Publications by authors named "Lushan Wang"

85 Publications

Orientated Immobilization of FAD-Dependent Glucose Dehydrogenase on Electrode by Carbohydrate-Binding Module Fusion for Efficient Glucose Assay.

Int J Mol Sci 2021 May 24;22(11). Epub 2021 May 24.

Shandong Provincial Key Laboratory of Biosensors, Biology Institute, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250103, China.

The discovery or engineering of fungus-derived FAD-dependent glucose 1-dehydrogenase (FAD-GDH) is especially important in the fabrication and performance of glucose biosensors. In this study, a novel FAD-GDH gene, phylogenetically distantly with other FAD-GDHs from species, was identified. Additionally, the wild-type GDH enzyme, and its fusion enzyme (GDH-NL-CBM2) with a carbohydrate binding module family 2 (CBM2) tag attached by a natural linker (NL), were successfully heterogeneously expressed. In addition, while the GDH was randomly immobilized on the electrode by conventional methods, the GDH-NL-CBM2 was orientationally immobilized on the nanocellulose-modified electrode by the CBM2 affinity adsorption tag through a simple one-step approach. A comparison of the performance of the two electrodes demonstrated that both electrodes responded linearly to glucose in the range of 0.12 to 40.7 mM with a coefficient of determination R > 0.999, but the sensitivity of immobilized GDH-NL-CBM2 (2.1362 × 10 A/(M*cm)) was about 1-fold higher than that of GDH (1.2067 × 10 A/(M*cm)). Moreover, a lower detection limit (51 µM), better reproducibility (<5%) and stability, and shorter response time (≈18 s) and activation time were observed for the GDH-NL-CBM2-modified electrode. This facile and easy immobilization approach used in the preparation of a GDH biosensor may open up new avenues in the development of high-performance amperometric biosensors.
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http://dx.doi.org/10.3390/ijms22115529DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8197230PMC
May 2021

Comparative molecular dynamics simulations identify a salt-sensitive loop responsible for the halotolerant activity of GH5 cellulases.

J Biomol Struct Dyn 2021 May 27:1-11. Epub 2021 May 27.

State Key Laboratory of Microbial Technology, Shandong University, Qingdao, China.

Halotolerant glycoside hydrolases (GH) have broad application potentials in biorefinery industries. Elucidating the structure-activity relationship underlying the halotolerant catalysis is essential to design superior biocatalysts. Here, we performed molecular dynamics simulations to investigate the structural dynamics of two GH5 cellulases, namely the halotolerant Cel5R and non-halotolerant Cel5A. Through characterizing the physical properties at different salt concentrations, the results revealed that the overall structures of Cel5R and Cel5A were marginally affected by the increase in salt concentrations. However, a salt-sensitive loop was identified from both Cel5R and Cel5A based on its significantly increased flexibility at high salt concentrations. Importantly, compared to Cel5A the salt-sensitive loop of Cel5R engaged more sodium ions and water molecules around the active site of the enzyme. Besides, the unique residue motif of the salt-sensitive loop in Cel5R formed more intramolecular hydrogen bonds, stabilizing the active architecture of Cel5R at high salt concentrations. Collectively, the structural and dynamic differences may contribute to the various catalytic halotolerance of Cel5R and Cel5A. These findings provide mechanistic insight into the halotolerant catalysis and will guide the ration design of GH5 cellulases with improved catalytic properties.Communicated by Ramaswamy H. Samy.
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http://dx.doi.org/10.1080/07391102.2021.1930167DOI Listing
May 2021

Synergistic mechanism of GH11 xylanases with different action modes from Aspergillus niger An76.

Biotechnol Biofuels 2021 May 10;14(1):118. Epub 2021 May 10.

State Key Laboratory of Microbial Technology, Institute of Microbial Technology, Shandong University, Qingdao, 266237, Shandong, China.

Background: Xylan is the most abundant hemicellulose polysaccharide in nature, which can be converted into high value-added products. However, its recalcitrance to breakdown requires the synergistic action of multiple enzymes. Aspergillus niger, possessing numerous xylan degrading isozyme-encoding genes, are highly effective xylan degraders in xylan-rich habitats. Therefore, it is necessary to explore gene transcription, the mode of action and cooperation mechanism of different xylanase isozymes to further understand the efficient xylan-degradation by A. niger.

Results: Aspergillus niger An76 encoded a comprehensive set of xylan-degrading enzymes, including five endo-xylanases (one GH10 and four GH11). Quantitative transcriptional analysis showed that three xylanase genes (xynA, xynB and xynC) were up-regulated by xylan substrates, and the order and amount of enzyme secretion differed. Specifically, GH11 xylanases XynA and XynB were initially secreted successively, followed by GH10 xylanase XynC. Biochemical analyses displayed that three GH11 xylanases (XynA, XynB and XynD) showed differences in catalytic performance and product profiles, possibly because of intricate hydrogen bonding between substrates and functional residues in the active site architectures impacted their binding capacity. Among these, XynB had the best performance in the degradation of xylan and XynE had no catalytic activity. Furthermore, XynA and XynB showed synergistic effects during xylan degradation.

Conclusions: The sequential secretion and different action modes of GH11 xylanases were essential for the efficient xylan degradation by A. niger An76. The elucidation of the degradation mechanisms of these xylanase isozymes further improved our understanding of GH-encoding genes amplification in filamentous fungi and may guide the design of the optimal enzyme cocktails in industrial applications.
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http://dx.doi.org/10.1186/s13068-021-01967-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8112042PMC
May 2021

Continuous production of fructooligosaccharides by recycling of the thermal-stable β-fructofuranosidase produced by Aspergillus niger.

Biotechnol Lett 2021 Jun 11;43(6):1175-1182. Epub 2021 Feb 11.

State Key Laboratory of Microbial Technology, Institute of Microbial Technology, Shandong University, Qingdao, 266237, People's Republic of China.

Objective: To achieve continuous production of fructooligosaccharides (FOS) by recycling of the mycelial cells containing the thermal-stable β-fructofuranosidase in Aspergillus niger without immobilization.

Results: The thermal-stable β-fructofuranosidase FopA-V1 was successfully expressed in A. niger ATCC 20611 under the control of the constitutive promoter PgpdA. The engineered A. niger strain FV1-11 produced the β-fructofuranosidase with improved thermostability, which remained 91.2% of initial activity at 50 °C for 30 h. Then its mycelial β-fructofuranosidase was recycled for the synthesis of FOS. It was found that the enzyme still had 79.3% of initial activity after being reused for six consecutive cycles, whereas only 62.3% β-fructofuranosidase activity was detected in the parental strain ATCC 20611. Meanwhile, the FOS yield of FV1-11 after six consecutive cycles reached 57.1% (w/w), but only 51.0% FOS yield was detected in ATCC 20611.

Conclusions: The thermal-stable β-fructofuranosidase produced by A. niger can be recycled to achieve continuous synthesis of FOS with high efficiency, providing a powerful and economical strategy for the industrial production of FOS.
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http://dx.doi.org/10.1007/s10529-021-03099-wDOI Listing
June 2021

Extracellular protease production regulated by nitrogen and carbon sources in Trichoderma reesei.

J Basic Microbiol 2021 Feb 4;61(2):122-132. Epub 2021 Jan 4.

State Key Laboratory of Microbial Technology, Institute of Microbial Technology, Shandong University, Qingdao, P.R. China.

The filamentous fungus Trichoderma reesei is an important producer of industrial enzymes, and possesses abundant extracellular protease genes based on the genome sequence data. However, the production of extracellular proteases remains poorly understood. Here, protease production was extensively investigated on different carbon (glucose and lactose) and nitrogen sources ((NH ) SO , NaNO , peptone, and corn steep liquor). It was found that protease production was dominantly regulated by nitrogen sources. Organic nitrogen sources were beneficial for protease production, while the preferred nitrogen source (NH ) SO inhibited the expression of proteases. As for carbon sources, lactose was a more effective inducer than glucose for protease production. The protease activity was further examined by protease inhibitors, which suggested that protease activity was predominantly inhibited by phenylmethanesulfonyl fluoride (PMSF) and slightly suppressed by ethylenediaminetetraacetic acid (EDTA). Moreover, proteomic analysis revealed a total of 29 extracellular proteases, including 13 serine proteases, 6 aspartic proteases, and 10 metalloproteases. In addition, seven proteases were found to be present among all conditions. These results showed the regulatory profile of extracellular protease production in Trichoderma reesei grown on various carbon and nitrogen sources, which will facilitate the development of T. reesei to be an effective workhorse for enzyme or high-value protein production in industry.
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http://dx.doi.org/10.1002/jobm.202000566DOI Listing
February 2021

A highly efficient protein degradation system in Bacillus sp. CN2: a functional-degradomics study.

Appl Microbiol Biotechnol 2021 Jan 2;105(2):707-723. Epub 2021 Jan 2.

State Key Laboratory of Microbial Technology, Institute of Microbial Technology, Shandong University, 72 Binhai Rd, Qingdao, 266237, Shandong, China.

A novel protease-producing Bacillus sp. CN2 isolated from chicken manure composts exhibited a relatively high proteolytic specific activity. The strain CN2 degradome consisted of at least 149 proteases and homolog candidates, which were distributed into 4 aspartic, 30 cysteine, 55 metallo, 56 serine, and 4 threonine proteases. Extracellular proteolytic activity was almost completely inhibited by PMSF (phenylmethylsulfonyl fluoride) rather than o-P, E-64, or pepstatin A, suggesting that strain CN2 primarily secreted serine protease. More importantly, analysis of the extracellular proteome of strain CN2 revealed the presence of a highly efficient protein degradation system. Three serine proteases of the S8 family with different active site architectures firstly fragmented protein substrates which were then degraded to smaller peptides by a M4 metalloendopeptidase that prefers to degrade hydrophobic peptides and by a S13 carboxypeptidase. Those enzymes acted synergistically to degrade intact substrate proteins outside the cell. Furthermore, highly expressed sequence-specific intracellular aminopeptidases from multiple families (M20, M29, and M42) accurately degraded peptides into oligopeptides or amino acids, thus realizing the rapid acquisition and utilization of nitrogen sources. In this paper, a systematic study of the functional-degradome provided a new perspective for understanding the complexity of the protease hydrolysis system of Bacillus, and laid a solid foundation for further studying the precise degradation of proteins with the cooperative action of different family proteases. KEY POINTS: • Bacillus sp. CN2 has relatively high proteolytic specific activity. • Bacillus sp. CN2 harbors a highly efficient protein degradation system. • The site-specific endopeptidases were secreted extracellular, while the sequence-specific aminopeptidases played a role in the cell.
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http://dx.doi.org/10.1007/s00253-020-11083-zDOI Listing
January 2021

A sequence embedding method for enzyme optimal condition analysis.

BMC Bioinformatics 2020 Nov 10;21(1):512. Epub 2020 Nov 10.

School of Software, Shandong University, Shunhua Road, Jinan, 250101, China.

Background: An enzyme activity is influenced by the external environment. It is important to have an enzyme remain high activity in a specific condition. A usual way is to first determine the optimal condition of an enzyme by either the gradient test or by tertiary structure, and then to use protein engineering to mutate a wild type enzyme for a higher activity in an expected condition.

Results: In this paper, we investigate the optimal condition of an enzyme by directly analyzing the sequence. We propose an embedding method to represent the amino acids and the structural information as vectors in the latent space. These vectors contain information about the correlations between amino acids and sites in the aligned amino acid sequences, as well as the correlation with the optimal condition. We crawled and processed the amino acid sequences in the glycoside hydrolase GH11 family, and got 125 amino acid sequences with optimal pH condition. We used probabilistic approximation method to implement the embedding learning method on these samples. Based on these embedding vectors, we design a computational score to determine which one has a better optimal condition for two given amino acid sequences and achieves the accuracy 80% on the test proteins in the same family. We also give the mutation suggestion such that it has a higher activity in an expected environment, which is consistent with the previously professional wet experiments and analysis.

Conclusion: A new computational method is proposed for the sequence based on the enzyme optimal condition analysis. Compared with the traditional process that involves a lot of wet experiments and requires multiple mutations, this method can give recommendations on the direction and location of amino acid substitution with reference significance for an expected condition in an efficient and effective way.
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http://dx.doi.org/10.1186/s12859-020-03851-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7653822PMC
November 2020

Novel Synergistic Mechanism for Lignocellulose Degradation by a Thermophilic Filamentous Fungus and a Thermophilic Actinobacterium Based on Functional Proteomics.

Front Microbiol 2020 11;11:539438. Epub 2020 Sep 11.

State Key Laboratory of Microbial Technology, Microbial Technology Institute, Shandong University, Qingdao, China.

Effective artificial microbial consortia containing microorganisms with desired biological functions have the potential to optimize the lignocellulose-based bioindustry. was a dominant actinobacterium in high-temperature corn stalk composts, but it was unable to grow alone in corn stalk solid medium. Interestingly, showed good growth and secreted enzymes when cocultured with . grew firstly during the initial stage, whereas dominated the system subsequently during cocultivation. The secretome indicated that mainly degraded xylan by expressing a GH11 xylanase (g4601.t1, GenBank AAB94633.1; with relative secretion of 4.95 ± 0.65%). was induced by xylan mainly to secrete a xylanase from GH11 family (W8GGR4, GenBank AHK22788.1; with relative secretion of 8.71 ± 3.83%) which could rapidly degrade xylan to xylo-oligosaccharide (XOS) and xylose within 2 min, while high concentrations (>0.5%, w/v) of XOS or xylose suppressed the growth of ; which may be the reason why unable to grow alone in corn stalk solid medium. However, could utilize the XOS and xylose produced by xylanases secreted by . During the synergistic degradation of lignocellulose by and , xylan was rapidly consumed by , the residual cellulose could specifically induced to express a GH10 xylanase with a CBM2 domain (Q47KR6, GenBank AAZ56956.1; with relative secretion of 5.03 ± 1.33%) and 6 cellulases (2 exocellulases and 4 endocellulases). Moreover, increased the secretion of cellulases from by 19-25%. The order of and was consistent with the multilayered structures of lignocellulose and could be regulated by different concentrations of XOS and xylose. The novel synergism of and gave a new sight for revealing more synergetic relationships in natural environments and exploring efficient microbial inoculants and enzyme cocktails for lignocellulose degradation.
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http://dx.doi.org/10.3389/fmicb.2020.539438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7518101PMC
September 2020

Diverse phylogeny and morphology of magnetite biomineralized by magnetotactic cocci.

Environ Microbiol 2021 02 10;23(2):1115-1129. Epub 2020 Oct 10.

Key Laboratory of Earth and Planetary Physics, Institute of Geology and Geophysics, Innovation Academy for Earth Science, Chinese Academy of Sciences, Beijing, China.

Magnetotactic bacteria (MTB) are diverse prokaryotes that produce magnetic nanocrystals within intracellular membranes (magnetosomes). Here, we present a large-scale analysis of diversity and magnetosome biomineralization in modern magnetotactic cocci, which are the most abundant MTB morphotypes in nature. Nineteen novel magnetotactic cocci species are identified phylogenetically and structurally at the single-cell level. Phylogenetic analysis demonstrates that the cocci cluster into an independent branch from other Alphaproteobacteria MTB, that is, within the Etaproteobacteria class in the Proteobacteria phylum. Statistical analysis reveals species-specific biomineralization of magnetosomal magnetite morphologies. This further confirms that magnetosome biomineralization is controlled strictly by the MTB cell and differs among species or strains. The post-mortem remains of MTB are often preserved as magnetofossils within sediments or sedimentary rocks, yet paleobiological and geological interpretation of their fossil record remains challenging. Our results indicate that magnetofossil morphology could be a promising proxy for retrieving paleobiological information about ancient MTB.
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http://dx.doi.org/10.1111/1462-2920.15254DOI Listing
February 2021

Insight into the effect of nitrogen-rich substrates on the community structure and the co-occurrence network of thermophiles during lignocellulose-based composting.

Bioresour Technol 2021 Jan 10;319:124111. Epub 2020 Sep 10.

Institute of Ecology and Biodiversity, School of Life Sciences, Shandong University, Qingdao 266237, PR China. Electronic address:

Thermophilic microorganisms play vital roles in the composting process. To elucidate how raw materials affect thermophilic microbial community composition and their interactions, the succession of thermophilic bacterial and fungal communities were monitored in reed straw co-composting with four common nitrogen-rich substrates. The results of high-throughput sequencing showed that raw materials and composting process significantly changed bacterial and fungal community composition. Firmicutes and Actinobacteria drove the assembly of bacterial communities, while Ascomycetes drove the assembly of fungal communities. Network analysis indicated that during the composting process, the addition of nitrogen-rich sources abundant in easily degradable substances promoted the complexity of thermophilic microbial network. Moreover, microorganisms mainly exhibited synergistic effects, and inter-kingdom competition was more intense than intra-kingdom competition. Notably, rare species play essential roles in maintaining the network construction. Our findings provided novel insights into thermophilic microbial community assembly and their co-occurrence networks during the composting process.
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http://dx.doi.org/10.1016/j.biortech.2020.124111DOI Listing
January 2021

Simulations of octapeptin-outer membrane interactions reveal conformational flexibility is linked to antimicrobial potency.

J Biol Chem 2020 11 10;295(47):15902-15912. Epub 2020 Sep 10.

Biomedicine Discovery Institute, Infection & Immunity Program, Department of Microbiology, Monash University, Melbourne, Victoria, Australia. Electronic address:

The octapeptins are lipopeptide antibiotics that are structurally similar to polymyxins yet retain activity against polymyxin-resistant Gram-negative pathogens, suggesting they might be used to treat recalcitrant infections. However, the basis of their unique activity is unclear because of the difficulty in generating high-resolution experimental data of the interaction of antimicrobial peptides with lipid membranes. To elucidate these structure-activity relationships, we employed all-atom molecular dynamics simulations with umbrella sampling to investigate the conformational and energetic landscape of octapeptins interacting with bacterial outer membrane (OM). Specifically, we examined the interaction of octapeptin C4 and FADDI-115, lacking a single hydroxyl group compared with octapeptin C4, with the lipid A-phosphoethanolamine modified OM of Octapeptin C4 and FADDI-115 both penetrated into the OM hydrophobic center but experienced different conformational transitions from an unfolded to a folded state that was highly dependent on the structural flexibility of their respective N-terminal fatty acyl groups. The additional hydroxyl group present in the fatty acyl group of octapeptin C4 resulted in the molecule becoming trapped in a semifolded state, leading to a higher free energy barrier for OM penetration. The free energy barrier for the translocation through the OM hydrophobic layer was ∼72 kcal/mol for octapeptin C4 and 62 kcal/mol for FADDI-115. Our results help to explain the lower antimicrobial activity previously observed for octapeptin C4 compared with FADDI-115 and more broadly improve our understanding of the structure-function relationships of octapeptins. These findings may facilitate the discovery of next-generation octapeptins against polymyxin-resistant Gram-negative 'superbugs.'
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http://dx.doi.org/10.1074/jbc.RA120.014856DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7681018PMC
November 2020

Molecular dynamics simulations informed by membrane lipidomics reveal the structure-interaction relationship of polymyxins with the lipid A-based outer membrane of Acinetobacter baumannii.

J Antimicrob Chemother 2020 12;75(12):3534-3543

Biomedicine Discovery Institute, Infection & Immunity Program and Department of Microbiology, Monash University, Melbourne, Australia.

Background: MDR bacteria represent an urgent threat to human health globally. Polymyxins are a last-line therapy against life-threatening Gram-negative 'superbugs', including Acinetobacter baumannii. Polymyxins exert antimicrobial activity primarily via permeabilizing the bacterial outer membrane (OM); however, the mechanism of interaction between polymyxins and the OM remains unclear at the atomic level.

Methods: We constructed a lipid A-based OM model of A. baumannii using quantitative membrane lipidomics data and employed all-atom molecular dynamics simulations with umbrella sampling techniques to elucidate the structure-interaction relationship and thermodynamics governing the penetration of polymyxins [B1 and E1 (i.e. colistin A) representing the two clinically used polymyxins] into the OM.

Results: Polymyxin B1 and colistin A bound to the A. baumannii OM by the initial electrostatic interactions between the Dab residues of polymyxins and the phosphates of lipid A, competitively displacing the cations from the headgroup region of the OM. Both polymyxin B1 and colistin A formed a unique folded conformation upon approaching the hydrophobic centre of the OM, consistent with previous experimental observations. Polymyxin penetration induced reorientation of the headgroups of the OM lipids near the penetration site and caused local membrane disorganization, thereby significantly increasing membrane permeability and promoting the subsequent penetration of polymyxin molecules into the OM and periplasmic space.

Conclusions: The thermodynamics governing the penetration of polymyxins through the outer leaflet of the A. baumannii OM were examined and novel structure-interaction relationship information was obtained at the atomic and membrane level. Our findings will facilitate the discovery of novel polymyxins against MDR Gram-negative pathogens.
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http://dx.doi.org/10.1093/jac/dkaa376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7662177PMC
December 2020

Outer Membranes of Polymyxin-Resistant with Phosphoethanolamine-Modified Lipid A and Lipopolysaccharide Loss Display Different Atomic-Scale Interactions with Polymyxins.

ACS Infect Dis 2020 10 15;6(10):2698-2708. Epub 2020 Sep 15.

Biomedicine Discovery Institute, Infection & Immunity Program, Department of Microbiology, Monash University, Melbourne, VIC 3800, Australia.

Resistance to the last-line polymyxins is increasingly reported in multidrug-resistant Gram-negative pathogens, including , which develops resistance via either lipid A modification (e.g., with phosphoethanolamine [pEtN]) or even lipopolysaccharide (LPS) loss in the outer membrane (OM). Considering these two different mechanisms, quantitative membrane lipidomics data were utilized to develop three OM models representing polymyxin-susceptible and -resistant strains. Through all-atom molecular simulations with enhanced sampling techniques, the effect of lipid A-pEtN modification and LPS loss on the action of colistin (i.e., polymyxin E) was examined for the first time, with a focus on the dynamics and energetics of colistin penetration into these OMs. Lipid A-pEtN modification improved the OM stability, impeding the penetration of colistin into the OM; this differed from the current literature that lipid A-pEtN modification confers resistance by diminishing the initial interaction with polymyxins. In contrast, the LPS deficiency significantly reduced the negative charges on the OM surface, diminishing the binding of colistin. Moreover, both lipid A-pEtN modification and LPS loss also constituted colistin resistance through disturbing the conformational transitions of the colistin molecule. Collectively, atomic-scale interactions between polymyxins and different bacterial OMs are very different and the findings may facilitate the discovery of new-generation polymyxins against Gram-negative 'superbugs'.
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http://dx.doi.org/10.1021/acsinfecdis.0c00330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7554230PMC
October 2020

The contribution of specific subsites to catalytic activities in active site architecture of a GH11 xylanase.

Appl Microbiol Biotechnol 2020 Oct 31;104(20):8735-8745. Epub 2020 Aug 31.

State Key Laboratory of Microbial Technology, Institute of Microbial Technology, Shandong University, 72 Binhai Rd, Qingdao, 266237, Shandong, China.

Background: Xylanase with high specific activity plays a crucial role in hemicellulose biodegradation and has important industrial application. The amino acids located in the active site determine the enzyme biological characterization. In this study, structure bioinformatics analysis and alanine screening experiments were performed to explore the roles of amino acids at each subsite of the GH11 xylanase active site.

Results: There are highly conserved amino acids at - 2 to + 1 subsites, and the network of the interactions is concentrated near the catalytic sites (E86, E178). However, the amino acids at relatively distal subsites, especially at the + 2 and + 3 subsites, are few but diverse. Alanine substitution of amino acids in the active site architecture exerted different impacts on catalytic efficiency. Interestingly, mutants Y180A at the + 2 subsite and Y96A at the + 3 subsite had reduced enzymatic activities by almost 95%, which indicate that these two aromatic residues are necessary for the catalysis of substrates in addition to the highly conserved residues at the - 2 and + 1 subsites. Moreover, in these two subsites, aromatic amino acids with different side-chain properties also affected enzyme activity. The mutants Y180W and Y96W showed 6.2% and 12.8% increase in specific activities by comparison with wild-type enzyme at 50 °C, respectively.

Conclusion: We elucidated the interaction between amino acids and substrates in the active site, which will aid understanding of the protein-ligand interaction in enzyme engineering.

Key Points: • Xylanase of GH11 family is a good industrial candidate. • The roles of residues at each subsite of GH11 xylanase active site are explored. • The two aromatic residues at the + 2 and + 3 subsites are necessary for the catalysis. • Y180W and Y96W increased the enzymatic activity by 6.2% and 12.8% at low temperature.
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http://dx.doi.org/10.1007/s00253-020-10865-9DOI Listing
October 2020

Penicillium oxalicum putative methyltransferase Mtr23B has similarities and differences with LaeA in regulating conidium development and glycoside hydrolase gene expression.

Fungal Genet Biol 2020 10 19;143:103445. Epub 2020 Aug 19.

State Key Laboratory of Microbial Technology, Microbial Technology Institute, Shandong University, No. 72 Binhai Road, Qingdao 266237, China; National Glycoengineering Research Center, Shandong University, No. 72 Binhai Road, Qingdao 266237, China. Electronic address:

Putative methyltranferase LaeA and LaeA-like proteins, which are conserved in many filamentous fungi, regulate the sporogenesis and biosynthesis of secondary metabolites. In this study, we reported the biological function of a LaeA-like methyltransferase, Penicillium oxalicum Mtr23B, which contains a methyltransf_23 domain and an S-adenosylmethionine binding domain, in controlling spore pigment formation and in the expression of secondary metabolic gene cluster and glycoside hydrolase genes. Additionally, we compared Mtr23B and LaeA, and determined their similarities and differences in terms of their roles in regulating the above biological processes. mtr23B had the highest transcriptional level among the 12 members of the methyltransf_23 family in P. oxalicum. The colony color of Δmtr23B (deletion of mtr23B) was lighter than that of ΔlaeA, although Δmtr23B produced ~ 19.2-fold more conidia than ΔlaeA. The transcriptional levels of abrA, abrB/yA, albA/wA, arpA, arpB, and aygA, which are involved in the dihydroxynaphtalene-melanin pathway, decreased in Δmtr23B. However, Mtr23B had a little effect on brush-like structures and conidium formation, and had a different function from LaeA. Mtr23B extensively regulated glycoside hydrolase gene expression. The absence of Mtr23B remarkably repressed prominent cellulase- and amylase-encoding genes in the whole culture period, while the effect of LaeA mainly occurred in the later phases of prolonged batch cultures. Similar to LaeA, Mtr23B was involved in the expression of 10 physically linked regions containing secondary metabolic gene clusters; the highest regulatory activities of Mtr23B and LaeA were observed in BrlA-dependent cascades. Although LaeA interacted with VeA, Mtr23B did not interact with VeA directly. We assumed that Mtr23B regulates cellulase and amylase gene transcription by interacting with the CCAAT-binding transcription factor HAP5 and chromatin remodeling complex.
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http://dx.doi.org/10.1016/j.fgb.2020.103445DOI Listing
October 2020

Insights into the cellulose degradation mechanism of the thermophilic fungus based on integrated functional omics.

Biotechnol Biofuels 2020 12;13:143. Epub 2020 Aug 12.

State Key Laboratory of Microbial Technology, Microbial Technology Institute, Shandong University, No. 72 Jimo Binhai Road, Qingdao, 266237 Shandong People's Republic of China.

Background: Lignocellulose is the most abundant and renewable biomass resource on the planet. Lignocellulose can be converted into biofuels and high-value compounds; however, its recalcitrance makes its breakdown a challenge. Lytic polysaccharide monooxygenases (LPMOs) offer tremendous promise for the degradation of recalcitrant polysaccharides. , having many LPMO-coding genes, is a dominant thermophilic fungus in cellulose-rich and self-heating habitats. This study explores the genome, secretomes and transcript levels of specific genes of .

Results: The genome of encoded a comprehensive set of cellulose- and xylan-degrading enzymes, especially 18 AA9 LPMOs that belonged to different subfamilies. Extracellular secretomes showed that arabinose and microcrystalline cellulose (MCC) could specifically induce the secretion of carbohydrate-active enzymes (CAZymes), especially AA9 LPMOs, by under different carbon sources. Temporal analyses of secretomes and transcripts revealed that arabinose induced the secretion of xylanases by , which was obviously different from other common filamentous fungi. MCC could efficiently induce the specific secretion of LPMO2s, possibly because the insert in loop3 on the substrate-binding surface of LPMO2s strengthened its binding capacity to cellulose. LPMO2s, cellobio hydrolases (CBHs) and cellobiose dehydrogenases (CDHs) were cosecreted, forming an efficient cellulose degradation system of oxidases and hydrolases under thermophilic conditions.

Conclusions: The specific expression of LPMO2s and cosecretion of hydrolases and oxidases by the thermophilic fungus play an important role in cellulose degradation. This insight increases our understanding of the cellulose degradation under thermophilic conditions and may inspire the design of the optimal enzyme cocktails for more efficient exploration of biomass resources in industrial applications.
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http://dx.doi.org/10.1186/s13068-020-01783-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7425565PMC
August 2020

Polymyxins Bind to the Cell Surface of Unculturable and Cause Unique Dependent Resistance.

Adv Sci (Weinh) 2020 Aug 8;7(15):2000704. Epub 2020 Jun 8.

Infection & Immunity Program Biomedicine Discovery Institute and Department of Microbiology Monash University Melbourne 3800 Australia.

Multidrug-resistant is a top-priority pathogen globally and polymyxins are a last-line therapy. Polymyxin dependence in (i.e., nonculturable on agar without polymyxins) is a unique and highly-resistant phenotype with a significant potential to cause treatment failure in patients. The present study discovers that a polymyxin-dependent strain possesses mutations in both (lipopolysaccharide biosynthesis) and (reactive oxygen species scavenging) genes. Correlative multiomics analyses show a significantly remodeled cell envelope and remarkably abundant phosphatidylglycerol in the outer membrane (OM). Molecular dynamics simulations and quantitative membrane lipidomics reveal that polymyxin-dependent growth emerges only when the lipopolysaccharide-deficient OM distinctively remodels with ≥ 35% phosphatidylglycerol, and with "patch" binding on the OM by the rigid polymyxin molecules containing strong intramolecular hydrogen bonding. Rather than damaging the OM, polymyxins bind to the phosphatidylglycerol-rich OM and strengthen the membrane integrity, thereby protecting bacteria from external reactive oxygen species. Dependent growth is observed exclusively with polymyxin analogues, indicating a critical role of the specific amino acid sequence of polymyxins in forming unique structures for patch-binding to bacterial OM. Polymyxin dependence is a novel antibiotic resistance mechanism and the current findings highlight the risk of 'invisible' polymyxin-dependent isolates in the evolution of resistance.
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http://dx.doi.org/10.1002/advs.202000704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7403960PMC
August 2020

Structure-Interaction Relationship of Polymyxins with the Membrane of Human Kidney Proximal Tubular Cells.

ACS Infect Dis 2020 08 17;6(8):2110-2119. Epub 2020 Jul 17.

Biomedicine Discovery Institute, Infection & Immunity Program and Department of Microbiology, Monash University, Clayton, Melbourne, Victoria 3800, Australia.

Multidrug-resistant Gram-negative bacteria are a serious global threat to human health. Polymyxins are increasingly used in patients as a last-line therapy to treat infections caused by these life-threatening 'superbugs'. Unfortunately, polymyxin-induced nephrotoxicity is the major dose-limiting factor and understanding its mechanism is crucial for the development of novel, safer polymyxins. Here, we undertook the first all-atom molecular dynamics simulations of the interaction between four naturally occurring polymyxins A, B, M and colistin A (representative structural variations of the polymyxin core structure) and the membrane of human kidney proximal tubular cells. All polymyxins inserted spontaneously into the hydrophobic region of the membrane where they were retained, although their insertion abilities varied. Polymyxin A completely penetrated into the hydrophobic region of the membrane with a unique folded conformation, whereas the other three polymyxins only inserted their fatty acyl tails into this region. Furthermore, local membrane defects and increased water penetration were induced by each polymyxin, which may represent the initial stage of cellular membrane damage. Finally, the structure-interaction relationship of polymyxins was investigated based on atomic interactions at the cell membrane level. The hydrophobicity at positions 6/7 and stereochemistry at position 3 regulated the interactions of polymyxins with the cell membrane. Collectively, our results provide new mechanistic insights into polymyxin-induced nephrotoxicity at the atomic level and will facilitate the development of new-generation polymyxins.
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http://dx.doi.org/10.1021/acsinfecdis.0c00190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7485602PMC
August 2020

Insights Into the Role of Exposed Surface Charged Residues in the Alkali-Tolerance of GH11 Xylanase.

Front Microbiol 2020 8;11:872. Epub 2020 May 8.

State Key Laboratory of Microbial Technology, Institute of Microbial Technology, Shandong University, Qingdao, China.

Thermostable and alkaline- or acid-stable xylanases are more advantageous in agricultural and industrial fields. In this study, a rational structure-based design was conducted based on a thermostable GH11 xylanase XynA from to improved pH-tolerance. Four mutant enzymes (P1, P2, P3, and P4) and five variants (N1, N2, N3, N4, and N5) were constructed by substituting surface charged residue combinations using site-directed mutagenesis. Compared to the native enzyme, two mutants P1 and P2 showed higher acid tolerance, especially at pH 3.0, presented 50 and 40% of their maximum activity, respectively. In addition, four mutants N1, N2, N3 and N4 had higher tolerance than the native enzyme to alkaline environments (pH 7.0-9.0). At pH 9.0, the residual activities of N1, N2, N3, and N4 were 86, 78, 77, and 66%, respectively. In summary, an improved pH-tolerance design principle is being reported.
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http://dx.doi.org/10.3389/fmicb.2020.00872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7225583PMC
May 2020

TGFβ regulates NK1R-Tr to affect the proliferation and apoptosis of breast cancer cells.

Life Sci 2020 Sep 5;256:117674. Epub 2020 May 5.

Department of Clinical Laboratory, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin's Clinical Research Center for Cancer, Tianjin Key Laboratory of Cancer Prevention and Therapy, Key Laboratory of Breast Cancer Prevention and Therapy of Educational Ministry, Tianjin Medical University, Tianjin, China. Electronic address:

Objectives: TGFβ promotes cancer aggressiveness in advanced stages. NK1R-Tr expression in advanced breast cancer has a pro-carcinogenic effect. In this study, we aimed to investigate the effect of the association of TGFβ with NK1R-Tr expression on the proliferation and apoptosis of breast cancer cells.

Methods: Immunohistochemical staining and Western blot analysis were used to detect TGFβ and NK1R-Tr in breast cancer and paracancerous tissue samples. MDA-MB-231 and BT549 cells were stimulated with TGFβ after NK1R knockdown or treated with the NK1R antagonist aprepitant, and the effects of TGFβ and NK1R-Tr on proliferation and apoptosis were detected by CCK-8, colony formation and flow cytometry assays. In vivo xenograft models were used to further verify the effects of NK1R-Tr and TGFβ. The regulatory effects of Smad4 on NK1R promoter activity were confirmed by ChIP and dual-luciferase reporter assays.

Results: The expression levels of TGFβ and NK1R-Tr were higher in breast cancer tissues than in adjacent tissues and were positively correlated in human breast cancer tissues. NK1R knockdown or aprepitant treatment in MDA-MB-231 and BT549 cells attenuated the effects of TGFβ on cell proliferation. The proportion of cells in G2/M phase significantly increased, the expression of cyclin B1 decreased, and the expression of P21 increased; these effects were weakened by TGFβ treatment. Apoptosis in breast cancer cells was significantly increased. In vivo xenograft models were used to further verify that NK1R-Tr and TGFβ promoted tumour growth. After TGFβ treatment, the binding capacity of Smad4 to the NK1R promoter, as well as luciferase activity, was enhanced.

Conclusions: The expression levels of TGFβ and NK1R-Tr were higher in breast cancer tissues than in normal tissues, and both were correlated with a poor patient prognosis. TGFβ and NK1R-Tr promoted cell proliferation and inhibited apoptosis, and TGFβ regulated the expression of NK1R-Tr via Smad4.
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http://dx.doi.org/10.1016/j.lfs.2020.117674DOI Listing
September 2020

Regulating polymyxin resistance in Gram-negative bacteria: roles of two-component systems PhoPQ and PmrAB.

Future Microbiol 2020 04 6;15:445-459. Epub 2020 Apr 6.

Biomedicine Discovery Institute & Department of Microbiology, Monash University, Melbourne 3800, Australia.

Polymyxins (polymyxin B and colistin) are last-line antibiotics against multidrug-resistant Gram-negative pathogens. Polymyxin resistance is increasing worldwide, with resistance most commonly regulated by two-component systems such as PmrAB and PhoPQ. This review discusses the regulatory mechanisms of PhoPQ and PmrAB in mediating polymyxin resistance, from receiving an external stimulus through to activation of genes responsible for lipid A modifications. By analyzing the reported nonsynonymous substitutions in each two-component system, we identified the domains that are critical for polymyxin resistance. Notably, for PmrB 71% of resistance-conferring nonsynonymous mutations occurred in the HAMP (present in histidine kinases, adenylate cyclases, methyl accepting proteins and phosphatase) linker and DHp (dimerization and histidine phosphotransfer) domains. These results enhance our understanding of the regulatory mechanisms underpinning polymyxin resistance and may assist with the development of new strategies to minimize resistance emergence.
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http://dx.doi.org/10.2217/fmb-2019-0322DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7236789PMC
April 2020

Aerobic composting as an effective cow manure management strategy for reducing the dissemination of antibiotic resistance genes: An integrated meta-omics study.

J Hazard Mater 2020 03 23;386:121895. Epub 2019 Dec 23.

State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong 250100, China. Electronic address:

Livestock manure is considered as an important source for spreading antibiotic resistance genes (ARGs) into the environment, and therefore poses a direct threat to public health. Whereas the effects of reused manure on soil microbial communities and ARGs have been studied extensively, comprehensive characterizations of microbial communities and ARGs of manure produced by different management methods are not well understood. Here, we analyzed the fate of microbial communities and ARGs of cow manure treated by three conventional management strategies: aerobic composting, mechanical drying and precipitation, applying an integrated-omics approach combining metagenomics and metaproteomics. Integrated-omics demonstrated that composted manure contained the lowest diversity of microbial community and ARGs compared with manure treated by other two strategies. Quantitative PCR methods revealed that the abundances of ARGs were reduced by over 83 % after composting for 14 days, regardless of the season. Besides, the potential ARG hosts Acinetobacter and Pseudomonas dominating mechanical drying process were sharply decreased in abundances after composting. The significant co-occurrence networks among bacteria, ARGs and transposase gene tnpA-01 in composting samples indicated the important role of these bacteria in the dissemination of ARGs. These findings offer insight into potential strategies to control the spread of ARGs during livestock manure reuse.
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http://dx.doi.org/10.1016/j.jhazmat.2019.121895DOI Listing
March 2020

Binding Process and Free Energy Characteristics of Cellulose Chain into the Catalytic Domain of Cellobiohydrolase .

J Phys Chem B 2019 10 10;123(42):8853-8860. Epub 2019 Oct 10.

School of Biological Sciences , Nanyang Technological University , 637551 Singapore.

It was observed in experiments that the catalytic domain (CD) of () hydrolyzes crystalline cellulose in a processive manner, but the underlying binding mechanism is still unknown. Here, through replica-exchange molecular dynamics simulations, we find that the loading and sucking-in process of the cellulose chain into CD is entropy-driven and enthalpy-unfavorable, which firmly relate to the desolvation of the binding channel of CD. During the loading process, hydrophobic interactions play a dominant role because several aromatic residues have been identified to guide the cellulose chain processing. At the active site, a transition from enthalpy- to entropy-driven is detected for the driving force. Such a finding reveals the indispensability of the catalytic reaction of the glycosidic bond to provide the energy to drive the movements of the cellulose chain. Our study reveals the interaction pictures between the cellulose chain and at the atomic level, which helps better understand the catalytic mechanism of .
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http://dx.doi.org/10.1021/acs.jpcb.9b05023DOI Listing
October 2019

Normal transcription of cellulolytic enzyme genes relies on the balance between the methylation of H3K36 and H3K4 in .

Biotechnol Biofuels 2019 20;12:198. Epub 2019 Aug 20.

1National Glycoengineering Research Center, Shandong University, Qingdao, 266237 China.

Background: Enzymatic hydrolysis of lignocellulose by fungi is a key step in global carbon cycle and biomass utilization. Cellulolytic enzyme production is tightly controlled at a transcriptional level. Here, we investigated the roles of different histone lysine methylation modifications in regulating cellulolytic enzyme gene expression, as histone lysine methylation is an important process of chromatin regulation associated with gene transcription.

Results: Set1 and Set2 in , orthologs of Set1 and Set2 in budding yeast, were associated with the methylation of histone H3 lysine 4 (H3K4) and lysine 36 (H3K36). Cellulolytic enzyme production was extensively upregulated by the disruption of Set2, but was significantly downregulated by the disruption of Set1. We revealed that the activation of cellulolytic enzyme genes was accompanied by the increase of H3K4me3 signal, as well as the decrease of H3K36me1 and H3K36me3 signal on specific gene loci. The repression of cellulolytic enzyme genes was accompanied by the absence of global H3K4me1 and H3K4me2. An increase in the H3K4me3 signal by disruption was eliminated by the further disruption of and accompanied by the repressed cellulolytic enzyme genes. The active or repressed genes were not always associated with transcription factors.

Conclusion: H3K4 methylation is an active marker of cellulolytic enzyme production, whereas H3K36 methylation is a marker of repression. A crosstalk occurs between H3K36 and H3K4 methylation, and Set2 negatively regulates cellulolytic enzyme production by antagonizing the Set1-H3K4me3 pathway. The balance of H3K4 and H3K36 methylation is required for the normal transcription of cellulolytic enzyme genes. These results extend our previous understanding that cellulolytic enzyme gene transcription is primarily controlled by transcription factors.
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http://dx.doi.org/10.1186/s13068-019-1539-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6700826PMC
August 2019

Three glycoside hydrolase family 12 enzymes display diversity in substrate specificities and synergistic action between each other.

Mol Biol Rep 2019 Oct 29;46(5):5443-5454. Epub 2019 Jul 29.

National Glycoengineering Research Center, Shandong University, No. 72 Binhai Road, Qingdao, 266237, China.

PoCel12A, PoCel12B, and PoCel12C are genes that encode glycoside hydrolase family 12 (GH12) enzymes in Penicillium oxalicum. PoCel12A and PoCel12B are typical GH12 enzymes that belong to fungal subfamilies 12-1 and 12-2, respectively. PoCel12C contains a low-complexity region (LCR) domain, which is not found in PoCel12A or PoCel12B and independent of fungal subfamily 12-1 or 12-2. Recombinant enzymes (named rCel12A, rCel12B and rCel12C) demonstrate existing diversity in the substrate specificities. Although most members in GH family 12 are typical endoglucanases and preferentially hydrolyze β-1,4-glucan (e.g., carboxymethylcellulose), recombinant PoCel12A is a non-typical endo-(1-4)-β-glucanase; it preferentially hydrolyzes mix-linked β-glucan (barley β-glucan, β-1,3-1,4-glucan) and slightly hydrolyzes β-1,4-glucan (carboxymethylcellulose). Recombinant PoCel12B possesses a significantly high activity against xyloglucan. A specific activity of rCel12B toward xyloglucan (239 µmol/min/mg) is the second-highest value known. Recombinant PoCel12C shows low activity toward β-glucan, carboxymethylcellulose, or xyloglucan. All three enzymes can degrade phosphoric acid-swollen cellulose (PASC). However, the hydrolysis products toward PASC by enzymes are different: the main hydrolysis products are cellotriose, cellotetraose, and cellobiose for rCel12A, rCel12B, and rCel12C, correspondingly. A synergistic action toward PASC among rCel12A and rCel12B is observed, thereby suggesting a potential application for preparing enzyme cocktails used in lignocellulose hydrolysis.
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http://dx.doi.org/10.1007/s11033-019-04999-xDOI Listing
October 2019

A novel thermostable chitinolytic machinery of sp. F-3 consisting of chitinases with different action modes.

Biotechnol Biofuels 2019 3;12:136. Epub 2019 Jun 3.

State Key Laboratory of Microbial Technology, Microbial Technology Institute, Shandong University, No. 72 Jimo Binhai Road, Qingdao, 266237 Shandong People's Republic of China.

Background: The biodegradation of chitin is an important part of the carbon and nitrogen cycles in nature. Speeding up the biotransformation of chitin substrates can not only reduce pollution, but also produce high value-added products. However, this process is strictly regulated by the catalytic efficiency of the chitinolytic machinery. Therefore, it is necessary to study the mode of action and compound mechanisms of different chitin-degrading enzymes in depth to improve the catalytic efficiency of the chitinolytic machinery.

Results: The thermophilic bacterium sp. F-3 showed comparatively high chitin degradation activities. To elucidate the mechanism underlying chitin hydrolysis, six chitin degradation-related enzymes were identified in the extracellular proteome of sp. F-3, including three chitinases (Chi18A, Chi18B, and Chi18C) from the GH18 family, one GH19 chitinase (Chi19A), one GH20 β--acetylhexosaminidase (GH20A), and one lytic polysaccharide monooxygenase (LPMO10A) from the AA10 family. All were upregulated by chitin. The heterologously expressed hydrolases could withstand temperatures up to 70 °C and were stable at pH values of 4 to 11. Biochemical analyses displayed that these chitin degradation-related enzymes had different functions and thus showed synergistic effects during chitin degradation. Furthermore, based on structural bioinformatics data, we speculated that the different action modes among the three GH18 chitinases may be caused by loop differences in their active site architectures. Among them, Chi18A is probably processive and mainly acts on polysaccharides, while Chi18B and Chi18C are likely endo-non-processive and displayed higher activity on the degradation of chitin oligosaccharides. In addition, proteomic data and synergy experiments also indicated the importance of LPMO10A, which could promote the activities of the hydrolases and increase the monosaccharide content in the reaction system, respectively.

Conclusions: In this article, the chitinolytic machinery of a thermophilic species was studied to explore the structural basis for the synergistic actions of chitinases from different GH18 subfamilies. The elucidation of the degradation mechanisms of these thermophilic chitinases will lay a theoretical foundation for the efficient industrialized transformation of natural chitin.
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http://dx.doi.org/10.1186/s13068-019-1472-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6545677PMC
June 2019

Prediction of Driver Modules via Balancing Exclusive Coverages of Mutations in Cancer Samples.

Adv Sci (Weinh) 2019 Feb 18;6(4):1801384. Epub 2018 Dec 18.

Department of Bioinformatics and Genomics College of Computing and Informatics The University of North Carolina at Charlotte 9201 University City Blvd Charlotte NC 28223 USA.

Mutual exclusivity of cancer driving mutations is a frequently observed phenomenon in the mutational landscape of cancer. The long tail of rare mutations complicates the discovery of mutually exclusive driver modules. The existing methods usually suffer from the problem that only few genes in some identified modules cover most of the cancer samples. To overcome this hurdle, an efficient method UniCovEx is presented via identifying mutually exclusive driver modules of balanced exclusive coverages. UniCovEx first searches for candidate driver modules with a strong topological relationship in signaling networks using a greedy strategy. It then evaluates the candidate modules by considering their coverage, exclusivity, and balance of coverage, using a novel metric termed exclusive entropy of modules, which measures how balanced the modules are. Finally, UniCovEx predicts sample-specific driver modules by solving a minimum set cover problem using a greedy strategy. When tested on 12 The Cancer Genome Atlas datasets of different cancer types, UniCovEx shows a significant superiority over the previous methods. The software is available at: https://sourceforge.net/projects/cancer-pathway/files/.
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http://dx.doi.org/10.1002/advs.201801384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6382311PMC
February 2019

New sesquiterpenoid glycoside from the rhizomes of .

Nat Prod Res 2020 Apr 8;34(8):1138-1145. Epub 2019 Jan 8.

School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, People's Republic of China.

Six sesquiterpenoids and four lignans (-) were isolated from the -BuOH extract of the rhizomes of . Among them, the new sesquiterpenoid glycoside named (4 , 5, 7)-hinesolone-11---ᴅ-glucopyranoside (), along with three known compounds (-) were first obtained from this genus. All the isolates were elucidated by spectroscopic analyses and chemical methods, and the absolute configurations were assigned by electronic circular dichroism spectroscopy technique. In addition, the cytotoxic bioassay of compound was evaluated and results showed it had no significant antitumor activity against human cancer cell lines MCF-7, HepG-2 and Hela.
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http://dx.doi.org/10.1080/14786419.2018.1553170DOI Listing
April 2020

MiR-34b/c-5p and the neurokinin-1 receptor regulate breast cancer cell proliferation and apoptosis.

Cell Prolif 2019 Jan 17;52(1):e12527. Epub 2018 Oct 17.

Department of Clinical Laboratory, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin's Clinical Research Center for Cancer, Tianjin Key Laboratory of Cancer Prevention and Therapy, Key Laboratory of Breast Cancer Prevention and Therapy of Educational Ministry, Tianjin Medical University, Tianjin, China.

Objectives: MiR-34 is a tumour suppressor in breast cancer. Neurokinin-1 receptor (NK1R), which is the predicted target of the miR-34 family, is overexpressed in many cancers. This study investigated the correlation and clinical significance of miR-34 and NK1R in breast cancer.

Materials And Methods: Western blotting, quantitative reverse transcription-PCR (qRT-PCR) and luciferase assays were conducted to analyse the regulation of NK1R by miR-34 in MDA-MB-231, MCF-7, T47D, SK-BR-3 and HEK-293 T cells. MiR-34b/c-5p, full-length NK1R (NK1R-FL) and truncated NK1R (NK1R-Tr) expression in fifty patients were quantified by qRT-PCR and correlated with their clinicopathological parameters. CCK-8 assays, colony formation assays and flow cytometry were used to measure cell proliferation and apoptosis in MDA-MB-231 and MCF-7 cells transfected with miR-34b/c-5p or NK1R-siRNA and before treatment with or without Substance P (SP), an endogenous peptide agonists of NK1R. The effect of NK1R antagonist aprepitant was also investigated. In vivo xenograft models were used to further verify the regulation of NK1R by miR-34b/c-5p.

Results: Expression levels of miR-34b/c-5p and NK1R-Tr, but not NK1R-FL, were associated with enhanced malignant potential, such as tumour stage and Ki67 expression. The overexpression of miR-34b/c-5p or NK1R silencing potently suppressed cell proliferation and induced G2/M phase arrest and the apoptosis of MDA-MB-231 and MCF-7 cells. The NK1R antagonist aprepitant had similar effects. In vivo studies confirmed that miR-34b/c-5p overexpression or NK1R silencing reduced the tumorigenicity of breast cancer. In addition, SP rescued the effects of miR-34b/c-5p overexpression or NK1R silencing on cell proliferation and apoptosis in vitro and in vivo assays.

Conclusions: MiR-34b/c-5p and NK1R contribute to breast cancer cell proliferation and apoptosis and are potential targets for breast cancer therapeutics.
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http://dx.doi.org/10.1111/cpr.12527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6430481PMC
January 2019

Integrated Functional-Omics Analysis of Thermomyces lanuginosus Reveals its Potential for Simultaneous Production of Xylanase and Substituted Xylooligosaccharides.

Appl Biochem Biotechnol 2019 Apr 28;187(4):1515-1538. Epub 2018 Sep 28.

The State Key Laboratory of Microbial Technology, Shandong University, 27 Shandanan Road, 250100, Jinan, People's Republic of China.

Thermophiles have several beneficial properties for the conversion of biomass at high temperatures. Thermomyces lanuginosus is a thermophilic filamentous fungus that was shown to secrete 40 glycoside hydrolases and 25 proteases when grown on different carbon sources. Among the 13 identified glycoside hydrolases with high expression levels, 9 were reduced sugar glycosidases (RSGs) belonging to seven GH families, and 7 of the 10 identified proteases were exopeptidases belonging to six different protease families. High expression of RSGs and exopeptidases may allow the fungus to efficiently degrade oligosaccharides and oligopeptides in saprophytic habitats. There were no xylan side chain-degrading enzymes predicted in the genome of T. lanuginosus, and only one thermophilic GH11 xylanase (g4601.t1) and one GH43 xylosidase (g3706.t1) were detected by liquid chromatography-mass spectrometry/mass spectrometry when T. lanuginosus grown on xylan, which led to the accumulation of substituted xylooligosaccharides (SXOS) during corncob xylan degradation where SXOS output made up more than 8% of the total xylan. The SXOS are beneficial prebiotics and important inducers for enzymes secretion of microorganisms. Thus, T. lanuginosus exhibits distinct advantages in utilizing cheap raw materials producing one thermostable xylanase and the high value-added SXOS as well as microbial inoculants to compost by batch fermentation.
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http://dx.doi.org/10.1007/s12010-018-2873-5DOI Listing
April 2019