Heterologously Expressed Cellobiose Dehydrogenase Acts as Efficient Electron-Donor of Lytic Polysaccharide Monooxygenase for Cellulose Degradation in Trichoderma reesei.

The conversion of lignocellulosic biomass to second-generation biofuels through enzymes is achieved at a high cost. Filamentous fungi through a combination of oxidative enzymes can easily disintegrate the glycosidic bonds of cellulose. The combination of cellobiose dehydrogenase (CDH) with lytic polysaccharide monooxygenases (LPMOs) enhances cellulose degradation in many folds. CDH increases cellulose deconstruction via coupling the oxidation of cellobiose to the reductive activation of LPMOs by catalyzing the addition of oxygen to C-H bonds of the glycosidic linkages. Fungal LPMOs show different regio-selectivity (C1 or C4) and result in oxidized products through modifications at reducing as well as nonreducing ends of the respective glucan chain. T. reesei LPMOs have shown great potential for oxidative cleavage of cellobiose at C1 and C4 glucan bonds, therefore, the incorporation of heterologous CDH further increases its potential for biofuel production for industrial purposes at a reduced cost. We introduced CDH of Phanerochaete chrysosporium (PcCDH) in Trichoderma reesei (which originally lacked CDH). We purified CDH through affinity chromatography and analyzed its enzymatic activity, electron-donating ability to LPMO, and the synergistic effect of LPMO and CDH on cellulose deconstruction. The optimum temperature of the recombinant PcCDH was found to be 45 °C and the optimum pH of PcCDH was observed as 4.5. PcCDH has high cello-oligosaccharide kcat, Km, and kcat/Km values. The synergistic effect of LPMO and cellulase significantly improved the degradation efficiency of phosphoric acid swollen cellulose (PASC) when CDH was used as the electron donor. We also found that LPMO undergoes auto-oxidative inactivation, and when PcCDH is used an electron donor has the function of a C1-type LPMO electron donor without additional substrate increments. This work provides novel insights into finding stable electron donors for LPMOs and paves the way forward in discovering efficient CDHs for enhanced cellulose degradation.

Use of alternative protein sources for fishmeal replacement in the diet of largemouth bass (Micropterus salmoides). Part II: effects of supplementation with methionine or taurine on growth, feed utilization, and health.

Fishmeal has long been a staple protein feedstuff for fish, but its global shortage and high price have prompted its replacement with alternative sustainable sources. In this experiment involving largemouth bass (a carnivorous fish), a new mixture of feedstuffs (45% poultry byproduct meal, 30% soybean meal, 15% blood meal, and 10% krill shrimp meal) was added to low (14.5%) fishmeal diets along with 0.0%, 0.5% taurine, 0.5% methionine, or 0.5% taurine plus 0.5% methionine (dry matter basis). The positive control diet [65.3% fishmeal (46% crude protein on dry matter basis)] and all low-fishmeal diets contained 40% true protein and 10% lipids. There were 3 tanks per treatment group (20 fish/tank). Fish with the mean initial body weight of 16.6 g were fed to satiety twice daily. Compared with the unsupplemented low-fishmeal group, supplementing either 0.5% methionine or 0.5% methionine plus 0.5% taurine to the low-fishmeal diet improved (P < 0.05) the growth, feed utilization, retention of dietary protein and lipids, and health of largemouth bass, reduced (P < 0.05) the occurrence of black skin syndrome from ~ 40 to ~ 10%. Histological sections of tissues from the fish with black skin syndrome showed retina degeneration, liver damage, and enteritis in the intestine. Compared with methionine supplementation, supplementing 0.5% taurine alone to the low-fishmeal diet did not affect the growth or feed efficiency of fish and had less beneficial effects (P < 0.05) on ameliorating the black skin syndrome. These results indicated that: (a) the basal low-fishmeal diet was inadequate in methionine or taurine; and (b) dietary supplementation with methionine was an effective method to improve the growth performance, feed efficiency, and health of largemouth bass. Further studies are warranted to understand the pathogenesis of the black skin syndrome in largemouth bass.

Use of alternative protein sources for fishmeal replacement in the diet of largemouth bass (Micropterus salmoides). Part I: effects of poultry by-product meal and soybean meal on growth, feed utilization, and health.

Five isonitrogenous and isocaloric diets [containing 54, 30, 15, 10, and 5% fishmeal crude-protein (CP), dry matter (DM) basis] were prepared by replacing fishmeal with poultry by-product meal plus soybean meal to feed juvenile largemouth bass (LMB, with an initial mean body weight of 4.9 g) for 8 weeks. All diets contained 54% CP and 13% lipids. There were four tanks of fish per treatment group (15 fish/tank). The fish were fed twice daily with the same feed intake (g/fish) in all the dietary groups. Results indicated that the inclusion of 15% fishmeal protein in the diet is sufficient for LMB growth. However, some of the fish that were fed diets containing ≤ 15% fishmeal CP had black skin syndrome (characterized by skin darkening and retinal degeneration, as well as intestinal and liver atrophies and structural abnormalities). The concentrations of taurine, methionine, threonine and histidine in serum were reduced (P < 0.05) in fish fed the diets containing 5, 10 and 15% fishmeal CP, compared with the 30 and 54% fishmeal CP diets. Interestingly, the concentrations of tyrosine and tryptophan in serum were higher in fish fed diets with ≤ 15% fishmeal CP than those in the 54% fishmeal CP group. These results indicated that 15% fishmeal CP in the diet containing poultry by-product meal and soybean meal was sufficient for the maximum growth and feed efficiency in LMB but inadequate for their intestinal, skin, eye, and liver health. A reduction in dietary methionine and taurine content and the possible presence of antinutritional factors in the fishmeal replacements diets containing high inclusion levels of soybean meal may contribute to black skin syndrome in LMB. We recommend that the diets of juvenile LMB contain 30% fishmeal CP (DM basis).

Effects of dietary starch and lipid levels on the protein retention and growth of largemouth bass (Micropterus salmoides).

Protein accretion in some fish species is affected by dietary lipids, starch and their interactions, but this aspect of nutrition is largely unknown in largemouth bass (LMB). Therefore, we designed six experimental diets with three starch levels (5%, 10%, and 15%; dry matter  basis) and two lipid levels (10% and 12.5%; dry matter basis) to evaluate the effects of dietary starch and lipid levels on the protein retention, growth, feed utilization, and liver histology of LMB. There were three tanks (18 fish per tank, ~ 4.85 g per fish) per dietary treatment group and the trial lasted for 8 weeks. Fish were fed to apparent satiation twice daily. Results indicated that increasing the dietary starch level from 5 to 15% reduced (P < 0.05) absolute feed intake (AFI; - 9.0%, - 15% and - 14% on days 14-28, 28-42, and 42-56, respectively) and weight gains (- 4.4% and - 6.5% on days 42 and 56, respectively) of LMB. Increasing the dietary lipid level from 10 to 12.5% reduced (P < 0.05) AFI (- 9.7%, - 11.7% and - 11.9% on days 14-28, 28-42; and 42-56, respectively), weight gains (- 4.2%, - 5.9% and - 6.9% on days 28, 42 and 56, respectively), and survival rate (by a 5.6% unit) of LMB. The retention of dietary protein and some amino acids in the body was affected by dietary starch or lipid levels and their interactions. The viscerosomatic index (VSI), hepatosomatic index (HSI), and intraperitoneal fat ratio (IPFR) increased with increasing the dietary starch level from 5 to 15%. Compared with 10% lipids, 12.5% lipids in diets increased IPFR but had no effect on VSI or HSI. The concentrations of glucose in serum increased with increasing the dietary starch level from 5 to 15% at 4 to 24 h after feeding, with the effect of dietary lipids being time-dependent. Compared with a 5%-starch diet, fish fed a diet with 10%- or 15%-starch exhibited an enlarged and pale liver with excessive glycogen. Based on these findings, we recommend dietary lipid and starch levels to be 10% and < 10%, respectively, for juvenile LMB to maximize the retention of dietary protein in their bodies.

Effects of dietary protein and lipid levels on the growth performance, feed utilization, and liver histology of largemouth bass (Micropterus salmoides).

The reported requirements of largemouth bass (LMB, which is native to North America) for dietary protein and lipids varied substantially among previous studies, and this fish fed current formulated diets exhibit poor growth performance and pale liver syndrome. Because amino acids and lipids are known to affect hepatic metabolism and function in mammals, it is imperative to understand the impacts of these dietary macronutrients on the growth and liver morphology of LMB. In this study, we designed six isocaloric diets to determine the effects of different dietary crude protein (CP; 40%, 45%, and 50%; dry matter basis) and lipid levels (7.5% and 10%; dry matter basis) on fat and glycogen deposits, as well as hepatosis in LMB. There were four tanks (12 fish per tank, an average initial weight of 18.4 g/fish) per dietary treatment group and the trial lasted for 8 weeks. Fish were fed to apparent satiation three times daily. Results indicated that LMB fed the 45% or 50% CP diet grew faster (P < 0.05), had less (P < 0.05) glycogen in the liver and smaller (P < 0.05) hepatocyte sizes than fish fed the 40% CP diet, but there was no difference in weight gain or feed efficiency between the 45% and 50% CP diets. The hepatic lipid content did not differ between LMB fed the 40% and 45% CP diets, and the values for these two groups were 29% lower (P < 0.05) than those for LMB fed the 50% CP diet. Compared with the 40% CP group, LMB fed the 45% or 50% CP diet had 8-12% lower content of total minerals, phosphorus, and calcium in the body. Increasing the dietary lipid level from 7.5 to 10% enhanced the weight gains (+ 15%) and feed efficiency (+ 22%), as well as the retention of dietary protein (+ 18%), energy (+ 25%), and phosphorus (+ 7.6%) in the body. No fatty liver occurred in any group of LMB (with hepatic lipid concentrations being < 2%, wet weight basis). Based on these growth, metabolic and histologic data, we recommend dietary CP and lipids levels to be 45% and 10%, respectively, for juvenile LMB.

Genomic characterization and expression profiling of the lytic polysaccharide monooxygenases AA9 family in thermophilic fungi Thermothelomyces fergusii in response to carbon source media.

Enhancing enzyme activity and stability in biomass degradation can improve substrate saccharification and, increases biorefinery efficiency. For the first time, we identified 20 lytic polysaccharide monooxygenases (LPMOs) AA9 genes in the genome of Thermothelomyces fergusii. Our results showed that TfAA9 was categorized into LPMOs1, LPMOs2, and LPMOs3 subgroups based on protein diversity. Protein- 3D structure analysis showed strong interactions between Myceliophthora thermophila AA9 proteins and 17 TfAA9 proteins. Gene ontology analysis indicated a high enrichment of cellulase activity in TfAA9 genes. KEGG pathways analysis revealed the role of TfAA9 proteins in the endohydrolysis of 1,4-beta-D-glucosidic linkages in cellulose. Numerous TfAA9s gene transcripts were up-regulated on avicel, cellobiose, and glucose, with a higher proportion on avicel. Protein concentration, endoglucanase, and cellulase activity were also boosted on avicel. However, limited fungal biomass was observed on avicel, despite the abundance of AA9 LPMOs in the T. fergusii genome. These findings expand our understanding of fungal AA9 genes and their role in lignocellulolytic degradation. The disparity between biomass and enzymatic activity suggests screening TfAA9 genes for highly active enzymes and redundant genes via heterologous expression. In short, functional characterization of these genes could contribute to improving the saccharification process of industrial raw materials.

Promoter regulation and genetic engineering strategies for enhanced cellulase expression in Trichoderma reesei.

BACKGROUND: Trichoderma reesei has extraordinary potential for high-level protein production at large scales, and it need to be further explored through genetic engineering tools to obtain a thorough understanding of its cellular physiology. Understanding the genetic factors involved in the intrinsic regulatory network is crucial; without this information, there would be restrictions in expressing genes of interest. Past and present studies are concentrated on the application and expansion of novel expression systems using synthetic biology concepts. These approaches involve either using previously established promoters that are strong or genetically engineered promoters. Genomic and transcriptomic methods have also been employed to isolate strong promoters and expression systems such as light-inducible expression systems, copper-inducible expression systems, L-methionine inducible promoters, and Tet-On expression system etc. AIMS OF REVIEW: In this review, we will highlight various research endeavors related to tunable and constitutive promoters; the role of different promoters in homologous and heterologous protein expression; the identification of innovative promoters, and strategies that may be beneficial for future research aimed at improving and enhancing protein expression in T. reesei. KEY SCIENTIFIC CONCEPTS OF THE REVIEW: The characterization of new promoters and implementation of novel expression systems that will result in a significant extension of the molecular toolbox that is accessible for the genetic engineering of innovative strains of T. reesei. Genetically engineered strong inducible promoters such as Pcbh1 through replacement of transcriptional repressors (cre1, ace1) with transcriptional activators (xyr1, ace2, ace3, hap2/3/5) and synthetic expression systems can result in elevated production of endoglucanases (EGLs), ß-glucosidases (BGLs), and cellobiohydrolases (CBHs). Strong constitutive promoters such as Pcdna1 can be converted into genetically engineered synthetic hybrid promoters by integrating the activation region of strong inducible promoters, which can allow the induction and expression of cellulases even on repressing media. More efforts are necessary to identify innovative promoters and novel expression strategies for the enhanced expression of desirable proteins at industrial scales.