using MS as the substrate. Treatment by the extracted cellulolytic enzyme with L. bulgaricus enhanced unfavorable conditions for MS fungal contamination, i.e., the production of lactic acid, a lowered pH, and increased L. bulgaricus colony-forming units, compared to the addition of enzyme extract or L. bulgaricus alone.

EFFECT OF NUTRIENTS ON CELLULOLYTIC AND PECTOLYTIC ENZYME PRODUCTION OF ALTERNARIA ALTERNATA A LEAF SPOT PATHOGEN OF TEAK (TECTONA GRANDIS L.F.)

Alternaria alternata is a potential pathogen of Tectona grandis L.f., was isolated from diseased Tectona grandis L.f. leaves from Nashik and used for the present study. Pathogen was grown on the Czapek-Dox liquid medium substituting or adding different carbon, nitrogen to study cellulolytic and pectolytic enzyme production and total phenol production. The activity of enzyme was observed on the 8th day of incubation period. A great extent of growth variation was observed on different carbon, nitrogen. Among the carbon source) the maximum loss in percentage viscosity maximum in lactose and fructose. While minimum in glucose and dextrose as compared to other nitrogen source. From nitrogen source the cellulolytic enzyme activity was maximum in control and cobalt nitrate followed by similar activity in potassium nitrate and nickel nitrate. While minimum cellulolytic enzyme activity was seen in barium nitrate.. Variation was also observe in pectolytic enzyme activity. the cellulolytic enzyme activity was maximum in glucose while minimum in dextrose as compared to lactose, control and fructose.. From nitrogen source the cellulose activity was maximum in potasium nitrate and minimum in cobalt nitrate as compared to nickel nitrate, barium nitrate and control.