Complete mitochondrial genomes of edible mushrooms: features, evolution, and phylogeny.

Edible mushrooms are an important food source with high nutritional and medicinal value. They are a useful source for studying phylogenetic evolution and species divergence. The exploration of the evolutionary relationships among these species conventionally involves analyzing sequence variations within their complete mitochondrial genomes, which range from 31,854 bp (Cordyceps militaris) to 197,486 bp (Grifolia frondosa). The study of the complete mitochondrial genomes of edible mushrooms has emerged as a critical field of research, providing important insights into fungal genetic makeup, evolution, and phylogenetic relationships. This review explores the mitochondrial genome structures of various edible mushroom species, highlighting their unique features and evolutionary adaptations. By analyzing these genomes, robust phylogenetic frameworks are constructed to elucidate mushrooms lineage relationships. Furthermore, the exploration of different variations of mitochondrial DNA presents novel opportunities for enhancing mushroom cultivation biotechnology and medicinal applications. The mitochondrial genomic features are essential for improving agricultural practices and ensuring food security through improved crop productivity, disease resistance, and nutritional qualities. The current knowledge about the mitochondrial genomes of edible mushrooms is summarized in this review, emphasising their significance in both scientific research and practical applications in bioinformatics and medicine.

A Novel Bacillus amyloliquefaciens Specifically Improving the Solubility and Antioxidant Activities of Edible Bird's Nest.

Edible bird's nest (EBN), a most highly priced and valuable foodstuff, contains high percentage of proteins and carbohydrates. However, proteins adhering to these carbohydrates make the EBN hard and tough, which need to be boiled as the bird's nest soup to make the Chinese cuisine. To overcome the hard and tough texture of EBN and improve the digestion degrees, the present study screened and identified a probiotic strain Bacillus amyloliquefaciens YZW02 from 5-year stored EBN sample completely solubilizing EBN for the first time. The 24-h B. amyloliquefaciens fermented EBN contained 20.30-21.48 mg/mL of the soluble protein contents with a recovery rate of 98-100%, DPPH radical scavenging rate of 84.76% and ABTS radical scavenging capacity of 41.05%. The mixed fermentation of B. amyloliquefaciens YZW02 and Bacillus natto BN1 were further applied to improve the low-MW peptide percentages and antioxidant activities. The mixed-fermentation of B. natto BN1 with 4-h cultured B. amyloliquefaciens YZW02 had the lowest percentage (82.23%) of >12-kDa proteins/peptides and highest percentages of 3-12 kDa, 1-3 kDa and 0.1-1 kDa peptides of 8.6% ± 0.08, 7.57% ± 0.09, 1.77% ± 0.05 and 0.73% ± 0.05, with the highest DPPH, ABTS and •OH scavenging capacity of 90.23%, 46.45% and 49.12%, respectively. These findings would provide an efficient strategy for improving the solubility and antioxidant activities of EBNs.

Recent insights into glucans biosynthesis and engineering strategies in edible fungi.

Fungal α/ß-glucans have significant importance in cellular functions including cell wall structure, host-pathogen interactions and energy storage, and wide application in high-profile fields, including food, nutrition, and pharmaceuticals. Fungal species and their growth/developmental stages result in a diversity of glucan contents, structures and bioactivities. Substantial progresses have been made to elucidate the fine structures and functions, and reveal the potential molecular synthesis pathway of fungal α/ß-glucans. Herein, we review the current knowledge about the biosynthetic machineries, including: precursor UDP-glucose synthesis, initiation, elongation/termination and remodeling of α/ß-glucan chains, and molecular regulation to maximally produce glucans in edible fungi. This review would provide future perspectives to biosynthesize the targeted glucans and reveal the catalytic mechanism of enzymes associated with glucan synthesis, including: UDP-glucose pyrophosphate phosphorylases (UGP), glucan synthases, and glucanosyltransferases in edible fungi.

The ß-1,3-glucan synthase gene GFGLS2 plays major roles in mycelial growth and polysaccharide synthesis in Grifola frondosa.

ß-1,3-Glucans are well-known biological and health-promoting compounds in edible fungi. Our previous results characterized a glucan synthase gene (GFGLS) of Grifola frondosa for the first time to understand its role in mycelial growth and glucan biosynthesis. In the present study, we identified and functionally reannotated another glucan synthase gene, GFGLS2, based on our previous results. GFGLS2 had a full sequence of 5944 bp including 11 introns and 12 exons and a coding information for 1713 amino acids of a lower molecular weight (195.2 kDa) protein with different conserved domain sites than GFGLS (5927 bp with also 11 introns and a coding information for 1781 aa). Three dual-promoter RNA-silencing vectors, pAN7-iGFGLS-dual, pAN7-iGFGLS2-dual, and pAN7-CiGFGLS-dual, were constructed to downregulate GFGLS, GFGLS2, and GFGLS/GFGLS2 expression by targeting their unique exon sequence or conserved functional sequences. Silencing GFGLS2 resulted in higher downregulation efficiency than silencing GFGLS. Cosilencing GFGLS and GFGLS2 had a synergistic downregulation effect, with slower mycelial growth and glucan production by G. frondosa. These findings indicated that GFGLS2 plays major roles in mycelial growth and polysaccharide synthesis and provides a reference to understand the biosynthesis pathway of mushroom polysaccharides. KEY POINTS: • The 5944-bp glucan synthase gene GFGLS2 of G. frondosa was cloned and reannotated • GFGLS2 showed identity and significant differences with the previously identified GFGLS • GFGLS2 played a major role in fermentation and glucan biosynthesis.

Improved mycelia and polysaccharide production of Grifola frondosa by controlling morphology with microparticle Talc.

BACKGROUND: Mushroom showed pellet, clump and/or filamentous mycelial morphologies during submerged fermentation. Addition of microparticles including Talc (magnesium silicate), aluminum oxide and titanium oxide could control mycelial morphologies to improve mycelia growth and secondary metabolites production. Here, effect of microparticle Talc (45 µm) addition on the mycelial morphology, fermentation performance, monosaccharide compositions of polysaccharides and enzymes activities associated with polysaccharide synthesis in G. frondosa was well investigated to find a clue of the relationship between polysaccharide biosynthesis and morphological changes. RESULTS: Addition of Talc decreased the diameter of the pellets and increased the percentage of S-fraction mycelia. Talc gave the maximum mycelial biomass of 19.25 g/L and exo-polysaccharide of 3.12 g/L at 6.0 g/L of Talc, and mycelial polysaccharide of 0.24 g/g at 3.0 g/L of Talc. Talc altered the monosaccharide compositions/percentages in G. frondosa mycelial polysaccharide with highest mannose percentage of 62.76 % and lowest glucose percentage of 15.22 % followed with the corresponding changes of polysaccharide-synthesis associated enzymes including lowest UDP-glucose pyrophosphorylase (UGP) activity of 91.18 mU/mg and highest UDP-glucose dehydrogenase (UGDG) and GDP-mannose pyrophosphorylase (GMPPB) activities of 81.45 mU/mg and 93.15 mU/mg. CONCLUSION: Our findings revealed that the presence of Talc significantly changed the polysaccharide production and sugar compositions/percentages in mycelial and exo-polysaccharides by affecting mycelial morphology and polysaccharide-biosynthesis related enzymes activities of G. frondosa.

Ultrasound-Assisted Extraction of Polysaccharides from Volvariella volvacea: Process Optimization and Structural Characterization.

The aims of the present study were to optimize the operational parameters to maximize the yield of ultrasound-assisted polysaccharide extraction from Volvariella volvacea (straw mushroom) fruiting bodies by using for the first time one-factor-at-a-time and three-level Box-Behnken factorial designs. A maximum polysaccharide yield of 8.28 ± 0.23% was obtained under the optimized conditions of ultrasound power of 175 W, extraction temperature of 57 °C, extraction time of 33 min, and the ratio of liquid to raw material of 25:1, respectively. Compared to the hot-water extraction, the ultrasound-assistance favored the extraction of polysaccharides from V. volvacea for its higher polysaccharide yield and efficiency. Further preliminary polysaccharide structural characterization indicated that ultrasound treatment affected the monosaccharide compositions and ratios, and molecular weight range of polysaccharides extracted from V. volvacea.

Grifola frondosa polysaccharides: A review on structure/activity, biosynthesis and engineering strategies.

Polysaccharides are the main polymers in edible fungi Grifola frondosa, playing a crucial role in the physiology and representing the healthy benefits for humans. Recent efforts have well elucidated the fine structures and biological functions of G. frondosa polysaccharides. The recently-rapid developments and increasing availability in fungal genomes also accelerated the better understanding of key genes and pathways involved in biosynthesis of G. frondosa polysaccharides. Herein, we provide a brief overview of G. frondosa polysaccharides and their activities, and comprehensively outline the complex process, genes and proteins corresponding to G. frondosa polysaccharide biosynthesis. The regulation strategies including strain improvement, process optimization and genetic engineering were also summarized for maximum production of G. frondosa polysaccharides. Some remaining unanswered questions in describing the fine synthesis machinery were also pointed out to open up new avenues for answering the structure-activity relationship and improving polysaccharide biosynthesis in G. frondosa. The review hopefully presents a reasonable full picture of activities, biosynthesis, and production regulation of polysaccharide in G. frondosa.

Overall quality changes and deterioration mechanism of fragrant rapeseed oils during 6-Month storage.

The strong-fragrant rapeseed oil (SFRO) is a popular rapeseed oil in China with a low refining degree only degumming with hot water, which remarkably affects its storage stability. The present study compared the overall changes of physical/chemical/nutrient quality of FROs at various temperatures, light wavelengths and headspace volumes. Results showed that red light (680 nm) had a most significant adverse effect on the overall quality of SFRO with the higher correlation coefficients to PV and TOTOX of 0.71 and 0.70, and lower correlation coefficients to chlorophyll and tocopherol of -0.95 and -0.53, respectively. Further studies revealed that red light accelerated the oxidation of fragrant rapeseed oils by degrading chlorophyll to initiate the photo-oxidation process and synthesize high amount of secondary oxidation products including aliphatic and aromatic oxidized compounds from linolenic acid. These findings provided a reference to control the deterioration of FROs by preventing the transmittance of red light.

Biochemical and structural characterization of a glucan synthase GFGLS2 from edible fungus Grifola frondosa to synthesize ß-1, 3-glucan.

BACKGROUND: Grifola frondosa is a Basidiomycete fungus belonging to the family of Grifolaceae and the order of Polyporales. ß-Glucans are the main polymers in G. frondosa, playing a crucial role in the physiology and representing the healthy benefits for humans. The membrane-integrated ß-1, 3-glucan synthase (GLS) is responsible for glucan synthesis, cell wall assembly, differentiation and growth of the edible fungi. However, the structural/catalytic characteristics and mechanisms of ß-1, 3-glucan synthases in G. frondosa are still unknown due to their extremely complex structures with multi-transmembranes and large molecular masses. RESULTS: Herein, a ß-1, 3-glucan synthase (GFGLS2) was purified and identified from the cultured mycelia with a specific activity of 60.01 pmol min-1 µg-1 for the first time. The GFGLS2 showed a strict specificity to UDP-glucose with a Vmax value of 1.29 ± 0.04 µM min-1 at pH 7.0 and synthesized ß-1, 3-glucan with a maximum degree of polymerization (DP) of 62. Sequence Similarity Network (SSN) analysis revealed that GFGLS2 has a close relationship with others in Ganoderma sinense, Trametes coccinea, Polyporus brumalis, and Trametes pubescens. With the assistance of 3D structure modelling by AlphaFold 2, molecular docking and molecular dynamics simulations, the central hydrophilic domain (Class III) in GFGLS2 was the main active sites through binding the substrate UDP-glucose to 11 amino acid residues via hydrogen bonds, π-stacking and salt bridges. CONCLUSIONS: The biochemical, 3D structural characterization and potential catalytic mechanism of a membrane-bound ß-1, 3-glucan synthase GFGLS2 from cultured mycelia of G. frondosa were well investigated and would provide a reasonable full picture of ß-1, 3-glucan synthesis in fungi.

A novel bacteriophage KSL-1 of 2-Keto-gluconic acid producer Pseudomonas fluorescens K1005: isolation, characterization and its remedial action.

BACKGROUND: Bacteriophages have the destructive damage on the industrial bioprocess. 2-Keto-gluconic acid (2KGA) producing bacteria had also been attacked and lysed by bacteriophages which lowered the glucose consumption and 2KGA yield and even stopped the fermentation process. In this study, we presented the characteristics of a novel virulent bacteriophage specifically infecting Pseudomonas fluorescens K1005 and proposed an efficient remedial action for this phage infection to reduce the production loss. RESULTS: The phage KSL-1 of Pseudomonas fluorescens K1005 was isolated from abnormal 2KGA fermentation broth. It belonged to the Siphoviridae family with a hexagonal head diameter of about 99 nm and a non-contractile tail of about 103 nm × 39 nm. The genome size of phage KSL-1 was estimated to be approximately 53 kbp. Its optimal MOI to infect P. fluorescens K1005 was about 0.001. One-step growth curve gave its latent and burst periods of 90 min and 75 min with a burst size of 52 phage particles per infected cell. This phage was stable with a pH range of 7.0-10.0, and sensitive to thermal treatment. Finally, a simple remedial action was proposed by feeding fresh seed culture. Compared with the infected 2KGA fermentation, the remedial experiments restored 2KGA fermentation performance by increasing the produced 2KGA concentration to 159.89 g/L and shortening the total fermentation time of 80 h with the productivity and yield of 2.0 g/L.h and 0.89 g/g. The obtained data proved that this method was effective to combat the phage infections problems during the 2KGA fermentation. CONCLUSION: The phage KSL-1 was a novel bacteriophage specifically infecting Pseudomonas fluorescens K1005. The remedial action of feeding fresh seed culture to the infected broth was an easily-operating and effective method to maintain a high 2KGA yield and avoid the draft of infected broth.

Functional Characterization and Structural Basis of the ß-1,3-Glucan Synthase CMGLS from Mushroom Cordyceps militaris.

ß-1,3-Glucan synthases play key roles in glucan synthesis, cell wall assembly, and growth of fungi. However, their multi-transmembrane domains (over 14 TMHs) and large molecular masses (over 100 kDa) significantly hamper understanding of their catalytic characteristics and mechanisms. In the present study, the 5841-bp gene CMGLS encoding the 221.7 kDa membrane-bound ß-1,3-glucan synthase CMGLS in Cordyceps militaris was cloned, identified, and structurally analyzed. CMGLS was partially purified with a specific activity of 87.72 pmol/min/µg, a purification fold of 121, and a yield of 10.16% using a product-entrapment purification method. CMGLS showed a strict specificity to UDP-glucose with a Km value of 84.28 µM at pH 7.0 and synthesized ß-1,3-glucan with a maximum degree of polymerization (DP) of 70. With the assistance of AlphaFold and molecular docking, the 3D structure of CMGLS and its binding features with substrate UDP-glucose were proposed for the first time to our knowledge. UDP-glucose potentially bound to at least 11 residues via hydrogen bonds, π-stacking ,and salt bridges, and Arg 1436 was predicted as a key residue directly interacting with the moieties of glucose, phosphate, and the ribose ring on UDP-glucose. These findings would open an avenue to recognize and understand the glucan synthesis process and catalytic mechanism of ß-1,3-glucan synthases in mushrooms.

Simultaneous identification and quantification of canrenone and 11-α-hydroxy-canrenone by LC-MS and HPLC-UVD.

A procedure for simultaneous identification and quantification of canrenone and its biotransformed product 11-α-hydroxy-canrenone by high-performance liquid chromatography with ultraviolet detector (HPLC-UVD) and mass spectrometry (LC-MS) methods was proposed. The optimal determination variables on the HPLC-UVD or LC-MS coupled with a ZORBAX Eclipse XDB-C18 column (150 mm × 4.6 mm, 5 µm) were set as follows: detection wavelength of 280 nm, mobile phase of water and methanol gradient elution, temperature for the chromatographic column of 30°C, flow rate of mobile phase of 0.8 mL/min, sample injection volume of 5 µL, and elution time of 40 min. The MS conditions were set as follows: the flow rate of sheath gas, aux gas, and sweep gas were kept at 35 arb, 5 arb, and 0 arb, respectively. The temperature of capillary was held at 300°C, and capillary voltage was set at 30.00 V. Tube lens were performed at 100.00 V. The proposed method was validated by linearity (r² ≥ 0.9910), average recovery (94.93%, RSD1.21%), precision (RSD ≤ 1.31%), limit of detection, and limit of quantification (LOD 0.1~0.12 mg/L, LOQ 0.5~0.67 mg/L), which proved to be affordable for simultaneously determining canrenone and its bio-transformed product 11-α-hydroxy-canrenone.

Changes of structures and biosynthesis/hydrolysis-associated genes expression of glucans at different Volvariella volvacea maturity stages.

In the present study, effects of maturity stage on structural characteristics and biosynthesis/hydrolysis-associated genes expression of glucans from Volvariella volvacea fruit body were well investigated. Elongation and pileus expansion stages decreased total soluble carbohydrate and protein contents to 17.09 mg/g and 8.33 mg/g, and significantly accumulated the total amino acids contents to 32.37 mg/g. Yields of crude polysaccharides significantly increased to 8.12% at egg stage and decreased to 3.72% at pileus expansion stage. Purified VVP I-a and VVP I-b were proved to be α-glucans. The maturity process affected the monosaccharide compositions, decreased the molecular weights of VVP I-a and VVP I-b with decreased transcription levels of glucan biosynthesis-associated enzyme genes vvugp and vvgls and increased glucan hydrolysis-associated glucanase gene vvexg2 expression with no significant effects on backbone structures including glycosidic linkages and configurations. The findings would benefit for understanding change patterns of V. volvacea glucan structures and their biosynthesis/hydrolysis-associated genes expression at maturity stages.

The role of Rho1 gene in the cell wall integrity and polysaccharides biosynthesis of the edible mushroom Grifola frondosa.

Grifola frondosa polysaccharides, especially ß-glucans, showed the significant antitumor, hypoglycemic, and immune-stimulating activities. In the present study, a predominant regulatory subunit gfRho1p of ß-1,3-glucan synthase in G. frondosa was identified with a molecular weight of 20.79 kDa and coded by a putative 648-bp small GTPase gene gfRho1. By constructing mutants of RNA interference and over-expression gfRho1, the roles of gfRho1 in the growth, cell wall integrity and polysaccharide biosynthesis were well investigated. The results revealed that defects of gfRho1 slowed mycelial growth rate by 22% to 33%, reduced mycelial polysaccharide and exo-polysaccharide yields by 4% to 7%, increased sensitivity to cell wall stress, and down-regulated gene transcriptions related to PKC-MAPK signaling pathway in cell wall integrity. Over-expression of gfRho1 improved mycelial growth rate and polysaccharide production of G. frondosa. Our study supports that gfRho1 is an essential regulator for polysaccharide biosynthesis, cell growth, cell wall integrity and stress response in G. frondosa.

Two-Stage Semi-Continuous 2-Keto-Gluconic Acid (2KGA) Production by Pseudomonas plecoglossicida JUIM01 From Rice Starch Hydrolyzate.

A two-stage semi-continuous strategy for producing 2-keto-gluconic acid (2KGA) by Pseudomonas plecoglossicida JUIM01 from rice starch hydrolyzate (RSH) has been developed. The initial glucose concentration (140 g/L) was selected for first-stage fermentation due to its highest 2KGA productivity of 7.58 g/(L⋅h), cell weight of 3.91 g/L, and residual glucose concentration of 25.00 g/L. Followed by removing 70.0% (v/v) of the first-stage broth and feeding 400.0 g/L of glucose to the second-stage fermentor, a total of 50680.0 g glucose was consumed, and 50005.20 g 2KGA was obtained with a yield of 0.9867 g/g by P. plecoglossicida JUIM01 after a 3-cycle two-stage semi-continuous fermentation. Our results indicated that the developed two-stage semi-continuous fermentation could be industrially applied due to its high 2KGA concentration, 2KGA yield and operation efficiency.

UDP-glucose pyrophosphorylase gene affects mycelia growth and polysaccharide synthesis of Grifola frondosa.

To elucidate potential roles of UDP-glucose pyrophosphorylase (UGP) in mycelial growth and polysaccharide synthesis of Grifola frondosa, a putative 2036-bp UDP-glucose pyrophosphorylase gene gfugp encoding a 53.17-kDa protein was cloned and re-annotated. Two dual promoter RNA silencing vectors of pAN7-iUGP-P-dual and pAN7-iUGP-C-dual were constructed to down-regulate gfugp expression by targeting its promoter or conserved functional sequences, respectively. Results showed that silence of gfugp promoter sequence had a higher down-regulating efficiency with slower mycelial growth and polysaccharide production than those of conserved sequence. The monosaccharide compositions/percentages of mycelial and exo-polysaccharides significantly changed with the increase of galactose and arabinose contents possibly due to block of UDP-glucose supply by gfugp silence and alteration of sugar metabolism via up-regulation of UDP-glucose-4-epimerase (gfuge) and UDP-xylose-4-epimerase (gfuxe) transcription. Our findings would provide a reference to know the biosynthesis pathway of mushroom polysaccharides and improve their production by metabolic regulation.

Regulation of HSP27 on NF-kappaB pathway activation may be involved in metastatic hepatocellular carcinoma cells apoptosis.

BACKGROUND: During the process of metastasis, cells are subjected to various apoptotic stimuli. Aberrant expression of apoptotic regulators often contribute to cell metastasis. Heat shock protein 27(HSP27) is confirmed as an apoptosis regulator, but its antiapoptotic mechanism in metastatic hepatocellular carcinoma (HCC) cells remains unclear. METHODS: Levels of HSP27 protein and its phosphorylation in Hep3B, MHCC97L to MHCC97H cells with different metastatic potentials were determined by western blot analysis. MHCC97H cells were transfected with specific small interference RNA (siRNA) against HSP27. The in vitro migration and invasion potentials of cells were evaluated by Transwell assay. The apoptosis ratio of MHCC97H cells was analyzed by TUNEL staining and Flow Cytometry. Alteration of signal transduction pathway after HSP27 knockdown in MHCC97H cells was evaluated through a Human Q Series Signal Transduction in Cancer Gene Array analysis. Nuclear NF-kappaB contentration and endogenous IKK activity were demonstrated by ELISA assay. The association of IKKalpha, IKKbeta, IkappaBalpha with HSP27 and the association between IKKbeta and IKKalpha in MHCC97H cells were determined by co-immunoprecipitation assay followed by western blot analysis. RESULTS: HSP27 protein and its phosphorylation increased in parallel with enhanced metastatic potentials of HCC cells. siRNA-mediated HSP27 knockdown in MHCC97H significantly suppressed cells migration and invasion in vitro and induced cell apoptosis; the prominently altered signal transduction pathway was NF-kappaB pathway after HSP27 knockdown in MHCC97H cells. Furthermore, inhibition of HSP27 expression led to a significant decrease of nuclear NF-kappaB contentration and endogenous IKK activity. In addition, HSP27 was associated with IKKalpha, IKKbeta, IkappaBalpha in three HCC cells above. ELISA assay and western blot analysis also showed a decrease of the association between IKKbeta and IKKalpha, the association between phosphor-HSP27 and IKK complex, and an increase of total IkappaBalpha but reducing tendency of phosphor-IkappaBalpha when HSP27 expression was efficiently knocked down in MHCC97H cells. CONCLUSION: Altogether, these findings revealed a possible effect of HSP27 on apoptosis in metastatic HCC cells, in which HSP27 may regulate NF-kB pathway activation.

Functions of a Glucan Synthase Gene GFGLS in Mycelial Growth and Polysaccharide Production of Grifola frondosa.

Glucan synthase (GLS) gene is known to be involved in the fungal biosynthesis of cell wall, differentiation, and growth. In the present study, a glucan synthase gene (GFGLS) in the edible mushroom Grifola frondosa with a full sequence of 5927 bp encoding a total of 1781 amino acids was cloned and characterized for the first time. GFGLSp is a membrane protein containing two large transmembrane domains connected with a hydrophilic cytoplasmic domain. With a constructed dual promoter RNA silencing vector pAN7-gfgls-dual, a GFGLS-silencing transformant iGFGLS-3 had the lowest GFGLS transcriptional expression level (26.1%) with a shorter length and thinner appearance of the mycelia, as well as decreased mycelial biomass and exo-polysaccharide production of 5.02 and 0.38 g/L, respectively. Further analysis indicated that GFGLS silence influenced slightly the monosaccharide compositions and ratios of mycelial and exo-polysaccharide. These findings suggest that GFGLS could affect mycelial growth and polysaccharide production by downregulating the glucan synthesis.

A Membrane-Bound Gluconate Dehydrogenase from 2-Keto-D-Gluconic Acid Industrial Producing Strain Pseudomonas plecoglossicida JUIM01: Purification, Characterization, and Gene Identification.

The membrane-bound gluconate dehydrogenase (mGADH) is a critical enzyme for 2-keto-D-gluconic acid (2KGA) production in Pseudomonas plecoglossicida JUIM01. The purified native flavin adenine dinucleotide-dependent mGADH (FAD-mGADH) was consisted of a gamma subunit, a flavoprotein subunit, and a cytochrome c subunit with molecular mass of ~ 27, 65, and 47 kDa, respectively. The specific activity of FAD-mGADH was determined as 90.71 U/mg at optimum pH and temperature of 6.0 and 35 °C. The Km and Vmax values of calcium D-gluconate were 0.631 mM and 0.734 mM/min. The metal ions Mg2+ and Mn2+ showed slight positive effects on FAD-mGADH activity. On the other hand, a 3868-bp-length gad gene cluster was amplified and expressed in Escherichia coli BL21(DE3). The recombinant protein showed the same molecular weight and enzyme activity as the native FAD-mGADH, which confirmed it as a FAD-mGADH encoding gene. The flavoprotein subunit and the cytochrome c subunit containing a putative FAD-binding motif and three possible heme-binding motifs concluded from alignment results of mGADHs. This study characterized the native and recombinant FAD-mGADH and would provide the basis for further genetic modification of Pseudomonas plecoglossicida JUIM01 with the intention of 2KGA productivity improvement.

Purification, characterization and gene identification of a membrane-bound glucose dehydrogenase from 2-keto-d-gluconic acid industrial producing strain Pseudomonas plecoglossicida JUIM01.

The membrane-bound glucose dehydrogenase (mGDH) is a rate-limiting enzyme for the industrial production of 2-keto-d-gluconic acid (2KGA) from glucose. In this study, mGDH was firstly purified from a 2KGA industrial producing strain Pseudomonas plecoglossicida JUIM01. The purified mGDH exhibited a specific activity of 16.85 U/mg and was identified as monomeric membrane-bound PQQ-dependent dehydrogenase with a molecular mass of ~87 kDa. The Km and Vmax value of d-glucose were 0.042 mM and 14.620 µM/min, and the optimal pH and temperature were of 6.0 and 35 °C with favorable acid resistance and poor heat tolerance. Ca2+/Mg2+ showed a significantly positive effect on mGDH activity with 20% increase, whereas EDTA/EGTA had a negative influence, and Ca2+ was essential for enzyme activity. Furthermore, a 2412 bp-length gcd was amplified by genome walking technique and heterologously expressed in Escherichia coli. Bioinformatics analysis and heterologous expression further confirmed it as a mGDH encoding gene. mGDH contained binding sites of Ca2+, cofactor PQQ and polypeptide binding sites concluded from alignment results of mGDHs from different genera. This study would lay the foundation for improving 2KGA productivity through further strain modification.