Feasibility of raw glycerol conversion into single cell oil by zygomycetes under non-aseptic conditions.

The use of plant oils as feedstock for the biodiesel manufacture has many drawbacks, thus, the interest has turned to single cell oil (SCO) as an alternative. However, the production of SCO is still too expensive, mainly due to the low oil productivity and the high cost of medium sterilization required. In this work raw glycerol was converted into SCO by oleaginous Zygomycetes under non-aseptic conditions on selective (i.e., containing essential oils and/or antibiotics) nitrogen limited media. The obtained data showed that although bacterial populations inhibited the fungal growth, lipid accumulation remained unaffected by the presence of bacteria in the growth medium compared to control experiments (conducted under aseptic conditions). Therefore, a two-stage process was developed in which growth was performed under aseptic conditions (1st stage) followed by lipid accumulation performed under non-aseptic conditions (2nd stage) in the presence of thyme essential oil as an antibacterial agent. Large amounts of lipids were accumulated inside the mycelia, yielding around 13% wt/wt of oil per glycerol consumed.

Morphological and metabolic shifts of Yarrowia lipolytica induced by alteration of the dissolved oxygen concentration in the growth environment.

Yarrowia lipolytica, an ascomycete with biotechnological potential, is able to form either yeast cells or hyphae and pseudohyphae in response to environmental conditions. This study shows that the morphology of Y. lipolytica, cultivated in batch cultures on hydrophilic (glucose and glycerol) and hydrophobic (olive oil) media, was not affected by the nature of the carbon source, nor by the nature or the concentration of the nitrogen source. By contrast, dissolved oxygen concentration (DOC) should be considered as the major factor affecting yeast morphology. Specifically, when growth occurred at low or zero DOC the mycelial and/or pseudomycelial forms predominated over the yeast form independently of the carbon and nitrogen sources used. Experimental data obtained from a continuous culture of Y. lipolytica on glycerol, being used as carbon and energy source, demonstrated that the mycelium-to-yeast form transition occurs when DOC increases from 0.1 to 1.5 mg l(-1). DOC also affected the yeast physiology, as the activity of enzymes implicated in lipid biosynthesis (i.e. ATP-citrate lyase, malic enzyme) was upregulated at high DOC whereas the activity of enzymes implicated in glycerol assimilation (such as glycerol dehydrogenase and kinase) remained fundamentally unaffected in the cell-free extract.

Data on cellular lipids of Yarrowia lipolytica grown on fatty substrates.

Yarrowia lipolytica, which is model oleaginous yeast with high industrial interest, was cultivated on fatty substrates. Data concerning fatty acid composition of both substrate and yeast lipids and comparisons of the experimental data with model predictions presented in "Biomodification of fats and oils and scenarios of adding value on renewable fatty materials through microbial fermentations: Modelling and trials with Yarrowia lipolytica" (Vasiliadou et al., 2018) were provided. Furthermore, the total yeast lipids were fractionated into their main fractions, that is, phospholipids, glucolipids plus sphingolipids and neutral lipids, and the fatty acid composition of each lipid fraction was reported.

Publisher Correction: An integrated bacterial system for the discovery of chemical rescuers of disease-associated protein misfolding.

In the version of this Article originally published, in Fig. 1c-e, on the x axes, the lines labelled 'Aß42' and 'Aß42(F19S;L34P)' grouped the data incorrectly; the line labelled Aß42 should have grouped the data for Random 1-2 and Clones 1-10, and the line labelled Aß42(F19S;L34P) should have only grouped the data for Random 1-2 on the right end of the plots and blots. These figures have now been corrected in all versions of the Article.

An integrated bacterial system for the discovery of chemical rescuers of disease-associated protein misfolding.

Protein misfolding and aggregation are common pathological features of several human diseases, including Alzheimer's disease and type 2 diabetes. Here, we report an integrated and generalizable bacterial system for the facile discovery of chemical rescuers of disease-associated protein misfolding. In this system, large combinatorial libraries of macrocyclic molecules are biosynthesized in Escherichia coli cells and simultaneously screened for their ability to rescue pathogenic protein misfolding and aggregation using a flow cytometric assay. We demonstrate the effectiveness of this approach by identifying drug-like, head-to-tail cyclic peptides that modulate the aggregation of the Alzheimer's disease-associated amyloid ß peptide. Biochemical, biophysical and biological assays using isolated amyloid ß peptide, primary neurons and various established Alzheimer's disease nematode models showed that the selected macrocycles potently inhibit the formation of neurotoxic amyloid ß peptide aggregates. We also applied the system to the identification of misfolding rescuers of mutant Cu/Zn superoxide dismutase-an enzyme linked with inherited forms of amyotrophic lateral sclerosis. Overall, the system enables the identification of molecules with therapeutic potential for rescuing the misfolding of disease-associated polypeptides.

Microbial oils as food additives: recent approaches for improving microbial oil production and its polyunsaturated fatty acid content.

In this short review, we summarize the latest research in the production of polyunsaturated microbial oils that are of interest in food technology. The current research targets the productivity of oleaginous microorganisms, as well as the biosynthesis of particular polyunsaturated fatty acids (PUFAs). The most important efforts target the efficiency of the oleaginous machinery, via overexpression of key-enzymes involved in lipid biosynthesis, as well as the minimization of lipid degradation, by repressing genes involved in the ß-oxidation pathway. The production of specific PUFAs is approached by homologous or heterologous expression of specific desaturases and elongases involved in PUFA biosynthesis in oleaginous microorganisms. New perspectives, such as the production of triacylglycerols of specific structure and the employment of adaptive experimental evolution for creating robust oleaginous strains able to produce PUFAs are also discussed.

Microalgal lipids biochemistry and biotechnological perspectives.

In the last few years, there has been an intense interest in using microalgal lipids in food, chemical and pharmaceutical industries and cosmetology, while a noteworthy research has been performed focusing on all aspects of microalgal lipid production. This includes basic research on the pathways of solar energy conversion and on lipid biosynthesis and catabolism, and applied research dealing with the various biological and technical bottlenecks of the lipid production process. In here, we review the current knowledge in microalgal lipids with respect to their metabolism and various biotechnological applications, and we discuss potential future perspectives. The committing step in fatty acid biosynthesis is the carboxylation of acetyl-CoA to form malonyl-CoA that is then introduced in the fatty acid synthesis cycle leading to the formation of palmitic and stearic acids. Oleic acid may also be synthesized after stearic acid desaturation while further conversions of the fatty acids (i.e. desaturations, elongations) occur after their esterification with structural lipids of both plastids and the endoplasmic reticulum. The aliphatic chains are also used as building blocks for structuring storage acylglycerols via the Kennedy pathway. Current research, aiming to enhance lipogenesis in the microalgal cell, is focusing on over-expressing key-enzymes involved in the earlier steps of the pathway of fatty acid synthesis. A complementary plan would be the repression of lipid catabolism by down-regulating acylglycerol hydrolysis and/or ß-oxidation. The tendency of oleaginous microalgae to synthesize, apart from lipids, significant amounts of other energy-rich compounds such as sugars, in processes competitive to lipogenesis, deserves attention since the lipid yield may be considerably increased by blocking competitive metabolic pathways. The majority of microalgal production occurs in outdoor cultivation and for this reason biotechnological applications face some difficulties. Therefore, algal production systems need to be improved and harvesting systems need to be more effective in order for their industrial applications to become more competitive and economically viable. Besides, a reduction of the production cost of microalgal lipids can be achieved by combining lipid production with other commercial applications. The combined production of bioactive products and lipids, when possible, can support the commercial viability of both processes. Hydrophobic compounds can be extracted simultaneously with lipids and then purified, while hydrophilic compounds such as proteins and sugars may be extracted from the defatted biomass. The microalgae also have applications in environmental biotechnology since they can be used for bioremediation of wastewater and to monitor environmental toxicants. Algal biomass produced during wastewater treatment may be further valorized in the biofuel manufacture. It is anticipated that the high microalgal lipid potential will force research towards finding effective ways to manipulate biochemical pathways involved in lipid biosynthesis and towards cost effective algal cultivation and harvesting systems, as well.

The olive mill wastewater as substrate for single cell oil production by Zygomycetes.

The conversion of olive mill wastewater (OMW) into high added value lipids containing polyunsaturated fatty acids (PUFA), in parallel with a significant phenolic removal by selected strains of Zygomycetes, is reported here for the first time. The growth of Mortierella isabellina, Mortierella ramanniana, Cunninghamella echinulata, Mucor sp., Thamnidium elegans and Zygorhynchus moelleri on solidified media was not significantly affected by the presence of OMW used in the growth medium up to 50% (v/v). Kinetic parameter values and conversion yields, estimated using a mathematical model which was fitted on the experimental data originated from submerged cultures, shows the ability of some Zygomycetes (i.e. T. elegans and Z. moelleri) to grow on OMW and accumulate storage material, i.e. lipids rich in PUFA, and these findings open new perspectives in OMW management and valorization. In liquid media containing OMW as sole carbon source, T. elegans and Z. moelleri produced 4.4 and 3.5g/L cell mass in surface (SC) and submerged (SMC) cultures, respectively, containing around 60% (w/w) of lipids. Oleic and palmitic acids were the predominant fatty acids. Gamma-linolenic acid was found in high percentages (up to 17.7%, w/w) in the lipid of Z. moelleri, in SMC with OMW as sole carbon source, while PUFA biosynthesis was not favored in SC.

Biochemical activities in Chlorella sp. and Nannochloropsis salina during lipid and sugar synthesis in a lab-scale open pond simulating reactor.

Chlorella sp. and Nannochloropsis salina cultivated in a lab-scale open pond simulating reactor grew well and produced 350-500mgL(-1) of biomass containing approximately 40% and 16% of lipids, respectively, while different trends in storage material (lipid and sugar) synthesis were identified for the two strains. In continuous culture the highest biomass and lipid productivity was respectively 0.7 and 0.06mgL(-1)h(-1) at D=0.0096h(-1), for Chlorella sp. and 0.8 and 0.09mgL(-1)h(-1) at D=0.007h(-1) for N. salina. The major polyunsaturated fatty acid (PUFA) in the lipid of Chlorella sp. was α-linolenic acid, found at a percentage of 23.0%, w/w, while N. salina synthesized eicosapentaenoic acid at a percentage of 27.0%, w/w. Glycolipids plus sphingolipids were predominant and richer in PUFA, compared to neutral lipids and phospholipids. Activities of some key enzymes, such as pyruvate dehydrogenase (PDC), ATP-citrate lyase (ATP:CL), malic enzyme (ME) and NAD-isocitrate dehydrogenase (ICDH), which are implicated in acetyl-CoA and NADPH biosynthesis, were studied in cells grown in batch and continuous modes. PDC involved in the conversion of pyruvate to acetyl-CoA presented a constant activity in all growth phases. The high ATP:CL activity observed in algal cells, combined with low or zero ICDH activity, indicated the algae ability to generate acetyl-CoA from sugar via citrate. However, the lipogenic capacity of the strains under investigation seemed to be restricted by the low ME activity resulting to limited NADPH synthesis.

Lipids containing polyunsaturated fatty acids synthesized by zygomycetes grown on glycerol.

Several strains of Zygomycetes cultivated on glycerol produced mycelia rich in lipids containing higher amounts of neutral lipids (NL) than glycolipids plus sphingolipids and phospholipids (P), while biosynthesis of P in Mortierella ramanniana, Mucor sp., and Cunninghamella echinulata occurred though NL accumulation process was in progress. Polyunsaturated fatty acids (PUFA) concentration gradually decreased in all lipid fractions of M. ramanniana during growth. In contrast, in C. echinulata concentration of both linoleic and γ-linolenic acids increased with time, especially in P. Taking for granted that the main function of PUFA is associated to their participation in mycelial membranes, we could suppose that biosynthesis of these fatty acids is associated to mycelial growth. However, this is accurate only for some Zygomycetes, e.g., M. ramanniana. On the contrary, PUFA biosynthesis in C. echinulata persists after growth cessation, suggesting that in this species biosynthetic ability is not a strictly growth-associated process. Phosphatidyl-inositol and phosphatidyl-choline were the major P classes in C. echinulata and M. ramanniana, respectively. In M. ramanniana, a decrease of PUFA concentration was noticed even when mycelia were incubated in low temperature (conditions that normally favor PUFA biosynthesis), indicating that PUFA biosynthesis in this fungus is associated to primary metabolism.