Evaluation of the food grade expression systems NICE and pSIP for the production of 2,5-diketo-D-gluconic acid reductase from Corynebacterium glutamicum

2,5-diketo-D-gluconic acid reductase (2,5-DKG reductase) catalyses the reduction of 2,5-diketo-D-gluconic acid (2,5-DKG) to 2-keto-L-gulonic acid (2-KLG), a direct precursor (lactone) of L-ascorbic acid (vitamin C). This reaction is an essential step in the biocatalytic production of the food supplement vitamin C from D-glucose or D-gluconic acid. As 2,5-DKG reductase is usually produced recombinantly, it is of interest to establish an efficient process for 2,5-DKG reductase production that also satisfies food safety requirements. In the present study, three recently described food grade variants of the Lactobacillales based expression systems pSIP (Lactobacillus plantarum) and NICE (Lactococcus lactis) were evaluated with regard to their effictiveness to produce 2,5-DKG reductase from Corynebacterium glutamicum. Our results indicate that both systems are suitable for 2,5-DKG reductase expression. Maximum production yields were obtained with Lb. plantarum/pSIP609 by pH control at 6.5. With 262 U per litre of broth, this represents the highest heterologous expression level so far reported for 2,5-DKG reductase from C. glutamicum. Accordingly, Lb. plantarum/pSIP609 might be an interesting alternative to Escherichia coli expression systems for industrial 2,5-DKG reductase production

New Plasmids for Fusarium Transformation Allowing Positive-Negative Selection and Efficient Cre-loxP Mediated Marker Recycling

In filamentous fungi such as Fusarium graminearum, disruption of multiple genes of interest in the same strain (e.g., to test for redundant gene function) is a difficult task due to the limited availability of reliable selection markers. We have created a series of transformation vectors that allow antibiotic-based selection of transformants and subsequent negative selection for marker removal using thymidine kinase fusions combined with the Cre-loxP system. The fusion genes contain commonly used C-terminal drug resistance markers, either nptII (G418), nat1 (nourseothricin), or hph (hygromycin B). These resistance genes are fused to the sequence encoding Herpes simplex virus thymidine kinase (HSVtk). Despite the presence of the 1 kb HSVtk gene (about ∼30% increase in total marker size), there is only a slight reduction in transformation efficiency on a molar basis. The fusion genes expressed under the Trichoderma pyruvate kinase (PKI) promoter also confer antibiotic resistance in Escherichia coli, allowing straightforward construction of disruption plasmids. For removal of the loxP flanked resistance cassettes, protoplasts of transformants are directly treated with purified Cre recombinase protein. Loss of the HSVtk containing cassette is selected by restoration of resistance to 5-fluoro-2-deoxyuridine (FdU). As a proof of principle, we demonstrated the efficiency of the HSVtk-based marker removal in Fusarium by reversing the disruption phenotype of the gene responsible for production of the red pigment aurofusarin. We first disrupted the FgPKS12 gene via integration of the loxP-flanked HSVtk-nptII cassette into the promoter or the first intron, thereby generating transformants with a white mycelium phenotype. Using Cre recombinase and FdU, the selection marker was subsequently removed, and the resulting transformants regained red pigmentation despite the remaining loxP site. We also found that it is possible to remove several unselected loxP-flanked cassettes with a single Cre protein treatment, as long as one of them contains a negative selectable HSVtk cassette. The negative selection system can also be used to introduce allele swaps into strains without leaving marker sequences, by first disrupting the gene of interest and then complementing the deletion in situ with genomic DNA containing a different allele

Heterologous expression of Oenococcus oeni malolactic enzyme in Lactobacillus plantarum for improved malolactic fermentation

Lactobacillus plantarum is involved in a multitude of food related industrial fermentation processes including the malolactic fermentation (MLF) of wine. This work is the first report on a recombinant L. plantarum strain successfully conducting MLF. The malolactic enzyme (MLE) from Oenococcus oeni was cloned into the lactobacillal expression vector pSIP409 which is based on the sakacin P operon of Lactobacillus sakei and expressed in the host strain L. plantarum WCFS1. Both recombinant and wild-type L. plantarum strains were tested for MLF using a buffered malic acid solution in absence of glucose. Under the conditions with L-malic acid as the only energy source and in presence of Mn2+ and NAD+, the recombinant L. plantarum and the wild-type strain converted 85% (2.5 g/l) and 51% (1.5 g/l), respectively, of L-malic acid in 3.5 days. Furthermore, the recombinant L. plantarum cells converted in a modified wine 15% (0.4 g/l) of initial L-malic acid concentration in 2 days. In conclusion, recombinant L. plantarum cells expressing MLE accelerate the malolactic fermentation

In vivo contribution of deoxynivalenol-3-β-D-glucoside to deoxynivalenol exposure in broiler chickens and pigs: oral bioavailability, hydrolysis and toxicokinetics

Crossover animal trials were performed with intravenous and oral administration of deoxynivalenol-3-β-D-glucoside (DON3G) and deoxynivalenol (DON) to broiler chickens and pigs. Systemic plasma concentrations of DON, DON3G and de-epoxy-DON were quantified using liquid chromatography-tandem mass spectrometry. Liquid chromatography coupled to high-resolution mass spectrometry was used to unravel phase II metabolism of DON. Additionally for pigs, portal plasma was analysed to study presystemic hydrolysis and metabolism. Data were processed via tailor-made compartmental toxicokinetic models. The results in broiler chickens indicate that DON3G is not hydrolysed to DON in vivo. Furthermore, the absolute oral bioavailability of DON3G in broiler chickens was low (3.79 ± 2.68 %) and comparable to that of DON (5.56 ± 2.05 %). After PO DON3G administration to pigs, only DON was detected in plasma, indicating a complete presystemic hydrolysis of the absorbed fraction of DON3G. However, the absorbed fraction of DON3G, recovered as DON, was approximately 5 times lower than after PO DON administration, 16.1 ± 5.4 compared with 81.3 ± 17.4 %. Analysis of phase II metabolites revealed that biotransformation of DON and DON3G in pigs mainly consists of glucuronidation, whereas in chickens predominantly conjugation with sulphate occurred. The extent of phase II metabolism is notably higher for chickens than for pigs, which might explain the differences in sensitivity of these species to DON. Although in vitro studies demonstrate a decreased toxicity of DON3G compared with DON, the species-dependent toxicokinetic data and in vivo hydrolysis to DON illustrate the toxicological relevance and consequently the need for further research to establish a tolerable daily intake

Development and Validation of an LC-MS/MS Based Method for the Determination of Deoxynivalenol and Its Modified Forms in Maize

The Fusarium mycotoxin deoxynivalenol (DON) is a common contaminant of cereals and is often co-occurring with its modified forms DON-3-glucoside (D3G), 3-acetyl-DON (3ADON) or 15-acetyl-DON (15ADON). A stable-isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) based method for their determination in cereals was developed and validated for maize. Therefore, 13C-labelled D3G was enzymatically produced using 13C-DON and [13C6Glc]-sucrose and used as an internal standard (IS) for D3G, while uniformly 13C labelled IS was used for the other mycotoxins. Baseline separation was achieved for the critical peak pair DON/D3G, while 3ADON/15ADON could not be fully baseline separated after testing various reversed phase, fluorinated phase and chiral LC columns. After grinding, weighing and extracting the cereal samples, the raw extract was centrifuged and a mixture of the four 13C-labelled ISs was added directly in a microinsert vial. The subsequent analytical run took 7 min, followed by negative electrospray ionization and selected reaction monitoring on a triple quadrupole MS. Maize was used as a complex cereal model matrix for validation. The use of the IS corrected the occurring matrix effects efficiently from 76 to 98% for D3G, from 86 to 103% for DON, from 68 to 100% for 15ADON and from 63 to 96% for 3ADON

Synthesis of Mono- and Di-Glucosides of Zearalenone and α-/β-Zearalenol by Recombinant Barley Glucosyltransferase HvUGT14077

Zearalenone (ZEN) is an estrogenic mycotoxin occurring in Fusarium-infected cereals. Glucosylation is an important plant defense mechanism and generally reduces the acute toxicity of mycotoxins to humans and animals. Toxicological information about ZEN-glucosides is limited due to the unavailability of larger amounts required for animal studies. HvUGT14077, a recently-validated ZEN-conjugating barley UDP-glucosyltransferase was expressed in Escherichia coli, affinity purified, and characterized. HvUGT14077 possesses high affinity (Km = 3 µM) and catalytic efficiency (kcat/Km = 190 s−1·mM−1) with ZEN. It also efficiently glucosylates the phase-I ZEN-metabolites α-zearalenol and β-zearalenol, with kcat/Km of 40 and 74 s−1·mM−1, respectively. HvUGT14077 catalyzes O-glucosylation at C-14 and C-16 with preference of 14-glucoside synthesis. Furthermore, relatively slow consecutive formation of 14,16-di-glucosides was observed; their structures were tentatively identified by mass spectrometry and for ZEN-14,16-di-glucoside confirmed by nuclear magnetic resonance spectroscopy. Recombinant HvUGT14077 allowed efficient preparative synthesis of ZEN-glucosides, yielding about 90% ZEN-14-glucoside and 10% ZEN-16-glucoside. The yield of ZEN-16-glucoside could be increased to 85% by co-incubation with a β-glucosidase highly selective for ZEN-14-glucoside. Depletion of the co-substrate UDP-glucose was counteracted by a sucrose synthase based regeneration system. This strategy could also be of interest to increase the yield of minor glucosides synthesized by other glucosyltransferases

Occurrence of Deoxynivalenol, Nivalenol, and Their Glucosides in Korean Market Foods and Estimation of Their Population Exposure through Food Consumption

Major type B trichothecene mycotoxins, including deoxynivalenol (DON), nivalenol (NIV), and their respective glucoside conjugates, deoxynivalenol-3-β-D-glucose (DON3G) and nivalenol-3-β-D-glucose (NIV3G), are present in food products, such as cereals, legumes, and their processed products. Thus, here, DON, NIV, and their 3-β-D-glucosides were monitored in 506 Korean market foods, and exposure to these mycotoxins was estimated in the population consuming these foods. The accuracy and precision of our method, which simultaneously determined four toxins, were 80.1–106.5% and 0.3–12.4%, in four representative food matrices assessed. The incidences of DON, DON3G, NIV, and NIV3G among all food samples tested were 13%, 8%, 12%, and 5%, respectively. The glucoside conjugate with free toxin was found to have the maximum co-occurrence of 49%. The estimated daily intakes of DON, DON3G, NIV, and NIV3G through food intake under four different scenarios were 0.019–0.102, 0.004–0.089, 0.007–0.094, and 0.002–0.095 μg kg−1 body weight (b.w.) day−1, respectively, which are lower than the established health-based guidance values. Overall, our results suggest that the estimated exposure of the Korean population to type B trichothecenes, namely, DON, NIV, and their 3-β-D-glucoside conjugates, may not pose a potential health risk

Metabolism of Zearalenone and Its Major Modified Forms in Pigs

The Fusarium mycotoxin zearalenone (ZEN) can be conjugated with polar molecules, like sugars or sulfates, by plants and fungi. To date, the fate of these modified forms of ZEN has not yet been elucidated in animals. In order to investigate whether ZEN conjugates contribute to the total ZEN exposure of an individual, ZEN (10 µg/kg b.w.) and equimolar amounts of two of its plant metabolites (ZEN-14-O-β-glucoside, ZEN-16-O-β-glucoside) and of one fungal metabolite (ZEN-14-sulfate) were orally administered to four pigs as a single bolus using a repeated measures design. The concentrations of ZEN, its modified forms and its mammalian metabolites ZEN-14-glucuronide, α-zearalenol (α-ZEL) and α-ZEL-14-glucuronide in excreta were analyzed by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) based methods. The biological recovery of ZEN in urine was 26% ± 10%, the total biological recovery in excreta was 40% ± 8%. Intact ZEN-14-sulfate, ZEN-14-O-β-glucoside and ZEN-16-O-β-glucoside were neither detected in urine nor in feces. After ZEN-14-sulfate application, 19% ± 5% of the administered dose was recovered in urine. In feces, no ZEN metabolites were detected. The total biological recoveries of ZEN-14-O-β-glucoside and ZEN-16-O-β-glucoside in the form of their metabolites in urine were 19% ± 11% and 13% ± 7%, respectively. The total biological recoveries in urine and feces amounted to 48% ± 7% and 34 ± 3%. An explanation for the low biological recoveries could be extensive metabolization by intestinal bacteria to yet unknown metabolites. In summary, ZEN-14-sulfate, ZEN-14-O-β-glucoside, and ZEN-16-O-β-glucoside were completely hydrolyzed in the gastrointestinal tract of swine, thus contributing to the overall toxicity of ZEN