Plant-associated microorganisms fulfill important functions for their hosts. Whereas promotion of plant growth and health is well-studied, little is known about the impact of microorganisms on plant ...or fruit flavor. To analyze the production of volatiles of grape-associated microorganisms, samples of grapes of the red cultivar ‘Blaufraenkisch’ were taken during harvest time from four different vineyards in Burgenland (Austria). The production of volatiles was analyzed for the total culturable microbial communities (bacteria, yeasts, fungi) found on and in the grapes as well as for single isolates. The microbial communities produced clearly distinct aroma profiles for each vineyard and phylogenetic group. Furthermore, half of the grape-associated microorganisms produced a broad spectrum of volatile organic compounds. Exemplarily, the spectrum was analyzed more in detail for three single isolates of Paenibacillus sp., Sporobolomyces roseus, and Aureobasidium pullulans. Well-known and typical flavor components of red wine were detected as being produced by microbes, for example, 2-methylbutanoic acid, 3-methyl-1-butanol, and ethyl octanoate.
The current demand to cut back on the use of plastic materials has brought a major boost to the search for bio-based alternatives. Not only are plastic bags and primary food packaging under scrutiny ...here, but also those materials used as functional barriers to reduce, for example, the migration of mineral oil hydrocarbons (MOH) from recycled paper and board packaging. Most of the barriers now in use are synthetic, often have only moderate barrier functionalities and in addition reduce the environmentally-friendly character of cellulose-based materials. Against this background, bio-based polymers have been evaluated in terms of their functional barrier properties. Chitosan was found to be among the best performers in these materials. In this study, the behavior of a lab-made chitosan acetate film was compared with conventionally produced polymer films. The two-sided migration experiment described recently was used to determine the barrier properties of the tested materials. This not only allowed to test the intrinsic migration of the films and the permeation through them, but also to simulate real packaging situations by using a recycled paper as donor for MOH. The migrated fractions were determined using gas-chromatography-based techniques. While the conventionally produced polymer films showed only moderate barrier function, excellent results were seen for the biopolymer. It reduced the migration from the recycled paper to not detectable, singling it out as a good alternative to conventional materials.
Chocolate is a highly appreciated food that develops its characteristic flavors in large part during the roasting of cacao beans. Many functional classes have been noted for their importance to ...chocolate flavor, including volatile organic sulfur compounds (VSCs). Despite this, the effect of roasting on the concentration of VSCs has never been thoroughly assessed. Here, we studied the effects of roasting temperature, time, and cacao origin on the formation of VSCs. Twenty-seven 100% chocolate samples made from cacao from three different origins and roasted according to an I-optimal experimental design were analyzed by comprehensive gas chromatography with sulfur-selective detection (GCxGC-SCD). For two compounds, dimethyl disulfide and dimethyl trisulfide, the effects of roasting time, roasting temperature, and cacao origin were modelled using response surface methodology and semi-quantified relative concentration. Overall, roasting increased the number of sulfur-containing volatiles present in chocolate, with a total of 28 detected, far more than previously thought. Increased roasting time and especially roasting temperature were found to significantly increase the concentration of VSCs (
< 0.05), while cacao origin effects were only seen for dimethyl disulfide (
< 0.05). The identity of most VSCs remains tentative, and more research is needed to unravel the impact of these volatiles on flavor perception in chocolate.
The sesquiterpenoid (+)-nootkatone is a highly demanded and highly valued aroma compound naturally found in grapefruit, pummelo or Nootka cypress tree. Extraction of (+)-nootkatone from plant ...material or its production by chemical synthesis suffers from low yields and the use of environmentally harmful methods, respectively. Lately, major attention has been paid to biotechnological approaches, using cell extracts or whole-cell systems for the production of (+)-nootkatone. In our study, the yeast Pichia pastoris initially was applied as whole-cell biocatalyst for the production of (+)-nootkatone from (+)-valencene, the abundant aroma compound of oranges. Therefore, we generated a strain co-expressing the premnaspirodiene oxygenase of Hyoscyamus muticus (HPO) and the Arabidopsis thaliana cytochrome P450 reductase (CPR) that hydroxylated extracellularly added (+)-valencene. Intracellular production of (+)-valencene by co-expression of valencene synthase from Callitropsis nootkatensis resolved the phase-transfer issues of (+)-valencene. Bi-phasic cultivations of P. pastoris resulted in the production of trans-nootkatol, which was oxidized to (+)-nootkatone by an intrinsic P. pastoris activity. Additional overexpression of a P. pastoris alcohol dehydrogenase and truncated hydroxy-methylglutaryl-CoA reductase (tHmg1p) significantly enhanced the (+)-nootkatone yield to 208mgL−1 cell culture in bioreactor cultivations. Thus, metabolically engineered yeast P. pastoris represents a valuable, whole-cell system for high-level production of (+)-nootkatone from simple carbon sources.
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•A self-sufficient, whole-cell system for (+)-nootkatone production in Pichia pastoris.•Metabolic engineering enhanced (+)-nootkatone yield to 208mgL−1 cell culture.•P. pastoris is proposed as promising, novel host for sesquiterpenoid production.
The addition of water to non‐activated carbon–carbon double bonds catalyzed by fatty acid hydratases (FAHYs) allows for highly regio‐ and stereoselective oxyfunctionalization of renewable oil ...feedstock. So far, the applicability of FAHYs has been limited to free fatty acids, mainly owing to the requirement of a carboxylate function for substrate recognition and binding. Herein, we describe for the first time the hydration of oleic acid (OA) derivatives lacking this free carboxylate by the oleate hydratase from Elizabethkingia meningoseptica (OhyA). Molecular docking of OA to the OhyA 3D‐structure and a sequence alignment uncovered conserved amino acid residues at the entrance of the substrate channel as target positions for enzyme engineering. Exchange of selected amino acids gave rise to OhyA variants which showed up to an 18‐fold improved conversion of OA derivatives, while retaining the excellent regio‐ and stereoselectivity in the olefin hydration reaction.
Redefining the substrate spectrum: The highly regio‐ and stereoselective hydration of oleic acid derivatives by an oleate hydratase is possible. The carboxylate of a free fatty acid—previously considered essential—is not mandatory for the conversion, which thus expands hydration biocatalysis beyond inferred restrictions.
The triterpenoid (+)‐ambrein is the major component of ambergris, a coprolite of the sperm whale that can only be rarely found on shores. Upon oxidative degradation of (+)‐ambrein, several fragrance ...molecules are formed, amongst them (−)‐ambrox, one of the highest valued compounds in the perfume industry. In order to generate a Saccharomyces cerevisiae whole‐cell biocatalyst for the production of (+)‐ambrein, intracellular supply of the squalene was enhanced by overexpression of two central enzymes in the mevalonate and sterol biosynthesis pathway, namely the N‐terminally truncated 3‐hydroxy‐3‐methylglutaryl‐CoA reductase 1 (tHMG) and the squalene synthase (ERG9). In addition, another key enzyme in sterol biosynthesis, squalene epoxidase (ERG1) was inhibited by an experimentally defined amount of the inhibitor terbinafine in order to reduce flux of squalene towards ergosterol biosynthesis while retaining sufficient activity to maintain cell viability and growth. Heterologous expression of a promiscuous variant of Bacillus megaterium tetraprenyl‐β‐curcumene cyclase (BmeTC‐D373C), which has been shown to be able to catalyse the conversion of squalene to 3‐deoxyachillol and then further to (+)‐ambrein resulted in production of these triterpenoids in S. cerevisiae for the first time. Triterpenoid yields are comparable with the best microbial production chassis described in literature so far, the methylotrophic yeast Pichia pastoris. Consequently, we discuss similarities and differences of these two yeast species when applied for whole‐cell (+)‐ambrein production.
Sterols exert a profound influence on numerous cellular processes, playing a crucial role in both health and disease. However, comprehending the effects of sterol dysfunction on cellular physiology ...is challenging. Consequently, numerous processes affected by impaired sterol biosynthesis still elude our complete understanding. In this study, we made use of yeast strains that produce cholesterol instead of ergosterol and investigated the cellular response mechanisms on the transcriptome as well as the lipid level. The exchange of ergosterol for cholesterol caused the downregulation of phosphatidylethanolamine and phosphatidylserine and upregulation of phosphatidylinositol and phosphatidylcholine biosynthesis. Additionally, a shift towards polyunsaturated fatty acids was observed. While the sphingolipid levels dropped, the total amounts of sterols and triacylglycerol increased, which resulted in 1.7-fold enlarged lipid droplets in cholesterol-producing yeast cells. In addition to internal storage, cholesterol and its precursors were excreted into the culture supernatant, most likely by the action of ABC transporters Snq2, Pdr12 and Pdr15. Overall, our results demonstrate that, similarly to mammalian cells, the production of non-native sterols and sterol precursors causes lipotoxicity in
, mainly due to upregulated sterol biosynthesis, and they highlight the different survival and stress response mechanisms on multiple, integrative levels.
Despite similarities of cellular membranes in all eukaryotes, every compartment displays characteristic and often unique features which are important for the functions of the specific organelles. In ...the present study, we biochemically characterized the plasma membrane of the methylotrophic yeast Pichia pastoris with emphasis on the lipids which form the matrix of this compartment. Prerequisite for this effort was the design of a standardized and reliable isolation protocol of the plasma membrane at high purity. Analysis of isolated plasma membrane samples from P. pastoris revealed an increase of phosphatidylserine and a decrease of phosphatidylcholine compared to bulk membranes. The amount of saturated fatty acids in the plasma membrane was higher than in total cell extracts. Ergosterol, the final product of the yeast sterol biosynthetic pathway, was found to be enriched in plasma membrane fractions, although markedly lower than in Saccharomyces cerevisiae. A further characteristic feature of the plasma membrane from P. pastoris was the enrichment of inositol phosphorylceramides over neutral sphingolipids, which accumulated in internal membranes. The detailed analysis of the P. pastoris plasma membrane is discussed in the light of cell biological features of this microorganism especially as a microbial cell factory for heterologous protein production.
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•A new protocol for the isolation of the plasma membrane from P. pastoris is presented.•Plasma membrane fractions are characterized by a typical lipid pattern.•Plasma membrane fractions are enriched in saturated fatty acids.•Molecular species of sphingolipids from the plasma membrane were analyzed.
•The interactions between paper samples and volatile compounds were investigated using three experimental setups.•. Adsorption and permeation experiments with paper samples and permeation experiments ...with paper samples and MPPO were used.•The test setup was complex and many factors influenced the results.•Paper cannot entirely be considered as a permeable barrier.
Paper is used in many different applications, due to its numerous favourable characteristics. An inherent property of paper is its porosity. One of the main effects of this porosity is that it allows the possibility of mass transport through it. In this work, we describe a study that was carried out to simulate the transport of substances relevant for food contact applications through paper using adsorption/desorption experiments and permeation experiments in migration cells. The results of the experiments are discussed, first in terms of the compounds that were transferred through the test paper samples and second in terms of the ability to use these methods to simulate transport processes. The results of the individual experiments were combined and used to explain similarities and differences in the sample behaviour. We show that the methods correlated well with one another and especially the polar compounds interacted strongly with the paper samples.
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