Characterization of the off-flavor from Pichia pastoris GS115 during the overexpression of an α-l-rhamnosidase.

The off-flavor of Pichia pastoris strains is a negative characteristic of proteins overexpressed with this yeast. In the present study, P. pastoris GS115 overexpressing an α-l-rhamnosidase was taken as the example to characterize the off-flavor via sensory evaluation, gas chromatography-mass spectrometer, gas chromatography-olfaction, and omission test. The result showed that the off-flavor was due to the strong sweaty note, and moderate metallic and plastic notes. Four volatile compounds, that is, tetramethylpyrazine, 2,4-di-tert-butylphenol, isovaleric acid, and 2-methylbutyric acid, were identified to be major contributors to the sweaty note. Dodecanol and 2-acetylbutyrolactone were identified to be contributors to the metallic and plastic notes, respectively. It is the first study on the off-flavor of P. pastoris strains, helping understand metabolites with off-flavor of this yeast. Interestingly, it is the first study illustrating 2-acetylbutyrolactone and dodecanol with plastic and metallic notes, providing new information about the aromatic contributors of biological products. IMPORTANCE: The methylotrophic yeast Pichia pastoris is an important host for the industrial expression of functional proteins. In our previous studies, P. pastoris strains have been sniffed with a strong off-flavor during the overexpression of various functional proteins, limiting the application of these proteins. Although many yeast strains have been reported with off-flavor, no attention has been paid to characterize the off-flavor in P. pastoris so far. Considering that P. pastoris has advantages over other established expression systems of functional proteins, it is of interest to identify the compounds with off-flavor synthesized in the overexpression of functional proteins with P. pastoris strains. In this study, the off-flavor synthesized from P. pastoris GS115 was characterized during the overexpression of an α-l-rhamnosidase, which helps understand the aromatic metabolites with off-flavor of P. pastoris strains. In addition, 2-acetylbutyrolactone and dodecanol were newly revealed with plastic and metallic notes, enriching the aromatic contributors of biological products. Thus, this study is important for understanding the metabolites with off-flavor of P. pastoris strains and other organisms, providing important knowledge to improve the flavor of products yielding with P. pastoris strains and other organisms. ONE-SENTENCE SUMMARY: Characterize the sensory and chemical profile of the off-flavor produced by one strain of P. pastoris in vitro.

The Analysis of the Glycosyltransferase Gene Function From a Novel Granaticin Producer, Streptomyces Vilmorinianum. YP1.

Glycosylation is common among the synthesis of natural product and imparts the bioactivity for natural product. As for granaticin, a natural product with great bioactivity, glycosylation is an unusual sugar attachment and remains enigmatic. Orf14 in the gra cluster is the predicted glycosyltransferase but without being identified. Recently, we isolated and identified a novel granaticin producer Streptomyces vilmorinianum YP1. Orf14 gene in gra cluster of YP1 is knocked out and complemented. The instrumental analysis of the blue product synthesized by orf14-deficient mutant exhibits the none-granaticin detection and deglycosylated intermediates accumulation. The bioactivity and stability test suggests the weaker or none antibacterial activity and cytotoxicity of this blue product with greater ultraviolet stability and thermostability than granaticin and derivatives produced by YP1. All the result indicates that orf14 encodes glycosyltransferase and glycosylation played an important role in the bioactivity of granaticin. Meanwhile, the blue pigment, deglycosylated intermediates, has favorable processing characteristics. Our finding supplies the function of orf14 and glycosylation, but also indicates a promising candidate of edible blue pigment applicated in food industry.

Determination of aflatoxin B1 by novel nanofiber-packed solid-phase extraction coupled with a high performance liquid chromatography-fluorescence detector.

A novel analytical proposal based on nanofiber-packed solid-phase extraction coupled with high performance liquid chromatography-fluorescence detector (HPLC-FLD) has been successfully developed for determining aflatoxin B1 (AFB1) in foods. Four types of nanofibers, including polystyrene (PS) nanofibers, polypyrrole (PPY) nanofibers, polystyrene-acrylic resin (PS-AR) nanofibers, and polystyrene-polyvinyl pyrrolidone (PS-PVP) nanofibers, were fabricated by electrospinning and utilized to prepare a home-made extraction device. In this study, the factors of different fibers, namely, fiber dosage, pH of extraction solution, type of salt ion, concentration of salt ion, and volume of the eluent were optimized. Under optimized conditions, the method showed good linearity in the range of 0.1-40 ng mL-1 with a correlation coefficient greater than 0.999 and good inter-day accuracy (90.8-112.7% recovery) and precision (1.8-3.6% intra-day RSDs, 2.6% inter-day RSD), and the limit of detection (LOD) was 0.05 ng mL-1. Due to its cost-effective, time-saving, environmentally friendly, and simple performance, it has the potential to be utilized to determine aflatoxins in complicated matrices.

Purification, identification and properties of a new blue pigment produced from Streptomyces sp. A1013Y.

A novel blue pigment was first isolated from Streptomyces sp. A1013Y. The purified component was identified as 4,8,13-trihydroxy-6,11-dione-trihydrogranaticins A (TDTA). Its physical properties were found to be: Molecular weight 462 Da; Color value, E0.1%1cm580 nm = 80; Solubility, it dissolved in organic solvents. In addition, the color of TDTA changed with pH but was found to be relatively stable between 20 and 100 °C, from pH 3 to pH 11, and under UV-light or darkness. TDTA's functional properties was as follows: TDTA showed excellent free radical scavenging properties, IC50 41.04 µg/mL and 13.75 µg/mL using 2, 20-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-n-(3, 2-ethyl-benzothiazole-6-sulfonic acid) ammonium salt (ABTS) respectively. TDTA might be a promising source of natural pigment and bioactive compound used as additive in food industry.

Detection of ß-agonists in pork tissue with novel electrospun nanofibers-based solid-phase extraction followed ultra-high performance liquid chromatography/tandem mass spectrometry.

A selective analytical method based on packed-fiber solid-phase extraction and ultra-high performance liquid chromatography-tandem mass spectrometry (PFSPE-UPLC-MS/MS) has been developed for determination of six ß-agonists (clorprenaline, bambuterol, clenbuterol, brombuterol, mabuterol, and penbuterol) in pork tissue. Polystyrene-polymeric crown ether (PS-PCE) composite nanofibers were fabricated by electrospinning and utilized to prepare the homemade extraction columns. With optimal conditions, all analytes were separated very well and the blank pork did not disturb the determination, and the linearity is good in a range of 5.0µg/kg-25.0µg/kg. The recoveries were 79.3-110.1%. RSDs for intra-day were in the range of 1.5-10.5% and RSDs for inter-day were 4.7-11.8%. Above all, only 5mg of sorbent and 200µL of elution solvent were favorable to directly extract all analytes in a complex matrix. The method is simple and cost-effective, and has the potential to be applied to quantitatively analyze the concentrations of polar species in food samples containing complex matrix.

New model for the genesis and maturation of viroplasms induced by fijiviruses in insect vector cells.

Plant reoviruses are thought to replicate and assemble within cytoplasmic, nonmembranous structures called viroplasms. Here, we established continuous cell cultures of the white-backed planthopper (Sogatella furcifera Horváth) to investigate the mechanisms for the genesis and maturation of the viroplasm induced by Southern rice black-streaked dwarf virus (SRBSDV), a fijivirus in the family Reoviridae, during infection of its insect vector. Electron and confocal microscopy revealed that the viroplasm consisted of a granular region, where viral RNAs and nonstructural proteins P6 and P9-1 accumulated, and a filamentous region, where viral RNAs, progeny cores, viral particles, as well as nonstructural proteins P5 and P6 accumulated. Our results suggested that the filamentous viroplasm matrix was the site for the assembly of progeny virions. Because viral RNAs were produced by assembled core particles within the filamentous viroplasm matrix, we propose that these viral RNAs might be transported to the granular viroplasm matrix. P5 formed filamentous inclusions and P9-1 formed granular inclusions in the absence of viral infection, suggesting that the filamentous and granular viroplasm matrices were formed primarily by P5 and P9-1, respectively. P6 was apparently recruited in the whole viroplasm matrix by direct interaction with P9-1 and P5. Thus, the present results suggested that P5, P6, and P9-1 are collectively required for the genesis and maturation of the filamentous and granular viroplasm matrix induced by SRBSDV infection. Based on these results, we propose a new model to explain the genesis and maturation of the viroplasms induced by fijiviruses in insect vector cells.

Poly-3,4-ethylenedioxythiophene nanoclusters for high effective solid phase extraction.

Solid phase extraction (SPE) has emerged as the widely used technique for sample preparation in the analytical process. Recent research trends of SPE are toward developing novel adsorbents to enrich the analytes simply and effectively. In this study, we proposed the poly-3,4-ethylenedioxythiophene (PEDOT) nanoclusters as the SPE adsorbent. During the application, only a small amount of PEDOT nanoclusters was needed and placed in a pipet tip with glass wool on either side. Without complex preparation, the target analytes could be directly extracted from the sample onto the extraction material and eluted in this lab-in-a-pipet-tip system. The efficiency of this approach was demonstrated by detecting 20 kinds of sulfonamides (SAs) in honey with ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS). All the analytes were detected by multiple reaction monitoring (MRM) mode. The established method was extensively validated by determining the linearity (R(2)≥0.991), average recovery (88.4-105.0%) and precision (relative standard deviation ≤9.60%). Low detection limits (0.5-4 µg kg(-1)), wide linearity (10-250 µg kg(-1)) and short sample pretreatment time (20 min) were achieved under the optimized conditions. The absolute recoveries of the SAs at high level ranged from 71.1% to 91.4%. Due to its simplicity, selectivity and sensitivity, our new method has potential applications in quantitative analysis of the target compounds in complex samples.