Direct evolution of an alkaline fungal laccase to degrade tetracyclines.

A fungal laccase-mediator system capable of high effectively oxidizing tetracyclines under a wide pH range will benefit environmental protection. This study reported a directed evolution of a laccase PIE5 to improve its performance on tetracyclines oxidization at alkaline conditions. Two mutants, namely MutA (D229N/A244V) and MutB (N123A/D229N/A244V) were obtained. Although they shared a similar optimum pH and temperature as PIE5, the two mutants displayed approximately 2- and 5-fold higher specific activity toward the mediators ABTS and guaiacol at pHs 4.0 to 6.5, respectively. Simultaneously, their catalytic efficiency increased by 8.0- and 6.4-fold compared to PIE5. At a pH range of 5-8 and 28 °C, MutA or MutB at a final concentration of 2.5 U·mL-1 degraded 77 % and 81 % of 100 mg·L-1 tetracycline within 10 min, higher than PIE5 (45 %). Furthermore, 0.1 U·mL-1 MutA or MutB completely degraded 100 mg·L-1 chlortetracycline within 6 min in the presence of 0.1 mM ABTS. At pH 8.0, MutA degraded tetracycline and chlortetracycline following the routine pathways were reported previously based on LC-MS analysis.

Two Zn2Cys6-type transcription factors respond to aromatic compounds and regulate the expression of laccases in the white-rot fungus Trametes hirsuta.

White-rot fungi differentially express laccases when they encounter aromatic compounds. However, the underlying mechanisms are still being explored. Here, proteomics analysis revealed that in addition to increased laccase activity, proteins involved in sphingolipid metabolism and toluene degradation as well as some cytochrome P450s (CYP450s) were differentially expressed and significantly enriched during 48 h of o-toluidine exposure, in Trametes hirsuta AH28-2. Two Zn2Cys6-type transcription factors (TFs), TH8421 and TH4300, were upregulated. Bioinformatics docking and isothermal titration calorimetry assays showed that each of them could bind directly to o-toluidine and another aromatic monomer, guaiacol. Binding to aromatic compounds promoted the formation of TH8421/TH4300 heterodimers. TH8421 and TH4300 silencing in T. hirsuta AH28-2 led to decreased transcriptional levels and activities of LacA and LacB upon o-toluidine and guaiacol exposure. EMSA and ChIP-qPCR analysis further showed that TH8421 and TH4300 bound directly with the promoter regions of lacA and lacB containing CGG or CCG motifs. Furthermore, the two TFs were involved in direct and positive regulation of the transcription of some CYP450s. Together, TH8421 and TH4300, two key regulators found in T. hirsuta AH28-2, function as heterodimers to simultaneously trigger the expression of downstream laccases and intracellular enzymes. Monomeric aromatic compounds act as ligands to promote heterodimer formation and enhance the transcriptional activities of the two TFs.IMPORTANCEWhite-rot fungi differentially express laccase isoenzymes when exposed to aromatic compounds. Clarification of the molecular mechanisms underlying differential laccase expression is essential to elucidate how white-rot fungi respond to the environment. Our study shows that two Zn2Cys6-type transcription factors form heterodimers, interact with the promoters of laccase genes, and positively regulate laccase transcription in Trametes hirsuta AH28-2. Aromatic monomer addition induces faster heterodimer formation and rate of activity. These findings not only identify two new transcription factors involved in fungal laccase transcription but also deepen our understanding of the mechanisms underlying the response to aromatics exposure in white-rot fungi.

Gongronella sp. w5 hydrolyzes plant sucrose and releases fructose to recruit phosphate-solubilizing bacteria to provide plants with phosphorus.

The mechanisms of how plant-beneficial rhizospheric fungi interact with the soil microbial community to promote plant growth by facilitating their phosphorus acquisition are poorly understood. This work supported that a Mucoromycotina fungus, Gongronella sp. w5 (w5), could promote phosphorus uptake of Medicago truncatula by increasing the available phosphorus (P) in the soil. The abundance of phosphate-solubilizing bacteria (PSB) and the activity of alkaline phosphatase (ALP) in alfalfa rhizosphere soil increased after w5 inoculation. Further analysis showed that w5 donated a portion of ALP activity and also stimulated the PSB to secrete ALP during plant-w5-PSB interaction to help release more available P in the rhizosphere of M. truncatula. Unlike most plant-beneficial rhizospheric fungi that mainly acquire hexoses from plants, w5 gained sucrose directly from the host plant and then recruited PSB to aid P acquisition by hydrolyzing sucrose and releasing mainly fructose to induce PSB to secrete ALP. IMPORTANCE: This work supported that after absorbing plant sucrose, Gongronella sp. w5 mainly releases sucrose hydrolysis product fructose into the environment. Fructose was used as a carbon source and signaling molecules to induce PSB to co-produce higher alkaline phosphatase activity, releasing soil-available phosphorus and promoting M. truncatula growth. This is the first report that plant-beneficial fungi could directly metabolize sucrose from plants and then recruit PSB to aid P acquisition by providing fructose. Our findings revealed the diversity in pathways of plant-fungi-PSB interactions on soil P acquisition and deepened our understanding of the cooperation of growth-promoting microorganisms in plant rhizosphere.

From waste to protein: a new strategy of converting composted distilled grain wastes into animal feed.

Distilled grain waste (DGW) is rich in nutrients and can be a potential resource as animal feed. However, DGW contains as much as 14% lignin, dramatically reducing the feeding value. White-rot fungi such as Pleurotus ostreatus could preferentially degrade lignin with high efficiency. However, lignin derivatives generated during alcohol distillation inhibit P. ostreatus growth. Thus, finding a new strategy to adjust the DGW properties to facilitate P. ostreatus growth is critical for animal feed preparation and DGW recycling. In this study, three dominant indigenous bacteria, including Sphingobacterium thermophilum X1, Pseudoxanthomonas byssovorax X3, and Bacillus velezensis 15F were chosen to generate single and compound microbial inoculums for DGW composting to prepare substrates for P. ostreatus growth. Compared with non-inoculated control or single microbial inoculation, all composite inoculations, especially the three-microbial compound, led to faster organic metabolism, shorter composting process, and improved physicochemical properties of DGW. P. ostreatus growth assays showed the fastest mycelial colonization (20.43 µg·g-1 ergosterol) and extension (9 mm/d), the highest ligninolytic enzyme activities (Lac, 152.68 U·g-1; Lip, 15.56 U·g-1; MnP, 0.34 U·g-1; Xylanase, 10.98 U·g-1; FPase, 0.71 U·g-1), and the highest lignin degradation ratio (30.77%) in the DGW sample after 12 h of composting with the three-microbial compound inoculation when compared to other groups. This sample was relatively abundant in bacteria playing critical roles in amino acid, carbohydrate, energy metabolism, and xenobiotic biodegradation, as suggested by metagenomic analysis. The feed value analysis revealed that P. ostreatus mycelia full colonization in composted DGW led to high fiber content retention and decreased lignin content (final ratio of 5% lignin) but elevated protein concentrations (about 130 g·kg-1 DM). An additional daily weight gain of 0.4 kg/d was shown in cattle feeding experiments by replacing 60% of regular feed with it. These findings demonstrate that compound inoculant consisting of three indigenous microorganisms is efficient to compost DGW and facilitate P. ostreatus growth. P. ostreatus decreased the lignin content of composted DGW during its mycelial growth, improving the quality of DGW for feeding cattle.

A cost-saving, safe, and highly efficient natural mediator for laccase application on aflatoxin detoxification.

Laccase mediators possess advantage of oxidizing substrates with high redox potentials, such as aflatoxin B1 (AFB1). High costs of chemically synthesized mediators limit laccase industrial application. In this study, thin stillage extract (TSE), a byproduct of corn-based ethanol fermentation was investigated as the potential natural mediator of laccases. Ferulic acid, p-coumaric acid, and vanillic acid were identified as the predominant phenolic compounds of TSE. With the assistance of 0.05 mM TSE, AFB1 degradation activity of novel laccase Glac1 increased by 17 times. The promoting efficiency of TSE was similar to ferulic acid, but superior to vanillic acid and p-coumaric acid, with 1.2- and 1.3-fold increases, respectively. After Glac1-TSE treatment, two oxidation products were identified. Ames test showed AFB1 degradation products lost mutagenicity. Meanwhile, TSE also showed 1.3-3.0 times promoting effect on laccase degradation activity in cereal flours. Collectively, a safe and highly efficient natural mediator was obtained for aflatoxin detoxification.

Enhanced extracellular production of laccase in Coprinopsis cinerea by silencing chitinase gene.

Laccase, a copper-containing polyphenol oxidase, is an important green biocatalyst. In this study, Laccase Lcc5 was homologous recombinantly expressed in Coprinopsis cinerea and a novel strategy of silencing chitinase gene expression was used to enhance recombinant Lcc5 extracellular yield. Two critical chitinase genes, ChiEn1 and ChiE2, were selected by analyzing the transcriptome data of C. cinerea FA2222, and their silent expression was performed by RNA interference (RNAi). It was found that silencing either ChiEn1 or ChiE2 reduced sporulation and growth rate, and increased cell wall sensitivity, but had no significant effect on mycelial branching. Among them, the extracellular laccase activity of the ChiE2-silenced engineered strain Cclcc5-antiChiE2-5 and the control Cclcc5-13 reached the highest values (38.2 and 25.5 U/mL, respectively) at 250 and 150 rpm agitation speeds, corresponding to productivity of 0.35 and 0.19 U/mL·h, respectively, in a 3-L fermenter culture. Moreover, since Cclcc5-antiChiE2-5 could withstand greater shear forces, its extracellular laccase activity was 2.6-fold higher than that of Cclcc5-13 when the agitation speed was all at 250 rpm. To our knowledge, this is the first report of enhanced recombinant laccase production in C. cinerea by silencing the chitinase gene. This study will pave the way for laccase industrial production and accelerate the development of a C. cinerea high-expression system. KEY POINTS: • ChiEn1 and ChiE2 are critical chitinase genes in C. cinerea FA2222 genome. • Chitinase gene silencing enhanced the tolerance of C. cinerea to shear forces. • High homologous production of Lcc5 is achieved by fermentation in a 3-L fermenter.

Structural insights into curdlan degradation via a glycoside hydrolase containing a disruptive carbohydrate-binding module.

BACKGROUND: Degradation via enzymatic processes for the production of valuable ß-1,3-glucooligosaccharides (GOS) from curdlan has attracted considerable interest. CBM6E functions as a curdlan-specific ß-1,3-endoglucanase, composed of a glycoside hydrolase family 128 (GH128) module and a carbohydrate-binding module (CBM) derived from family CBM6. RESULTS: Crystallographic analyses were conducted to comprehend the substrate specificity mechanism of CBM6E. This unveiled structures of both apo CBM6E and its GOS-complexed form. The GH128 and CBM6 modules constitute a cohesive unit, binding nine glucoside moieties within the catalytic groove in a singular helical conformation. By extending the substrate-binding groove, we engineered CBM6E variants with heightened hydrolytic activities, generating diverse GOS profiles from curdlan. Molecular docking, followed by mutation validation, unveiled the cooperative recognition of triple-helical ß-1,3-glucan by the GH128 and CBM6 modules, along with the identification of a novel sugar-binding residue situated within the CBM6 module. Interestingly, supplementing the CBM6 module into curdlan gel disrupted the gel's network structure, enhancing the hydrolysis of curdlan by specific ß-1,3-glucanases. CONCLUSIONS: This study offers new insights into the recognition mechanism of glycoside hydrolases toward triple-helical ß-1,3-glucans, presenting an effective method to enhance endoglucanase activity and manipulate its product profile. Furthermore, it discovered a CBM module capable of disrupting the quaternary structures of curdlan, thereby boosting the hydrolytic activity of curdlan gel when co-incubated with ß-1,3-glucanases. These findings hold relevance for developing future enzyme and CBM cocktails useful in GOS production from curdlan degradation.

Sphingomicrobium clamense sp. nov., Isolated from Sediment of Clam Island Beach in China.

A Gram-stain-negative, non-flagellated, aerobic, ovoid or rod-shaped bacterium with motility, designated B8T, was isolated from the sediment of Clam Island beach, Liaoning province, China. The optimum growth of strain B8T occurred at 35 oC, pH 7.0, and in the presence of 4.0-5.0% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain B8T formed a distinct lineage within the genus Sphingomicrobium and was closely related to Sphingomicrobium nitratireducens O-35T (98.3% sequence similarity), Sphingomicrobium aestuariivivum KCTC 42286T (96.9%), and Sphingomicrobium astaxanthinifaciens JCM 18551T (96.5%). The digital DNA-DNA hybridization and average nucleotide identity values between strain B8T and closely related strains were lower than 21.0% and 78.0%, much lower than the cutoff values of 70.0% and 95.0%, respectively, for bacterial species delineation. The dominant respiratory quinone of strain B8T was ubiquinone-10. The major fatty acids were Sum In Feature 8 (C18:1ω7c and/or C18:1ω6c), Sum In Feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C17:1ω6c, C18:1 2-OH, and C16:0. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, sphingoglycolipid, glycolipids, and four unknown polar lipids. The DNA G + C content of strain B8T was 63.9%. Based on the phenotypic, phylogenetic, and chemotaxonomic analyses, strain B8T is considered a new species of Sphingomicrobium, for which the name Sphingomicrobium clamense sp. nov. is proposed. The type strain is B8T (= CGMCC 1.19486T = KCTC 92052T).

An apoptosis-inducing factor controls programmed cell death and laccase expression during fungal interactions.

Apoptotic-like programmed cell death (PCD) is one of the main strategies for fungi to resist environmental stresses and maintain homeostasis. The apoptosis-inducing factor (AIF) has been shown in different fungi to trigger PCD through upregulating reactive oxygen species (ROS). This study identified a mitochondrial localized AIF homolog, CcAIF1, from Coprinopsis cinerea monokaryon Okayama 7. Heterologous overexpression of CcAIF1 in Saccharomyces cerevisiae caused apoptotic-like PCD of the yeast cells. Ccaif1 was increased in transcription when C. cinerea interacted with Gongronella sp. w5, accompanied by typical apoptotic-like PCD in C. cinerea, including phosphatidylserine externalization and DNA fragmentation. Decreased mycelial ROS levels were observed in Ccaif1 silenced C. cinerea transformants during cocultivation, as well as reduction of the apoptotic levels, mycelial growth, and asexual sporulation. By comparison, Ccaif1 overexpression led to the opposite phenotypes. Moreover, the transcription and expression levels of laccase Lcc9 decreased by Ccaif1 silencing but increased firmly in Ccaif1 overexpression C. cinerea transformants in coculture. Thus, in conjunction with our previous report that intracellular ROS act as signal molecules to stimulate defense responses, we conclude that CcAIF1 is a regulator of ROS to promote apoptotic-like PCD and laccase expression in fungal-fungal interactions. In an axenic culture of C. cinerea, CcAIF1 overexpression and H2O2 stimulation together increased laccase secretion with multiplied production yield. The expression of two other normally silent isozymes, Lcc8 and Lcc13, was unexpectedly triggered along with Lcc9. KEY POINTS: • Mitochondrial CcAIF1 induces PCD during fungal-fungal interactions • CcAIF1 is a regulator of ROS to trigger the expression of Lcc9 for defense • CcAIF1 overexpression and H2O2 stimulation dramatically increase laccase production.

A starch-binding domain of α-amylase (AmyPG) disrupts the structure of raw starch.

Most raw starch-digesting enzymes possess at least one non-catalytic starch-binding domain (SBD), which enhances enzymatic hydrolysis of insoluble starch granules. Previous studies of SBD-starch interaction mainly focus on binding affinity for substrates, while the mechanism involved disruption of starch granules remains partially understood. Raw starch-digesting α-amylases AmyPG and AmyP were from Photobacterium gaetbulicola and an uncultured marine bacterium, respectively. Here, comparative studies on the two α-amylases and their SBDs (SBDPG and SBDAmyP) with high sequence identity were carried out. The degradation capacity of AmyPG towards raw starch was approximately 2-fold higher than that of AmyP, which was due to the stronger disruptive ability of SBDPG rather than the binding ability. Two non-binding amino acids (K626, T618) of SBDPG that specifically support the disruptive ability were first identified using affinity gel electrophoresis, amylose­iodine absorbance spectra, and differential scanning calorimetry. The mutants SBDPG-K626A and SBDPG-T618A exhibited stronger disruptive ability, while the corresponding mutants of AmyPG enhanced the final hydrolysis degree of raw starch. The results confirmed that the disruptive ability of SBD can independently affect raw starch hydrolysis. This advancement in the functional characterization of SBDs contributes to a better understanding of enzyme-starch granule interactions, pushing forward designs of raw starch-digesting enzymes.

Improving the cellobiose hydrolysis activity of glucose-stimulating ß-glucosidase Bgl2A.

ß-Glucosidases with high catalytic activity and glucose tolerant properties possess promising applications in lignocellulose-based industries. To obtain enzymes possessing these properties, a semi-rational strategy was employed to engineer the glucose-stimulating ß-glucosidase Bgl2A for high cellobiose hydrolysis activity. A total of 18 mutants were constructed. A22S, V224D, and A22S/V224D exhibited high specific activities of 272.06, 237.60, and 239.29 U/mg toward cellobiose, which were 2.5- to 2.8-fold of Bgl2A. A22S, V224D, and A22S/V224D exhibited increased kcat values, which were 2.7- to 3.1-fold of Bgl2A. A22S and V224D maintained glucose-stimulating property, whereas A22S/V224D lost it. Using 150 g/L cellobiose as the substrate, the amount of glucose produced by A22S was the highest, yielding 129.70 g/L glucose after 3 h reaction at 35 °C. The synergistic effects of the engineered enzymes with commercial cellulase on hydrolyzing cellulose were investigated. Supplemented with the commercial cellulase and A22S, the highest glucose amount of 23.30 g/L was yielded from cellulose with hydrolysis rate of 21.02 %. Given its high cellobiose hydrolysis activity and glucose-stimulating properties, A22S can be used as a component of enzyme cocktail to match mesophilic cellulases for efficient cellulose hydrolysis.

Seryl-tRNA Synthetase Shows a Noncanonical Activity of Upregulating Laccase Transcription in Trametes hirsuta AH28-2 Exposed to Copper Ion.

The function of Seryl-tRNA synthetase in fungi during gene transcription regulation beyond translation has not been reported. Here, we report a seryl-tRNA synthetase, ThserRS, which can negatively regulate laccase lacA transcription in Trametes hirsuta AH28-2 under exposure to copper ion. ThserRS was obtained through yeast one-hybrid screening using a bait sequence of lacA promoter (-502 to -372 bp). ThserRS decreased while lacA increased at the transcription level in T. hirsuta AH28-2 in the first 36 h upon CuSO4 induction. Then, ThserRS was upregulated, and lacA was downregulated. ThserRS overexpression in T. hirsuta AH28-2 resulted in a decrement in lacA transcription and LacA activity. By comparison, ThserRS silencing led to increased LacA transcripts and activity. A minimum of a 32-bp DNA fragment containing two putative xenobiotic response elements could interact with ThserRS, with a dissociation constant of 919.9 nM. ThserRS localized in the cell cytoplasm and nucleus in T. hirsuta AH28-2 and was heterologously expressed in yeast. ThserRS overexpression also enhanced mycelial growth and oxidative stress resistance. The transcriptional level of several intracellular antioxidative enzymes in T. hirsuta AH28-2 was upregulated. Our results demonstrate a noncanonical activity of SerRS that acts as a transcriptional regulation factor to upregulate laccase expression at an early stage after exposure to copper ions. IMPORTANCE Seryl-tRNA synthetase is well known for the attachment of serine to the corresponding cognate tRNA during protein translation. In contrast, its functions beyond translation in microorganisms are underexplored. We performed in vitro and cell experiments to show that the seryl-tRNA synthetase in fungi with no UNE-S domain at the carboxyl terminus can enter the nucleus, directly interact with the promoter of the laccase gene, and negatively regulate the fungal laccase transcription early upon copper ion induction. Our study deepens our understanding of the Seryl-tRNA synthetase noncanonical activities in microorganisms. It also demonstrates a new transcription factor for fungal laccase transcription.

Enhanced extracellular production of raw starch-degrading α-amylase in Bacillus subtilis through expression regulatory element modification and fermentation optimization.

BACKGROUND: Raw starch-degrading α-amylase (RSDA) can hydrolyze raw starch at moderate temperatures, thus contributing to savings in starch processing costs. However, the low production level of RSDA limits its industrial application. Therefore, improving the extracellular expression of RSDA in Bacillus subtilis, a commonly used industrial expression host, has great value. RESULTS: In this study, the extracellular production level of Pontibacillus sp. ZY raw starch-degrading α-amylase (AmyZ1) in B. subtilis was enhanced by expression regulatory element modification and fermentation optimization. As an important regulatory element of gene expression, the promoter, signal peptide, and ribosome binding site (RBS) sequences upstream of the amyZ1 gene were sequentially optimized. Initially, based on five single promoters, the dual-promoter Pveg-PylB was constructed by tandem promoter engineering. Afterward, the optimal signal peptide SPNucB was obtained by screening 173 B. subtilis signal peptides. Then, the RBS sequence was optimized using the RBS Calculator to obtain the optimal RBS1. The resulting recombinant strain WBZ-VY-B-R1 showed an extracellular AmyZ1 activity of 4824.2 and 41251.3 U/mL during shake-flask cultivation and 3-L fermenter fermentation, which were 2.6- and 2.5-fold greater than those of the original strain WBZ-Y, respectively. Finally, the extracellular AmyZ1 activity of WBZ-VY-B-R1 was increased to 5733.5 U/mL in shake flask by optimizing the type and concentration of carbon source, nitrogen source, and metal ions in the fermentation medium. On this basis, its extracellular AmyZ1 activity was increased to 49082.1 U/mL in 3-L fermenter by optimizing the basic medium components as well as the ratio of carbon and nitrogen sources in the feed solution. This is the highest production level reported to date for recombinant RSDA production. CONCLUSIONS: This study represents a report on the extracellular production of AmyZ1 using B. subtilis as a host strain, and achieved the current highest expression level. The results of this study will lay a foundation for the industrial application of RSDA. In addition, the strategies employed here also provide a promising way for improving other protein production in B. subtilis.

Maribacter aquimaris sp. nov., isolated from seawater adjacent to Fildes Peninsula, Antarctica.

A novel Gram-stain-negative, aerobic, and rod-shaped bacterium with gliding motility, named strain ANRC-HE7T, was isolated from the seawater of Biological Bay adjacent to Fildes Peninsula, Antarctica. The optimal growth of this strain occurred at 28 °C, pH 7.5, and in the presence of 1.0% (w/v) NaCl. Strain ANRC-HE7T can produce amylase and harbors gene clusters involved in cellulose degradation. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain ANRC-HE7T formed a distinct lineage within the genus Maribacter and was closely related to Maribacter luteus RZ05T (98.4% sequence similarity), Maribacter polysiphoniae LMG 23671T (98.3%), and Maribacter arenosus CAU 1321T (97.3%). However, digital DNA-DNA hybridization and average nucleotide identity values between strain ANRC-HE7T and closely related strains were 17.4-49.1% and 70.9-92.7%, much lower than the cutoff values of 70% and 95%, respectively. On the other hand, strain ANRC-HE7T shared characteristics with most type strains within the genus. Its respiratory quinone was MK-6. The major fatty acids were iso-C15:0, summed feature 3 (C16:1 ω7c and/or C16:1 ω6c), and anteiso-C15:0. The major polar lipids were phosphatidylethanolamine, two unidentified aminolipids, four unidentified phospholipids, and five unidentified glycolipids. The DNA G + C content of strain ANRC-HE7T was 40.1%. Based on the results of the biochemical, phylogenetic, and chemotaxonomic analyses, strain ANRC-HE7T is suggested to represent a novel species of the genus Maribacter, for which the name Maribacter aquimaris sp. nov. is proposed. The type strain is ANRC-HE7T (= MCCC 1K03787T = KCTC 72532T).

Structures of the NDP-pyranose mutase belonging to glycosyltransferase family 75 reveal residues important for Mn2+ coordination and substrate binding.

Members of glycosyltransferase family 75 (GT75) not only reversibly catalyze the autoglycosylation of a conserved arginine residue with specific NDP-sugars but also exhibit NDP-pyranose mutase activity that reversibly converts specific NDP-pyranose to NDP-furanose. The latter activity provides valuable NDP-furanosyl donors for glycosyltransferases and requires a divalent cation as a cofactor instead of FAD used by UDP-D-galactopyranose mutase. However, details of the mechanism for NDP-pyranose mutase activity are not clear. Here we report the first crystal structures of GT75 family NDP-pyranose mutases. The novel structures of GT75 member MtdL in complex with Mn2+ and GDP, GDP-D-glucopyranose, GDP-L-fucopyranose, GDP-L-fucofuranose, respectively, combined with site-directed mutagenesis studies, reveal key residues involved in Mn2+ coordination, substrate binding, and catalytic reactions. We also provide a possible catalytic mechanism for this unique type of NDP-pyranose mutase. Taken together, our results highlight key elements of an enzyme family important for furanose biosynthesis.

Isoenzyme N-Acyl-l-Amino Acid Amidohydrolase NA Increases Ochratoxin A Degradation Efficacy of Stenotrophomonas sp. CW117 by Enhancing Amidohydrolase ADH3 Stability.

Ochratoxin A (OTA) is a potent mycotoxin mainly produced by toxicogenic strains of Aspergillus spp. and seriously contaminates foods and feedstuffs. OTA detoxification strategies are significant to food safety. A superefficient enzyme ADH3 to OTA hydrolysis was isolated from the difunctional strain Stenotrophomonas sp. CW117 in our previous study. Here, we identified a gene N-acyl-l-amino acid amidohydrolase NA, which is an isoenzyme of ADH3. However, it is not as efficient a hydrolase as ADH3. The kinetic constant showed that the catalytic efficiency of ADH3 (Kcat/Km = 30,3938 s-1 · mM-1) against OTA was 29,113 times higher than that of NA (Kcat/Km = 10.4 s-1 · mM-1), indicating that ADH3 was the overwhelming superior detoxifying gene in CW117. Intriguingly, when gene na was knocked out from the CW117 genome, degradation activity of the Δna mutant was significantly reduced at the first 6 h, suggesting that the two enzymes might have an interactive effect on OTA transformation. Gene expressions and Western blotting assay showed that the Δna mutant and wild-type CW117 showed similar adh3 expression levels, but na deficiency decreased ADH3 protein level in CW117. Collectively, isoenzyme NA was identified as a factor that improved the stability of ADH3 in CW117 but not as a dominant hydrolase for OTA transformation. IMPORTANCE Ochratoxin A (OTA) is a potent mycotoxin mainly produced by toxicogenic strains of Aspergillus spp. and seriously contaminates foods and feedstuffs. Previous OTA detoxification studies mainly focused on characterizations of degradation strains and detoxifying enzymes. Here, we identified a gene N-acyl-l-amino acid amidohydrolase NA from strain CW117, which is an isoenzyme of the efficient detoxifying enzyme ADH3. Isoenzyme NA was identified as a factor that improved the stability of ADH3 in CW117 and, thus, enhanced the degradation activity of the strain. This is the first study on an isoenzyme improving the stability of another efficient detoxifying enzyme in vivo.

Sedimentimonas flavescens gen. nov., sp. nov., isolated from sediment of Clam Island, Liaoning Province.

A novel Gram-stain negative, aerobic and ovoid to short rod shaped bacterium with a single polar flagellum, named strain B57T, was isolated from sediment of Clam Island, Liaoning Province, China. The optimal growth of this strain was found to occur at 37 °C, pH 6-6.5, and in the presence of 2% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain B57T forms a distinct lineage within the family Rhodobacteraceae, sharing high 16S rRNA gene sequence similarity with Sinirhodobacter populi sk2b1T (97.4%). The average amino acid identity of B57T and the closely related species were lower than the threshold level for genus delineation. The dominant respiratory quinone of strain B57T was identified as Q-10. The major fatty acids were found to be Summed Feature 8 (C18:1ω7c and/or C18:1ω6c), Summed Feature 3 (C16:1ω7c and/or C16:1ω6c) and C16: 0. The polar lipids were identified as phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, diphosphatidylglycerol, two unidentified phospholipids, one unidentified glycolipid, and one unidentified lipid. The DNA G + C content of strain B57T was determined to be 64.1 mol%. Based on the biochemical, phylogenetic and chemotaxonomic analysis, strain B57T is concluded to represent a novel species of a novel genus, for which the name Sedimentimonas flavescens gen. nov., sp. nov.is proposed. The type strain is B57T (= CGMCC1.19488T = KCTC 92053T).

Enhanced extracellular raw starch-degrading α-amylase production in Bacillus subtilis by promoter engineering and translation initiation efficiency optimization.

BACKGROUND: A raw starch-degrading α-amylase from Pontibacillus sp. ZY (AmyZ1), previously screened by our laboratory, showed a promising application potential for starch-processing industries. However, the AmyZ1 secretory production still under investigation, which seriously restricts its application in the starch-processing industry. On the other hand, Bacillus subtilis is widely used to achieve the extracellular expression of target proteins. RESULTS: AmyZ1 secretory production was achieved in B. subtilis and was enhanced by promoter engineering and translation initiation efficiency optimization. First, based on the different phase-dependent promoters, the dual-promoter PspoVG-PspoVG142 was constructed by combining dual-promoter engineering and promoter modification. The corresponding strain BZd34 showed an extracellular AmyZ1 activity of 1437.6 U/mL during shake flask cultivation, which was 3.11-fold higher than that of the original strain BZ1 (PgroE). Then, based on translation initiation efficiency optimization, the best strain BZd343 containing optimized 5'-proximal coding sequence (opt3) produced the highest extracellular α-amylase activity of 1691.1 U/mL, which was 3.65-fold higher than that of the strain BZ1. Finally, cultivation of BZd343 in 3-L fermenter exhibited an extracellular AmyZ1 activity of 14,012 U/mL at 48 h, with productivity of 291.9 U/mL·h. CONCLUSIONS: This is the first report of recombinant expression of AmyZ1 in B. subtilis and the expression level of AmyZ1 represents the highest raw starch-degrading α-amylase level in B. subtilis to date. The high-level expression of AmyZ1 in this work provides a foundation for its industrial production. The strategies used in this study also provide a strategic reference for improving the secretory expression of other enzymes in B. subtilis.

ThhspA1 is involved in lacA transcriptional regulation of Trametes hirsuta AH28-2 exposed to o-toluidine.

White rot fungi, especially Trametes spp., respond to a wide range of aromatic compounds and dramatically enhance laccase activity, while the activation mechanisms remain to be elucidated. Here, we show that an Hsp70 homolog named ThhspA1 regulates the transcription of laccase LacA in Trametes hirsuta AH28-2 when confronted with o-toluidine. ThhspA1 is pulled down by lacA promoter sequence from the nuclear mixture extracted from T. hirsuta AH28-2 induced by 2 mM o-toluidine. Silencing of ThhspA1 results in a sharp decrease in lacA transcripts and laccase activity in vivo. By comparison, ThhspA1 overexpression does not affect lacA transcription, and laccase activity shows slight enhancement or remains unchanged upon induction with o-toluidine. Electrophoretic mobility shift assays suggest a direct interaction between ThhspA1 and the lacA promoter region. Further investigation shows that the integrity of ThhspA1 is critical since its substrate binding domain (SBD) and nucleotide-binding domain (NBD) are both necessary for DNA binding, with a higher affinity of SBD than NBD based on fluorescence polarization assay. Our results demonstrate that ThhspA1 functions as an aromatic-stress-related DNA binding transcriptional factor required for LacA expression.

A Superefficient Ochratoxin A Hydrolase with Promising Potential for Industrial Applications.

As the most seriously controlled mycotoxin produced by Aspergillus spp. and Penicillium spp., ochratoxin A (OTA) results in various toxicological effects and widely contaminates agro-products. Biological detoxification is the highest priority regarding OTA in food and feed industry, but currently available detoxification enzymes have relatively low effectiveness in terms of time and cost. Here we show a superefficient enzyme, ADH3, identified from Stenotrophomonas acidaminiphila that has a strong ability to transform OTA into nontoxic ochratoxin-α by acting as an amidohydrolase. Recombinant ADH3 (1.2 µg/mL) completely degrades 50 µg/L OTA within 90 s, while the other most efficient OTA hydrolases available take several hours. The kinetic constant showed that rADH3 (Kcat/Km) catalytic efficiency was 56.7 to 35,000 times higher than those of previous hydrolases rAfOTase, rOTase, and commercial carboxypeptidase A (CPA). Protein structure-based assay suggested that ADH3 has a preference for hydrophobic residues to form a larger hydrophobic area than other detoxifying enzymes at the cavity of the catalytic sites, and this structure allows OTA easier access to the catalytic sites. In addition, ADH3 shows considerable temperature adaptability to exert hydrolytic function at the temperature down to 0°C or up to 70°C. Collectively, we report a superefficient OTA detoxifying enzyme with promising potential for industrial applications. IMPORTANCE Ochratoxin A (OTA) can result in various toxicological effects and widely contaminates agro-products and feedstuffs. OTA detoxifications by microbial strains and bio-enzymes are significant to food safety. Although previous studies showed OTA could be transformed through several pathways, the ochratoxin-α pathway is recognized as the most effective one. However, the most currently available enzymes are not efficient enough. Here, a superefficient hydrolase, ADH3, which can completely transform 50 µg/L OTA into ochratoxin-α within 90 s was screened and characterized. The hydrolase ADH3 shows considerable temperature adaptability (0 to 70°C) to exert the hydrolytic function. Findings of this study supplied an efficient OTA detoxifying enzyme and predicted the superefficient degradation mechanism, laying a foundation for future industrial applications.