Metabolic mechanism of Cr(VI) pollution remediation by Alicycliphilus denitrificans Ylb10.

Cr(VI) is a well-known toxic pollutant and its remediation has attracted great attention. It is important to continuously discover and explore new high-efficiency Cr(VI) reducing bacteria to further improve the efficiency of Cr(VI) pollution remediation. In this paper, metabolic mechanism of Cr(VI) reduction in a new highly efficient Cr(VI) reducing bacterium, Alicycliphilus denitrificans Ylb10, was investigated. The results showed that Ylb10 could tolerate and completely reduce 450 mg/L Cr(VI). Cr(VI) can be reduced in the intracellular compartment, membrane and the extracellular compartment, with the plasma membrane being the main active site for Cr(VI) reduction. With the addition of NADH, the reduction efficiency of cell membrane components for Cr(VI) increased 2.3-fold. The omics data analysis showed that sulfite reductase CysJ, thiosulfate dehydrogenase TsdA, nitrite reductase NrfA, nitric oxide reductase NorB, and quinone oxidoreductase ChrR play important roles in the reduction of Cr(VI), in the intracellular, and the extracellular compartment, and the membrane of Ylb10, and therefore Cr(VI) was reduced by the combined action of several reductases at these three locations.

[DNA assembly by multi-fragment digestion/ligation and homologous recombination].

To develop an accurate and efficient protocol for multi-fragment assembly and multi-site mutagenesis, we integrated and optimized the common multi-fragment assembly methods and validated the established method by using fructose-1,6-diphosphatase 1 (FBP1) with 4 mutant sites. The fragments containing mutations were assembled by introducing mutant sites and Bsa I recognition sequences. After digestion/ligation, the ligated fragment was amplified with the primers containing overlap region to the linearized vector. The amplified fragment was ligated to the linearized vector and the ligation product was transformed into Escherichia coli. After screening and sequencing, the recombinant plasmid with 4 mutant sites was obtained. This protocol overcame the major defects of Gibson assembly and Golden Gate assembly, serving as an efficient solution for multi-fragment assembly and multi-site mutagenesis.

Optimization of Hydrogenobyrinic Acid Synthesis in a Cell-Free Multienzyme Reaction by Novel S-Adenosyl-methionine Regeneration.

Hydrogenobyrinic acid, a modified tetrapyrrole composed of eight five-carbon compounds, is a key intermediate and central framework of vitamin B12. Synthesis of hydrogenobyrinic acid requires eight S-adenosyl-methionine working as the methyl group donor catalyzed by 12 enzymes including six methyltransferases, causing the great shortage of S-adenosyl-methionine and accumulation of S-adenosyl-homocysteine, which is uneconomic and unsustainable for the cascade reaction. Here, we report a cell-free synthetic system for producing hydrogenobyrinic acid by integrating 12 enzymes using 5-aminolevulininate as a substrate and develop a novel S-adenosyl-methionine regeneration system to steadily supply S-adenosyl-methionine and avoid the accumulated inhibition of S-adenosyl-homocysteine by consuming a cheaper substrate (l-methionine and polyphosphate). By combination of the reaction system optimization and S-adenosyl-methionine regeneration, the titer of hydrogenobyrinic acid was improved from 0.61 to 29.39 mg/L in a 12 h reaction period, representing an increase of 48.18-fold, raising an efficient and rapidly evolutional alternative method to produce high-value-added compounds and intermediate products.

Engineered Phomopsis liquidambaris with Fhb1 and Fhb7 Enhances Resistance to Fusarium graminearum in Wheat.

Fusarium head blight is one of the most serious diseases caused by Fusarium graminearum in wheat. Here, we developed a new way to prevent and control Fusarium head blight by introducing the resistance genes Fhb1 and Fhb7 into the endophytic fungus Phomopsis liquidambaris, named PL-Fhb1 and PL-Fhb7, respectively, which could colonize wheat. The wheat seedlings were preinoculated with PL-Fhb1 and PL-Fhb7 to enhance the resistance against deoxynivalenol (DON) and PL-Fhb1 and PL-Fhb7 inhibited the growth of F. graminearum by 73% and 49%, respectively. The incidence rate of diseased spikes decreased to 35.2% and 45.4%, and the corresponding DON levels for wheat grains decreased from 13.2 to 1.79 µg/g and from 13.2 µg/g to 0.39 µg/g when the leaves were preinoculated with PL-Fhb1 and PL-Fhb7 after overwintering, respectively. The incidence rates of diseased spikes decreased to 25.7% and 34.7%, and the DON levels for wheat grains decreased from 17.48 µg/g to 1.23 µg/g and from 17.48 µg/g to 0 µg/g when the wheat flowers were inoculated with PL-Fhb1 and PL-Fhb7, and the wheat flowers were subsequently infected with F. graminearum, respectively. It was confirmed that DON was transformed into DON-glutathione (GSH) by PL-Fhb7 using high-performance liquid chromatography-mass spectrometry (HPLC-MS). However, PL-Fhb1 may have increased plant immunity and enhanced the resistance to F. graminearum. This study indicates that engineered endophytes can improve the resistance to Fusarium head blight and presents a new method for the biological control of Fusarium head blight.

Enrichment and characterization of an effective hexavalent chromium-reducing microbial community YEM001.

Chromium (Cr) is one of the most widely used heavy metals in industrial processes, resulting in water and soil pollution that seriously threaten environmental safety. In this paper, we have directionally enriched a Cr(VI)-reducing bacterial community YEM001 from no-Cr(VI) polluted pond sedimental sludge by selectively growing it in Cr(VI)-containing media. This community could effectively reduce Cr(VI) in laboratory rich media containing different concentrations of Cr(VI), such as 61% reduction at 435 mg/L Cr(VI), 85% reduction at 355 mg/L Cr(VI), and complete reduction at 269 mg/L Cr(VI) in 93.5 h. It was also able to completely reduce 100 mg/L and 300 mg/L Cr(VI) in landfill leachate and natural sludge in 48 h, respectively. Optimal pH for Cr(VI) reduction of the YEM001 is between 7 and 8 and the best efficiency for Cr(VI) reduction occurs at 30 °C. Metagenomic data demonstrated that the YEM001 community was composed of multiple bacteria, including well-known Cr(VI)-reducing bacteria and non-Cr(VI)-reducing bacteria. Delftia, Comamonas, Alicycliphilus, Acidovorax, Bacillus, and Clostridioides account for 83% of total community abundance. The stability of the composition of the YEM001 community and its Cr(VI)-reducing activity allows for its application in bioremediation of environmental Cr(VI) pollution.

Activation of Naringenin and Kaempferol through Pathway Refactoring in the Endophyte Phomopsis Liquidambaris.

Abundant gene clusters of natural products are observed in the endophytic fungus Phomopsis liquidambaris; however, most of them are silent. Herein, a plug-and-play DNA assembly tool has been applied for flavonoid synthesis in P. liquidambaris. A shuttle plasmid was constructed based on S. cerevisiae, E. coli, and P. liquidambaris with screening markers URA, Amp, and hygR, respectively. Each fragment or cassette was successively assembled by overlap extension PCR with at least 40-50 bp homologous arms in S. cerevisiae for generating a new vector. Seven native promoters were screened by the DNA assembly based on the fluorescence intensity of the mCherry reporter gene in P. liquidambaris, and two of them were new promoters. The key enzyme chalcone synthase was the limiting step of the pathway. The naringenin and kaempferol pathways were refactored and activated with the titers of naringenin and kaempferol of 121.53 mg/L and 75.38 mg/L in P. liquidambaris using fed-batch fermentation, respectively. This study will be efficient and helpful for the biosynthesis of secondary metabolites.

Evaluation of a recombinant MIC3 based latex agglutination test for the rapid serodiagnosis of Toxoplasma gondii infection in swines.

The entire gene encoding microneme protein 3 (MIC3) from Toxoplasma gondii was cloned into the plasmid pGEX-KG and subsequently expressed in Escherichia coli as a glutathione-S-transferase (GST) fusion protein. The recombinant MIC3 (rMIC3) was purified and evaluated in a latex agglutination test (LAT) as the diagnostic antigen for the detection of antibodies to T. gondii in pig sera. The specificity, stability, and reproducibility of the test were examined. No agglutination was found when the sensitized latex beads were mixed with phosphate-buffered saline (PBS), borate-buffered saline (BBS), normal saline, and negative serum samples. There was no cross-reactivity with the standard positive sera of other pathogens. But intense agglutination occurred with T. gondii antibody positive serum samples. In our study, the coincidence rate of tested positive-sera of the LAT with rMIC3-sensitized latex particles and the ELISA with rSAG1 was up to 92.8%, T. gondii specific antibodies were detected by the LAT in all piglets that were experimentally infected with T. gondii tachyzoites from 8 to 42 days after infection. Our results indicated that the rMIC3 based latex agglutination test appears to be suitable for the detection of T. gondii antibodies at the early stage of infection.

Synthesis of rebaudioside D, using glycosyltransferase UGTSL2 and in situ UDP-glucose regeneration.

Steviol glycosides from Stevia rebaudiana leaves are used in stevia-based sweeteners for their intense sweetness and low calories. Rebaudioside D is present in leaves in minute quantities (∼0.4-0.5% w/w total dry weight), but it is ∼350 times sweeter than sucrose, and sweeter than the more abundant rebaudioside A and stevioside. In the present study, pathways for rebaudioside D synthesis and UDP-glucose recycling were developed by coupling recombinant UDP-glucosyltransferase UGTSL2 from Solanum lycopersicum and sucrose synthase StSUS1 from Solanum tuberosum. Reaction parameters, including substrate ratio, sucrose concentration, temperature, crude extract concentration, and reaction time, were evaluated, and 17.4 g/l of rebaudioside D (yield = 74.6%) was obtained from 20 g/l of rebaudioside A after 20 h, using UDP or UDP-glucose in recombinant cell crude extracts. Extending the reaction time generated rebaudioside M2 from further glycosylation of rebaudioside D. Km values for UGTSL2 indicated a higher affinity for rebaudioside D than for rebaudioside A.

A new cinnamic acid derivative from plant-derived endophytic fungus Pyronema sp.

Ten secondary metabolites (1-10) including a new 4-hydroxycinnamic acid derivatives, methyl 2-{(E)-2-[4-(formyloxy)phenyl]ethenyl}-4-methyl-3-oxopentanoate (1), and nine known compounds (2-10) were isolated from an EtOAc extract derived from a solid rice medium of endophytic fungal strain Pyronema sp. (A2-1 & D1-2). Their structures were elucidated from NMR and HRMS data. All the compounds were tested for antibacterial activity against Mycobacterium marinum ATCCBAA-535. Compounds 1, 8 and 9 exhibited moderate antibiotic activity with IC50 of 64, 43 and 32 µM, respectively.

Characterization of Microbial Communities in Chinese Rice Wine Collected at Yichang City and Suzhou City in China.

Two typical microbial communities from Chinese rice wine fermentation collected in Yichang city and Suzhou city in China were investigated. Both communities could ferment glutinous rice to rice wine in 2 days. The sugar and ethanol contents were 198.67 and 14.47 mg/g, respectively, for rice wine from Yichang city, and 292.50 and 12.31 mg/g, respectively, for rice wine from Suzhou city. Acetic acid and lactic acid were the most abundant organic acids. Abundant fungi and bacteria were detected in both communities by high-throughput sequencing. Saccharomycopsis fibuligera and Rhizopus oryzae were the dominant fungi in rice wine from Suzhou city, compared with R. oryzae, Wickerhamomyces anomalus, Saccharomyces cerevisiae, Mucor indicus, and Rhizopus microsporus in rice wine from Yichang city. Bacterial diversity was greater than fungal diversity in both communities. Citrobacter was the most abundant genus. Furthermore, Exiguobacterium, Aeromonas, Acinetobacter, Pseudomonas, Enterobacter, Bacillus, and Lactococcus were highly abundant in both communities.

Biotransformation of (-)-(10E,15S)-10,11-Dehydrocurvularin.

(-)-(10E,15S)-10,11-Dehydrocurvularin (1), produced from an associated-fungus of Scolopendra subspinipes mutilans on a gram scale, was microbiologically converted to curvularin (2) and 5-methoxycurvularin (3) by Antrodiella semisupina in 61% totally isolated yield. The structures of these compounds were elucidated on the basis of spectroscopic and mass spectrometric analysis. The undescribed assignments of 1H and 13C NMR spectral data for 5-methoxycurvularin (2) has now been explicitly provided. The cytotoxic activities of compounds 1-3 against four human cancer cell lines were evaluated. Dehydrocurvularin (1) showed moderate cytotoxicity against Caski and Hep-G2, and curvularin (2) was selectively cytotoxic against MDA-MB-231.

The effect of temperature on the structure and function of a cellulose-degrading microbial community.

The purpose of this study was to investigate the effect of temperature on the structure and straw degradation capability of a microbial community grown from wheat straw compost. Two cellulolytic microbial communities, WDC1 and WDC2, were obtained from compost. The communities had been cultured under 50 and 60 °C by continuous enrichment, respectively. The wheat straw degradation capabilities were 45.69 % (WDC1) and 59.5 % (WDC2). By changing the culture temperatures, two new stable communities were obtained: WDC1-6N (WDC1, cultivated at 60 °C for eight generations) and WDC2-5N (WDC2, cultivated at 50 °C for eight generations). The wheat straw degradation capabilities for the new communities were 59.75 and 52.60 %, respectively. The results showed that compared to 50 °C, the wheat straw degradation capability of the communities cultured at 60 °C was stronger. Sequencing of selected denaturing gradient gel electrophoresis (DGGE) bands and analysis of DGGE profiles indicated that the WDC2 structure was significantly different from the structure of WDC1. This was so even though the two communities were enriched from the same compost. With the change of culture temperature, the community structures underwent significant transitions. Included communities were thermophilic, anaerobic bacteria, and any cellulolytic bacteria (e.g., Clostridium thermocellum) that were active and abundant at conditions under 60 °C. These results have the potential to significantly aid in the enrichment of a cellulose-degrading community from the environment and to enhance the community capability to conduct straw biotransformation.