Rational Design of a Yeast-derived 3',5'-bisphosphate Nucleotidase with Improved Substrate Specificity.

In recent years, a convenient phosphatase-coupled sulfotransferase assay method has been proven to be applicable to most sulfotransferases. The central principle of the method is that phosphatase specifically degrades 3'-phosphoadenosine-5'-phosphate (PAP) and leaves 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Our group previously acquired a yeast 3',5'-bisphosphate nucleotidase (YND), which showed a higher catalytic activity for PAP than PAPS and could be a potential phosphatase for the sulfotransferase assay. Here, we obtained a beneficial mutant of YND with markedly improved substrate specificity towards PAP via rational design. Of 9 chosen mutation sites in the active site pocket, the mutation G236D showed the best specificity for PAP. After optimization of the reaction conditions, the mutant YNDG236D displayed a 4.8-fold increase in the catalytic ratio PAP/PAPS compared to the wild-type. We subsequently applied YNDG236D to the assay of human SULT1A1 and SULT1A3 with their known substrate 1-naphthol, indicating that the mutant could be used to evaluate sulfotransferase activity by colorimetry. Analysis of the MD simulation results revealed that the improved substrate specificity of the mutant towards PAP may stem from a more stable protein conformation and the changed flexibility of key residues in the entrance of the substrate tunnel. This research will provide a valuable reference for the development of efficient sulfotransferase activity assays.

Regulator DegU can remarkably influence alkaline protease AprE biosynthesis in Bacillus licheniformis 2709.

Alkaline protease AprE, produced by Bacillus licheniformis 2709 is an important edible hydrolase, which has potential applications in nutrient acquisition and medicine. The expression of AprE is finely regulated by a complex transcriptional regulation system. However, there is little study on transcriptional regulation mechanism of AprE biosynthesis in Bacillus licheniformis, which limits system engineering and further enhancement of AprE. Here, the severely depressed expression of aprE in degU and degS deletion mutants illustrated that the regulator DegU and its phosphorylation played a crucial part in AprE biosynthesis. Further electrophoretic mobility shift assay (EMSA) in vitro indicated that phosphorylated DegU can directly bind to the regulatory region though the DNase I foot-printing experiments failed to observe protected region. The plasmid-mediated overexpression of degU32 (Hy) obviously improved the yield of AprE by 41.6 % compared with the control strain, which demonstrated the importance of phosphorylation state of DegU on the transcription of aprE in vivo. In this study, the putative binding sequence of aprE (5'-TAAAT……AAAAT…….AACAT…TAAAA-3') located upstream -91 to -87 bp, -101 to -97 bp, -195 to -191 bp, -215 to -211 bp of the transcription start site (TSS) in B. licheniformis was computationally identified based on the DNA-binding sites of DegU in Bacillus subtilis. Overall, we systematically investigated the influence of the interplay between phosphorylated DegU and its cognate DNA sequence on expression of aprE, which not only contributes to the further AprE high-production in a genetically modified host in the future, but also significantly increases our understanding of the aprE transcription mechanism.

Heterologous expression and fibrillary characterization of the microtubule-binding domain of tau associated with tauopathies.

BACKGROUND: Neurofibrillary tangles (NFTs) are one of the most common pathological characteristics of Alzheimer's disease. The NFTs are mainly composed of hyperphosphorylated microtubule-associated tau. Thus, recombinant tau is urgently required for the study of its fibrillogenesis and its associated cytotoxicity. METHODS AND RESULTS: Heterologous expression, purification, and fibrillation of the microtubule-binding domain (MBD) of tau (tauMBD) were performed. The tauMBD was heterologously expressed in E. coli. Ni-chelating affinity chromatography was then performed to purify the target protein. Thereafter, tauMBD was systematically identified using the SDS-PAGE, western blot and MALDI-TOF MS methods. The aggregation propensity of the tauMBD was explored by both the thioflavin T fluorescence and atomic force microscopy experiments. CONCLUSIONS: The final yield of the recombinant tauMBD was ~ 20 mg L-1. It is shown that TauMBD, in the absence of an inducer, self-assembled into the typical fibrils at a faster rate than wild-type tau. Finally, the in vitro cytotoxicity of tauMBD aggregates was validated using PC12 cells. The heterologously expressed tau in this study can be further used in the investigation of the biophysical and cellular cytotoxic properties of tau.

Heterologous expression and characterization of an M4 family extracellular metalloprotease for detergent application.

Proteolytic enzymes stand out as the most widely employed category utilized in manufacturing industry. A new protease was separated from Planococcus sp.11815 strain and named as nprS-15615 in this research. The gene of this protease has not been reported, and its enzymatic properties have been studied for the first time. To enhance enzyme production, the Planococcus sp. protease gene was expressed in Bacillus licheniformis 2709. The expression level of nprS-15615 was observed under the control of regulatory elements PaprE. nprS-15615 protease activity reached 1186.24±32.87 U/mL after 48 hours of cultivation in shake flasks which was nearly four times the output of the original bacteria (291.38±25.73U/mL). The optimum temperature and pH of the recombinant protease were 30 ℃ and 8.0, respectively.The enzyme exhibited the highest capacity for hydrolyzing casein and demonstrated resilience towards a NaCl concentration of 10.0% (wt/v). Furthermore, in the presence of 0.5% surfactants, the recombinant protease activity can maintain above 75%, and with the existence of 0.5% liquid detergents, there was basically no loss of enzyme activity which indicated that nprS-15615 had good compatibility with surfactants and liquid detergents. In addition, npS-15615 performed well in the washing experiment, and the washing effect at 20 ℃ can be significantly improved by adding crude enzyme solution in the washing process.

Site-Directed Mutation of Salicylate Decarboxylase Gene and Mechanism of Ginkgo Acid Decarboxylation.

Ginkgo seed is an important Chinese medicine and food resource in China, but the toxicity of ginkgo acid in it limits its application. Previous studies have found that salicylic acid decarboxylase (Sdc) has a decarboxylation degradation effect on ginkgo acid. In order to improve the decarboxylation ability of Sdc to Ginkgo acid, 11 residues of the Sdc around the substrate (salicylic acid) were determined as mutation targets according to the analysis of crystal structure of Sdc (PDB ID:6JQX), from Trichosporon moniliiforme WU-0401, and a total of 30 single point mutant enzymes and one compound mutant enzyme were obtained. With Ginkgo acid C15:1 as the substrate, it was found from activity assay that Sdc-Y64T and Sdc-P191A had higher decarboxylation activity, which increased by 105.18% and 116.74% compared with that of wild type Sdc, respectively. The optimal pH for Sdc Y64T and Sdc-P191A to decarboxylate Ginkgo acid C15:1 was 5.5, which is the same as the wild type Sdc. The optimal temperature of Sdc-P191A was 50 °C, which was consistent with that of the wild type Sdc, but the optimal temperature of the mutant Sdc-Y64T was 40 °C, which was 10 °C lower than that of wild type Sdc.

Ulvan inhibits α-synuclein fibrillation and disrupts the mature fibrils: In vitro and in vivo studies.

Misfolding and aggregation of α-synuclein (α-syn) play a key role in the pathogenesis of Parkinson's disease (PD). Herein, the inhibitory effect of ulvan on α-syn fibrillogenesis was studied using thioflavin T fluorescence and atomic force microscope assays. It is shown that ulvan could inhibit α-syn fibrillogenesis in a dose-dependent manner. Based on the circular dichroism results, it is found that ulvan delays greatly the conformational transition from its initial random coil to ß-sheet rich structure. The protective effect of ulvan against celllular death induced by α-syn aggregates was investigated by MTT colorimetric and cellular staining methods. It is found that ulvan protects greatly PC12 cells from α-syn fibril-induced cytotoxicity. In addition, ulvan disaggregates preformed α-syn fibrils and reduces cytotoxicity in a dose-dependent manner. Thereafter, the inhibitory effects of ulvan against α-syn fibrillogenesis were probed using Caenorhabditis elegans model NL5901 expressing human α-syn. It is found that ulvan extends the lifespan of NL5901 and recovers the lipid deposition by reducing the accumulation of α-syn. Finally, the molecular interactions between ulvan and α-syn pentamer was also explored using molecular docking. These findings suggest that ulvan can be pursued as a novel candidate drug for treatment of PD.

Construction of an alkaline protease overproducer strain based on Bacillus licheniformis 2709 using an integrative approach.

Bacillus licheniformis 2709 is a potential cell factory for the production of alkaline protease AprE, which has important value in industrial application but still lacks sufficient production capacity. To address this problem, we investigated the effects of the secretory viscous materials on the synthesis of AprE, which might seriously affect the industrial fermentation. Furthermore, an iterative chromosomal integration strategy at various chromosomal loci was implemented to achieve stable high-level expression of AprE in B. licheniformis 2709. The host was genetically modified by disrupting the native pgs cluster controlling the biosynthesis of viscous poly-glutamic acid identified in the study by GC/MS, generating a mutant with significantly higher biomass and better bioreactor performance. We further enhanced the expression of alkaline protease by integrating two additional aprE expression cassettes into the genome, generating the integration mutant BL ∆UEP-3 with three aprE expression cassettes, whose AprE enzyme activity in shake flasks reached 25,736 ± 997 U/mL, which was 136% higher than that of the original strain, while the aprE transcription level increased 4.05 times. Thus, an AprE high-yielding strain with excellent fermentation traits was engineered, which was more suitable for bulk-production. Finally, the AprE titer was further increased in a 5-L fermenter, reaching 57,763 ± 1039 U/mL. In summary, genetic modification is an enabling technology for enhancing enzyme production by eliminating the unfavorable characteristics of the host and optimizing the expression of aprE through iterative chromosomal integration. We believe that the protocol developed in this study provides a valuable reference for chromosomal overexpression of proteins or bioactive molecules in other Bacillus species.

Molecular basis for the inhibitory effects of 5-hydroxycyclopenicillone on the conformational transition of Aß40 monomer.

Previous studies have indicated that 5-hydroxycyclopenicillone (HCP), an active compound derived from marine sponge, could inhibit oligomerization of amyloid ß-protein (Aß). However, the molecular basis for the interaction between HCP and Aß remains unclear. Herein, all-atom molecular dynamics (MD) simulations were used to explore the conformational conversion of an Aß40 monomer at different concentrations (0-40 mM) of HCP at the atomic level. It is confirmed that the conformational transition of the Aß40 monomer is prevented by HCP in a concentration-dependent manner in silico. In 40 mM HCP solution, the initial α-helix-rich conformation of Aß40 monomer is kept under the action of HCP. The intra-peptide hydrophobic collapse and D23-K28 salt bridge are prevented by HCP. Moreover, it is indicated that the non-polar binding energy dominates the binding between HCP and Aß40 monomer as evaluated by molecular mechanics Poisson-Boltzmann surface area method. And, the residues of F4, Y10, V12, L17 and L34 in Aß40 might contribute to the binding energy in HCP-Aß40 complex. All these results elucidate the molecular mechanism underlying the inhibitory effects of HCP against the conformational transformation of Aß40, providing a support that HCP may be developed as a potential anti-Aß compound for the treatment of Aß-related diseases.Communicated by Ramaswamy H. Sarma.

Transcriptome based functional identification and application of regulator AbrB on alkaline protease synthesis in Bacillus licheniformis 2709.

Bacillus licheniformis 2709 is the major alkaline protease producer, which has great potential value of industrial application, but how the high-producer can be regulated rationally is still not completely understood. It's meaningful to understand the metabolic processes during alkaline protease production in industrial fermentation medium. Here, we collected the transcription database at various enzyme-producing stages (preliminary stage, stable phase and decline phase) to specifically research the synthesized and regulatory mechanism of alkaline protease in B. licheniformis. The RNA-sequencing analysis showed differential expression of numerous genes related to several processes, among which genes correlated with regulators were concerned, especially the major differential gene abrB on enzyme (AprE) synthesis was investigated. It was further verified that AbrB is a repressor of AprE by plasmid-mediated over-expression due to the severely descending enzyme activity (11,300 U/mL to 2695 U/mL), but interestingly it is indispensable for alkaline protease production because the enzyme activity of the null abrB mutant was just about 2279 U/mL. Thus, we investigated the aprE transcription by eliminating the theoretical binding site (TGGAA) of AbrB protein predicated by computational strategy, which significantly improved the enzyme activity by 1.21-fold and gene transcription level by 1.77-fold in the mid-log phase at a cultivation time of 18 h. Taken together, it is of great significance to improve the production strategy, control the metabolic process and oriented engineering by rational molecular modification of regulatory network based on the high throughput sequencing and computational prediction.

Insight into enzyme-catalyzed aziridine formation mechanism in ficellomycin biosynthesis.

Ficellomycin is an aziridine-containing antibiotic, produced by Streptomyces ficellus. Based on the newly identified ficellomycin gene cluster and the assigned functions of its genes, a possible pathway for aziridine ring formation in ficellomycin was proposed, which is a complex process involving at least 3 enzymatic steps. To obtain support for the proposed mechanism, the targeted genes encoding sulfate adenylyltransferase, adenylsulfate kinase, and a putative sulfotransferase were respectively disrupted and the subsequent analysis of their fermentation products revealed that all the three genes were involved in aziridine formation. To further confirm the mechanism, the key gene encoding a putative sulfotransferase was over expressed in Escherichia coli Rosseta (DE3). Enzyme assays indicated that the expressed sulfotransferase could specifically transfer a sulfo group from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) onto the hydroxyl group of (R)-(-)-2-pyrrolidinemethanol. This introduces a good leaving group in the form of the sulfated hydroxyl moiety, which is then converted into an aziridine ring through an intramolecular nucleophilic attack by the adjacent secondary amine. The sulfation/intramolecular cyclization reaction sequence maybe a general strategy for aziridine biosynthesis in microorganisms. Discovery of this mechanism revealed an enzyme-catalyzed route for the synthesis of aziridine-containing reagents and provided an important insight into the functional diversity of sulfotransferases.

Spo0A can efficiently enhance the expression of the alkaline protease gene aprE in Bacillus licheniformis by specifically binding to its regulatory region.

The expression of enzymes in Bacillus licheniformis, such as the valuable extracellular alkaline protease AprE, is highly regulated by a complex transcriptional regulation mechanism. Here, we found that the transcript abundance of aprE varies >343-fold in response to the supply of nutrients or to environmental challenges. To identify the underlying regulatory mechanism, the core promoter of aprE and several important upstream regulatory regions outside the promoter were firstly confirmed by 5'-RACE and mutagenesis experiments. The specific proteins that bind to the identified sequences were subsequently captured by DNA pull-down experiments, which yielded the transcriptional factors (TFs) Spo0A, CggR, FruR, YhcZ, as well as fragments of functionally unassigned proteins. Further electrophoretic mobility shift assay (EMSA) and DNase I foot-printing experiments indicated that Spo0A can directly bind to the region from -92 to -118 nucleotides upstream of the transcription start site, and the deletion of this specific region drastically decreased the production of AprE. Taken together, these results indicated that the expression of aprE was mainly regulated by the interplay between Spo0A and its cognate DNA sequence, which was successfully applied to overproduce AprE in a genetically modified host harboring three aprE expression cassettes. The DNA binding proteins may serve to increase the efficiency of transcription by creating an additional binding site for RNA polymerase. The discovery of this mechanism significantly increases our understanding of the aprE transcription mechanism, which is of great importance for AprE overproduction.

Optimized expression and enhanced production of alkaline protease by genetically modified Bacillus licheniformis 2709.

BACKGROUND: Bacillus licheniformis 2709 is extensively applied as a host for the high-level production of heterologous proteins, but Bacillus cells often possess unfavorable wild-type properties, such as production of viscous materials and foam during fermentation, which seriously influenced the application in industrial fermentation. How to develop it from a soil bacterium to a super-secreting cell factory harboring less undomesticated properties always plays vital role in industrial production. Besides, the optimal expression pattern of the inducible enzymes like alkaline protease has not been optimized by comparing the transcriptional efficiency of different plasmids and genomic integration sites in B. licheniformis. RESULT: Bacillus licheniformis 2709 was genetically modified by disrupting the native lchAC genes related to foaming and the eps cluster encoding the extracellular mucopolysaccharide via a markerless genome-editing method. We further optimized the expression of the alkaline protease gene (aprE) by screening the most efficient expression system among different modular plasmids and genomic loci. The results indicated that genomic expression of aprE was superior to plasmid expression and finally the transcriptional level of aprE greatly increased 1.67-fold through host optimization and chromosomal integration in the vicinity of the origin of replication, while the enzyme activity significantly improved 62.19% compared with the wild-type alkaline protease-producing strain B. licheniformis. CONCLUSION: We successfully engineered an AprE high-yielding strain free of undesirable properties and its fermentation traits could be applied to bulk-production by host genetic modification and expression optimization. In summary, host optimization is an enabling technology for improving enzyme production by eliminating the harmful traits of the host and optimizing expression patterns. We believe that these strategies can be applied to improve heterologous protein expression in other Bacillus species.

Semi-rational mutagenesis of an industrial Streptomyces fungicidicus strain for improved enduracidin productivity.

The biosynthesis of the valuable antibiotic enduracidin by Streptomyces fungicidicus TXX3120 is a complex multistep process. To identify the rate-limiting step of the entire biosynthetic process, we carried out a deep RNA sequencing towards the mycelia of TXX3120 at different fermentation stages. Comparative RNA-seq analysis indicated that the expression level of the endC gene during the enduracidin production phase was evidently lower than that of the other relevant genes to enduracidin biosynthesis. This result was further confirmed by quantitative RT-PCR, and the giant non-ribosomal peptide synthase (NRPS) encoded by endC was predicated to be the rate-limiting enzyme in enduracidin biosynthesis. To increase the expression of endC during the enduracidin production phase, a reporter-based selection system was developed by genetically replacing the initial part of the endC gene with a thiostrepton resistance gene (tsr), which will then act as a selectable marker to report the expression level of the rate-limiting gene endC, thereby facilitating the selection of enduracidin-overproducing mutants following random mutagenesis. After one round of mutagenesis, thiostrepton resistance selection, and restoration of the endC gene, three mutant strains with improved endC expression levels were obtained. Their highest enduracidin titers reached 9780.54, 9272.46, and 8849.06 U/mL, respectively representing 2.31-, 2.19-, and 2.09-fold of the initial industrial strain TXX3120. Our research provides a useful strategy for the rational breeding of industrial strains that synthesize complex natural products.

Identification of two novel highly inducible promoters from Bacillus licheniformis by screening transcriptomic data.

Bacillus licheniformis TCCC11148 is an important industrial strain used to produce alkaline protease. In this study, the transcriptome of B. licheniformis TCCC11148 was analyzed by high throughput RNA sequencing (RNA-Seq) to identify genes that are expressed differentially in the different phases were detected using RNA-Seq. In total, 440 differentially expressed genes between the 12 h and 48 h groups were identified, including 267 up- and 173 downregulated genes. Additionally, 198 differentially expressed genes were identified in the 48 h vs. the 60 h group, including 182 up- and 16 downregulated genes. To screen for novel inducible promoters, an alkaline protease reporter gene was used to test 24 promoters from 66 candidate genes with obviously higher expression levels (RPKM values) than the control group based on the transcriptome data of B. licheniformis in different phases. Gene 707, related to coenzyme transport and metabolism, and gene 1004, related to posttranslational modification were identified as likely having inducible promoters. The expression level of recombinant strains with reporter genes under the control of promoters p707 and p1004 were 8 times higher than that of the control group. This study contributes a method for finding useful inducible promoters for industrial use based on transcriptomic data.

Optimization of alkaline protease production by rational deletion of sporulation related genes in Bacillus licheniformis.

BACKGROUND: Our laboratory has constructed a Bacillus licheniformis strain that secretes alkaline protease (AprE) with excellent enzymatic properties. B. licheniformis is generally regarded as safe and has a high industrial exoenzyme secretion capacity, but the host retains some undomesticated characteristic that increase its competitiveness and survival, such as spore-formation, which increases the requirements and difficulties in industrial operations (e.g. sterilization and enzyme activity control). Furthermore, the influence of sporulation on alkaline protease production in B. licheniformis has not been elucidated in detail. RESULT: A series of asporogenic variants of the parent strain were constructed by individually knocking out the master regulator genes (spo0A, sigF and sigE) involved in sporulation. Most of the variants formed abortively disporic cells characterized by asymmetric septa at the poles and unable to survive incubation at 75 °C for 10 min. Two of them (ΔsigF and ΔsigE) exhibited superior characteristics in protease production, especially improving the expression of the aprE gene. Under the currently used fermentation conditions, the vegetative production phase of ΔsigF can be prolonged to 72 h, and the highest protease production of ΔsigF reached 29,494 ± 1053 U/mL, which was about 19.7% higher than that of the wild-type strain. CONCLUSION: We first constructed three key sporulation-deficient strain to investigate the effect of sporulation on alkaline protease synthesis. The sigF mutant retained important industrial properties such as facilitating the sterilization process, a prolonged stable phase of enzyme production and slower decreasing trend, which will be superior in energy conservation, simpler operations and target product controlling effect. In summary, the work provides a useful industrial host with preferable characteristics and a novel strategy to enhance the production of protease.

[Characterization of a novel bidirectional promoter in Bacillus subtilis].

Based on the transcriptome analysis data of a Bacillus licheniformis, a novel bidirectional promoter was identified from the strain and its transcriptional strength was analyzed. The expression level of a Bacillus clausii derived alkaline protease gene driven by the bidirectional promoter was studied by using the known strong constitutive promoter pShuttle-09 as a control. Three recombinant expression vectors and the corresponding recombinant bacteria were constructed. Under the control of the new promoter pLA and its reverse promoter pLB, the alkaline protease expression level respectively reached 164 U/mL and 111 U/mL. The results indicated that the transcription strength of pLA was significantly higher than that of pShuttle-09 and pLB, and both the pLA and pLB promoters could initiate the expression of the alkaline protease. Thus, it provides a new expression element for the heterogenous genes in Bacillus sp. and a new idea for the co-expression of two genes in one prokaryotic strain.

Rational design of a Yarrowia lipolytica derived lipase for improved thermostability.

To improve the thermostability of the lipase LIP2 from Yarrowia lipolytica, molecular dynamics (MD) simulations at various temperatures were used to investigate the common fluctuation sites of the protein, which are considered to be thermally weak points. Two of these residues were selected for mutations to improve the enzyme's thermostability, and the variants predicted by MD simulations to have improved thermostability were expressed in Pichia pastoris GS115 for further investigations. According to the proline rule, the high fluctuation site S115 or V213 was replaced with proline residue, the two lipase mutants S115P and V213P were obtained. The mutant V213P exhibited evidently enhanced thermostability with an approximately 70% longer half-life at 50 °C than that of the parent LIP2 expressed in P. pastoris. The temperature optimum of V213P was 42 °C, which was about 5.0 °C higher than that of the parent LIP2, while its specific catalytic activity was comparable to that of the parent and reached 876.5 U/mg. The improved thermostability of V213P together with its high catalytic efficiency indicated that the rational design strategy employed here can be efficiently applied for structure optimization of industrially important enzymes.

Engineered variants of a lipase from Yarrowia lipolytica with improved trypsin resistance for enzyme replacement therapy.

To improve the proteolytic stability of the lipase LIP2 from Yarrowia lipolytica, the peptide bonds susceptible to trypsin in LIP2 were analyzed by tandem mass spectrometry and redesigned by site-directed mutagenesis. Different variants of the enzyme were expressed in Pichia pastoris GS115 and their biochemical properties were subsequently investigated. Although most of the variants were still cleaved by trypsin, some of them did show an evident increase of resistance against proteolytic degradation. The most stable mutant was LIP2-C5, in which five trypsin-cleavage sites were replaced by non-preferred amino acids. Upon incubation with human trypsin for 80 min at 37°C, the mutant LIP2-C5 was found to retain >70% of its initial activity, compared to only 10% for the wild-type.

Development and application of a CRISPR/Cas9 system for Bacillus licheniformis genome editing.

A highly efficient genome editing system for Bacillus licheniformis was developed based on single-plasmid CRISPR/Cas9. For highly efficient genome editing the shuttle vector pWH1520 was selected to construct the knockout plasmids. A construct harboring a pS promoter driving cas9 endonuclease expression, a strong pLY-2 promoter driving the transcription of a single guide RNA was demonstrated as being the most effective. To verify the feasibility of the method the uprT gene coding uracil phosphoribosyltransferase was selected as the reporter gene. The efficiency of introducing nucleotide point mutations and single gene deletion reached an editing efficiency of up to 99.2% and 97.3%, respectively. After a upp-deficient strain was engineered, the system and strain were applied to introduce genomic deletions of another two genes, amyL and chiA (encoding amylase and chitinase, respectively) with about 90% deletion efficiency. As two native extracellular proteins with relatively high secretion in the host, amylase and chitinase can hamper the secretion and expression of alkaline protease. It was demonstrated that the mutant with deletions of the two genes effectively improved the alkaline protease yield by 24.8%. The results illustrated that the establishment of a CRISPR/Cas9 system for Bacillus licheniformis is of significance, and confirmed the system's high efficiency. The system provides support for effective molecular modification and metabolic regulation of Bacillus licheniformis, and offers promise for applications in genetic modification of other industrially relevant Bacillus species.

An acid-stable ß-glucosidase from Aspergillus aculeatus: Gene expression, biochemical characterization and molecular dynamics simulation.

ß-Glucosidases hydrolyze terminal, non-reducing ß-d-glucosyl residues and thereby release ß-d-glucose. They have applications in the production of biofuels, beverages and pharmaceuticals. In this study, a ß-glucosidase derived from Aspergillus aculeatus (BGLA) was expressed, characterized, and the molecular mechanism of its acid denaturation was comprehensively probed. BGLA exhibited maximal activity at pH 5.0-6.0. Its optimal temperature was 70 °C. Its enzyme activity was enhanced by Mg2+, Ca2+ and Ba2+, while Cu2+, Mn2+, Zn2+, Fe2+ and Fe3+ had a negative effect. BGLA showed activity on a broad range of substrates including salicin, cellobiose, arbutin, geniposide and polydatin. Finally, the acid-denaturation mechanism of BGLA was probed using molecular dynamics (MD) simulations. The results of simulation at pH 2.0 imply that the contact number, solvent accessible surface area and number of hydrogen bonds in BGLA decreased greatly. Moreover, the distance between the residues Asp280 and Glu509 that are part of the active site increased, which eventually destroyed the enzyme's catalytic activity. These MD results explain the molecular mechanism of acid denaturation of BGLA, which will greatly benefit the rational design of more acid-stable ß-glucosidase variants in the future.