Constructing a cellulosic yeast host with an efficient cellulase cocktail.

Cellulose is a renewable feedstock for green industry. It is therefore important to develop a technique to construct a host with a high cellulolytic efficiency to digest cellulose. In this study, we developed a convenient host-engineering technique to adjust the expression levels of heterologous genes in the host by promoter rearrangement and gene copy number adjustment. Using genes from different glycoside hydrolase (GH) families including GH2, GH3, GH5, GH6, GH7, and GH12 from Aspergillus niger, Trichoderma reesei, and Neocallimastix patriciarum, we constructed a cellulolytic Kluyveromyces marxianus with eight cellulase gene-cassettes that produced a cellulase cocktail with a high cellulolytic efficiency, leading to a significant reduction in enzyme cost in a rice straw saccharification process. Our technique can be used to design a host that can efficiently convert biomass feedstock to biofuel.

Efficiency enhancement of a new cellulase cocktail at low enzyme loading for high solid digestion of alkali catalyzed atmospheric glycerol organosolvent pre-treated sugarcane bagasse.

The acquisition during biomass saccharification of elevated levels of fermentable sugars with lower cellulase concentration is central to ensuring an economically viable and industrially relevant hydrolytic process. Thus, using a new cellulase preparation (LT4) at low cellulase loading (2 mg protein/g dried substrate), this study assessed the possible boosting effect of integrating accessory enzymes and additives on high-solids hydrolysis of sugarcane bagasse via fed-batch feeding. Hydrolysis which commenced with initial 8% solids loading and subsequent substrate feeding of 4% solids at 6 h, 18 h, and 24 h respectively, proved optimal for the 20% high-solids saccharification producing 158 g/L total sugars and 83% glucose yield after 72 h with the combined optimized additives and accessory enzymes. The results obtained indicate that the integration of accessory enzymes and additives offers a benignant approach to minimizing the enzyme load and cost of high solids saccharification of lignocellulosic heteropolymers while also boosting enzyme hydrolytic performance.

A novel thermostable cellulase cocktail enhances lignocellulosic bioconversion and biorefining in a broad range of pH.

Lignocellulose is the most abundant biomass in nature, and the effective biorefining of them is dependent upon enzymes with high catalytic activity and stability in extreme pH and high temperatures. Due to the molecular constraints for a single enzyme, obtaining a more excellent active pH range can be more easily achievable through the simultaneous activity of two or more enzymes in a cocktail. To address this, we attempted to develop a cocktail of novel thermostable cellulases with high hydrolytic ability and stability. Two cellulases were mined, identified, cloned, and expressed from the camel rumen microbiota. The PersiCel1 demonstrated its maximum relative activity at the pH of 8, and the temperature of 60 °C and the PersiCel2 was optimally active at the pH of 5 and the temperature of 50 °C. Furthermore, utilization of the enzyme cocktail implies the synergistic relationship and significantly increased the saccharification yield of lignocellulosic substrates up to 71.7% for sugar-beet pulp (active pH range of 4-9) and 138.7% for rice-straw (active pH range of 5-8), compared to maximum hydrolysis of Persicel1 or PersiCel2 separately at 55 °C. Our results indicate the probable applicability of PersiCel1, PersiCel2, and their cocktail in numerous industries, specifically biorefineries and lignocellulose bioconversion based technologies.

A myxobacterial LPMO10 has oxidizing cellulose activity for promoting biomass enzymatic saccharification of agricultural crop straws.

Myxobacteria are soil microorganisms with the ability to break down biological macromolecules due to the secretion of a large number of extracellular enzymes, but there has been no research report on myxobacterial lytic polysaccharide monooxygenases (LPMOs). In this study, two LPMO10s, ViLPMO10A and ViLPMO10B, from myxobacterium Vitiosangium sp. GDMCC 1.1324 were characterized. Of which, ViLPMO10B is a C1-oxidizing cellulose-active LPMO. Moreover, ViLPMO10B could decrease the degree of polymerization of crop straws cellulose and synergize with commercial cellulase to promote the saccharification. When the weight ratio of commercial cellulase to ViLPMO10B was 9:1, the conversion efficiency of corn stalk, sugarcane bagasse, and rice straw into reducing sugar was improved by 17%, 16%, and 22%, respectively, compared with commercial cellulase without ViLPMO10B. These results indicate that ViLPMO10B has the potential to be a component of a high-efficient cellulase cocktail and has application value in the saccharification of agricultural residual biomasses.

Efficient and Supplementary Enzyme Cocktail from Actinobacteria and Plant Biomass Induction.

Actinobacteria plays a key role in the cycling of organic matter in soils. They secret biomass-degrading enzymes that allow it to produce the unique metabolites that originate in plant biomass. Although past studies have focused on these unique metabolites, a large-scale screening of Actinobacteria is yet to be reported to focus on their biomass-degrading ability. In the present study, a rapid and simple method is constructed for a large-scale screening, and the novel resources that form the plant biomass-degrading enzyme cocktail are identified from 850 isolates of Actinobacteria. As a result, Nonomuraea fastidiosa secretes a biomass degrading enzyme cocktail with the highest enzyme titer, although cellulase activities are lower than a commercially available enzyme. So the rich accessory enzymes are suggested to contribute to the high enzyme titer for a pretreated bagasse with a synergistic effect. Additionally, an optimized cultivation method of biomass induction caused to produce the improved enzyme cocktail indicated strong enzyme titers and a strong synergistic effect. Therefore, the novel enzyme cocktails are selected via the optimized method for large-scale screening, and then the enzyme cocktail can be improved via the optimized production with biomass-induction.

Degradation of corn stover by fungal cellulase cocktail for production of polyhydroxyalkanoates by moderate halophile Paracoccus sp. LL1.

Bioprocessing of lignocellulose as a renewable resource for fuels, chemicals or value added products is a necessity to fulfil demands of petroleum products. This study aims to convert corn stover to polyhydroxyalkanoates (PHA). Corn stover was hydrolyzed to crude sugars by an on-site prepared cellulase cocktail from co-culture of Trichoderma reesei and Aspergillus niger. The potent PHA producer, Paracoccus sp. LL1, was isolated from Lonar Lake, India and could accumulate PHA up to 72.4% of its dry cell weight. PHA production reached 9.71 g/L from corn stover hydrolysate containing 40 g/L sugar mixture. The PHA synthase gene (phaC) sequence of the isolate showed 79% identity with the phaC gene of Paracoccus seriniphilus (E71) strain from the NCBI database. The nature/type of PHA was found to be poly(3-hydroxybutyrate) by Fourier transform infrared spectroscopy.

Reducing non-productive adsorption of cellulase and enhancing enzymatic hydrolysis of lignocelluloses by noncovalent modification of lignin with lignosulfonate.

Four fractions of one commercial sodium lignosulfonate (SXP) with different molecular weight (MW) and anionic polymers were studied to reduce non-productive adsorption of cellulase on bound lignin in a lignocellulosic substrate. SXP with higher MW had stronger blocking effect on non-productive adsorption of a commercial Trichoderma reesi cellulase cocktail (CTec2) on lignin measured by quartz crystal microgravimetry with dissipation monitoring. Linear anionic aromatic polymers have strong blocking effect, but they would also reduce CTec2 adsorption on cellulose to decrease the enzymatic activity. The copolymer of lignin and polyethylene glycol (AL-PEG1000) has strong enhancement in enzymatic hydrolysis of lignocelluloses, because it not only improves the cellulase activity to cellulose, but also blocks the non-productive cellulase adsorption on lignin. Apart from improving the cellulase activity to cellulose, the enhancements of enzymatic hydrolysis of lignocellulose by adding AL-PEG1000 and SXPs are the result of the decreased cellulase non-productive adsorption on lignin.