Engineering yeast for high-level production of diterpenoid sclareol.

The diterpenoid sclareol is an industrially important precursor for alternative sustainable supply of ambergris. However, its current production from plant extraction is neither economical nor environmental-friendly, since it requires laborious and cost-intensive purification procedures and plants cultivation is susceptible to environmental factors. Engineering cell factories for bio-manufacturing can enable sustainable production of natural products. However, stringent metabolic regulation poses challenges to rewire cellular metabolism for overproduction of compounds of interest. Here we used a modular approach to globally rewire the cellular metabolism for improving sclareol production to 11.4 g/L in budding yeast Saccharomyces cerevisiae, the highest reported diterpenoid titer in microbes. Metabolic flux analysis showed that modular balanced metabolism drove the metabolic flux toward the biosynthesis of targeted molecules, and transcriptomic analysis revealed that the expression of central metabolism genes was shaped for a new balanced metabolism, which laid a foundation in extensive metabolic engineering of other microbial species for sustainable bio-production.

Changes in protease activity during fermentation of fish sauce and their correlation with antioxidant activity.

BACKGROUND: Antioxidant activity has been found in fermented fish sauce. In this experiment, the properties of endogenous protease and antioxidant activity were studied in anchovy sauce during fermentation. The correlation between protease activity and antioxidant activity in fermented anchovy sauce was analyzed using the partial least squares (PLS) method. RESULTS: The results showed that at least four proteases were present in the endogenous enzyme solution, and the optimum pH values were 2.5, 5.5, 9.0, and 12.5, respectively. The maximum inhibition rate of endogenous protease, from high to low, was: serine protease inhibitor > trypsin inhibitor > aspartic protease inhibitor (pepsin inhibitor) > cysteine protease inhibitor > metalloprotease inhibitor. At the sixth month of fermentation, fish sauce had stronger trypsin, pepsin-like activity, and antioxidant activity. At the ninth month of fermentation, the cathepsin activity was greater. A model correlating changes in protease activity with antioxidant activity suggested that the trypsin and serine protease were the main factors affecting antioxidant activity. CONCLUSION: This study reports a model correlating changes in protease activity with the antioxidant activity of fish sauce. It lays a foundation for further exploration of the formation of antioxidant substances and antioxidant effects during the process of fish sauce fermentation. © 2021 Society of Chemical Industry.

Identification, taste characterization and molecular docking study of novel umami peptides from the Chinese anchovy sauce.

BACKGROUND: Fish sauce has a subtle flavor with prominent umami and salty taste, and is accompanied by a certain sweetness and bitterness. In order to identify a wider range of umami peptides, Chinese southern and northern anchovy sauce were selected for the study. RESULTS: Seventeen peptides were obtained by separation and purification, and their taste activity was predicted. Through the taste characterization and descriptive analysis, it was found that the synthesized peptides were umami and umami-enhancing peptides. Seventeen umami peptides were simulated and embedded into the umami receptor T1R1/T1R3 by inserting into the Venus flytrap domain (VFTD) of the T1R3 subunit; the interaction forces were mainly hydrogen bonding, electrostatic interaction, van der Waals force and hydrophobic interaction. According to the docking interaction energies, long-chain peptides may be easier to bind to the receptor than short-chain peptides. Asp196, Glu128 and Glu197 were the main binding sites for docking, and could affect umami synergism. CONCLUSION: For the first time, novel umami peptides in Chinese anchovy sauce have been reported. This study is helpful for discovering umami marine resource peptides, and can provide a basis for further understanding the flavor system of anchovy sauce. © 2020 Society of Chemical Industry.

Leaching and retention of dissolved metals in particulate loaded pervious concrete columns.

This study examined metal leaching and retention in pervious concrete with or without embedded particulate matter. Particulate matter was collected from an adjacent parking lot and from a nearby parking garage as examples of weathered and un-weathered particulate matter. Particle size distributions were similar, but metal content was 3-35-fold higher and organic matter content was 3-fold higher in the parking garage particulate matter compared to the parking lot particulate matter. Replicate columns were established with either no particulate added as the control, or 20 g of parking lot or parking garage particulate matter. Synthetic rainwater was passed through the columns at variable rainfall intensity or fixed intensity to assess leaching. Metals were leached at higher concentrations from the parking garage particulate amended column, but from all columns less than 1% of the metal mass leached. Rainfall intensity did not have a large effect on leached metal concentrations, only varying effluent by about 2-fold. Synthetic stormwater with elevated dissolved Cu, Zn, Cd and Pb concentrations was passed through the same columns and metal removal efficiencies were on the order of 85-95%, 30-95%, 60-90%, and 95+% for each metal, respectively. After loading the column with a year's worth of stormwater metal exposure, removal efficiencies in the no particulate and parking lot particulate amended columns decreased, while parking garage particulate amended columns performed similarly with a small drop in Cu and Pb removal efficiencies. Generally, columns with no particulate and parking lot particulate amendments performed similarly, suggesting the pervious concrete is responsible for the majority of the initial metal retention. The parking garage particulate amended columns retained more metals from stormwater, perhaps due to an increase in pH that promoted surface precipitation as hydroxides or carbonate species on the pervious concrete, or due to complexation in the higher concentrations of organic matter and iron oxides in the particulate matter. Overall, metal retention was aided by the presence of organic matter in the particulate matter, but the pervious concrete itself was more important than particulates for metal retention. A strategy to increase metal retention and removal from the environment could involve amending pervious concrete with mixtures of well-defined sorbents to enhance metal retention.

Enzymatic hydrolysis of 7-xylosyltaxanes by an extracellular xylosidase from Cellulosimicrobium cellulans.

OBJECTIVES: To find extracellular biocatalysts that can specifically and efficiently remove the C-7 xylosyl group from 7-xylosyltaxanes. RESULTS: A Cellulosimicrobium cellulans strain F16 that can remove the C-7 xylosyl group from 7-xylosyltaxanes was isolated from the root soil of an old Taxus yunnanensis tree. Using corn cob as sole carbon source, the maximum 7-xylosyl-10-deacetylpaclitaxel ß-xylosidase activity of 9.6 U l(-1) was achieved. The ß-xylosidase could be trapped by a ceramic tubular membrane (pore size 50 nm), and exhibited an apparent molecular weight much greater than 500 kDa. Under optimized conditions, 3.75 l cell-free culture medium transformed 2 grams 7-xylosyltaxane mixtures to their corresponding aglycones within 3 h, with a conversion >98%. CONCLUSION: This is the first report that C. cellulans can produce extracellular ß-xylosidases capable of removing the C-7 xylosyl group from 7-xylosyltaxanes.

Four amino acids play an important role in the allergenicity of hemocyanin allergen.

To identify the key amino acids (AAs) affecting the allergenicity of hemocyanin (HC) allergens from Chinese mitten crabs, in this study, two epitopes, P1-SHFTGSKSNPEQR and P2-LSPGANTITR were employed and four potential key AAs (P1: F3 and N9 and P2: N6 and R10) were predicted. Mast cell and mouse models revealed that four mutants induced lower levels of immunoglobulin E (IgE) and Th2 type cytokines (15.47-49.89 %), proving that F3, N9, N6, and R10 were the key AAs of two epitopes. Mutants reduce allergic responses via the Th2 pathway. However, the roles of every key AA affecting allergenicity were different (P1-F3 > N9 and P2-N6 > R10). In addition, lower transport and higher efflux were observed in the mutants during transport absorption by Caco-2 cells. The allergenicity of HC was stronger when the transport absorption efficiency of epitopes and mutants was higher and their efflux was lower. Our study provides a novel method for revealing the allergenic molecular mechanisms of food allergens.

Simultaneous Identification and Species Differentiation of Major Allergen Tropomyosin in Crustacean and Shellfish by Infrared Spectroscopic Chemometrics.

To solve the lack of rapid and accurate methods for allergen identification and traceability, an infrared spectroscopic chemometric analytical model (IR-CAM) was established by combining infrared spectroscopy with principal component and cluster analysis. By comparing the second derivative infrared (SD-IR) spectra of 5 proteins and 14 crustaceans and shellfish tropomyosin (TM), 8 shared peaks and unique fingerprint peaks in the amide III region were found for crabs, shrimps, and shellfish. Based on the unique fingerprint peaks coexisting with shared peaks, allergen TM in crustaceans and shellfish could be identified within 10 min (cf. ELISA âˆ¼ 4 h). Concurrently, the species differentiation of TM at the Class/Family level was achieved based on IR-CAM. Validation by fermented aquatic products TM (n = 60) demonstrated that the developed IR-CAM could simultaneously identify and differentiate TM in crustaceans and shellfish accurately. It could be applied for allergen detection and traceability of aquatic products on an antibody-free basis.

Molecular and allergenic properties of natural hemocyanin from Chinese mitten crab (Eriocheir sinensis).

Hemocyanin in crustaceans is an allergen for humans. However, little information was available on its molecular, structural and allergenic properties. In this study, the purified natural protein was identified as Eriocheir sinensis HC by LC-MS/MS, which was allergenic because its reaction with the serum IgE of crustacean patients. Results of the molecular properties showed that, HC was resistant to trypsin digestion, but not a heat-stable protein. Boiling (55.05 ± 3.50 %) and steaming (66.84 ± 1.65 %) induced an increase in ß-sheet and decreased allergenicity of HC. By comparing the amino acid sequences of eight crustaceans, HC was found to be highly conserved. Five epitopes of HC were identified and validated by murine sensitization model, and two of them (P3 and P10) were exactly as the predicted by six types of bioinformatics. Multiple bioinformatics analysis combining with murine sensitization model seemed to be effective way for identification of allergenic epitopes.

Changes in taste substances during fermentation of fish sauce and the correlation with protease activity.

Anchovy sauce shows different taste profiles under different fermentation time. The change rules of free amino acids was measured by amino acid analyzer, and other taste substances, such as nucleotides and organic acids in anchovy sauce under different fermentation time were also investigated. Moreover, the correlation between protease activity and taste substances in anchovy sauce fermentation was analyzed by orthogonal partial least squares. Throughout the fermentation process, the taste substances in anchovy sauce increased during early months and then decreased as time increased. The content of amino acid nitrogen, TCA-soluble peptides, 5'-nucleotides (AMP, GMP, IMP) and organic acids (lactic acid, succinic acid) in anchovy sauce increased by 26%, 33%, (45%, 35%, 68%) and (27%, 2%) respectively in comparison with 6 months fermentation. Total amino acid content reached its maximum after 18 months fermentation. Results of electronic tongue demonstrated that the umami of anchovy sauce after 12 months fermentation increased by 17% in comparison with 6 months fermentation. A model correlating changes in protease activity with taste formation suggested that protease activity impacted the content of Ala, Glu, Lys, Asp, Leu, TCA-soluble peptides and succinic acid. This study can provide empirical evidence to guide the efficient processing of anchovy sauce.

Characterization and structure-activity relationship of novel umami peptides isolated from Thai fish sauce.

Fish sauce has a prominent umami flavor. In this study, umami peptides were isolated, purified and identified from Thai fish sauce, and their structure-activity relationships were analyzed. Six novel umami peptides were characterized and verified by using sensory evaluation and a electronic tongue. Molecular docking with T1R1/T1R3 receptors showed that the interaction forces were mainly hydrogen bonds, electrostatic interaction and van der Waals force. In the constructed three dimensional quantitative structure-activity relationship model (3D-QSAR) model, the regression coefficient (R2) for the degree of dispersion between the predicted molecular and the experimental values of the six peptides was 0.976. The association between the structure and activity of umami peptides was revealed through 3D-QSAR. Results showed that the spatial effect was significant for long chain peptides.

Rapid qualitative and quantitative determination of seven valuable taxanes from various Taxus species by UFLC-ESI-MS and UFLC-DAD.

The distribution and level of yew constituents vary with species and tissues. In this study, a rapid and valid method incorporating ultra-fast liquid chromatography (UFLC) with MS and UV detection was developed for simultaneous determination of paclitaxel and its six semisynthesis precursors in needles and hair roots from various Taxus species. All target analytes could be identified by comparing their retention times as well as UV and MS spectra with authentic standards, while seven valuable taxanes in botanical samples can be rapidly determined by UFLC-DAD with excellent sensitivity. Analysis of more than one hundred yew samples from nine species showed significant variations in distribution and content of seven evaluated taxanes. Thus, different developmental schemes should be used for better utilization of various yew resources.

Deoxynojirimycin enhanced the transglycosylation activity of a glycosidase from the China white jade snail.

A beta-D-glycosidase (G I) from the China white jade snail showed non-Michaelis-Menten mode in catalyzing the reaction using pNPGlu and pNPFuc as the substrate and monitoring the released pNP. We determined quantitatively both the transglycosidic and hydrolytic products of pNPGlu and pNPFuc solvolysis for the detailed kinetic analysis on G I-catalyzed hydrolysis and transglycosylation reaction. The inhibition kinetic studies using deoxynojirimycin (DNJ) and butanol as inhibitors were preceded. DNJ only inhibited competitively the hydrolysis of cellobiose and pNPGlu while "activated" the transglycosylation of pNPGlu and pNPFuc. This was evident from the increased V(max)tr value with no change of the apparent K(m)tr. In contrast, butanol exhibited a competitive inhibition to the transglycosylation reaction and non-competitive inhibition to the hydrolysis. The results indicated that the non-Michaelis-Menten kinetic behavior was caused by the co-occurrence of substrate transglycosylation reaction. This study provided a simple method to increase the transglycosylation yield by using DNJ to inhibit hydrolysis.

Acceptor specificity and transfer efficiency of a beta-glycosidase from the Chinese white jade snail.

The acceptor specificity and transfer potential of a beta-D-glycosidase (G I), which had been purified from the China white jade snail, were further investigated by using various sugars as acceptors. G I had broad monosaccharide acceptor specificity for its transglycosylation activity. More specifically, it efficiently catalyzed the transfer of the beta-D-fucosyl, beta-D-glucosyl or beta-D-galactosyl moiety from the corresponding p-nitrophenyl-beta-D-glycopyranosides to various monosaccharides. The transfucosylation efficiency of G I was studied by using p-nitrophenyl-beta-D-fucopyranoside (pNPFuc) as the donor and glucose and xylose as the acceptors. The yields under conditions of non-initial velocity were 88% for glucose and 93% for xylose. The transfer product with glucose as the acceptor was isolated and identified as beta-fucosyl-1,6-glucose by an NMR analysis. The data from these analyses indicate that G I had broad acceptor specificity and high efficiency for transglycosylation. These uncommon properties of G I could make it a valuable biocatalyst for the synthesis of various disaccharides.

Size characterization of dissolved metals and organic matter in source waters to streams in developed landscapes.

Individual and mixed water samples from wastewater treatment plant effluents, stormwater runoff, streams from developed areas were characterized with respect to organic matter concentration and spectral properties and metal concentration and size distribution. In addition, asymmetric flow-field flow fractionation coupled to inductively coupled plasma mass spectrometry was used to measure concentration, size distribution and association of metals in the colloidal size range. Results reveal that Fe, Cu, Zn and Pb in the colloidal size range were mainly associated with the less than 5 nm, or less than 10 kDa size range. Cu was most strongly associated with organic matter, while Zn and Pb were mixed between Fe and organic matter. Effluent showed higher binding capacity for metals, while stormwater, even with higher organic matter concentrations showed more exchangeable metals. Upon mixing of source waters, colloidal metal concentrations and size distributions were conserved.

Self-sustained reduction of multiple metals in a microbial fuel cell-microbial electrolysis cell hybrid system.

A self-sustained hybrid bioelectrochemical system consisting of microbial fuel cell (MFC) and microbial electrolysis cell (MEC) was developed to reduce multiple metals simultaneously by utilizing different reaction potentials. Three heavy metals representing spontaneous reaction (chromium, Cr) and unspontaneous reaction (lead, Pb and nickel, Ni) were selected in this batch-mode study. The maximum power density of the MFC achieved 189.4 mW m(-2), and the energy recovery relative to the energy storage circuit (ESC) was ∼ 450%. At the initial concentration of 100 mg L(-1), the average reduction rate of Cr(VI) was 30.0 mg L(-1) d(-1), Pb(II) 32.7 mg L(-1) d(-1), and Ni(II) 8.9 mg L(-1) d(-1). An electrochemical model was developed to predict the change of metal concentration over time. The power output of the MFC was sufficient to meet the requirement of the ESC and MEC, and the "self-sustained metal reduction" was achieved in this hybrid system.

Functional and structural properties of a novel cellulosome-like multienzyme complex: efficient glycoside hydrolysis of water-insoluble 7-xylosyl-10-deacetylpaclitaxel.

Cellulosome is a kind of multienzyme complex that displays high activity, selectivity, and stability. Here, we report a novel, non-cellulolytic, cellulosome-like multienzyme complex that produced by the Cellulosimicrobium cellulans wild-type strain F16 isolated from soil microflora. This multienzyme complex, with excellent catalytic efficiency of kcat 13.2 s(-1) to remove the C-7 xylosyl group from 7-xylosyl-10-deacetylpaclitaxel (10-DAXP), has an outstanding tolerance against organic solvents and an excellent general stability, with the long half-life of 214 hours. This cellulosome-like multienzyme complex has a novel structure distinct from the well-documented ones. The key catalytic subunit responsible for the ß-xylosidase activity against 10-DAXP is identified to be a novel protein, indicating a new glycoside hydrolase (GH) family. The pioneering work described here offers a novel nanoscale biocatalyst for the production of biofuels and chemicals from renewable plant-based natural resources.

[Industrial preparative chromatography purification of 10-deacetylpaclitaxel, the enzymatic product of 7-xylosyl-10-deacetylpaclitaxel].

A scheme for industrial preparative chromatography purification of 10-deacetylpaclitaxel (10-DAP), the semi-synthesized precursor of anticancer drug paclitaxel was developed. 7-Xylosyl-10-deacetylpaclitaxel (10-DAXP) is the most abundant constitute in the needles of Taxus Chinensis, a specific yew species distributed in China. 10-DAXP has been recognized as a good material to convert into 10-DAP, the most ideal precursor of paclitaxel. The partially purified extract from yew needles which mainly contains 10-DAXP (> 60%) and other two minor 7-xylosyl-10-deacetyltaxanes including 7-xylosyl-10-deacetylcephalomannine (10-DAXC) and 7-xylosyl-10-deacetylpaclitaxel C (10-DAXP C), was used as the starting material. The total scheme can be divided into four steps. Firstly, the starting material was hydrolyzed by beta-xylosidase to remove the C-7 xylosyl group completely; and then the hydrolyzed products mainly containing 10-DAP were eluted on a column packed with resin to get crude 10-DAP (with the purity of 20.5%) with high yield (96.3%). The crude 10-DAP was purified by a column packed with normal phase, and then by a reversed-phase preparative chromatography with ODS as the solid phase. After these two steps, the purity of the aim product 10-DAP was 96% with the overall yield of 79.7%. This novel scheme was suitable for large-scale purification of 10-DAP from 10-DAXP.

Effective dechlorination of HCB by nanoscale Cu/Fe particles.

We previously reported that microscale Cu/Fe bimetal could be used for the dechlorination of hexachlorobenzene (HCB), a representative polychlorinated persistent organic pollutant (POPs). But slow reduction rate and rather incomplete dechlorination were reached. In this study, HCB dechlorination by nanoscale Fe and Cu/Fe was evaluated. It was found that HCB reduction by nanoscale Fe was rather slow, and the reduction was significantly increased by nanoscale Cu/Fe. Near complete reduction of HCB was obtained by nanoscale Cu/Fe for 48 h treatment. HCB was quickly dechlorinated to PeCB, TeCBs, TCBs and DCBs without selectivity via a stepwise process. The reduction rate and dechlorination extent were much higher compared with microscale Cu/Fe. Lowering pH during reduction showed slightly negative influence on HCB reduction by nanoscale Cu/Fe due to retarded co-precipitation. A catalytic hydrogenation process on Cu surface through iron oxide film was suggested for the increased HCB reduction by Cu coating on nanoscale Fe. This study proved that using a much cheaper bimetallic iron of nanoscale Cu/Fe than nanoscale Pd/Fe could also achieve the effective dechlorination of HCB.

Quantitative structure-retention relationship studies for taxanes including epimers and isomeric metabolites in ultra fast liquid chromatography.

Seven pairs of epimers and one pair of isomeric metabolites of taxanes, each pair of which have similar structures but different retention behaviors, together with additional 13 taxanes with different substitutions were chosen to investigate the quantitative structure-retention relationship (QSRR) of taxanes in ultra fast liquid chromatography (UFLC). Monte Carlo variable selection (MCVS) method was adopted to choose descriptors. The selected four descriptors were used to build QSRR model with multi-linear regression (MLR) and artificial neural network (ANN) modeling techniques. Both linear and nonlinear models show good predictive ability, of which ANN model was better with the determination coefficient R(2) for training, validation and test set being 0.9892, 0.9747 and 0.9840, respectively. The results of 100 times' leave-12-out cross validation showed the robustness of this model. All the isomers can be correctly differentiated by this model. According to the selected descriptors, the three dimensional structural information was critical for recognition of epimers. Hydrophobic interaction was the uppermost factor for retention in UFLC. Molecules' polarizability and polarity properties were also closely correlated with retention behaviors. This QSRR model will be useful for separation and identification of taxanes including epimers and metabolites from botanical or biological samples.

[Purification and characterization of a beta-D-xylosidase from Leifsonia shinshuensis DICP 16].

A beta-D-xylosidase from Leifsonia shinshuensis DICP 16 was purified to apparent homogeneity using a combination of ammonium sulfate precipitation, DE 52 anion-exchange, Q-Sepharose Fast Flow anion-exchange, Toyopearl Butyl 650C hydrophobic-interaction and Sephacryl S-300 HR gel-permeation chromatography. The purified xylosidase consisted of two same subunits and had the relative molecular weight of 180 kD as determined by SDS-PAGE and gel-permeation chromatography. The maximal beta-D-xylosidase activity occurred at 55 degrees C and pH 7.0. It was stable at 45 degrees C and retained its original activity for 60 min. The stability declined rapidly when the temperature rose above 55 degrees C. The xylosidase was stable in the pH range from 6.0 to 11.0 for 20 h. At pH 7.0 and 45 degrees C the Km for p-nitrophenyl-beta-D-xylopyranoside (pNPX) was 1.04 mmol/L and the Vmx was 0.095 mmol nitrophenol/min/mg xylosidase. The enzyme was inhibited strongly by Fe2+ and Cu2+. It exhibited low levels of activity against other artificial substrates, compared to its activity against pNPX. When different natural xylosides were used as the substrates, the xylosidase showed distinct hydrolysis ability. It could hydrolyze 20-C, beta-(1-->6)-xyloside of ginsenoside Rb3 (G-Rb3) into ginsenoside Rd, but did not hydrolyze the other beta-D-glucosidic bonds of G-Rb3. Additionally, the xylosidase could not hydrolyze C-7 xylosyl-bearing taxanes.