Carbon source impact on Yarrowia lipolytica KKP 379 lipase production

The production of lipases by microorganisms is strongly influenced by the culture conditions. The optimum culture conditions for enzyme production are strain- and species-dependent. The aim of this study was to evaluate the impact of the carbon source used in the culture medium on the profile of lipases produced by Yarrowia lipolytica KKP 379. We observed a different pattern of extracellular and cell-bound lipase production, which was the highest in the early exponential phase. The extracellular lipase activity increased in the late exponential phase due to the lower accumulation of lipase molecules in cell walls. The best carbon source for extracellular lipase production by Y. lipolytica KKP 379 was olive oil. Glucose, dodecane and olive oil had a positive effect on biomass yield. Dodecane and/or glycerol utilization in microbiological lipase production was possible, but this process could not proceed without the addition of some activators such as olive oil in the cultivation medium.     

Biotransformation of 6,7-epoxygeraniol by fungi

The biotransformation of 6,7-epoxygeraniol by resting cells of selected fungi was investigated. The main product obtained from the transformation in Rhodotorula glutinis and R. marina cultures was 6,7-epoxynerol (5–48% of chloroform extracts), whereas Saccharomyces cerevisiae, Candida parapsilosis and C. kefyr reduced this substrate to 6,7-epoxycitronellol (30–33% of chloroform extracts). Cultures of Yarrowia lipolytica, Botrytis cinerea and S. cerevisiae promoted the cyclisation of 6,7-epoxygeraniol to 2-methyl-2-(2-hydroxyethyl)-5-(2-hydroxyprop-2-yl)tetrahydrofuran (11–99% of chloroform extracts). The biotransformation of 6,7-epoxynerol was also investigated. However, none of the tested micro-organisms converted this compound.     

Selection of autochthonous yeast strains able to degrade biphenyl

In order to assess the role of yeasts in the natural detoxification process of sediments polluted with biaryl compounds, indigenous yeast species able to degrade biphenyl (BP) were isolated and identified. The degradation ability of 24 strains of the genera Candida spp., Cryptococcus spp., Pichia spp., Rhodotorula spp., Trichosporon spp. and Yarrowia spp. was evaluated by the identification of the BP-metabolites, by HPLC analysis. 4-Hydroxybiphenyl was the main derivative in the Candida krusei, C. tenuis, C. tropicalis, Pichia haplophila, Rhodotorula glutinis, Trichosporon pullulans and Yarrowia lipolytica cultures. 3-Hydroxybiphenyl was detected in minor amounts in the culture supernatant of C. tropicalis, C. krusei strains and R. glutinis. Further hydroxylation led to 3,4-dihydroxy and 2,3-dihydroxybiphenyl; the former in C. tropicalis, C. krusei and R. glutinis cultures, and the latter only in the R. glutinis assays. The cleavage product 4-phenyl-2-pyrone-6-carboxylic acid, was observed in R. glutinis and Y. lipolytica cultures. The degradation ability of the R. glutinis isolates was noteworthy; as four hydrolxylated intermediates and a ring-cleavage product were obtained in both strain cultures. The species studied in this report were dominant in polluted sediments; furthermore, R. glutinis had been mentioned as able to degrade other aromatic hydrocarbons and had high relevance in bioremediation experiments.     

Functional characterization of extracellular chitinase encoded by the YlCTS1 gene in a dimorphic yeast Yarrowia lipolytica

The hemiascomycetes yeast Yarrowia lipolytica is a dimorphic yeast with alternating yeast and mycelia forms. Bioinformatic analysis revealed the presence of three putative chitinase genes, YlCTS1, YlCTS2, and YlCTS3, in the Y. lipolytica genome. Here, we demonstrated that the protein of YlCTS1 (YlCts1p), which contains an N-terminal secretion signal peptide, a long C-terminal Ser/Thr-rich domain, and a chitin-binding domain, is a homologue to Saccharomyces cerevisiae chitinase 1 (ScCts1p). Deletion of YlCTS1 remarkably reduced extracellular endochitinase activity in the culture supernatant of Y. lipolytica and enhanced cell aggregation, suggesting a role of YlCts1p in cell separation as ScCts1p does in S. cerevisiae. However, loss of YlCts1p function did not affect hyphal formation induced by fetal bovine serum addition. The mass of YlCts1p was dramatically decreased by jack bean α-mannosidase digestion but not by PNGase F treatment, indicating that YlCts1p is modified only by O-mannosylation without N-glycosylation. Moreover, the O-glycan profile of YlCts1p was identical to that of total cell wall mannoproteins, supporting the notion that YlCts1p can be used as a good model for studying O-glycosylation in this dimorphic yeast.     

Enhanced alpha-ketoglutaric acid production in Yarrowia lipolytica WSH-Z06 by regulation of the pyruvate carboxylation pathway

In previous research, a thiamine-auxotrophic yeast for alpha-ketoglutaric acid (KGA) overproduction was screened in our laboratory and named Yarrowia lipolytica WSH-Z06 (CCTCC no. M207143). However, the high concentration of by-products (mainly pyruvate) limited its application on an industrial scale. To enhance KGA production and reduce pyruvate (PA) accumulation, the pyruvate carboxylation pathway was regulated. By overexpressing the pyruvate carboxylase genes ScPYC1 from Saccharomyces cerevisiae and RoPYC2 from Rhizopus oryzae in Y. lipolytica WSH-Z06, the yields of KGA in Y. lipolytica-ScPYC1 and Y. lipolytica-RoPYC2 increased by 24.5 and 35.3?%, and the yields of PA decreased by 51.9 and 69.8?% in shake flasks, respectively. These changes in the expression levels and activities of key intracellular enzymes showed that enhancing the pyruvate carboxylation pathway had successfully redistributed the carbon flux from PA to KGA. Finally, by controlling the pH in a 3-L fermenter, the maximum concentration of KGA in Y. lipolytica-RoPYC2 reached 62.5?g?L^?1 with an evident decrease in PA yield from 35.2 to 13.5?g?L^?1.     

Cloning of the isocitrate lyase gene (ICL1) from Yarrowia lipolytica and characterization of the deduced protein

The ICL1 gene encoding isocitrate lyase was cloned from the dimorphic fungus Yarrowia lipolytica by complementation of a mutation (acuA3) in the structural gene of isocitrate lyase of Escherichia coli. The open reading frame of ICL1 is 1668 by long and contains no introns in contrast to currently sequenced genes from other filamentous fungi. The ICL1 gene encodes a deduced protein of 555 amino acids with a molecular weight of 62 kDa, which fits the observed size of the purified monomer of isocitrate lyase from Y. lipolytica. Comparison of the protein sequence with those of known pro- and eukaryotic isocitrate lyases revealed a high degree of homology among these enzymes. The isocitrate lyase of Y. lipolytica is more similar to those from Candida tropicalis and filamentous fungi than to Sacharomyces cerevisiae. This enzyme of Y. lipolytica has the putative glyoxysomal targeting signal S-K-L at the carboxy-terminus. It contains a partial repeat which is typical for eukaryotic isocitrate lyases but which is absent from the E. coli enzyme. Surprisingly, deletion of the ICL1 gene from the genome not only inhibits the utilization of acetate, ethanol, and fatty acids, but also reduces the growth rate on glucose.     

Sustainable conversion of coffee and other crop wastes to biofuels and bioproducts using coupled biochemical and thermochemical processes in a multi-stage biorefinery concept

The environmental impact of agricultural waste from the processing of food and feed crops is an increasing concern worldwide. Concerted efforts are underway to develop sustainable practices for the disposal of residues from the processing of such crops as coffee, sugarcane, or corn. Coffee is crucial to the economies of many countries because its cultivation, processing, trading, and marketing provide employment for millions of people. In coffee-producing countries, improved technology for treatment of the significant amounts of coffee waste is critical to prevent ecological damage. This mini-review discusses a multi-stage biorefinery concept with the potential to convert waste produced at crop processing operations, such as coffee pulping stations, to valuable biofuels and bioproducts using biochemical and thermochemical conversion technologies. The initial bioconversion stage uses a mutant Kluyveromyces marxianus yeast strain to produce bioethanol from sugars. The resulting sugar-depleted solids (mostly protein) can be used in a second stage by the oleaginous yeast Yarrowia lipolytica to produce bio-based ammonia for fertilizer and are further degraded by Y. lipolytica proteases to peptides and free amino acids for animal feed. The lignocellulosic fraction can be ground and treated to release sugars for fermentation in a third stage by a recombinant cellulosic Saccharomyces cerevisiae, which can also be engineered to express valuable peptide products. The residual protein and lignin solids can be jet cooked and passed to a fourth-stage fermenter where Rhodotorula glutinis converts methane into isoprenoid intermediates. The residues can be combined and transferred into pyrocracking and hydroformylation reactions to convert ammonia, protein, isoprenes, lignins, and oils into renewable gas. Any remaining waste can be thermoconverted to biochar as a humus soil enhancer. The integration of multiple technologies for treatment of coffee waste has the potential to contribute to economic and environmental sustainability.     

Functional analysis of recombinant human and <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> <Emphasis Type="Italic">O</Emphasis>-GlcNAc transferases expressed in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation is an important post-translational modification in many cellular processes. It is mediated by O-GlcNAc transferases (OGTs), which catalyze the addition of O-GlcNAc to serine or threonine residues of the target proteins. In this study, we expressed a putative Yarrowia lipolytica OGT (YlOGT), the only homolog identified in the subphylum Saccharomycotina through bioinformatics analysis, and the human OGT (hOGT) as recombinant proteins in Saccharomyces cerevisiae, and performed their functional characterization. Immunoblotting assays using antibody against O-GlcNAc revealed that recombinant hOGT (rhOGT), but not the recombinant YlOGT (rYlOGT), undergoes auto-O-GlcNAcylation in the heterologous host S. cerevisiae. Moreover, the rhOGT expressed in S. cerevisiae showed a catalytic activity during in vitro assays using casein kinase II substrates, whereas no such activity was obtained in rYlOGT. However, the chimeric human-Y. lipolytica OGT, carrying the human tetratricopeptide repeat (TPR) domain along with the Y. lipolytica catalytic domain (CTD), mediated the transfer of O-GlcNAc moiety during the in vitro assays. Although the overexpression of full-length OGTs inhibited the growth of S. cerevisiae, no such inhibition was obtained upon overexpression of only the CTD fragment, indicating the role of TPR domain in growth inhibition. This is the first report on the functional analysis of the fungal OGT, indicating that the Y. lipolytica OGT retains its catalytic activity, although the physiological role and substrates of YlOGT remain to be elucidated.     

The gluconeogenic enzyme fructose-1,6-bisphosphatase is dispensable for growth of the yeast Yarrowia lipolytica in gluconeogenic substrates

The genes encoding gluconeogenic enzymes in the nonconventional yeast Yarrowia lipolytica were found to be differentially regulated. The expression of Y. lipolytica FBP1 (YlFBP1) encoding the key enzyme fructose-1,6-bisphosphatase was not repressed by glucose in contrast with the situation in other yeasts; however, this sugar markedly repressed the expression of YlPCK1, encoding phosphoenolpyruvate carboxykinase, and YlICL1, encoding isocitrate lyase. We constructed Y. lipolytica strains with two different disrupted versions of YlFBP1 and found that they grew much slower than the wild type in gluconeogenic carbon sources but that growth was not abolished as happens in most microorganisms. We attribute this growth to the existence of an alternative phosphatase with a high K_m (2.3 mM) for fructose-1,6-bisphosphate. The gene YlFBP1 restored fructose-1,6-bisphosphatase activity and growth in gluconeogenic carbon sources to a Saccharomyces cerevisiae fbp1 mutant, but the introduction of the FBP1 gene from S. cerevisiae in the Ylfbp1 mutant did not produce fructose-1,6-bisphosphatase activity or growth complementation. Subcellular fractionation revealed the presence of fructose-1,6-bisphosphatase both in the cytoplasm and in the nucleus.     

Characterization of extracellular yeast peptide factors and their stress-protective effect on probiotic lactic acid bacteria

Protective effect of the extracellular peptide fraction (reactivating factors, RF) produced by yeasts of various taxonomic groups (Saccharomyces cerevisiae, Kluyveromyces lactis, Candida utilis, and Yarrowia lipolytica) on probiotic lactic acid bacteria (LAB) Lactobacillus casei, L. acidophilus, and L. reuteri under bile salt (BS)-induced stress was shown. RF of all yeasts were shown to be of peptide nature; the active component of the S. cerevisiae RF was identified as a combination of low-molecular polypeptides with molecular masses of 0.6 to 1.5 kDa. The protective and reactivating effects of the yeast factors were not species-specific and were similar to those of the Luteococcus japonicus subsp. casei RF. In BS-treated cells of the tester bacteria, a protective effect was observed after 10-min preincubation of the LAB cell suspension with yeast RF: the number of surviving cells (CFU) was 2 to 4.5 times higher than in the control. The reactivating effect was observed when RF was added to LAB cell suspensions not later than 15 min after stress treatment. It was less pronounced than the protector effect, with the CFU number 1 to 3 times that of the control. Both the protector and the reactivating effects were most pronounced in the S. cerevisiae and decreased in the row C. utilis > K. lactis > Y. lipolytica. The efficiency of protective action of yeast RF was found to depend on the properties of recipient LAB cells, with the L. casei strain being most sensitive to BS treatment. In both variants, the highest protective effect of RF (increase in the CFU number) was observed for L. acidophilus, while the least pronounced one was observed for L. casei. The reasons for application of the LAB strains combining high stress resistance and high response to stress-protecting metabolites, including RF factors, as probiotics, is discussed.     

Protective and reactivating effect of the protein exometabolite on yeast cells inactivated by the ultraviolet irradiation

It has been shown that Saccharomyces cerevisiae, Kluyveromyces lactis, and Candida utilis strains produce the protein exometabolites, which has a protective and reactivating effect on the ultraviolet irradiated yeast cells. The protective effect of the preliminary ultraviolet irradiated (activated) protein exometabolite of all strains increased 2–3 times, though its reactivating activity did not change. Yarrowia lipolytica yeast cells, isolated from the areas with the high daily irradiation, and Endomyces magnusii, the obligate fungi parasites, were characterized by the highest ultraviolet tolerance in comparison with the other strains. However, they did not produce the exometabolites with the antistress effect. Luteococcus casei reactivating factor demonstrated protective and reactivating cross-action in relation to the ultraviolet irradiated S. cerevisiae, K. lactis, and C. utilis cells and were inactive in relation to Y. lipolytica and E. magnusii. Using killer and nonkiller S. cerevisiae strain, it has been shown that the peptide exometabolite accumulation was not associated with toxin production.     

Sec14 family of lipid transfer proteins in yeasts

The hydrophobicity of lipids prevents their free movement across the cytoplasm. To achieve highly heterogeneous and precisely regulated lipid distribution in different cellular membranes, lipids are transported by lipid transfer proteins (LTPs) in addition to their transport by vesicles. Sec14 family is one of the most extensively studied groups of LTPs. Here we provide an overview of Sec14 family of LTPs in the most studied yeast Saccharomyces cerevisiae as well as in other selected non-Saccharomyces yeasts—Schizosaccharomyces pombe, Kluyveromyces lactis, Candida albicans, Candida glabrata, Cryptococcus neoformans, and Yarrowia lipolytica. Discussed are specificities of Sec14-domain LTPs in various yeasts, their mode of action, subcellular localization, and physiological function. In addition, quite few Sec14 family LTPs are target of antifungal drugs, serve as modifiers of drug resistance or influence virulence of pathologic yeasts. Thus, they represent an important object of study from the perspective of human health.     

Inhibitory effect of glucose and adenosine 3′,5′-monophosphate on the synthesis of inducible N-acetylglucosamine catabolic enzymes in yeast

Glucose can block the utilization of N-acetylglucosamine in Saccharomyces cerevisiae, a facultative aerobe, but not in Candida albicans, an obligatory aerobe. Furthermore, glucose represses the synthesis of the enzymes of the N-acetylglucosamine catabolic pathway in S. cerevisiae, but not in C. albicans. The results suggest that catabolite repression is present in S. cerevisiae, but not in C. albicans. Cyclic AMP added to S. cerevisiae cells maintained in a glucose medium cannot bring about their release from catabolite repression. On the contrary, the synthesis of inducible enzymes of N-acetylglucosamine pathway was inhibited by cyclic AMP in both the yeasts. This seems to indicate that cyclic AMP can penetrate into the yeast cells. Furthermore, cyclic AMP inhibits protein synthesis, suggesting that protein synthesis in yeast is under cyclic AMP control.     

Overexpression of malic enzyme (ME) of Mucor circinelloides improved lipid accumulation in engineered Rhodotorula glutinis

The oleaginous yeast Rhodotorula glutinis has been known to be a potential feedstock for lipid production. In the present study, we investigated the enhancement of expression of malic enzyme (ME; NADP⁺ dependent; EC 1.1.1.40) from Mucor circinelloides as a strategy to improve lipid content inside the yeast cells. The 26S rDNA and 5.8S rDNA gene fragments isolated from Rhodotorula glutinis were used for homologous integration of ME gene into R. glutinis chromosome under the control of the constitutively highly expressed gene phosphoglycerate kinase 1 to achieve stable expression. We demonstrated that by increasing the expression of the foreign ME gene in R. glutinis, we successfully improved the lipid content by more than twofold. At the end of lipid accumulation phrase (96 h) in the transformants, activity of ME was increased by twofold and lipid content of the yeast cells was increased from 18.74 % of the biomass to 39.35 %. Simultaneously, there were no significant differences in fatty acid profiles between the wild-type strain and the recombinant strain. Over 94 % of total fatty acids were C_16:0, C_18:0, C_16:1, C_18:1, and C_18:2. Our results indicated that heterologous expression of NADP⁺-dependent ME involved in fatty acid biosynthesis indeed increased the lipid accumulation in the oleaginous yeast R. glutinis.     

Oil production by oleaginous yeasts using the hydrolysate from pretreatment of wheat straw with dilute sulfuric acid

This paper explores the use of the hydrolysate from the dilute sulfuric acid pretreatment of wheat straw for microbial oil production. The resulting hydrolysate was composed of pentoses (24.3 g/L) and hexoses (4.9 g/L), along with some other degradation products, such as acetic acid, furfural, and hydroxymethylfurfural (HMF). Five oleaginous yeast strains, Cryptococcus curvatus, Rhodotorula glutinis, Rhodosporidium toruloides, Lipomyces starkeyi, and Yarrowia lipolytica, were evaluated by using this hydrolysate as substrates. The results showed that all of these strains could use the detoxified hydrolysate to produce lipids while except R. toruloides non-detoxified hydrolysate could also be used for the growth of all of the selective yeast strains. C. curvatus showed the highest lipid concentrations in medium on both the detoxified (4.2 g/L) and non-detoxified (5.8 g/L) hydrolysates. And the inhibitory effect studies on C. curvatus indicated HMF had insignificant impacts at a concentration of up to 3 g/L while furfural inhibited cell growth and lipid content by 72.0% and 62.0% at 1 g/L, respectively. Our work demonstrates that lipid production is a promising alternative to utilize hemicellulosic sugars obtained during pretreatment of lignocellulosic materials.