The consumers' demand for natural flavour and fragrances rises. To be natural, compounds have to result from the extraction of natural materials and/or to be transformed by natural means such as the use of enzymes or whole cells. Fungi are able to transform some fatty acids into lactones that can thus be natural. Although some parts of this subject have been reviewed several times, the present article proposes to review the different pathways utilised, the metabolic engineering strategies and some current concerns on the reactor application of the transformation including scaling up data. The main enzymatic steps are hydroxylation and β-oxidation in the traditional way, and lactone desaturation or Baeyer-Villiger oxidation. Although the pathway to produce γ-decalactone is rather well known, metabolic engineering strategies may result in significant improvements in the productivity. For the production of other lactones, a key step is the hydroxylation of fatty acids. Beside the biotransformation, increasing the production of the various lactones requires from biotechnologists to solve two main problems which are the toxicity of lactones toward the producing cell and the aeration of the emulsified reactor as the biochemical pathway is very sensitive to the level of available oxygen. The strategies employed to resolve these problems will be presented.
In hydrophobic compounds biotechnology, medium-chain-length metabolites often perturb cell activity. Their effect is usually studied in model conditions of growth in glucose media. Here, we study whether culture on lipids has an impact on the resistance of Yarrowia lipolytica to such compounds: Cells were cultured on glucose or oleate and submitted to gamma-dodecalactone. After a 60-min exposure to 3 g L(-1), about 80% of the glucose-grown cells (yeast extract peptone dextrose (YPD) cells) had lost their cultivability, 38% their membrane integrity, and 31% their reducing capacity as shown with propidium iodide and methylene blue, respectively. For oleate-grown cells, treatment at 6 g L(-1) did not alter cultivability despite some transient loss of membrane integrity from 3 g L(-1). It was shown with diphenylhexatriene and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene that oleate-grown cells had membranes more fluid and less sensitive to the lactone-induced fluidization. Analyses revealed also higher contents of ergosterol but, for YPD- and minimum-oleate-grown cells (YNBO cells), the addition of lactone provoked a decrease in the concentration of ergosterol in a way similar to the depletion by methyl-beta-cyclodextrin and an important membrane fluidization. Ergosterol depletion or incorporation increased or decreased, respectively, cell sensitivity to lactone. This study shows that the embedment of oleate moieties into membranes as well as higher concentrations of sterol play a role in the higher resistance to lactone of oleate-grown cells (YPO cells). Similar oleate-induced increase in resistance was also observed for Rhodotorula and Candida strains able to grow on oleate as the sole carbon source whereas Saccharomyces and Sporidiobolus cells were more sensitive after induction.
Based on the observation that shocks provoked by heat or amphiphilic compounds present some similarities, this work aims at studying whether cells grown on oleate (amphiphilic pre-stress) acquire a tolerance to heat shock. In rich media, changing glucose for oleate significantly enhanced the cell resistance to the shock, however, cells grown on a minimal oleate medium lost their ability to grow on agar with the same kinetic than glucose-grown cells (more than 7-log decrease in 18 min compared with 3-log for oleate-grown cells). Despite this difference in kinetics, the sequence of events was similar for oleate-grown cells maintained at 50°C with a (1) loss of ability to form colonies at 27°C, (2) loss of membrane integrity and (3) lysis (observed only for some minimal-oleate-grown cells). Glucose-grown cells underwent different changes. Their membranes, which were less fluid, lost their integrity as well and cells were rapidly inactivated. But, surprisingly, their nuclear DNA was not stained by propidium iodide and other cationic fluorescent DNA-specific probes but became stainable by hydrophobic ones. Moreover, they underwent a dramatic increase in membrane viscosity. The evolution of lipid bodies during the heat shock depended also on the growth medium. In glucose-grown cells, they seemed to coalesce with the nuclear membrane whereas for oleate-grown cells, they coalesced together forming big droplets which could be released in the medium. In some rare cases of oleate-grown cells, lipid bodies were fragmented and occupied all the cell volume. These results show that heat triggers programmed cell death with uncommon hallmarks for glucose-grown cells and necrosis for methyl-oleate-grown cells.
The yeast ecosystem of Nem chua, a Vietnamese traditional fermented sausage naturally rich in mediumchain-length lipid-derived flavouring compounds, was investigated to select biocatalysts able to produce the C10-fatty acid-derived aroma compound c-decalactone. The total number of yeast was about 5 9 10 4 to 4 9 10 5 CFU g À1 , and eighty four different species were identified from morphological, physiological and 26S rDNA characteristics, with Candida sake and Candida haemulonii being found in all samples. Six strains able to produce c-decalactone from castor oil were selected, of which three Yarrowia lipolytica strains were able to produce between 1 and 2 g L À1 in our study. The strains produced the same amount from the acyl substrate under the form of ester or free fatty acid. Every strain degraded the product at the end of the culture. These high productions make them good candidates for industrial processes and confirm that traditional fermented foods are interesting bioresources for biocatalysts.
<abstract> <p>Yeast are a powerful material for the encapsulation of compounds. Usually, yeast used as capsules are inactivated by the encapsulation treatment, which is stressful to cells. However, if kept active, cells can bring their own activity in addition to the encapsulated compound. We have observed previously that lipid-grown <italic>Yarrowia lipolytica</italic> were more resistant to encapsulation. The objective of the present study was to identify physiological markers involved in this resistance. Cells were cultured in the presence of glucose or methyl-oleate as the sole carbon source and submitted to a γ-dodecalactone stress. This paper focuses on the role of intracellular lipid droplets (LDs) and of the ergosteryl content to protect cells during the lactone treatment. Lipid-grown cells were more resistant to lactone and the presence of LDs before the shock increased significantly the resistance. The ergosteryl esters from the LD pool were hydrolysed to release ergosterol able to strenghten the plasma membrane during the shock. For cells devoid of LDs, membrane ergosterols were esterified concomitantly with LDs growth, resulting in a membrane weakening. By using <italic>pox3</italic>-mutant strains, which possesse numerous and small-sized LDs, we observed the original behaviour: these mutants showed no increased resistance and their LDs exploded in the cytoplasma during the shock. These results point out the role of LDs in cell resistance to amphiphilic stresses as a storage compartment as well as in ergosterol homeostasis.</p> </abstract>
This study investigated the possibility of using yeast fermentation to transform shrimp waste to chitin for further application. The white leg shrimp head was incubated with three yeast strains Yarrowia lipolytica, Candida tropicalis and Pichia kudriavzevii, in comparison with bacteria Bacillus subtilis and commercial protease Alcalase. The efficacy of fermentation was evaluated through deproteinization and demineralization levels after 2 days. A deproteinization of 80.9% was obtained when incubation with Y. lipolytica which was significantly higher than 76.9% and 65.6% obtained with Alcalase hydrolysis and B. subtilis incubation respectively. Besides, C. tropicalis and P. kudriavzevii expressed a similar low level on deproteinization (31.3-31.7%.) All three yeast showed a good demineralization in range 38.2-49.4% on shrimp head while B. subtilis could demineralize only 16.0%. This primary research shows a potential application of yeast fermentation in chitin recovery from shrimp waste.
Biohythane produced via dark fermentation is much greener than hythane that is generated using natural gas. Biohythane production using a single-stage system has potential to increase the economic viability since it requires fewer controls than a two-stage system that has individual acidogenic and methanogenic reactors. This single-stage system is an innovative method in producing biohythane. The present work investigated the performance of a mesophilic single-stage system with a batch mode operation to generate biohythane. The reactor was seeded with hydrogenic and methanogenic bacteria (HB and MB), which were entrapped in κ-carrageenan/ gelatin beads (2%/2% w/w) using the dripping method. The energy yield of 0.41 to 1.48 kJ g–1 glucose and the hydrogen content in biohythane (H2/(H2 + CH4)) of 0.35 to 0.69 were obtained. These results indicate that different biohythane compositions would be obtained by regulating the HB/MB bacteria concentration ratio, substrate concentration and cultivation pH. Moreover, a comparison of two-stages and single-stage systems as well as the challenges were also elucidated.
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