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.

Disruption of the Snf1 Gene Enhances Cell Growth and Reduces the Metabolic Burden in Cellulase-Expressing and Lipid-Accumulating Yarrowia lipolytica

Yarrowia lipolytica is known to be capable of metabolizing glucose and accumulating lipids intracellularly; however, it lacks the cellulolytic enzymes needed to break down cellulosic biomass directly. To develop Y. lipolytica as a consolidated bioprocessing (CBP) microorganism, we previously expressed the heterologous CBH I, CBH II, and EG II cellulase enzymes both individually and collectively in this microorganism. We concluded that the coexpression of these cellulases resulted in a metabolic drain on the host cells leading to reduced cell growth and lipid accumulation. The current study aims to build a new cellulase coexpressing platform to overcome these hinderances by (1) knocking out the sucrose non-fermenting 1 (Snf1) gene that represses the energetically expensive lipid and protein biosynthesis processes, and (2) knocking in the cellulase cassette fused with the recyclable selection marker URA3 gene in the background of a lipid-accumulating Y. lipolytica strain overexpressing ATP citrate lyase (ACL) and diacylglycerol acyltransferase 1 (DGA1) genes. We have achieved a homologous recombination insertion rate of 58% for integrating the cellulases-URA3 construct at the disrupted Snf1 site in the genome of host cells. Importantly, we observed that the disruption of the Snf1 gene promoted cell growth and lipid accumulation and lowered the cellular saturated fatty acid level and the saturated to unsaturated fatty acid ratio significantly in the transformant YL163t that coexpresses cellulases. The result suggests a lower endoplasmic reticulum stress in YL163t, in comparison with its parent strain Po1g ACL-DGA1. Furthermore, transformant YL163t increased in vitro cellulolytic activity by 30%, whereas the “total in vivo newly formed FAME (fatty acid methyl esters)” increased by 16% in comparison with a random integrative cellulase-expressing Y. lipolytica mutant in the same YNB-Avicel medium. The Snf1 disruption platform demonstrated in this study provides a potent tool for the further development of Y. lipolytica as a robust host for the expression of cellulases and other commercially important proteins.

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.

Enzymatic depolymerization of wheat straw polysaccharides

The purpose of this study is to develop a technology for enzymatic processing for depolymerization of polysaccharides in wheat straw to obtain the maximum yield of glucose and sorbitol. Cellulolytic enzymes endo-1,4-β-glucanase (EC 3.2.1.4) and cellobiose (1,3-β-glucosidase) (CF 3.2.1.21) were isolated and studied in local strains Tr. viride 121, which are grown under deep cultivation conditions. A technology has been developed for obtaining a complex preparation “Cellozyme G20x” with a high yield and specific activity of cellulase, xylanase, β-glucanase and pectinase, and a scheme for purification from cellulases by precipitation, ultrafiltration, and freeze drying is not inferior in efficiency to commercial preparations. The physicochemical properties of the preparation “Cellozyme G20x” have been studied, the optimal parameters of the action and stability of the enzyme preparation have been established. The efficiency of Cellozyme G20x for hydrolysis of straw polysaccharides was 35-40% in terms of glucose yield.

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.

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.