Improving the catalytic activity and thermostability of Aspergillus niger xylanase through computational design.

Xylanase plays the most important role in catalyzing xylan to xylose moieties. GH11 xylanases have been widely used in many fields, but most GH11 xylanases are mesophilic enzymes. To improve the catalytic activity and thermostability of Aspergillus niger xylanase (Xyn-WT), we predicted potential key mutation sites of Xyn-WT through multiple computer-aided enzyme engineering strategies. We introduce a simple and economical Ni affinity chromatography purification method to obtain high-purity xylanase and its mutants. Ten mutants (Xyn-A, Xyn-B, Xyn-C, E45T, Q93R, E45T/Q93R, A161P, Xyn-D, Xyn-E, Xyn-F) were identified. Among the ten mutants, four (Xyn-A, Xyn-C, A161P, Xyn-F) presented improved thermal stability and activity, with Xyn-F(A161P/E45T/Q93R) being the most thermally stable and active. Compared with Xyn-WT, after heat treatment at 55 °C and 60 °C for 10 min, the remaining enzyme activity of Xyn-F was 12 and 6 times greater than that of Xyn-WT, respectively, and Xyn-F was approximately 1.5 times greater than Xyn-WT when not heat treated. The pH adaptation of Xyn-F was also significantly enhanced. In summary, an improved catalytic activity and thermostability of the design variant Xyn-F has been reported.

Bilirubin oxidase expression and activity enhancement from Myrothecium verrucaria in Aspergillus species.

We constructed a new Aspergillus expression vector (pSENSU2512nid) under the control of the enolase promoter with 12 tandem repeats of cis-acting elements (region III) and the heat shock protein 12 (Hsp12) 5' untranslated region (UTR). Bilirubin oxidase (EC: 1.3.3.5) from Myrothecium verrucaria, which catalyzes the oxidation of bilirubin to biliverdin, was overexpressed in Aspergillus oryzae and A. niger. The productivity was estimated to be approximately 1.2 g/L in the culture broth, which was approximately 6-fold higher than that of recombinant bilirubin oxidase (BOD) expressed in Pichia pastoris (Komagataella phaffii). BOD was purified using hydrophobic interaction chromatography, followed by ion exchange chromatography. The specific activity of the purified BOD against 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) substrate was 57.6 U/mg and 66.4 U/mg for A. oryzae and A. niger, respectively. l-Ascorbic acid (4 mM) addition and storage under deoxygenated conditions for 3-7 d increased the specific activity of these Aspergillus-expressed BODs approximately 2.3-fold (154.1 U/mg). The BOD specific activity was enhanced by incubation at higher temperature (30-50 °C). Further characterization of the enzyme catalytic efficiency revealed that the Km value remained unchanged, whereas the kcat value improved 3-fold. In conclusion, this high-level of BOD expression meets the requirements for industrial-level production. Additionally, we identified an effective method to enhance the low specific activity during expression, making it advantageous for industrial applications.

Unveiling the synthesis of aromatic compounds in sauce-flavor Daqu from the functional microorganisms to enzymes.

Aromatic compounds serve as the primary source of floral and fruity aromas in sauce-flavor (Maotai flavor) baijiu, constituting the skeleton components of its flavor profile. Nevertheless, the formation mechanism of these compounds and key aroma-producing enzymes in sauce-flavor Daqu (fermentation agent, SFD) remain elusive. Here, we combined metagenomics, metaproteomics, metabolomics, and key enzyme activity to verify the biosynthesis pathway of aromatic compounds and to identify key enzymes, genes, and characteristic microorganisms in SFD. The results showed that the later period of fermentation was critical for the generation of aromatic compounds in SFD. In-situ verification was conducted on the potential key enzymes and profiles in various metabolites, providing comprehensive evidence for the main synthetic pathways of aromatic compounds in SFD. Notably, our results showed that primary amine oxidase (PrAO) and aldehyde dehydrogenase (ALDH) emerged as two key enzymes promoting aromatic compound synthesis. Additionally, two potential key functional genes regulating aromatics generation were identified during SFD fermentation through correlation analysis between proteins and relevant metabolites, coupled with in vitro amplification test. Furthermore, original functional strains (Aspergillus flavus-C10 and Aspergillus niger-IN2) exhibiting high PrAO and ALDH production were successfully isolated from SFD, thus validating the results of metagenomics and metaproteomics analyses. This study comprehensively elucidates the pathway of aromatic compound formation in SFD at the genetic, proteomic, enzymatic, and metabolomic levels, providing new ideas for the investigation of key flavor substances in baijiu. Additionally, these findings offer valuable insights into the regulatory mechanisms of aromatic compounds generation.

[Construction and culture condition optimization of a Saccharomyces cerevisiae strain for production of galactitol].

Galactitol, a rare sugar alcohol, has promising potential in the food industry and pharmaceutical field. The available industrial production methods rely on harsh hydrogenation processes, which incur high costs and environmental concerns. It is urgent to develop environmentally friendly and efficient biosynthesis technologies. In this study, a xylose reductase named AnXR derived from Aspergillus niger CBS 513.88 was identified and characterized for the enzymatic properties. AnXR exhibited the highest activity at 25 ℃ and pH 8.0, and it belonged to the NADPH-dependent aldose reductase family. To engineer a strain for galactitol production, we deleted the galactokinase (GAL1) gene in Saccharomyes cerevisiae by using the recombinant gene technology, which significantly reduced the metabolic utilization of D-galactose by host cells. Subsequently, we introduced the gene encoding AnXR into this modified strain, creating an engineered strain capable of catalyzing the conversion of D-galactose into galactitol. Furthermore, we optimized the whole-cell catalysis conditions for the engineered strain, which achieved a maximum galactitol yield of 12.10 g/L. Finally, we tested the reduction ability of the strain for other monosaccharides and discovered that it could produce functional sugar alcohols such as xylitol and arabinitol. The engineered strain demonstrates efficient biotransformation capabilities for galactitol and other functional sugar alcohols, representing a significant advancement in environmentally sustainable production practices.

Low frequency magnetic field assisted production of acidic protease by Aspergillus niger.

Acid protease is widely used in industries such as food processing and feed additives. In the study, low frequency magnetic field (LF-MF) as an aid enhances acid protease production by Aspergillus niger (A. niger). The study assessed mycelial biomass, the enzymic activity of the acidic protease and underlying mechanism. At low intensities, alternating magnetic field (AMF) is more effective than static magnetic fields (SMF). Under optimal magnetic field conditions, acid protease activity and biomass increased by 91.44% and 16.31%, as compared with the control, respectively. Maximum 19.87% increase in enzyme activity after magnetic field treatment of crude enzyme solution in control group. Transcriptomics analyses showed that low frequency alternating magnetic field (LF-AMF) treatment significantly upregulated genes related to hydrolases and cell growth. Our results showed that low-frequency magnetic fields can enhance the acid protease production ability of A. niger, and the effect of AMF is better at low intensities. The results revealed that the effect of magnetic field on the metabolic mechanism of A. niger and provided a reference for magnetic field-assisted fermentation of A. niger.

Evaluation of different glycerol fed-batch strategies in a lab-scale bioreactor for the improved production of a novel engineered ß-fructofuranosidase enzyme in Pichia pastoris.

The ß-fructofuranosidase enzyme from Aspergillus niger has been extensively used to commercially produce fructooligosaccharides from sucrose. In this study, the native and an engineered version of the ß-fructofuranosidase enzyme were expressed in Pichia pastoris under control of the glyceraldehyde-3-phosphate dehydrogenase promoter, and production was evaluated in bioreactors using either dissolved oxygen (DO-stat) or constant feed fed-batch feeding strategies. The DO-stat cultivations produced lower biomass concentrations but this resulted in higher volumetric activity for both strains. The native enzyme produced the highest volumetric enzyme activity for both feeding strategies (20.8% and 13.5% higher than that achieved by the engineered enzyme, for DO-stat and constant feed, respectively). However, the constant feed cultivations produced higher biomass concentrations and higher volumetric productivity for both the native as well as engineered enzymes due to shorter process time requirements (59 h for constant feed and 155 h for DO-stat feed). Despite the DO-stat feeding strategy achieving a higher maximum enzyme activity, the constant feed strategy would be preferred for production of the ß-fructofuranosidase enzyme using glycerol due to the many industrial advantages related to its enhanced volumetric enzyme productivity.

Antioxidant capacity of xylooligosaccharides generated from beechwood xylan by recombinant family GH10 Aspergillus niger xylanase A and insights into the enzyme's competitive inhibition by riceXIP.

In this study, the family GH10 xylanase AnXylA10 derived from Aspergillus niger JL15 strain was expressed in Pichia pastoris X33. The recombinant xylanase, reAnXylA10 exhibited optimal activity at 40 ℃ and pH 5.0. The hydrolysates generated from beechwood xylan using reAnXylA10 primarily consisted of xylobiose (X2) to xylohexaose (X6) and demonstrated remarkable antioxidant capacity. Furthermore, the rice xylanase inhibitory protein (riceXIP) was observed to competitively inhibit reAnXylA10, exhibiting an inhibition constant (Ki) of 140.6 nM. Molecular dynamics (MD) simulations of AnXylA10-riceXIP complex revealed that the α-7 helix (Q225-S238) of riceXIP intruded into the catalytic pocket of AnXylA10, thereby obstructing substrate access to the active site. Specifically, residue K226 of riceXIP formed robust interactions with E136 and E242, the two catalytic sites of AnXylA10, predominantly through high-occupied hydrogen bonds. Based on QTAIM, electron densities for the atom pairs K226riceXIP@HZ1-E136AnXylA10@OE2 and K226riceXIP@HZ3-E242AnXylA10@OE1 were determined to be 0.04628 and 0.02914 a.u., respectively. Binding free energy of AnXylA10-riceXIP complex was -59.0±7.6 kcal/mol, significantly driven by electrostatic and van der Waals forces. Gaining insights into the interaction between xylanase and its inhibitors, and mining the inhibition mechanism in depth, will facilitate the design of innovative GH10 family xylanases that are both highly efficient and resistant to inhibitors.

Enhancing Acid Resistance of Aspergillus niger Pectin Lyase through Surface Charge Design for Improved Application in Juice Clarification.

Pectin lyases (PNLs) can enhance juice clarity and flavor by degrading pectin in highly esterified fruits, but their inadequate acid resistance leads to rapid activity loss in juice. This study aimed to improve the acid resistance of Aspergillus niger PNL pelA through surface charge design. A modification platform was established by fusing pelA with a protein tag and expressing the fusion enzyme in Escherichia coli. Four single-point mutants were identified to increase the surface charge using computational tools. Moreover, the combined mutant M6 (S514D/S538E) exhibited 99.8% residual activity at pH 3.0. The M6 gene was then integrated into the A. niger genome using a multigene integration system to obtain the recombinant PNL AM6. Notably, AM6 improved the light transmittance of orange juice to 45.3%, which was 8.39 times higher than that of pelA. In conclusion, AM6 demonstrated the best-reported acid resistance, making it a promising candidate for industrial juice clarification.

Heterologous expression and characterization of mutant cellulase from indigenous strain of Aspergillus niger.

The purpose of current research work was to investigate the effect of mutagenesis on endoglucanase B activity of indigenous strain of Aspergillus niger and its heterologous expression studies in the pET28a+ vector. The physical and chemical mutagens were employed to incorporate mutations in A. niger. For determination of mutations, mRNA was isolated followed by cDNA synthesis and cellulase gene was amplified, purified and sequenced both from native and mutant A. niger. On comparison of gene sequences, it was observed that 5 nucleotide base pairs have been replaced in the mutant cellulase. The mutant recombinant enzyme showed 4.5 times higher activity (428.5 µmol/mL/min) as compared to activity of native enzyme (94 µmol/mL/min). The mutant gene was further investigated using Phyre2 and I-Tesser tools which exhibited 71% structural homology with Endoglucanase B of Thermoascus aurantiacus. The root mean square deviation (RMSD), root mean square fluctuation (RMSF), solvent accessible surface area (SASA), radius of gyration (Rg) and hydrogen bonds analysis were carried at 35°C and 50°C to explore the integrity of structure of recombinant mutant endoglucanase B which corresponded to its optimal temperature. Hydrogen bonds analysis showed more stability of recombinant mutant endoglucanase B as compared to native enzyme. Both native and mutant endoglucanase B genes were expressed in pET 28a+ and purified with nickel affinity chromatography. Theoretical masses determined through ExPaSy Protparam were found 38.7 and 38.5 kDa for native and mutant enzymes, respectively. The optimal pH and temperature values for the mutant were 5.0 and 50°C while for native these were found 4.0 and 35°C, respectively. On reacting with carboxy methyl cellulose (CMC) as substrate, the mutant enzyme exhibited less Km (0.452 mg/mL) and more Vmax (50.25 µmol/ml/min) as compared to native having 0.534 mg/mL as Km and 38.76 µmol/ml/min as Vmax. Among metal ions, Mg2+ showed maximum inducing effect (200%) on cellulase activity at 50 mM concentration followed by Ca2+ (140%) at 100 mM concentration. Hence, expression of a recombinant mutant cellulase from A. niger significantly enhanced its cellulytic potential which could be employed for further industrial applications at pilot scale.

Metabolic engineering of Aspergillus niger for accelerated malic acid biosynthesis by improving NADPH availability.

Microbial production of L-malic acid from renewable carbon sources has attracted extensive attention. The reduced cofactor NADPH plays a key role in biotransformation because it participates in both biosynthetic reactions and cellular stress responses. In this study, NADPH or its precursors nicotinamide and nicotinic acid were added to the fermentation medium of Aspergillus niger RG0095, which significantly increased the yield of malic acid by 11%. To further improve the titer and productivity of L-malic acid, we increased the cytoplasmic NADPH levels of A. niger by upregulating the NAD kinases Utr1p and Yef1p. Biochemical analyses demonstrated that overexpression of Utr1p and Yef1p reduced oxidative stress, while also providing more NADPH to catalyze the conversion of glucose into malic acid. Notably, the strain overexpressing Utr1p reached a malate titer of 110.72 ± 1.91 g L-1 after 108 h, corresponding to a productivity of 1.03 ± 0.02 g L-1 h-1. Thus, the titer and productivity of malate were increased by 24.5% and 44.7%, respectively. The strategies developed in this study may also be useful for the metabolic engineering of fungi to produce other industrially relevant bulk chemicals.

The pleiotropic phenotype of FlbA of Aspergillus niger is explained in part by the activity of seven of its downstream-regulated transcription factors.

Inactivation of flbA in Aspergillus niger results in thinner cell walls, increased cell lysis, abolished sporulation, and an increased secretome complexity. A total of 36 transcription factor (TF) genes are differentially expressed in ΔflbA. Here, seven of these genes (abaA, aslA, aslB, azf1, htfA, nosA, and srbA) were inactivated. Inactivation of each of these genes affected sporulation and, with the exception of abaA, cell wall integrity and protein secretion. The impact on secretion was strongest in the case of ΔaslA and ΔaslB that showed increased pepsin, cellulase, and amylase activity. Biomass was reduced of agar cultures of ΔabaA, ΔaslA, ΔnosA, and ΔsrbA, while biomass was higher in liquid shaken cultures of ΔaslA and ΔaslB. The ΔaslA and ΔhtfA strains showed increased resistance to H2O2, while ΔaslB was more sensitive to this reactive oxygen species. Together, inactivation of the seven TF genes impacted biomass formation, sporulation, protein secretion, and stress resistance, and thereby these genes explain at least part of the pleiotropic phenotype of ΔflbA of A. niger.

Conditional expression of FumA in Aspergillus niger enhances synthesis of L-malic acid.

Currently, the L-malic acid titer achieved through Aspergillus niger fermentation reaches 201 g/L, meeting industrial demands satisfactorily. However, the co-presence of structurally similar fumaric acid and succinic acid in fermentation products suggests a theoretical potential for further improvement in L-malic acid production. In the tricarboxylic acid cycle, fumarate reductase mediates the conversion of succinic acid to fumaric acid. Subsequently, fumarase catalyzes the conversion of fumaric acid to L-malic acid. Notably, both enzymatic reactions are reversible. Our investigation revealed that A. niger contains only one mitochondria-located fumarase FumA. Employing CRISPR-Cas9 technology, we performed a replacement of the fumA promoter with a doxycycline-induced promoter Tet. Under non-inducing condition, the conditional strain exhibited increased levels of fumaric acid and succinic acid. It strongly suggests that FumA mainly promotes the flow of fumaric acid to L-malic acid. Furthermore, a promoter PmfsA that is exclusively activated in a fermentation medium by calcium carbonate was identified through RNA-sequencing screening. Utilizing PmfsA to regulate fumA expression led to a 9.0% increase in L-malic acid titer, an 8.75% increase in yield (glucose to L-malic acid), and an 8.86% enhancement in productivity. This research serves as a significant step toward expediting the industrialization of L-malic acid synthesis via biological fermentation. Additionally, it offers valuable insights for the biosynthesis of other organic acids.IMPORTANCEThis study focuses on enhancing L-malic acid synthesis by modifying the tricarboxylic acid cycle within the mitochondria of Aspergillus niger. We emphasize the significant role of fumarase in converting fumaric acid into L-malic acid, enhancing our understanding of metabolic pathways in A. niger. The precise regulation of fumA is highlighted as a key factor in enhancing L-malic acid production. Furthermore, this research introduces a stringent conditional promoter (PmfsA), exclusively activated by CaCO3. The utilization of PmfsA for fumA expression resulted in heightened L-malic acid titers. The progress in metabolic engineering and bioprocess optimization holds promise for expediting industrial L-malic acid synthesis via biological fermentation. Moreover, it carries implications for the biosynthesis of various other organic acids.

Effective synthesis of high-content fructooligosaccharides in engineered Aspergillus niger.

BACKGROUND: Aspergillus niger ATCC 20611 is an industrially important fructooligosaccharides (FOS) producer since it produces the ß-fructofuranosidase with superior transglycosylation activity, which is responsible for the conversion of sucrose to FOS accompanied by the by-product (glucose) generation. This study aims to consume glucose to enhance the content of FOS by heterologously expressing glucose oxidase and peroxidase in engineered A. niger. RESULTS: Glucose oxidase was successfully expressed and co-localized with ß-fructofuranosidase in mycelia. These mycelia were applied to synthesis of FOS, which possessed an increased purity of 60.63% from 52.07%. Furthermore, peroxidase was expressed in A. niger and reached 7.70 U/g, which could remove the potential inhibitor of glucose oxidase to facilitate the FOS synthesis. Finally, the glucose oxidase-expressing strain and the peroxidase-expressing strain were jointly used to synthesize FOS, which content achieved 71.00%. CONCLUSIONS: This strategy allows for obtaining high-content FOS by the multiple enzymes expressed in the industrial fungus, avoiding additional purification processes used in the production of oligosaccharides. This study not only facilitated the high-purity FOS synthesis, but also demonstrated the potential of A. niger ATCC 20611 as an enzyme-producing cell factory.

Enhancing l-Malic Acid Production in Aspergillus niger via Natural Activation of sthA Gene Expression.

The efficient production of l-malic acid using Aspergillus niger requires overcoming challenges in synthesis efficiency and excessive byproduct buildup. This study addresses these hurdles, improving the activity of NADH-dependent malate dehydrogenase (Mdh) in the early stages of the fermentation process. By employing a constitutive promoter to express the Escherichia coli sthA responsible for the transfer of reducing equivalents between NAD(H) and NADP(H) in A. niger, the l-malic acid production was significantly elevated. However, this resulted in conidiation defects of A. niger, limiting industrial viability. To mitigate this, we discovered and utilized the PmfsA promoter, enabling the specific expression of sthA during the fermentation stage. This conditional expression strain showed similar phenotypes to its parent strain while exhibiting exceptional performance in a 5 L fermenter. Notably, it achieved a 65.5% increase in productivity, reduced fermentation cycle by 1.5 days, and lowered succinic acid by 76.2%. This work marks a promising advancement in industrial l-malic acid synthesis via biological fermentation, showcasing the potential of synthetic biology in A. niger for broader applications.

Cultivation of recombinant Aspergillus niger strains on dairy whey as a carbohydrate source.

Agricultural waste valorisation provides a sustainable solution to waste management, and combining waste utilisation with commodity production allows for responsible production processes. Recombinant Aspergillus niger D15 strains expressing fungal endoglucanases (Trichoderma reesei eg1 and eg2 and Aspergillus carneus aceg) were evaluated for their ability to utilise lactose as a carbon source to determine whether dairy waste could be used as a feedstock for enzyme production. The recombinant A. niger D15[eg1]PyrG, D15[eg2]PyrG, and D15[aceg]PyrG strains produced maximum endoglucanase activities of 34, 54, and 34 U/mL, respectively, on lactose and 23, 27, and 22 U/mL, respectively, on whey. The A. niger D15[eg2]PyrG strain was used to optimise the whey medium. Maximum endoglucanase activity of 46 U/mL was produced on 10% whey medium containing 0.6% NaNO3. The results obtained indicate that dairy whey can be utilised as a feedstock for recombinant enzyme production. However, variations in enzyme activities were observed and require further investigation.

Characterization of a recombinant Aspergillus niger GZUF36 lipase immobilized by ionic liquid modification strategy.

Enzyme immobilized on magnetic nanomaterials is a promising biocatalyst with efficient recovery under applied magnets. In this study, a recombinant extracellular lipase from Aspergillus niger GZUF36 (PEXANL1) expressed in Pichia pastoris GS115 was immobilized on ionic liquid-modified magnetic nano ferric oxide (Fe3O4@SiO2@ILs) via electrostatic and hydrophobic interaction. The morphology, structure, and properties of Fe3O4@SiO2@ILs and immobilized PEXANL1 were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, x-ray diffraction, vibration sample magnetometer, and zeta potential analysis. Under optimized conditions, the immobilization efficiency and activity recovery of immobilized PEXANL1 were 52 ± 2% and 122 ± 2%, respectively. The enzymatic properties of immobilized PEXANL1 were also investigated. The results showed that immobilized PEXANL1 achieved the maximum activity at pH 5.0 and 45 °C, and the lipolytic activity of immobilized PEXANL1 was more than twice that of PEXANL1. Compared to PEXANL1, immobilized PEXANL1 exhibited enhanced tolerance to temperature, metal ions, surfactants, and organic solvents. The operation stability experiments revealed that immobilized PEXANL1 maintained 86 ± 3% of its activity after 6 reaction cycles. The enhanced catalytic performance in enzyme immobilization on Fe3O4@SiO2@ILs made nanobiocatalysts a compelling choice for bio-industrial applications. Furthermore, Fe3O4@SiO2@ILs could also benefit various industrial enzymes and their practical uses. KEY POINTS: • Immobilized PEXANL1 was confirmed by SEM, FT-IR, and XRD. • The specific activity of immobilized PEXANL1 was more than twice that of PEXANL1. • Immobilized PEXANL1 had improved properties with good operational stability.

Improving glucose oxidase catalysis in Aspergillus niger via Vitreoscilla hemoglobin fusion protein.

Oxygen is crucial for converting glucose to gluconic acid catalyzed by glucose oxidase (Gox). However, industrial gluconic acid production faces oxygen supply limitations. To enhance Gox efficiency, Vitreoscilla hemoglobin (VHb) has been considered as an efficient oxygen transfer carrier. This study identified GoxA, a specific isoform of Gox in the industrial gluconic acid-producing strain of Aspergillus niger. Various forms of VHb expression in A. niger were tested to improve GoxA's catalytic efficiency. Surprisingly, the expression of free VHb, both intracellularly and extracellularly, did not promote gluconic acid production during shake flask fermentation. Then, five fusion proteins were constructed by linking Gox and VHb using various methods. Among these, VHb-GS1-GoxA, where VHb's C-terminus connected to GoxA's N-terminus via the flexible linker GS1, demonstrated a significantly higher Kcat/Km value (96% higher) than GoxA. Unfortunately, the expression of VHb-GS1-GoxA in A. niger was limited, resulting in a low gluconic acid production of 3.0 g/L. To overcome the low expression problem, single- and dual-strain systems were designed with tools of SpyCatcher/SpyTag and SnoopCatcher/SnoopTag. In these systems, Gox and VHb were separately expressed and then self-assembled into complex proteins. Impressively, the single-strain system outperformed the GoxA overexpression strain S1971, resulting in 23% and 9% higher gluconic acid production under 0.6 vvm and 1.2 vvm aeration conditions in the bioreactor fermentation, respectively. The successful construction of Gox and VHb fusion or complex proteins, as proposed in this study, presents promising approaches to enhance Gox catalytic efficiency and lower aerodynamic costs in gluconic acid production. KEY POINTS: • Overexpressing free VHb in A. niger did not improve the catalytic efficiency of Gox • The VHb-GS1-GoxA showed an increased Kcat/Km value by 96% than GoxA • The single-strain system worked better in the gluconic acid bioreactor fermentation.

Development of a two-enzyme system in Aspergillus niger for efficient production of N-acetyl-ß-D-glucosamine from powdery chitin.

A chitinase (PbChi70) from Paenibacillus barengoltzii was engineered by directed evolution to enhance its hydrolysis efficiency towards powder chitin. Through two rounds of screening, a mutant (mPbChi70) with a maximum specific activity of 73.21 U/mg was obtained, which is by far the highest value ever reported. The mutant gene was further transformed into Aspergillus niger FBL-B (ΔglaA) which could secrete high level of endogenously ß-N-acetylglucosaminidase (GlcNAcase), thus a two-enzyme expression system was constructed. The highest chitinase activity of 61.33 U/mL with GlcNAcase activity of 353.1 U/mL was obtained in a 5-L fermentor by high-cell density fermentation. The chitin-degrading enzyme cocktail was used for the bioconversion of GlcNAc from powder chitin directly, and the highest conversion ratio reached high up to 71.9 % (w/w) with GlcNAc purity ≥95 % (w/w). This study may provide an excellent chitinase as well as a double enzyme cocktail system for efficient biological conversion of chitin materials.

[Expression of ß-xylosidase An-xyl from Aspergillus niger and characterization of its xylose tolerance].

The hydrolysis of xylo-oligosaccharides catalyzed by ß-xylosidase plays an important role in the degradation of lignocellulose. However, the enzyme is easily inhibited by its catalytic product xylose, which severely limits its application. Based on molecular docking, this paper studied the xylose affinity of Aspergillus niger ß-xylosidase An-xyl, which was significantly differentially expressed in the fermentation medium of tea stalks, through cloning, expression and characterization. The synergistic degradation effect of this enzyme and cellulase on lignocellulose in tea stems was investigated. Molecular docking showed that the affinity of An-xyl to xylose was lower than that of Aspergillus oryzae ß-xylosidase with poor xylose tolerance. The Ki value of xylose inhibition constant of recombinant-expressed An-xyl was 433.2 mmol/L, higher than that of most ß-xylosidases of the GH3 family. The Km and Vmax towards pNPX were 3.6 mmol/L and 10 000 µmol/(min·mL), respectively. The optimum temperature of An-xyl was 65 ℃, the optimum pH was 4.0, 61% of the An-xyl activity could be retained upon treatment at 65 ℃ for 300 min, and 80% of the An-xyl activity could be retained upon treatment at pH 2.0-8.0 for 24 h. The hydrolysis of tea stem by An-xyl and cellulase produced 19.3% and 38.6% higher reducing sugar content at 2 h and 4 h, respectively, than that of using cellulase alone. This study showed that the An-xyl mined from differential expression exhibited high xylose tolerance and higher catalytic activity and stability, and could hydrolyze tea stem lignocellulose synergistically, which enriched the resource of ß-xylosidase with high xylose tolerance, thus may facilitate the advanced experimental research and its application.

CRISPR/Cas9 System-Mediated Multi-copy Expression of an Alkaline Serine Protease in Aspergillus niger for the Production of XOD-Inhibitory Peptides.

CRISPR/Cas9 system-mediated multi-copy expression of an alkaline serine protease (AoproS8) from Aspergillus oryzae was successfully built in Aspergillus niger. Furthermore, AoproS8 was continuously knocked in the glaA, amyA, and aamy gene loci in A. niger to construct multi-copy expression strains. The yield of the AoproS8 3.0 strain was 2.1 times higher than that of the AoproS8 1.0 strain. Then, a high protease activity of 11,023.2 U/mL with a protein concentration of 10.8 mg/mL was obtained through fed-batch fermentation in a 5 L fermenter. This is the first report on the high-level expression of alkaline serine proteases in A. niger. AoproS8 showed optimal activity at pH 9.0 and 40 °C. It was used for the production of xanthine oxidase (XOD)-inhibitory peptides from eight food processing protein by-products. Among them, the duck hemoglobin hydrolysates showed the highest XOD-inhibitory activity with an IC50 value of 2.39 mg/mL. Thus, our work provides a useful way for efficient expression of proteases in A. niger and high-value utilization of protein by-products.