Expression of POX2 gene and disruption of POX3 genes in the industrial Yarrowia lipolytica on the γ-decalactone production

The yeast Yarrowia lipolytica growing on methyl ricinoleate can produce γ-decalactone, the worthy aroma compound, which can exhibit fruity and creamy sensorial notes, and recognized internationally as a safe food additive. Unfortunately, the yield is poor because of lactone degradation by enzyme Aox3 (POX3 gene encoded), which was responsible for continuation of oxidation after C(10) level and lactone reconsumption. In this paper, we chose the industrial Y. lipolytica (CGMCC accession number 2.1405), which is the diploid strain as the starting strain and constructed the recombinant strain Tp-12 by targeting the POX3 locus of the wild type, one copy of POX3 was deleted by CRF1+POX2 insertion. The other recombinant strain Tpp-11, which was a null mutant possessing multiple copies of POX2 and disrupted POX3 genes on two chromosomes, was constructed by inserting XPR2+hpt into the other copy of POX3 of Tp-12. The growth ability of the recombinants was changed after genetic modification in the fermentation medium. The production of γ-decalactone was increased, resulting from blocking β-oxidation at the C(10) Aox level and POX2 overexpression. The recombinant strain Tpp-11 was stable. Because there was no reconsumption of γ-decalactone, the mutant strain could be grown in continuous fermentation of methyl ricinoleate to produce γ-decalactone.     

Peroxisomal β-oxidation activities and γ-decalactone production by the yeast Yarrowia lipolytica

γ-Decalactone is a peachy aroma compound resulting from the peroxisomal β-oxidation of ricinoleic acid by yeasts. The expression levels of acyl-CoA oxidase (gene deletion) and 3-ketoacyl-CoA thiolase activities (gene amplification on replicative plasmids) were modified in the yeast Yarrowia lipolytica. The effects of these modifications on β-oxidation were measured. Overexpression of thiolase activity did not have any effect on the overall β-oxidation activity. The disruption of one of the acyl-CoA oxidase genes resulted in an enhanced activity. The enhancement led to an increase of overall β-oxidation activity but reduced the γ-decalactone production rates. This seemed to indicate a non-rate-limiting role for β-oxidation in the biotransformation of ricinoleic acid to γ-decalactone by the yeast Yarrowia lipolytica. All strains produced and then consumed γ-decalactone. We checked the ability of the different strains to consume γ-decalactone in a medium containing the lactone as sole carbon source. The consumption of the strain overexpressing acyl-CoA oxidase activity was higher than that of the wild-type strain. We␣concluded that peroxisomal β-oxidation is certainly involved in γ-decalactone catabolism by the yeast Y.␣lipolytica. The observed production rates probably depend on an equilibrium between production and consumption of the lactone. Received: 13 June 1997 / Received revision: 2 October 1997 / Accepted: 14 October 1997     

Application of a Novel Oscillatory Flow Micro-bioreactor to the Production of γ-decalactone in a Two Immiscible Liquid Phase Medium

A novel micro-bioreactor based on the oscillatory flow technology was applied to the scale-down of the biotechnological production of γ-decalactone. A decrease up to 50% of the time required to obtain the maximum concentration of the compound was observed, when compared with other scaled-down platforms (stirred tank bioreactor or shake flask). A three-fold increase in γ-decalactone productivity was obtained by increasing oscillatory mixing intensity from Re_o ~482 to Re_o ~1447. This was presumably related to the effective contribution of the reactor geometry to enhanced mass transfer rates between the two immiscible liquid phases involved in the process by increasing the interfacial area. Revisions requested 11 October 2005; Revisions received 4 January 2006     

On the Wholesomeness of γ-Irradiated Potatoes

Fattening pigs and pigs for breeding have been fed γ-irradiated and non-irradiated control potatoes. The irradiation dose was 14–15 kilorad at the rates 175 r/sec. and 625 r/hr. Rats (Wistar) were also fed irradiated and control potatoes, but in this case the tubers were given a dose of about 200 kilorad. No unfavourable effects have been observed from the feeding with irradiated potatoes. All the facts indicate that the nutritional adequacy of the irradiated and the control tubers is equal. In some cases, the animals have even shown an improvement after feeding on irradiated potatoes which was not apparent after feeding on non-irradiated potatoes; for example there was a more rapid growth, somewhat higher fertility, and increased haemoglobin values. However, current experiments with larger groups of animals will provide more definite evidence in these questions.     

The enhancement of the oral bioavailability of γ-tocotrienol in mice by γ-cyclodextrin inclusion

Cyclodextrin (CD) is widely used in the pharmaceutical and nutritional fields to form an inclusion complex with lipophilic compounds for the improvement of their aqueous solubility, stability and diffusibility under physiological conditions. In this study, we investigated the effect of the γ-tocotrienol (γT3) inclusion complex with CD on its oral bioavailability. Five-week-old C57BL6 mice were fed a vitamin E-free diet for 28 days, followed by the oral administration of 2.79 mg of γT3-rich fraction (TRF) extracted from rice bran or the equivalent dose (14.5 mg) of a CD inclusion complex with TRF (TRF/CD). The levels of γT3 in sequentially collected plasma were determined by LC-MS/MS. The pharmacokinetic study revealed that the plasma concentrations of γT3 were increased and peaked at 6 or 3 h after the oral administration of TRF or TRF/CD, respectively (C_max values of 7.9±3.3 or 11.4±4.5 μM, respectively). The area under the curve of plasma γT3 concentration also showed a 1.4-fold increase in the group administered with TRF/CD compared with the TRF-only group. Furthermore, the mice that had received the TRF/CD tended to reduce the endotoxin shock induced by injection with lethal amounts of Escherichia coli lipopolysaccharide, compared with the mice that had received TRF alone. Taken together, our results suggest that the CD inclusion improved γT3 bioavailability, resulting in the enhancement of γT3 physiological activity, which would be a useful approach for the nutrition delivery system.     

Spectral Changes of the γ-Irradiated Glucose Solution

β-1,3-Xylanase was purified to gel electrophoretic homogeneity and 83-fold from a cell-free culture fluid of Vibrio sp. XY-214 by ammonium sulfate precipitation and successive chromatographies. The enzyme had a pl of 3.6 and a molecular mass of 52 kDa. The enzyme had the highest level of activity at pH 7.0 and 37°C. The enzyme activity was completely inhibited by Cu²⁺, Hg²⁺, and N-bromosuccinimide. The enzyme hydrolyzed β-1,3-xylan to produce mainly xylotriose and xylobiose but did not act on xylobiose, p-nitrophenyl-β-D-xyloside, β-1,4-xylan, β-1,3-glucan, or carboxymethyl cellulose.     

Effect of the three-dimensional organization of liver cells on the biogenesis of the γ-glutamyltransferase fraction pattern

Context Four gamma-glutamyltransferase (GGT) fractions with different molecular weights (big-, medium-, small- and free-GGT) are detectable in human plasma. Objective Verify if liver cells can release all four GGT fractions and if the spatial cell organization influences their release. Methods Hepatoma (HepG2) and melanoma (Me665/2/60) cells were cultured as monolayers or spheroids. GGT released in culture media was analysed by gel-filtration chromatography. Results HepG2 and Me665/2/60 monolayers released the b-GGT fraction, while significative levels of s-GGT and f-GGT were detectable only in media of HepG2-spheroids. Bile acids alone or in combination with papain promoted the conversion of b-GGT in s-GGT or f-GGT, respectively. Conclusions GGT is usually released as b-GGT, while s-GGT and f-GGT are likely to be produced in the liver extracellular environment by the combined action of bile acids and proteases.     

The Construction of γ-TIP Prokaryotic Expression Vector and Preparation of the γ- TIP Antibody

A prokaryotic expression vector, pGEX-TIP, was constructed from Arabidopsis thaliana (L.) Heynh. Employing PCR, 205 bp fragment near 3' end of γ-TIP cDNA, which has specific aquaporin activity, was amplified and cloned into pGEX-KG. Restriction endonuclease analysis and sequencing confirmed the correct construction, and 0.4 mmoL/L IPTC can induce high expression of GST-TIP fused protein which was about 50% in total of E. coli proteins. The IPTG induced E. coli was collected and ]ysed by supersonic treatment. The fusion protein was mainly recovered as an inclusion body. The expressed GST-TIP was purified by SDS-PAGE according to their molecular weight, which was about 32 kD. The purified protein was used to immune rabbits directly or was electrophoretically eluted before it was used for immunization. The highly qualified antibody for GST-TIP was obtained, which provides a very useful protein probe for the research on localization and function of aquaporins.     

Contribution of the γ-secretase subunits to the formation of catalytic pore of presenilin 1 protein

γ-Secretase is an intramembrane-cleaving protease related to the etiology of Alzheimer disease. γ-Secretase is a membrane protein complex composed of presenilin (PS) and three indispensable subunits: nicastrin, Aph-1, and Pen-2. PS functions as a protease subunit forming a hydrophilic catalytic pore structure within the lipid bilayer. However, it remains unclear how other subunits are involved in the pore formation. Here, we show that the hydrophilic pore adopted with an open conformation has already been formed by PS within the immature γ-secretase complex. The binding of the subunits induces the close proximity between transmembrane domains facing the catalytic pore. We propose a model in which the γ-secretase subunits restrict the arrangement of the transmembrane domains of PS during the formation of the functional structure of the catalytic pore.     

Involvement of sialic acid in the regulation of γ--aminobutyric acid uptake activity of γ-aminobutyric acid transporter 1

The γ-aminobutyric acid (GABA) transporters (GATs) have long been recognized for their key role in the uptake of neurotransmitters. The GAT1 belongs to the family of Na(+)- and Cl(-)-coupled transport proteins, which possess 12 putative transmembrane (TM) domains and three N-glycosylation sites on the extracellular loop between TM domains 3 and 4. Previously, we demonstrated that terminal trimming of N-glycans is important for the GABA uptake activity of GAT1. In this work, we examined the effect of deficiency, removal or oxidation of surface sialic acid residues on GABA uptake activity to investigate their role in the GABA uptake of GAT1. We found that the reduced concentration of sialic acid on N-glycans was paralleled by a decreased GABA uptake activity of GAT1 in Chinese hamster ovary (CHO) Lec3 cells (mutant defective in sialic acid biosynthesis) in comparison to CHO cells. Likewise, either enzymatic removal or chemical oxidation of terminal sialic acids using sialidase or sodium periodate, respectively, resulted in a strong reduction in GAT1 activity. Kinetic analysis revealed that deficiency, removal or oxidation of terminal sialic acids did not affect the K(m) GABA values. However, deficiency and removal of terminal sialic acids of GAT1 reduced the V(max) GABA values with a reduced apparent affinity for extracellular Na(+). Oxidation of cell surface sialic acids also strongly reduced V(max) without affecting both affinities of GAT1 for GABA and Na(+), respectively. These results demonstrated for the first time that the terminal sialic acid of N-linked oligosaccharides of GAT1 plays a crucial role in the GABA transport process.     

An improved method for the synthesis of γ-DDB

A mild and efficient method for the synthesis γ-DDB has been developed through anhydride-linker assisted intramolecular Ullmann reaction. Highly regioselective bromination of differentially protected gallate was realized by virtue of the introduction of NBS.     

Investigation of the early stages of human γD-crystallin aggregation process

Cataract, a major cause of visual impairment worldwide, is a common disease of the eye lens related to protein aggregation. Several factors including the exposure of ultraviolet irradiation and possibly acidic condition may induce the unfolding and subsequent aggregation of the crystallin proteins leading to crystalline lens opacification. Human γD-crystallin (HγDC), a 173 residue monomeric protein, abundant in the nucleus of the human eye lens, has been shown to aggregate and form amyloid fibrils under acidic conditions and that this aggregation route is thought to be a potential initiation pathway for the onset of age-related nuclear cataract. However, the underlying mechanism of fibril formation remains elusive. This report is aimed at examining the structural changes and possible amyloid fibril formation pathway of HγDC using molecular dynamics and molecular docking simulations. Our findings demonstrated that incubation of HγDC under the acidic condition redistributes the protein surface charges and affects the protein interaction with its surrounding solvent environment. This brings about a twist motion in the overall tertiary structure that gives rise to newly formed anti-parallel β-strands in the C-terminal flexible loop regions. The change in protein structural conformation also involves an alteration in specific salt-bridge interactions. Altogether, these findings revealed a plausible mechanism for amyloid fibril formation of HγDC that is important to the early stages of HγDC aggregation involved in cataractogenesis.