Biosynthesis of ß-nicotinamide mononucleotide from glucose via a new pathway in Bacillus subtilis.

Introduction: ß-nicotinamide mononucleotide (ß-NMN) is an essential precursor of nicotinamide adenine dinucleotide (NAD+) and plays a key role in supplying NAD+ and maintaining its levels. Existing methods for NMN production have some limitations, including low substrate availability, complex synthetic routes, and low synthetic efficiency, which result in low titers and high costs. Methods: We constructed high-titer, genetically engineered strains that produce NMN through a new pathway. Bacillus subtilis WB600 was used as a safe chassis strain. Multiple strains overexpressing NadE, PncB, and PnuC in various combinations were constructed, and NMN titers of different strains were compared via shake-flask culture. Results: The results revealed that the strain B. subtilis PncB1-PnuC exhibited the highest total and extracellular NMN titers. Subsequently, the engineered strains were cultured in a 5-L fermenter using batch and fed-batch fermentation. B. subtilis PncB1-PnuC achieved an NMN titer of 3,398 mg/L via fed-batch fermentation and glucose supplementation, which was 30.72% higher than that achieved via batch fermentation. Discussion: This study provides a safe and economical approach for producing NMN on an industrial scale.

[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.

Molecular simulations guide immobilization of lipase on nest-like ZIFs with regulatable hydrophilic/hydrophobic surface.

The catalytic performance of immobilized lipase is greatly influenced by functional support, which attracts growing interest for designing supports to achieve their promotive catalytic activity. Many lipases bind strongly to hydrophobic surfaces where they undergo interfacial activation. Herein, the behavioral differences of lipases with distinct lid structures on interfaces of varying hydrophobicity levels were firstly investigated by molecular simulations. It was found that a reasonable hydrophilic/hydrophobic surface could facilitate the lipase to undergo interfacial activation. Building on these findings, a novel "nest"-like superhydrophobic ZIFs (ZIFN) composed of hydrophobic ligands was prepared for the first time and used to immobilize lipase from Aspergillus oryzae (AOL@ZIFN). The AOL@ZIFN exhibited 2.0-folds higher activity than free lipase in the hydrolysis of p-Nitrophenyl palmitate (p-NPP). Especially, the modification of superhydrophobic ZIFN with an appropriate amount of hydrophilic tannic acid can significantly improve the activity of the immobilized lipase (AOL@ZIFN-TA). The AOL@ZIFN-TA exhibited 30-folds higher activity than free lipase, and still maintained 82% of its initial activity after 5 consecutive cycles, indicating good reusability. These results demonstrated that nanomaterials with rational arrangement of the hydrophilic/hydrophobic surface could facilitate the lipase to undergo interfacial activation and improve its activity, displaying the potential of the extensive application.

A newly discovered glycosyltransferase gene UGT88A1 affects growth and polysaccharide synthesis of Grifola frondosa.

Grifola frodosa polysaccharides, especially ß-D-glucans, possess significant anti-tumor, antioxidant and immunostimulatory activities. However, the synthesis mechanism remains to be elucidated. A newly discovered glycosyltransferase UGT88A1 was found to extend glucan chains in vitro. However, the role of UGT88A1 in the growth and polysaccharide synthesis of G. frondosa in vivo remains unclear. In this study, the overexpression of UGT88A1 improved mycelial growth, increased polysaccharide production, and decreased cell wall pressure sensitivity. Biomass and polysaccharide production decreased in the silenced strain, and the pressure sensitivity of the cell wall increased. Overexpression and silencing of UGT88A1 both affected the monosaccharide composition and surface morphology of G. frondosa polysaccharides and influenced the antioxidant activity of polysaccharides from different strains. The messenger RNA expression of glucan synthase (GLS), UTP-glucose-1-phosphate uridylyltransferase (UGP), and UDP-xylose-4-epimerase (UXE) related to polysaccharide synthesis, and genes related to cell wall integrity increased in the overexpression strain. Overall, our study indicates that UGT88A1 plays an important role in the growth, stress, and polysaccharide synthesis of G. frondosa, providing a reference for exploring the pathway of polysaccharide synthesis and metabolic regulation. KEY POINTS: •UGT88A1 plays an important role in the growth, stress response, and polysaccharide synthesis in G. frondosa. •UGT88A1 affected the monosaccharide composition, surface morphology and antioxidant activity of G. frondosa polysaccharides. •UGT88A1 regulated the mRNA expression of genes related to polysaccharide synthesis and cell wall integrity.

Effect of TEMPO-oxidized bacterial cellulose nanofibers stabilized Pickering emulsion of cinnamon essential oil on structure and properties of gelatin composite films.

Pure gelatin film often exhibits high hydrophilicity and a lack of antibacterial activity, hindering its practical application in the field of food preservation. To address these issues, we incorporated 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-oxidized bacterial cellulose (TOBC) nanofibers stabilized cinnamon essential oil (CEO) Pickering emulsions into the gelatin matrix to develop active food packaging films. The study revealed that the good distribution of emulsion droplets in the film matrix. While with increasing Pickering emulsion proportion, the microstructures of composite films were more heterogeneous, showing some pores or cavities. In addition, the insertion of TOBC-stabilized CEO emulsions could improve the elongation at break (EAB), water-resistance, UV blocking ability, and antibacterial activity of film, but reduced its tensile strength (TS) and water vapor barrier properties (WVP). Notably, the film prepared with 4 % TOBC-stabilized CEO Pickering emulsion demonstrated enhanced preservation of strawberries. Overall, the as-prepared gelatin-based active composite films have considerable potential for food packaging.

Construction of catalase@hollow silica nanosphere: Catalase with immobilized but not rigid state for improving catalytic performances.

Enzyme immobilization usually make use of nanomaterials to hold up biocatalysis stability in various unamiable reaction conditions, but also lead large discount on enzyme activity. Thus, there are abundant researches focus on how to deal with the relation of enzyme molecules and supports. In this work, a new state of highly active enzymes has been established through facile and novel in situ immobilization and soft template removal method to construct enzyme contained hollow silica nanosphere (catalase@HSN) biocatalysts where enzymes in the cavity exhibit "immobilized but not rigid state". The obtained catalase@HSN was characterized by transmission electron microscopy, scanning electron microscopy and confocal laser scanning microscopy et al. Catalase@HSN exhibits excellent activity (about 80 % activity recovery rate) and stability suffers from extreme pH, temperature, and organic solvents. Moreover, the reusability and storage stability of catalase@HSN also are satisfactory. This proposed strategy provides a facile method for preparing biocatalysts under mild conditions, facilitating the applications of immobilized enzyme in the fields of real biocatalytic industry with high apparent activity and passable stability.

NIR-triggered bacterial cellulose-based wound dressings for multiple synergistic therapy of infected wound.

Skin wounds are repaired by a complex series of events and overlapping phases in which bacterial infection and insufficient angiogenesis at the wound site delay the healing process. Thus, functional wound dressings with enhanced antibacterial activity and angiogenic capacity have attracted attention. Herein, bacterial cellulose (BC)-based dressings were successfully fabricated by functionalization with a polydopamine (PDA) coating and copper sulfide nanoparticles (CuS NPs). Under 808 nm laser illumination, the BC/PDA/CuS composite membranes exhibited outstanding adjustable photothermal and photodynamic activities as well as controlled Cu2+ release, endowing the composite membranes with synergetic antibacterial activity. Specially, a bactericidal efficiency of 99.7 % and 88.0 % for Staphylococcus aureus and Escherichia coli was achieved after treatment with BC/PDA/CuS5 sample under NIR irradiation (0.8 W/cm2, 10 min), respectively. Moreover, the BC/PDA/CuS5 composite membrane could enhance the angiogenesis due to the released Cu2+. In vivo experiments revealed that the BC/PDA/CuS5 composite membrane dressing could accelerate the wound closure process of the full-thickness skin defects with S. aureus by synergistically reducing inflammation, enhancing collagen deposition, and promoting vascularization under NIR irradiation. Additionally, the BC/PDA/CuS5 composite membrane exhibited high biocompatibility and biosafety. This work offers a new strategy to prepare multifunctional BC-based dressing for clinical wound healing.

Multifunctional Asymmetric Bacterial Cellulose Membrane with Enhanced Anti-Bacterial and Anti-Inflammatory Activities for Promoting Infected Wound Healing.

An asymmetric wound dressing acts as a skin-like structure serves as a protective barrier between a wound and its surroundings. It allows for the absorption of tissue fluids and the release of active substances at the wound site, thus speeding up the healing process. However, the production of such wound dressings requires the acquisition of specialized tools, expensive polymers, and solvents that contain harmful byproducts. In this study, an asymmetric bacterial cellulose (ABC) wound dressing using starch as a porogen has been developed. By incorporating silver-metal organic frameworks (Ag-MOF) and curcumin into the ABC membrane, the wound dressing gains antioxidant, reactive oxygen species (ROS) scavenging, and anti-bacterial activities. Compared to BC-based wound dressings, this dressing promotes efficient dissolution and controlled release of curcumin and silver ions. In a full-thickness skin defect model, wound dressing not only inhibits the growth of bacteria on infected wounds but also regulates the release of curcumin to reduce inflammation and promote the production of epithelium, blood vessels, and collagen. Consequently, this dressing provides superior wound treatment compared to BC-based dressing.

A chemo-biocatalyst based on glutamate oxidase-integrated biomimetic trimanganese tetraoxide as cascade composite nano-catalyst for synthesis of α­Ketoglutaric acid.

The combination of chemo- and biocatalysts to perform one-pot synthetic route has presented great challenges for decades. Herein, glutamate oxidase (GLOX) and trimanganese tetraoxide (Mn3O4) nanocrystals were combined for the first time by one-step biomineralization to construct a mimic multi-enzyme system (GLOX@Mn3O4) for chemoenzymatic synthesis of α­ketoglutaric acid (α­KG). Mn3O4 not only served as a support for the enzyme immobilization, but also contributed its catalytic activity to co-operate with natural enzymes for the cascade reactions. The as-synthesized chemo-enzyme catalysts with directly contacted catalytic sites of the enzyme and inorganic catalyst maximizes the substrate channeling effffects for in situ rapid decomposition of the oxidative intermediate, H2O2, during the enzymatic oxidation of sodium glutamate, thus relieving the inhibition of H2O2 accumulation for GLOX. Benefiting from the excellent stability and reusability of GLOX@Mn3O4, a nearly 100% conversion (99.7%) of l-glutamate to α-KG was achieved, over 4.7 times higher than that of the free GLOX system (21.2%). This work provides a feasibility for constructing a high-performance chemo-enzyme catalyst for cascade catalysis, especially for those reactions with toxic intermediates.

Co-production of 7-chloro-tryptophan and indole pyruvic acid based on an efficient FAD/FADH2 regeneration system.

Efficient FAD/FADH2 regeneration is vital for enzymatic biocatalysis and metabolic pathway optimization. Here, we constructed an efficient and simple FAD/FADH2 regeneration system through a combination of L-amino acid deaminase (L-AAD) and halogenase (CombiAADHa), which was applied for catalyzing the conversion of an L-amino acid to halide and an α-keto acid. For cell-free biotransformation, the optimal activity ratio of L-AAD and halogenase was set between 1:50 and 1:60. Within 6 h, 170 mg/L of 7-chloro-tryptophan (7-Cl-Trp) and 193 mg/L of indole pyruvic acid (IPA) were synthesized in the selected mono-amino acid system. For whole-cell biotransformation, 7-Cl-Trp and IPA synthesis was enhanced by 15% (from 96 to 110 mg/L) and 12% (from 115 to 129 mg/L), respectively, through expression fine-tuning and the strengthening of FAD/FADH2 supply. Finally, ultrasound treatment was applied to improve membrane permeability and adjust the activity ratio, resulting in 1.6-and 1.4-fold higher 7-Cl-Trp and IPA yields. The products were then purified. This system could also be applied to the synthesis of other halides and α-keto acids. KEY POINTS: • In this study, a whole cell FAD/FADH2 regeneration system co-expressing l-AAD and halogenase was constructed • This study found that the activity and ratio of enzyme and the concentration of cofactors had a significant effect on the catalytic process for the efficient co-production of 7-chlorotryptophan and indole pyruvate.

Improvement in bacterial cellulose production by co-culturing Bacillus cereus and Komagataeibacter xylinus.

Bacterial cellulose (BC) is a bio-produced nanostructure material widely used in biomedical, food, and paper-manufacturing industries. However, low production efficiency and high-cost have limited its industrial applications. This study aimed to examine the level of improvement in BC production by co-culturing Bacillus cereus and Komagataeibacter xylinus. The BC yield in corn stover enzymatic hydrolysate was found to be obviously enhanced from 1.2 to 4.4 g/L after the aforementioned co-culturing. The evidence indicated that acetoin (AC) and 2,3-butanediol (2,3-BD) produced by B. cereus were the key factors dominating BC increment. The mechanism underlying BC increment was that AC and 2,3-BD increased the specific activity of AC dehydrogenase and the contents of adenosine triphosphate (ATP) and acetyl coenzyme A (acetyl-CoA), thus promoting the growth and energy level of K. xylinus. Meanwhile, the immobilization of BC could also facilitate oxygen acquisition in B. cereus under static conditions. This study was novel in reporting that the co-culture could effectively enhance BC production from the lignocellulosic enzymatic hydrolysate.

Metagenomic analysis of the relationship between the microorganisms and the volatiles' development in the wines during spontaneous fermentation from the eastern foothills of the Ningxia Helan mountains in China.

BACKGROUND: The natural fermentation of multispecies microbial communities is responsible for unique flavors of winery regions of the eastern foothills of the Ningxia Helan Mountains in China. However, the participation of different microorganisms in the metabolic network for the development of important flavor substances is not clearly defined. Microbial population and diversity on different fermentation phases of Ningxia wine were analyzed by metagenomic sequencing approach. RESULTS: Gas chromatography-mass spectrometry and ion chromatography were used to identify flavor components, and 13 esters, 13 alcohols, nine aldehydes and seven ketones were detected in volatile substances with odor activity values > 1, and eight organic acids were detected as important flavor components in young wine. Thus, 52 238 predicted protein-coding genes from 24 genera were identified in the Kyoto Encyclopedia of Genes and Genomes level 2 pathways of global and overview maps, and the genes were primarily involved in amino acid metabolism and carbohydrate metabolism. Major microbial genera (Saccharomyces, Tatumella, Hanseniaspora, Lactobacillus, and Lachancea) were closely related to self-characteristic compound metabolism and further contributed to wine flavor. CONCLUSION: This study clarifies the different metabolic roles of microorganisms in flavor formation during Ningxia wine spontaneous fermentation. Saccharomyces, dominant fungi involved in glycolysis and pyruvate metabolism, produces not only ethanol but also two important precursors, pyruvate and acetyl-CoA, which are necessary for the tricarboxylic acid cycle, fatty acid metabolism, amino acid metabolism, and flavor formation. Lactobacillus and Lachancea, dominant bacteria involved in lactic acid metabolism. Tatumella, dominant bacteria involved in amino acid metabolism, fatty acid metabolism, and acetic acid metabolism to produce esters in the Shizuishan City region samples. These findings provide insights into the use of local functional strains to generate unique flavor formation, as well as improved stability and quality, in wine production. © 2023 Society of Chemical Industry.

Immobilization of lipase on hydrophobic MOF synthesized simultaneously with oleic acid and application in hydrolysis of natural oils for improving unsaturated fatty acid production.

The hydrolysis of natural oils (vegetable oils and fats) by lipase has significant applications in food and medicine. However, free lipases are usually sensitive to temperature, pH and chemical reagents in aqueous solutions, which hinders their widespread industrial application. Excitingly, immobilized lipases have been widely reported to overcome these problems. Herein, inspired by lipase interface activation, a hydrophobic Zr-MOF (UiO-66-NH2-OA) with oleic acid was synthesized for the first time in an emulsion consisting of oleic acid and water, and the Aspergillus oryzae lipase (AOL) was immobilized onto the UiO-66-NH2-OA through hydrophobic interaction and electrostatic interaction to obtain immobilized lipase (AOL/UiO-66-NH2-OA). 1H NMR and FT-IR data indicated that oleic acid was conjugated with the 2-amino-1,4-benzene dicarboxylate (BDC-NH2) by amidation reaction. As a result, the Vmax and Kcat values of AOL/UiO-66-NH2-OA were 179.61 µM﹒min-1 and 8.27 s-1, which were 8.56 and 12.92 times higher than those of the free enzyme, respectively, due to the interfacial activation. After treated at 70 °C for 120 min, the immobilized lipase maintained 52 % of its original activity, but free AOL only retained 15 %. Significantly, the yield of fatty acids by the immobilized lipase reached 98.3 % and still exceeded 82 % after seven times of recycling.

Synthesis, molecular docking, and binding Gibbs free energy calculation of ß-nitrostyrene derivatives: Potential inhibitors of SARS-CoV-2 3CL protease.

The outbreak of novel coronavirus disease 2019 (COVID-19), caused by the novel coronavirus (SARS-CoV-2), has had a significant impact on human health and the economic development. SARS-CoV-2 3CL protease (3CLpro) is highly conserved and plays a key role in mediating the transcription of virus replication. It is an ideal target for the design and screening of anti-coronavirus drugs. In this work, seven ß-nitrostyrene derivatives were synthesized by Henry reaction and ß-dehydration reaction, and their inhibitory effects on SARS-CoV-2 3CL protease were identified by enzyme activity inhibition assay in vitro. Among them, 4-nitro-ß-nitrostyrene (compound a) showed the lowest IC50 values of 0.7297 µM. To investigate the key groups that determine the activity of ß-nitrostyrene derivatives and their interaction mode with the receptor, the molecular docking using the CDOCKER protocol in Discovery Studio 2016 was performed. The results showed that the hydrogen bonds between ß-NO2 and receptor GLY-143 and the π-π stacking between the aryl ring of the ligand and the imidazole ring of receptor HIS-41 significantly contributed to the ligand activity. Furthermore, the ligand-receptor absolute binding Gibbs free energies were calculated using the Binding Affinity Tool (BAT.py) to verify its correlation with the activity of ß-nitrostyrene 3CLpro inhibitors as a scoring function. The higher correlation(r2=0.6) indicates that the absolute binding Gibbs free energy based on molecular dynamics can be used to predict the activity of new ß-nitrostyrene 3CLpro inhibitors. These results provide valuable insights for the functional group-based design, structure optimization and the discovery of high accuracy activity prediction means of anti-COVID-19 lead compounds.

Salicylic Acid and Jasmonic Acid Increase the Polysaccharide Production of Nostoc flagelliforme via the Regulation of the Intracellular NO Level.

To significantly improve the polysaccharide production of Nostoc flagelliforme, a total of 12 chemicals were evaluated for their effects on polysaccharide accumulation. The results showed that salicylic acid and jasmonic acid increased the accumulation of the polysaccharides in N. flagelliforme significantly, by more than 20%. Three polysaccharides, namely control-capsule polysaccharide, salicylic acid-capsule polysaccharide, and jasmonic acid-capsule polysaccharide, were extracted and purified from N. flagelliforme under normal, salicylic acid, and jasmonic acid culture conditions, respectively. Their chemical compositions slightly differed regarding the total sugar and uronic acid contents, with average molecular weights of 2.06 × 103, 2.16 × 103 and 2.04 × 103 kDa, respectively. They presented similar Fourier transform infrared spectra and no significant difference in antioxidant activity. It was revealed that the salicylic acid and jasmonic acid significantly increased the level of nitric oxide. By investigating the effects of the exogenous nitric oxide scavenger and nitric oxide donor on the nitric oxide levels and polysaccharide yield of N. flagelliforme, the results showed that the increase in intracellular nitric oxide levels might be an important factor promoting the accumulation of polysaccharides. These findings provide a theoretical foundation for enhancing the yield of secondary metabolites by regulating the intracellular nitric oxide levels.

Four polysaccharides isolated from Poria cocos mycelium and fermentation broth supernatant possess different activities on regulating immune response.

Four polysaccharide fractions were isolated and purified from the culture supernatant and mycelium of Poria cocos, and differences in their immunomodulatory activity were investigated. The average molecular weights of EPS-0M, EPS-0.1M, IPS-0M, and IPS-0.1M were 1.77 × 103, 2.01 × 103, 0.03 × 103 and 4.97 × 103 kDa, respectively. They all mainly consisted of 5 monosaccharides, including glucose, mannose, galactose, fucose and rhamnose, but with different molar ratios. At a dose of 50 µg/mL, EPS-0M, EPS-0.1M, and IPS-0.1M significantly increased the production of nitric oxide (NO), as well as the mRNA and protein levels of pro-inflammatory factors including interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß) in RAW264.7 cells, suggesting that they enhanced macrophage-mediated innate immunity. Moreover, based on the in vitro inflammation model of lipopolysaccharide (LPS)-stimulated RAW264.7 cells, EPS-0M, EPS-0.1M and IPS-0M but not IPS-0.1M could inhibit the LPS-induced excessive inflammatory response, including NO, IL-6, TNF-α, IL-1ß production and gene transcription. Interestingly, IPS-0M showed a relatively poor immunostimulatory effect, but had the strongest inhibitory effect against the LPS-induced RAW264.7 inflammatory response. Furthermore, our results indicate that the nuclear factor-kappa B (NF-κB) pathway is associated with the immunomodulatory effects of the polysaccharide samples on RAW264.7 cells. This study can provide a reference for the more targeted application of different polysaccharide components from Poria cocos for human health.

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.

Engineering allosteric inhibition of homoserine dehydrogenase by semi-rational saturation mutagenesis screening.

Allosteric regulation by pathway products plays a vital role in amino acid metabolism. Homoserine dehydrogenase (HSD), the key enzyme for the biosynthesis of various aspartate family amino acids, is subject to feedback inhibition by l-threonine and l-isoleucine. The desensitized mutants with the potential for amino acid production remain limited. Herein, a semi-rational approach was proposed to relieve the feedback inhibition. HSD from Corynebacterium glutamicum (CgHSD) was first characterized as a homotetramer, and nine conservative sites at the tetramer interface were selected for saturation mutagenesis by structural simulations and sequence analysis. Then, we established a high-throughput screening (HTS) method based on resistance to l-threonine analog and successfully acquired two dominant mutants (I397V and A384D). Compared with the best-ever reported desensitized mutant G378E, both new mutants qualified the engineered strains with higher production of CgHSD-dependent amino acids. The mutant and wild-type enzymes were purified and assessed in the presence or absence of inhibitors. Both purified mutants maintained >90% activity with 10 mM l-threonine or 25 mM l-isoleucine. Moreover, they showed >50% higher specific activities than G378E without inhibitors. This work provides two competitive alternatives for constructing cell factories of CgHSD-related amino acids and derivatives. Moreover, the proposed approach can be applied to engineering other allosteric enzymes in the amino acid synthesis pathway.

Enhancing bacterial cellulose production with hypoxia-inducible factors.

Komagataeibacter xylinus is an aerobic strain that produces bacterial cellulose (BC). Oxygen levels play a critical role in regulating BC synthesis in K. xylinus, and an increase in oxygen tension generally means a decrease in BC production. Fumarate nitrate reduction protein (FNR) and aerobic respiration control protein A (ArcA) are hypoxia-inducible factors, which can signal whether oxygen is present in the environment. In this study, FNR and ArcA were used to enhance the efficiency of oxygen signaling in K. xylinus, and globally regulate the transcription of the genome to cope with hypoxic conditions, with the goal of improving growth and BC production. FNR and ArcA were individually overexpressed in K. xylinus, and the engineered strains were cultivated under different oxygen tensions to explore how their overexpression affects cellular metabolism and regulation. Although FNR overexpression did not improve BC production, ArcA overexpression increased BC production by 24.0% and 37.5% as compared to the control under oxygen tensions of 15% and 40%, respectively. Transcriptome analysis showed that FNR and ArcA overexpression changed the way K. xylinus coped with oxygen tension changes, and that both FNR and ArcA overexpression enhanced the BC synthesis pathway. The results of this study provide a new perspective on the effect of oxygen signaling on growth and BC production in K. xylinus and suggest a promising strategy for enhancing BC production through metabolic engineering. KEY POINTS: • K. xylinus BC production increased after overexpression of ArcA • The young's modulus is enhanced by the ArcA overexpression • ArcA and FNR overexpression changed how cells coped with changes in oxygen tension.

A ß-glucan from Aureobasidium pullulans enhanced the antitumor effect with rituximab against SU-DHL-8.

ß-Glucans affect the immune system and have antitumor activity; therefore, they are being investigated as immunomodulators and chemotherapeutic adjuvants. In this study, we investigated a specific ß-glucan, exopolysaccharide (EPS-1) derived from Aureobasidium pullulans (CGMCC 20363), to investigate its impact on the efficacy of rituximab against diffuse large B cell lymphoma (SU-DHL-8 cells) in vitro and in vivo. The results show that compared to rituximab alone, EPS-1 enhanced the inhibition of SU-DHL-8, had antitumor effects in vivo, and improved the response of the immune system of the host. RNA sequencing results reveal that EPS-1 had a chemotactic effect on T cells through the JAK-STAT signaling pathway and recruited immune cells into tumor tissues. EPS-1 also played an antitumor role through the mitochondrial and death receptor Fas-related apoptotic pathways. In summary, EPS-1 may be an effective adjuvant to treat diffuse large B cell lymphoma in combination with rituximab.