Enhanced catalytic activities and modified substrate preferences for taxoid 10ß-O-acetyl transferase mutants by engineering catalytic histidine residues.

OBJECTIVES: Taxoid 10ß-O-acetyl transferase (DBAT) was redesigned to enhance its catalytic activity and substrate preference for baccatin III and taxol biosynthesis. RESULTS: Residues H162, D166 and R363 were determined as potential sites within the catalytic pocket of DBAT for molecular docking and site-directed mutagenesis to modify the activity of DBAT. Enzymatic activity assays revealed that the kcat/KM values of mutant H162A/R363H, D166H, R363H, D166H/R363H acting on 10-deacetylbaccatin III were about 3, 15, 26 and 60 times higher than that of the wild type of DBAT, respectively. Substrate preference assays indicated that these mutants (H162A/R363H, D166H, R363H, D166H/R363H) could transfer acetyl group from unnatural acetyl donor (e.g. vinyl acetate, sec-butyl acetate, isobutyl acetate, amyl acetate and isoamyl acetate) to 10-deacetylbaccatin III. CONCLUSION: Taxoid 10ß-O-acetyl transferase mutants with redesigned active sites displayed increased catalytic activities and modified substrate preferences, indicating their possible application in the enzymatic synthesis of baccatin III and taxol.

Production of antibacterial peptide from bee venom via a new strategy for heterologous expression.

Honey bee is important economic insect that not only pollinates fruits and crops but also provides products with various physiological activities. Bee venom is a functional agent that is widely applied in clinical treatment and pharmacy. Secapin is one of these agents that have a significant role in therapy. The functions of secapin from the bee venom have been documented, but little information is known about its heterologous expression under natural condition. Moreover, few scholars verified experimentally the functions of secapin from bee venom in vitro. In this study, we successfully constructed a heterologous expression vector, which is different from conventional expression system. A transgenic approach was established for transformation of secapin gene from the venom of Apis mellifera carnica (Ac-sec) into the edible fungi, Coprinus cinereus. Ac-sec was encoded by a 234 bp nucleotide that contained a signal peptide domain and two potential phosphorylation sites. The sequence exhibited highly homology with various secapins characterized from honey bee and related species. Southern blot data indicated that Ac-sec was present as single or multiple copy loci in the C. cinereus genome. By co-transformation and double-layer active assay, Ac-sec was expressed successfully in C. cinereus and the antibacterial activity of the recombinants was identified, showing notable antibacterial activities on different bacteria. Although Ac-sec is from the venom of Apidae, phylogenetic analysis demonstrated that Ac-sec was more closely related to that of Vespid than to bee species from Apidae. The molecular characteristics of Ac-sec and the potential roles of small peptides in biology were discussed.

Overproduction of geranylgeraniol in Coprinopsis cinerea by the expression of geranylgeranyl diphosphate synthase gene.

(E, E, E)-Geranylgeraniol (GGOH) is a valuable ingredient of many perfumes and a valuable precursor for synthesizing pharmaceuticals. In an attempt to increase the GGOH concentration in Coprinopsis cinerea, we demonstrated that the expression of geranylgeranyl diphosphate synthase (ggpps) gene isolated from Taxus x media could promote GGOH production. Furthermore, the concentrations of squalene and ergosterol were measured in the engineered strains. Expectedly, significant decreases of squalene and ergosterol levels were observed in those strains transformed with ggpps gene. This could be explained by the partial redirection of metabolic flux from squalene to GGOH, whose biosynthesis competes for the same precursor with squalene. This work suggested that the expression of ggpps in higher fungi was an effective method for bio-production of GGOH.

Molecular cloning of a laccase gene from Ganoderma lucidum and heterologous expression in Pichia pastoris.

A genomic laccase gene and cDNA were cloned from the white-rot fungi Ganoderma lucidum TR6. The genomic laccase gene contained 2086 bp with nine introns. The laccase cDNA had an open reading frame of 1563 bp. The deduced mature protein consisted of 520 amino acids. Both the genomic laccase gene and cDNA were expressed in the Pichia pastoris GS115. Laccase activities could be detected in transformants with laccase cDNA but not in transformants with genomic laccase gene. The highest activity value reached 685.8 U L(-1). The effects of temperature, pH and nitrogen source on laccase expression in P. pastoris were analyzed. The recombinant laccase was purified and the molecular mass was 73.4 KDa, a little bigger than native laccase. The optimal pH and temperature were specific at pH 3.5 and special range from 60 to 90 °C. The laccase was stable at pH 7.0 and temperature range of 20-30 °C. The Km and Vm values of this recombinant laccase for ABTS were 0.521 mM and 19.65 mM min(-1), respectively.

Cloning and characterization of squalene synthase gene from Poria cocos and its up-regulation by methyl jasmonate.

Squalene synthase (SQS) catalyzes the condensation of two molecules of farnesyl diphosphate to give presqualene diphosphate and the subsequent rearrangement to form squalene. The gene encoding squalene synthase was cloned from Poria cocos by degenerate PCR and inverse PCR. The open reading frame of the gene is 1,497 bp, which encodes 499 amino acid residues. A phylogenetic analysis revealed that P. cocos SQS belonged to the fungus group, and was more closely related to the SQS of Ganoderma lucidum than other fungi. The treatment of P. cocos with methyl jasmonate (MeJA) significantly enhanced the transcriptional level of P. cocos sqs gene and the content of squalene in P. cocos. The transcriptional level of sqs gene was approximately fourfold higher than the control sample and the squalene content reached 128.62 µg/g, when the concentration of MeJA was 300 µM after 72 h induction.

Individual and combined effects of α-amylase, xylanase, and cellulase on the breadmaking and nutritional properties of steamed bun enriched in wheat bran.

The present study investigates the effects of α-amylase (6 and 10 ppm), xylanase (70 and 120 ppm), and cellulase (35 and 60 ppm) on the physicochemical characteristics and nutritional quality of Chinese steamed bun (CSB) incorporated with 15% wheat bran (WB). Compared to the single enzyme, the combined enzymes improved the specific volume of CSB up to the highest value (2.50 mL/g) and decreased the hardness to the minimum value (299.61 g) when the concentration was 6, 120, 35 ppm. Additionally, the combined enzymes (6, 120, and 35 ppm) significantly (p < 0.05) decreased the total dietary fiber from 14.65% to 13.10% and hence increased the area under the reducing sugar release curve during in vitro digestion from 302.12 to 357.26 mg/g. Consequently, enzymes combination can significantly improve the quality of WB CSB, whereas reduce the nutritional value of WB CSB.

Effect of Enyzmes on the Quality and Predicting Glycaemic Response of Chinese Steamed Bread.

The present study investigates the individual and interactional effects of α-amylase (6 and 10 ppm), xylanase (70 and 120 ppm) and cellulase (35 and 60 ppm) on the physicochemical characteristics and nutritional quality of Chinese steamed bread (CSB) incorporated with 15% oat bran. As a result, the single enzyme can significantly improve the specific volume and texture of CSB. Compared to the single enzyme, the combined enzymes improved the specific volume of CSB up to the highest value (2.51 mL/g) and decreased the hardness to the minimum value (233.61 g) when the concentration was 6, 70 and 35 ppm. With respect to chemical and nutritional properties, the addition of single enzyme had no great changes, while the combined enzymes (6, 70 and 35 ppm) significantly (p < 0.05) decreased the total starch from 37.52 to 34.11% and hence increased the area under the reducing sugar release curve during 2 h in vitro digestion (AUC) from 344.61 to 371.26. Consequently, enzymes combination can significantly improve the quality of oat bran CSB whereas reduce the nutritional value of oat bran CSB.

Dual roles of nanocrystalline cellulose extracted from jute (Corchorus olitorius L.) leaves in resisting antibiotics and protecting probiotics.

Antibiotics can cure diseases caused by bacterial infections, but their widespread use can have some side effects, such as probiotic reduction. There is an urgent need for such agents that can not only alleviate the damage caused by antibiotics, but also maintain the balance of the gut microbiota. In this study, we first characterized the nanocrystalline cellulose (NCC) extracted from plant jute (Corchorus olitorius L.) leaves. Next, we evaluated the protective effect of jute NCC and cellulose on human model gut bacteria (Lacticaseibacillus rhamnosus and Escherichia coli) under antibiotic stress by measuring bacterial growth and colony forming units. We found that NCC is more effective than cellulose in adsorbing antibiotics and defending the gut bacteria E. coli. Interestingly, the low-dose jute NCC clearly maintained the balance of key gut bacteria like Snodgrassella alvi and Lactobacillus Firm-4 in bees treated with tetracycline and reduced the toxicity caused by antibiotics. It also showed a more significant protective effect on human gut bacteria, especially L. rhamnosus, than cellulose. This study first demonstrated that low-dose NCC performed satisfactorily as a specific probiotic to mitigate the adverse effects of antibiotics on gut bacteria.

Proximity ligation assay induced and DNAzyme powered DNA motor for fluorescent detection of thrombin.

A novel DNA motor for thrombin detection was described here based on proximity ligation assay (PLA) induced DNAzyme recycling cleavage. Fluorophore labeled DNA is modified on gold nanoparticles (AuNPs) and the fluorescent signal is quenched by AuNPs. The PLA between target thrombin and two aptamers induces the forming of Mg2+-dependent DNAzyme. The fluorophore labeled DNA is cleaved circularly by the DNAzyme, releasing the fluorescent fragment from AuNPs surface. The cleavage and rebinding process create a processive walking along AuNPs surface track. As a result, the fluorescent intensity recovers significantly. A good linear relationship is obtained between the ratio of fluorescence intensity and thrombin concentration in the range from 10 pM to 10 nM. The limit of detection is calculated to be 4 pM. These results are comparable or even better than other amplification based methods.

Optimization of Baccatin III Production by Cross-Linked Enzyme Aggregate of Taxoid 10ß-O-Acetyltransferase.

Taxoid 10ß-O-acetyltransferase (DBAT) is the key enzyme to produce baccatin III, a key precursor in paclitaxel synthesis, by acetyl group transfer from acetyl-CoA to the C10 hydroxyl of 10-deacetylbaccatin III. In this study, the recombinant DBAT (rDBAT) was immobilized by cross-linked enzyme aggregates (CLEAs). To further optimize the enzyme recovery, single-factor experiment and response surface methodology were applied. 60% ammonium sulfate as precipitant, 0.05% glutaraldehyde as fixing agent, pH 7.0, 2 h as cross-linking time, 30 °C as cross-linking temperature were confirmed to be the optimum conditions to prepare the CLEAs-rDBAT in single-factor experiment. In addition, 62% for ammonium sulfate saturation, 0.15% for glutaraldehyde, and pH 6.75 were confirmed to be the optimum conditions with averagely 73.9% activity recovery in 3 replications, which was consistent with the prediction of response surface methodology. After cross-linking, the optimum temperature of CLEAs-rDBAT rose up to 70 °C and CLEAs-rDBAT could be recycled for three times.

Activity Essential Residue Analysis of Taxoid 10ß-O-Acetyl Transferase for Enzymatic Synthesis of Baccatin.

Taxoid 10ß-O-acetyl transferase (DBAT) is a key enzyme in the biosynthesis of the famous anticancer drug paclitaxel, which catalyses the formation of baccatin III from 10-deacetylbaccatin III (10-DAB). However, the activity essential residues of the enzyme are still unknown, and the acylation mechanism from its natural substrate 10-deacetylbaccatin III and acetyl CoA to baccatin III remains unclear. In this study, the homology modelling, molecular docking, site-directed mutagenesis, and kinetic parameter determination of the enzyme were carried out. The results showed that the enzyme mutant DBATH162A resulted in complete loss of enzymatic activity, suggesting that the residue histidine at 162 was essential to DBAT activity. Residues D166 and R363 which were located in the pocket of the enzyme by homology modelling and molecular docking were also important for DBAT activity through the site-directed mutations. Furthermore, four amino acid residues including S31 and D34 from motif SXXD, D372 and G376 from motif DFGWG also played important roles on acylation. This was the first report of the elucidation of the activity essential residues of DBAT, making it possible for the further structural-based re-design of the enzyme for efficient biotransformation of baccatin III and paclitaxel.

[Combinational expression of geranylgeranyl diphosphate synthase and taxadiene synthase in Coprinopsis cinerea].

Taxa-4(5),11(12)-diene is the precursor for paclitaxel biosynthesis. The diterpenoid paclitaxel (marketed as Taxol), a plant secondary metabolite isolated from yew, is an effective drug widely used in the treatment of numerous cancers. However, further application of taxol has been restricted due to its low yield in plants and the difficulties in extraction. To increase the intact isoprene flux, we constructed the fusion gene plasmid pBgGGTS and individual cassette plasmid pBgGGgTS to enhance the expression levels of geranylgeranyl diphosphate synthase gene (ggpps) and a taxadiene synthase gene (ts) in Coprinopsis cinerea. These two plasmids were separately transformed into C. cinerea LT2 strain, resulting in several putative transformants. Putative transformants were determined by PCR technique, indicating that 5 out of 13 putative transformants transformed by pBgGGTS and 6 out of 13 putative transformants transformed by pBgGGgTS, respectively. Additionally, the Southern blotting analysis of these 10 transformants confirmed that both ggpps and ts gene were stably integrated into the genome of C. cinerea. Crude extracts from each of the transformants were analyzed. There is no difference in the mycelium extracts among the wild-type LT2 and two types of transformants. However, analysis of culture filtrates indicated that an additional GC peak was found at the retention time of 16.762 min which was absent in the wild type control. The mass fragmentation pattern of this peak had the same diagnostic ions with taxa-4(5),11(12)-diene. According to peak area, the amounts of taxa-4(5),11(12)-diene in each fermented broth were 44 ng/L (transformed with pBgGGgTS) and 30 ng/L (transformed with pBgGGTS), respectively. In conclusion, co-expression of the ggpps and ts gene could increase the taxadiene production in C. cinerea.