A High-Quality Reference Genome Assembly of Prinsepia uniflora (Rosaceae).

This study introduces a meticulously constructed genome assembly at the chromosome level for the Rosaceae family species Prinsepia uniflora, a traditional Chinese medicinal herb. The final assembly encompasses 1272.71 megabases (Mb) distributed across 16 pseudochromosomes, boasting contig and super-scaffold N50 values of 2.77 and 79.32 Mb, respectively. Annotated within this genome is a substantial 875.99 Mb of repetitive sequences, with transposable elements occupying 777.28 Mb, constituting 61.07% of the entire genome. Our predictive efforts identified 49,261 protein-coding genes within the repeat-masked assembly, with 45,256 (91.87%) having functional annotations, 5127 (10.41%) demonstrating tandem duplication, and 2373 (4.82%) classified as transcription factor genes. Additionally, our investigation unveiled 3080 non-coding RNAs spanning 0.51 Mb of the genome sequences. According to our evolutionary study, P. uniflora underwent recent whole-genome duplication following its separation from Prunus salicina. The presented reference-level genome assembly and annotation for P. uniflora will significantly facilitate the in-depth exploration of genomic information pertaining to this species, offering substantial utility in comparative genomics and evolutionary analyses involving Rosaceae species.

Efficient Secretory Production of Lytic Polysaccharide Monooxygenase BaLPMO10 and Its Application in Plant Biomass Conversion.

Lytic polysaccharide monooxygenases (LPMOs) can oxidatively break the glycosidic bonds of crystalline cellulose, providing more actionable sites for cellulase to facilitate the conversion of cellulose to cello-oligosaccharides, cellobiose and glucose. In this work, a bioinformatics analysis of BaLPMO10 revealed that it is a hydrophobic, stable and secreted protein. By optimizing the fermentation conditions, the highest protein secretion level was found at a IPTG concentration of 0.5 mM and 20 h of fermentation at 37 °C, with a yield of 20 mg/L and purity > 95%. The effect of metal ions on the enzyme activity of BaLPMO10 was measured, and it was found that 10 mM Ca2+ and Na+ increased the enzyme activity by 47.8% and 98.0%, respectively. However, DTT, EDTA and five organic reagents inhibited the enzyme activity of BaLPMO10. Finally, BaLPMO10 was applied in biomass conversion. The degradation of corn stover pretreated with different steam explosions was performed. BaLPMO10 and cellulase had the best synergistic degradation effect on corn stover pretreated at 200 °C for 12 min, improving reducing sugars by 9.2% compared to cellulase alone. BaLPMO10 was found to be the most efficient for ethylenediamine-pretreated Caragana korshinskii by degrading three different biomasses, increasing the content of reducing sugars by 40.5% compared to cellulase alone following co-degradation with cellulase for 48 h. The results of scanning electron microscopy revealed that BaLPMO10 disrupted the structure of Caragana korshinskii, making its surface coarse and poriferous, which increased the accessibility of other enzymes and thus promoted the process of conversion. These findings provide guidance for improving the efficiency of enzymatic digestion of lignocellulosic biomass.

Construction and characterization of a Myceliophthora thermophila lytic polysaccharide monooxygenase mutant S174C/A93C with improved thermostability.

Lytic polysaccharide monooxygenases (LPMOs) can oxidatively cleave the glycosidic bonds of crystalline polysaccharides, providing more accessible sites for polysaccharide hydrolases and promoting efficient conversion of biomass. In order to promote industrial applications of LPMOs, the stability of an LPMO of Myceliophthora thermophila C1 (MtC1LPMO) was improved by adding disulfide bonds in this study. Firstly, the structural changes of wild-type (WT) MtC1LPMO at different temperatures were explored using molecular dynamics simulations, and eight mutants were selected by combining the predicted results from Disulfide by Design (DBD), Multi agent stability prediction upon point mutations (Maestro) and Bridge disulfide (BridgeD) websites. Then, the enzymatic properties of the different mutants were determined after their expression and purification, and the mutant S174C/A93C with the highest thermal stability was obtained. The specific activities of unheated S174C/A93C and WT were 160.6 ± 1.7 U/g and 174.8 ± 7.5 U/g, respectively, while those of S174C/A93C and WT treated at 70 °C for 4 h were 77.7 ± 3.4 U/g and 46.1 ± 0.4 U/g, respectively. The transition midpoint temperature of S174C/A93C was 2.7 °C higher than that of WT. The conversion efficiency of S174C/A93C for both microcrystalline cellulose and corn straw was about 1.5 times higher than that of WT. Finally, molecular dynamics simulations revealed that the introduction of disulfide bonds increased the ß-sheet content of the H1-E34 region, thus improving the rigidity of the protein. Therefore, the overall structural stability of S174C/A93C was improved, which in turn improved its thermal stability.

Lytic polysaccharide monooxygenase - A new driving force for lignocellulosic biomass degradation.

Lytic polysaccharide monooxygenases (LPMOs) can catalyze polysaccharides by oxidative cleavage of glycosidic bonds and have catalytic activity for cellulose, hemicellulose, chitin, starch and pectin, thus playing an important role in the biomass conversion of lignocellulose. The catalytic substrates of LPMOs are different and the specific catalytic mechanism has not been fully elucidated. Although there have been many studies related to LPMOs, few have actually been put into industrial biomass conversion, which poses a challenge for their expression, regulation and application. In this review, the origin, substrate specificity, structural features, and the relationship between structure and function of LPMOs are described. Additionally, the catalytic mechanism and electron donor of LPMOs and their heterologous expression and regulation are discussed. Finally, the synergistic degradation of biomass by LPMOs with other polysaccharide hydrolases is reviewed, and their current problems and future research directions are pointed out.

The discovery and enzymatic characterization of a novel AA10 LPMO from Bacillus amyloliquefaciens with dual substrate specificity.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes, which can catalyze the oxidative cleavage of polysaccharide ß-1,4 glycosidic bonds to improve the hydrolysis efficiency of the substrate by other glycoside hydrolases. To improve the conversion efficiency of cellulose and chitin, a strain was screened from the soil of Yuelu Mountain in Hunan province, China. The gene sequence of a novel AA10 LPMO (BaLPMO10) was successfully cloned from the genome of the strain and heterologously expressed in E. coli BL21 (DE3). The optimal enzyme activity of BaLPMO10 was observed at pH 6.0 and 70 °C using 2,6-dimethoxyphenol as substrate, and its maximum specific activity was 91.4 U/g. When BaLPMO10 synergized with glycoside hydrolase to degrade microcrystalline cellulose and colloidal chitin, the reducing sugar content increased by 7% and 23%, respectively, compared to glycoside hydrolase alone. Moreover, the results of molecular docking and molecular dynamics simulation showed that the distance between BaLPMO10 and cellohexaose were further than that of BaLPMO10 and chitohexaose, and the number of hydrogen bonds between BaLPMO10 and cellohexaose were lower than that of BaLPMO10 and chitohexaose. Finally, the hot spot residues of BaLPMO10 interacting with chitohexaose/cellohexaose were identified.

Rational design of signal peptides for improved MtC1LPMO production in Bacillus amyloliquefaciens.

A high-throughput screening system was established by employing enhanced green fluorescent protein as a screenable fusion tag to evaluate the expression and secretion of a lytic polysaccharide monooxygenase (MtC1LPMO) using 20 Sec-type signal peptides (SPs) from Bacillus amyloliquefaciens 111018. Among these, 10 SPs were found to be better than the native SP of MtC1LPMO. The protein expression and secretion levels using SP12 (MNITNWAAILQLQSMALQSISNTGTASS) were the highest among all SPs, with 4.1- and 2.1-fold increases over the native SP, respectively. Then, the amino acids of the 10 best SPs were analyzed, and the results indicated that the most abundant amino acid of the N-region was K, those of the H-region were L, F, A and V, and the C-region contained an AXA motif. Additionally, we found that the protein expression level gradually improved along with the increasing folding free energies of the SP-encoding part of the mRNA. Finally, the SPs were rationally designed to improve the expression and secretion level of MtC1LPMO. An increased positive charge of the SP N-region was found to enhance the protein expression and secretion level, as long as the folding free energy of the mRNA did not change significantly.

Construction of the R17L mutant of MtC1LPMO for improved lignocellulosic biomass conversion by rational point mutation and investigation of the mechanism by molecular dynamics simulations.

To enhance the biomass conversion efficiency, the R17L mutant of the lytic polysaccharide monooxygenase (LPMO) MtC1LPMO with improved catalytic efficiency was constructed via rational point mutation based on the HotSpot Wizard 3.0 and dezyme web servers. Compared with the wild-type (WT) MtC1LPMO, R17L exhibited a 1.8-fold increase of specific activity and 1.92-fold increase of catalytic efficiency (kcat/Km). The degree of increase of the reducing sugar yield from microcrystalline cellulose and three plant biomass materials during synergistic hydrolysis using cellulase in combination with R17L was about 2 times higher than with the WT. Molecular dynamics simulations revealed that the R17L mutation reduced the stability of the region R18-I36, which then weakened the direct interactions between region N24-V31 and the substrate cellohexaose. Consequently, the deflection time of the cellohexaose conformation in R17L was prolonged compared to the WT, which enhanced its catalytic efficiency.

The one-pot synthesis of CuNi nanoparticles with a Ni-rich surface for the electrocatalytic methanol oxidation reaction.

The use of fuel cells is one of the most promising renewable energy strategies, but they still suffer from many limitations. The high mass enthalpy of hydrogen as a fuel comes at the cost of inconveniences and risks associated with storage, transportation and utilization, while the high performance of Pt catalysts in commercial fuel cells is limited by their high cost, low earth abundance, and poor stability as a result of CO intermediate poisoning. To circumvent these dilemmas, direct methanol fuel cells (DMFCs) were developed, using methanol as a fuel and Ni as the anode catalyst. Thanks to the condensed form of the fuel, DMFCs are considered as the most promising fuel-cell solution for portable electronic devices. Usually, other elements have to be introduced into Ni-based catalysts to modify the active sites to provide better alternatives to pristine Ni metal in terms of activity and stability. In this study, we provide a mild synthetic method for the preparation of CuNi alloy nanoparticles. The proper alloying ratio leads to the suitable modification of the electronic structure of Ni, which promotes the MOR catalytic reaction on the NiCu alloy. The NiCu alloy catalyst exhibits a mass current density of 1028 mA mgmetal-1 for the MOR at 1.55 V (vs. RHE), which is among the best values obtained from similarly prepared Ni-based catalysts.