Improvement of the Stability and Activity of an LPMO Through Rational Disulfide Bonds Design

Lytic polysaccharide monooxygenases (LPMOs) oxidatively break down the glycosidic bonds of crystalline polysaccharides, significantly improving the saccharification efficiency of recalcitrant biomass, and have broad application prospects in industry. To meet the needs of industrial applications, enzyme engineering is needed to improve the catalytic performance of LPMOs such as enzyme activity and stability. In this study, we engineered the chitin-active CjLPMO10A from Cellvibrio japonicus through a rational disulfide bonds design. Compared with the wild-type, the variant M1 (N78C/H116C) exhibited a 3-fold increase in half-life at 60°C, a 3.5°C higher T5015, and a 7°C rise in the apparent Tm. Furthermore, the resistance of M1 to chemical denaturation was significantly improved. Most importantly, the introduction of the disulfide bond improved the thermal and chemical stability of the enzyme without causing damage to catalytic activity, and M1 showed 1.5 times the specific activity of the wild-type. Our study shows that the stability and activity of LPMOs could be improved simultaneously by selecting suitable engineering sites reasonably, thereby improving the industrial adaptability of the enzymes, which is of great significance for applications.

Microwave Assisted Pretreatment of Szarvasi (Agropyron elongatum) Biomass to Enhance Enzymatic Saccharification and Direct Glucose Production

Biomass from perennial plants can be considered a carbon-neutral renewable resource. The tall wheatgrass hybrid Szarvasi-1 (Agropyron elongatum, hereafter referred to as “Szarvasi”) belongs to the perennial Poaceae representing a species, which can grow on marginal soils and produce large amounts of biomass. Several conventional and advanced pretreatment methods have been developed to enhance the saccharification efficiency of plant biomass. Advanced pretreatment methods, such as microwave-assisted pretreatment methods are faster and use less energy compared to conventional pretreatment methods. In this study, we investigated the potential of Szarvasi biomass as a biorefinery feedstock. For this purpose, the lignocellulosic structure of Szarvasi biomass was investigated in detail. In addition, microwave-assisted pretreatments were applied to Szarvasi biomass using different reagents including weak acids and alkali. The produced pulp, hydrolysates, and extracted lignin were quantitatively characterized. In particular, the alkali pretreatment significantly enhanced the saccharification efficiency of the pulp 16-fold compared to untreated biomass of Szarvasi. The acid pretreatment directly converted 25% of the cellulose into glucose without the need of enzymatic digestion. In addition, based on lignin compositional and lignin linkage analysis a lignin chemical model structure present in Szarvasi biomass could be established.

Inducer-Free Cellulase Production System Based on the Constitutive Expression of Mutated Xyr1 and Ace3 in the Industrial Fungus Trichoderma Reesei

Background: Trichoderma reesei (Hypocrea jecorina) is a filamentous fungus that can produce extremely high levels of protein; consequently, it is utilized as a host for the production of cellulase and hemicellulase cocktails for lignocellulosic biomass degradation. Several hyper-producer strains of T. reesei have been bred for use in industrial production, but they generally require inducers to achieve high production capacities. The most commonly used inducers are soluble sugars produced by the degradation of cellulose; however, the dependence on cellulose degradation is problematic because cellulose is insoluble and has poor handling properties as a carbon source. Furthermore, once cellulose is decomposed, little cellulase is produced, making it difficult to produce the enzyme continuously and efficiently. The aim of this study was to establish a simple, inducer-free, cellulase production system using glucose as the sole carbon source.Results: Here, we focused on transcription factors that regulate both cellulase and hemicellulase genes. First, we verified that the previously reported Xylanase regulator 1 (Xyr1) mutation had a glucose-blind phenotype in T. reesei, and confirmed that constitutive expression of the V821F mutation in Xyr1 produced high levels of proteins, especially hemicellulase and cellulase, even in inducer-free conditions. However, the majority of proteins were hemicellulases. To reproduce cellulase/hemicellulase production similar to those observed under induced conditions, an activator of cellulase expression 3 (Ace3) was expressed in Xyr1V821F expressed strain additionally. As a result, the T. reesei strain constitutively expressing Xyr1V821F and Ace3 exhibited a 1.5-fold increase than Xyr1V821F expressed only in protein productivity under inducer-free conditions. Notably, the enzyme composition significantly improved for cellulases ratio and similar to that induced by cellulose. Furthermore, the enzymes exhibited a high saccharification efficiency when compared to that of produced by the strain expressing only the mutated Xyr1.Conclusions: This work shows that the constitutive expression of mutated Xyr1 and Ace3 can increase cellulase and hemicellulase production in T. reesei without inducers. This inducer-free enzyme production method could provide an effective system to reduce costs and simplify production processes, and is expected to be applied in the production of various proteins.

Genetic Modification of KNAT7 Transcription Factor Expression Enhances Saccharification and Reduces Recalcitrance of Woody Biomass in Poplars

The precise role of KNAT7 transcription factors (TFs) in regulating secondary cell wall (SCW) biosynthesis in poplars has remained unknown, while our understanding of KNAT7 functions in other plants is continuously evolving. To study the impact of genetic modifications of homologous and heterologous KNAT7 gene expression on SCW formation in transgenic poplars, we prepared poplar KNAT7 (PtKNAT7) overexpression (PtKNAT7-OE) and antisense suppression (PtKNAT7-AS) vector constructs for the generation of transgenic poplar lines via Agrobacterium-mediated transformation. Since the overexpression of homologous genes can sometimes result in co-suppression, we also overexpressed Arabidopsis KNAT7 (AtKNAT7-OE) in transgenic poplars. In all these constructs, the expression of KNAT7 transgenes was driven by developing xylem (DX)-specific promoter, DX15. Compared to wild-type (WT) controls, many SCW biosynthesis genes downstream of KNAT7 were highly expressed in poplar PtKNAT7-OE and AtKNAT7-OE lines. Yet, no significant increase in lignin content of woody biomass of these transgenic lines was observed. PtKNAT7-AS lines, however, showed reduced expression of many SCW biosynthesis genes downstream of KNAT7 accompanied by a reduction in lignin content of wood compared to WT controls. Syringyl to Guaiacyl lignin (S/G) ratios were significantly increased in all three KNAT7 knockdown and overexpression transgenic lines than WT controls. These transgenic lines were essentially indistinguishable from WT controls in terms of their growth phenotype. Saccharification efficiency of woody biomass was significantly increased in all transgenic lines than WT controls. Overall, our results demonstrated that developing xylem-specific alteration of KNAT7 expression affects the expression of SCW biosynthesis genes, impacting at least the lignification process and improving saccharification efficiency, hence providing one of the powerful tools for improving bioethanol production from woody biomass of bioenergy crops and trees.

Physical–chemical properties of cell wall interface significantly correlated to the complex recalcitrance of corn straw

Background Tissue heterogeneity significantly influences the overall saccharification efficiency of plant biomass. However, the mechanisms of specific organ or tissue recalcitrance to enzymatic deconstruction are generally complicated and unclear. A multidimensional analysis of the anatomical fraction from 12 corn cultivars was conducted to understand the essence of recalcitrance. Results The results showed that leaf, leaf sheath, stem pith and stem rind of corn straw exhibited remarkable heterogeneity in chemical composition, physical structure and cell type, which resulted in the different saccharification ratio of cellulose. The high saccharification ratio ranging from 21.47 to 38.96% was in stem pith, whereas the low saccharification ratio ranging from 17.1 to 27.43% was in leaf sheath. High values of lignin, hemicelluloses, degree of polymerization and crystallinity index were critical for the increased recalcitrance, while high value of neutral detergent soluble and pore size generated weak recalcitrance. Interestingly, pore traits of cell wall, especial for microcosmic interface structure, seemed to be a crucial factor that correlated to cellulase adsorption and further affected saccharification. Conclusions Highly heterogeneity in cell wall traits influenced the overall saccharification efficiency of biomass. Furthermore, the holistic outlook of cell wall interface was indispensable to understand the recalcitrance and promote the biomass conversion. Graphic abstract

Arabidopsis GELP7 is plasma membrane-localized acetyl xylan esterase, and its overexpression improves saccharification efficiency.

Acetyl substitution on the xylan chain is critical for stable interaction with cellulose and other cell wall polymers in the secondary cell wall. Xylan acetylation pattern is governed by Golgi and extracellular localized acetyl xylan esterase (AXE). We investigated the role of Arabidopsis clade Id from the GDSL esterase/lipase or GELP family in polysaccharide deacetylation. The investigation of the AtGELP7 T-DNA mutant line showed a decrease in stem esterase activity and an increase in stem acetyl content. We further generated overexpressor AtGELP7 transgenic lines, and these lines showed a decrease in xylan acetylation in comparison with wild type plants. Therefore, we have named this enzyme as AtAXE1. The subcellular localization studies showed that the AtAXE1 enzyme is secreted out, associated with the plasma membrane and involved in xylan de-esterification post-synthesis. The cellulose digestibility was improved in AtAXE1 overexpressor lines without pre-treatment, after alkali and xylanases pre-treatment. Furthermore, we have also established that the AtGELP7 gene is upregulated in the overexpressor line of AtMYB46, which is a secondary cell wall specific transcription factor. This transcriptional regulation can drive AtGELP7 or AtAXE1 to perform de-esterification of xylan in a tissue-specific manner.

CRISPR-Knockout of CSE Gene Improves Saccharification Efficiency by Reducing Lignin Content in Hybrid Poplar

Caffeoyl shikimate esterase (CSE) has been shown to play an important role in lignin biosynthesis in plants and is, therefore, a promising target for generating improved lignocellulosic biomass crops for sustainable biofuel production. Populus spp. has two CSE genes (CSE1 and CSE2) and, thus, the hybrid poplar (Populus alba × P. glandulosa) investigated in this study has four CSE genes. Here, we present transgenic hybrid poplars with knockouts of each CSE gene achieved by CRISPR/Cas9. To knockout the CSE genes of the hybrid poplar, we designed three single guide RNAs (sg1–sg3), and produced three different transgenic poplars with either CSE1 (CSE1-sg2), CSE2 (CSE2-sg3), or both genes (CSE1/2-sg1) mutated. CSE1-sg2 and CSE2-sg3 poplars showed up to 29.1% reduction in lignin deposition with irregularly shaped xylem vessels. However, CSE1-sg2 and CSE2-sg3 poplars were morphologically indistinguishable from WT and showed no significant differences in growth in a long-term living modified organism (LMO) field-test covering four seasons. Gene expression analysis revealed that many lignin biosynthetic genes were downregulated in CSE1-sg2 and CSE2-sg3 poplars. Indeed, the CSE1-sg2 and CSE2-sg3 poplars had up to 25% higher saccharification efficiency than the WT control. Our results demonstrate that precise editing of CSE by CRISPR/Cas9 technology can improve lignocellulosic biomass without a growth penalty.

Gelatinization or Pasting? The Impact of Different Temperature Levels on the Saccharification Efficiency of Barley Malt Starch

Efficient enzymatic hydrolysis of cereal starches requires a proper hydrothermal pre-treatment. For malted barley, however, the exact initial temperature is presently unknown. Therefore, samples were micro-mashed according to accurately determined gelatinization and pasting temperatures. The impact on starch morphology, mash viscometry and sugar yields was recorded in the presence and absence of an amylase inhibitor to differentiate between morphological and enzymatic effects. Mashing at gelatinization onset temperatures (54.5–57.1 °C) led to negligible morphological and viscometric changes, whereas mashing at pasting onset temperatures (57.5–59.8 °C) induced significant starch granule swelling and degradation resulting in increased sugar yields (61.7% of upper reference limit). Complete hydrolysis of A-type and partial hydrolysis of B-type granules was achieved within only 10 min of mashing at higher temperatures (61.4–64.5 °C), resulting in a sugar yield of 97.5% as compared to the reference laboratory method mashing procedure (65 °C for 60 min). The results indicate that the beginning of starch pasting was correctly identified and point out the potential of an adapted process temperature control.