Mutation in yl-HOG1 represses the filament-to-yeast transition in the dimorphic yeast Yarrowia lipolytica.

BACKGROUND: Yarrowia lipolytica is a dimorphic fungus, which switches from yeast to filament form in response to environmental conditions. For industrial purposes it is important to lock cells in the yeast or filamentous form depending on the fermentation process. yl-Hog1 kinase is a key component of the HOG signaling pathway, responsible for activating the osmotic stress response. Additionally, deletion of yl-Hog1 leads to increased filamentation in Yarrowia lipolytica, but causes significant sensitivity to osmotic stress induced by a high concentration of a carbon source. RESULTS: In this study, we tested the effect of point mutations on the function of yl-Hog1 protein kinase. The targets of modification were the phosphorylation sites (T171A-Y173A) and the active center (K49R). Introduction of the variant HOG1-49 into the hog1∆ strain partially improved growth under osmotic stress, but did not recover the yeast-like shape of the cells. The HOG1-171/173 variant was not functional, and its introduction further weakened the growth of hog1∆ strains in hyperosmotic conditions. To verify a genetic modification in filament form, we developed a new system based on green fluorescent protein (GFP) for easier screening of proper mutants. CONCLUSIONS: These results provide new insights into the functions of yl-Hog1 protein in dimorphic transition and constitute a good starting point for further genetic modification of Y. lipolytica in filament form.

In-depth analysis of erythrose reductase homologs in Yarrowia lipolytica.

The unconventional yeast Yarrowia lipolytica produces erythritol as an osmoprotectant to adapt to osmotic stress. In this study, the array of putative erythrose reductases, responsible for the conversion of d-erythrose to erythritol, was analyzed. Single knockout and multiple knockout strains were tested for their ability to produce polyols in osmotic stress conditions. Lack of six of the reductase genes does not affect erythritol significantly, as the production of this polyol is comparable to the control strain. Deletion of eight of the homologous erythrose reductase genes resulted in a 91% decrease in erythritol synthesis, a 53% increase in mannitol synthesis, and an almost 8-fold increase in arabitol synthesis as compared to the control strain. Additionally, the utilization of glycerol was impaired in the media with induced higher osmotic pressure. The results of this research may shed new light on the production of arabitol and mannitol from glycerol by Y. lipolytica and help to develop strategies for further modification in polyol pathways in these microorganisms.

The Direct Anti-Virulence but Not Bactericidal Activity of Human Neutrophil Elastase against Moraxella catarrhalis.

Neutrophil elastase (NE) contributes to innate antibacterial defense at both the intracellular (phagocytosis) and extracellular (degranulation, NETosis) levels. Moraxella catarrhalis, a human respiratory pathogen, can exist in an inflammatory milieu which contains NE. No data are available on the action of NE against M. catarrhalis or on the counteraction of NE-dependent host defenses by this pathogen. Using time-kill assays we found that bacteria are able to survive and replicate in the presence of NE. Transmission electron microscopy and flow cytometry studies with NE-treated bacteria revealed that while NE admittedly destabilizes the outer membrane leaflet, it does not cause cytoplasmic membrane rupture, suggesting that the enzyme does not target components that are essential for cell integrity. Using LC-MS/MS spectroscopy we determined that NE cleaved at least three virulent surface proteins in outer membrane vesicles (OMVs) of M. catarrhalis, including OMP CD, McaP, and TbpA. The cleavage of OMP CD contributes to the significant decrease in resistance to serum complement in the complement-resistant strain Mc6. The cleavage of McaP did not cause any sensitization to erythromycin nor did NE disturb its drug action. Identifying NE as a novel but subtle anti-virulence agent together with its extracellularly not-efficient bactericidal activity against M. catarrhalis may facilitate the pathogen's existence in the airways under inflammation.

Lipid production from lignocellulosic biomass using an engineered Yarrowia lipolytica strain.

BACKGROUND: The utilization of industrial wastes as feedstock in microbial-based processes is a one of the high-potential approach for the development of sustainable, environmentally beneficial and valuable bioproduction, inter alia, lipids. Rye straw hydrolysate, a possible renewable carbon source for bioconversion, contains a large amount of xylose, inaccessible to the wild-type Yarrowia lipolytica strains. Although these oleaginous yeasts possesses all crucial genes for xylose utilization, it is necessary to induce their metabolic pathway for efficient growth on xylose and mixed sugars from agricultural wastes. Either way, biotechnological production of single cell oils (SCO) from lignocellulosic hydrolysate requires yeast genome modification or adaptation to a suboptimal environment. RESULTS: The presented Y. lipolytica strain was developed using minimal genome modification-overexpression of endogenous xylitol dehydrogenase (XDH) and xylulose kinase (XK) genes was sufficient to allow yeast to grow on xylose as a sole carbon source. Diacylglycerol acyltransferase (DGA1) expression remained stable and provided lipid overproduction. Obtained an engineered Y. lipolytica strain produced 5.51 g/L biomass and 2.19 g/L lipids from nitrogen-supplemented rye straw hydrolysate, which represents an increase of 64% and an almost 10 times higher level, respectively, compared to the wild type (WT) strain. Glucose and xylose were depleted after 120 h of fermentation. No increase in byproducts such as xylitol was observed. CONCLUSIONS: Xylose-rich rye straw hydrolysate was exploited efficiently for the benefit of production of lipids. This study indicates that it is possible to fine-tune a newly strain with as minimally genetic changes as possible by adjusting to an unfavorable environment, thus limiting multi-level genome modification. It is documented here the use of Y. lipolytica as a microbial cell factory for lipid synthesis from rye straw hydrolysate as a low-cost feedstock.

Brown seaweed hydrolysate as a promising growth substrate for biomass and lipid synthesis of the yeast yarrowia lipolytica.

Biomass of the brown algae Fucus vesiculosus and Saccharina latissima is a promising, renewable feedstock because of the high growth rate, accessibility and content of glucose and mannitol. Saccharification of seaweeds is a simple process due to the lack of lignocellulose in the cell wall. The high content of glucose and mannitol makes these seaweeds an attractive feedstock for lipid production in the yeast Yarrowia lipolytica. This study demonstrated that hydrolysates of brown algae biomass can be applied as a substrate for synthesis of yeast biomass and lipids without any supplementation. To increase the lipid titer in yeast biomass, we employed an engineered strain of Y. lipolytica overexpressing DGA1/DGA2. In consequence, the C/N ratio has a lower impact on lipid synthesis. Moreover, the applied substrates allowed for high synthesis of unsaturated fatty acids (UFA); the level exceeded 90% in the fatty acid pool. Oleic (C18:1) and linoleic acids (C18:2) achieved the highest content. The study showed that Y. lipolytica is able to grow on the seaweed hydrolysate and produces a high content of UFA in the biomass.

Production of PETase by engineered Yarrowia lipolytica for efficient poly(ethylene terephthalate) biodegradation.

There has been a growing interest in poly(ethylene terephthalate) PET degradation studies in the last few years due to its widespread use and large-scale plastic waste accumulation in the environment. One of the most promising enzymatic methods in the context of PET degradation is the use of PETase from Ideonella sakaiensis, which has been reported to be an efficient enzyme for hydrolysing ester bonds in PET. In our study, we expressed a codon-optimized PETase gene in the yeast Yarrowia lipolytica. The obtained strain was tested for its ability to degrade PET directly in culture, and a screening of different supplements that might raise the level of PET hydrolysis was performed. We also carried out long-term cultures with PET film, the surface of which was examined by scanning electron microscopy. The efficiency of PET degradation was tested based on the concentration of degradation products released, and the results showed that supplementation of the culture with olive oil resulted in 66 % higher release of terephthalic acid into the medium compared to the mutant culture without supplementation. The results indicate the possibility of ethylene glycol uptake by both strains, and, additionally, the PETase produced by the newly engineered strain hydrolyses MHET. The structure of the PET film after culture with the modified strain, meanwhile, had numerous surface defects, cracks, and deformations.

Metabolic engineering of Yarrowia lipolytica for poly(ethylene terephthalate) degradation.

Polyethylene terephthalate (PET) is the most widely used plastic, whose global production scale causes serious problems due to it being highly non-biodegradable. The present work provides a novel approach to plastic degradation studies, which involves direct degradation of PET in the culture of a modified Y. lipolytica yeast strain extracellularly producing cutinase from Fusarium solani. In this study, we successfully accomplished a scale-up of the degradation process in culture, which is promising from the perspective of wider application of the developed method in the future. Additionally, we tested the effect of various supplements, which may increase the PET degradation efficiency in the culture of the Y. lipolytica pAD CUT_FS strain. The ability of PET decomposition was verified by the amount of the released degradation products, such as terephthalic acid (TPA) and mono-(2-hydroxyethyl)-terephthalic acid (MHET), during cultivation. We observed that the quantities of TPA and MHET released during the PET degradation process were increasing daily, and were 1.51 gL-1 and 0.45 gL-1, respectively after 240 h of the bioreactor fermentation. Analysis of the PET film by electron microscopy indicated that there was abundant damage on the surface of the material. This study also demonstrated that the engineered Y. lipolytica strain is able to degrade PET at 28 °C during fermentation. The results obtained in this study using amorphous PET powder provide a wide range of possibilities for application of the cutinase-secreting strain of Y. lipolytica on the more difficult to degrade highly crystalline PET films, PET bottles and PET melts.

Identification of novel extracellular putative chitinase and hydrolase from Geomyces sp. B10I with the biodegradation activity towards polyesters.

Cold-adapted filamentous fungal strain Geomyces sp. B10I has been reported to decompose polyesters such as poly(e-caprolactone) (PCL), poly(butylene succinate) (PBS) and poly(butylene succinate-co-butylene adipate) (PBSA). Here, we identified the enzymes of Geomyces sp. B10I, which appear to be responsible for its biodegradation activity. We compared their amino acid sequences with sequences of well-studied fungal enzymes. Partial purification of an extracellular mixture of the two enzymes, named hydrGB10I and chitGB10I, using ammonium sulfate precipitation and ionic exchange chromatography gave 14.16-fold purity. The amino acid sequence of the proteins obtained from the MALDI-TOF analysis determined the molecular mass of 77.2 kDa and 46.5 kDa, respectively. Conserved domain homology analysis revealed that both proteins belong to the class of hydrolases; hydrGB10I belongs to the glycosyl hydrolase 81 superfamily, while chitGB10I contains the domain of the glycosyl hydrolase 18 superfamily. Phylogenetic analysis suggests a distinct nature of the hydrGB10I and chitGB10I of Geomyces sp. B10I when compared with other fungal polyester-degrading enzymes described to date.

Current Knowledge on Polyethylene Terephthalate Degradation by Genetically Modified Microorganisms.

The global production of polyethylene terephthalate (PET) is estimated to reach 87.16 million metric tons by 2022. After a single use, a remarkable part of PET is accumulated in the natural environment as plastic waste. Due to high hydrophobicity and high molecular weight, PET is hardly biodegraded by wild-type microorganisms. To solve the global problem of uncontrolled pollution by PET, the degradation of plastic by genetically modified microorganisms has become a promising alternative for the plastic circular economy. In recent years many studies have been conducted to improve the microbial capacity for PET degradation. In this review, we summarize the current knowledge about metabolic engineering of microorganisms and protein engineering for increased biodegradation of PET. The focus is on mutations introduced to the enzymes of the hydrolase class-PETase, MHETase and cutinase-which in the last few years have attracted growing interest for the PET degradation processes. The modifications described in this work summarize the results obtained so far on the hydrolysis of polyethylene terephthalate based on the released degradation products of this polymer.

The Overexpression of YALI0B07117g Results in Enhanced Erythritol Synthesis from Glycerol by the Yeast Yarrowia lipolytica.

The unconventional yeast Yarrowia lipolytica is used to produce erythritol from glycerol. In this study, the role of the erythrose reductase (ER) homolog YALI0B07117g in erythritol synthesis was analyzed. The deletion of the gene resulted in an increased production of mannitol (308%) and arabitol (204%) before the utilization of these polyols began. The strain overexpressing the YALI0B07117g gene was used to increase the erythritol yield from glycerol as a sole carbon source in batch cultures, resulting in a yield of 0.4 g/g. The specific consumption rate (qs) increased from 5.83 g/g/L for the WT strain to 8.49 g/g/L for the modified strain and the productivity of erythritol increased from 0.28 g/(L h) for the A101 strain to 0.41 g/(L h) for the modified strain. The application of the research may prove positive for shortening the cultivation time due to the increased rate of consumption of the substrate combined with the increased parameters of erythritol synthesis.

The potential of cold-adapted microorganisms for biodegradation of bioplastics.

Due to the extensive use of plastics, their quantity in the environment is constantly increasing, which creates a global problem. In the present study, we sought to isolate, test and identify Antarctic microorganisms which possess the ability to biodegrade bioplastics such as poly(ε-caprolactone) (PCL), poly(butylene succinate) (PBS) and poly(butylene succinate-co-butylene adipate) (PBSA) at low temperatures. 161 bacterial and 38 fungal isolates were isolated from 22 Antarctic soil samples. Among them, 92.16% of bacterial and 77.27% of fungal isolates formed a clear zone on emulsified PBSA, 98.04% and 81.82% on PBS and 100% and 77.27% on PCL as an additive to minimal medium at 20 °C. Based on the 16S and 18S rRNA sequences, bacterial strains were identified as species belonging to Pseudomonas and Bacillus and fungal strains as species belonging to Geomyces, Sclerotinia, Fusarium and Mortierella, while the yeast strain was identified as Hansenula anomala. In the biodegradation process conducted under laboratory conditions at 14, 20 and 28 °C, Sclerotinia sp. B11IV and Fusarium sp. B3'M strains showed the highest biodegradation activity at 20 °C (49.68% for PBSA and 33.7% for PCL, 45.99% for PBSA and 49.65% for PCL, respectively). The highest biodegradation rate for Geomyces sp. B10I was noted at 14 °C (25.67% for PBSA and 5.71% for PCL), which suggested a preference for lower temperatures (at 20 °C the biodegradation rate was only 11.34% for PBSA, and 4.46% for PCL). These data showed that microorganisms isolated from Antarctic regions are good candidates for effective plastic degradation at low temperatures.

HOG-Independent Osmoprotection by Erythritol in Yeast Yarrowia lipolytica.

Erythritol is a polyol produced by Yarrowia lipolytica under hyperosmotic stress. In this study, the osmo-sensitive strain Y. lipolytica yl-hog1Δ was subjected to stress, triggered by a high concentration of carbon sources. The strain thrived on 0.75 M erythritol medium, while the same concentrations of glucose and glycerol proved to be lethal. The addition of 0.1 M erythritol to the medium containing 0.75 M glucose or glycerol allowed the growth of yl-hog1Δ. Supplementation with other potential osmolytes such as mannitol or L-proline did not have a similar effect. To examine whether the osmoprotective effect might be related to erythritol accumulation, we deleted two genes involved in erythritol utilization, the transcription factor Euf1 and the enzyme erythritol dehydrogenase Eyd1. The strain eyd1Δ yl hog1Δ, which lacked the erythritol utilization enzyme, reacted to the erythritol supplementation significantly better than yl-hog1Δ. On the other hand, the strain euf1Δ yl-hog1Δ became insensitive to supplementation, and the addition of erythritol could no longer improve the growth of this strain in hyperosmotic conditions. This indicates that Euf1 regulates additional, still unknown genes involved in erythritol metabolism.

The influence of transketolase on lipid biosynthesis in the yeast Yarrowia lipolytica.

BACKGROUND: During the pentose phosphate pathway (PPP), two important components, NADPH and pentoses, are provided to the cell. Previously it was shown that this metabolic pathway is a source of reducing agent for lipid synthesis from glucose in the yeast Yarrowia lipolytica. Y. lipolytica is an attractive microbial host since it is able to convert untypical feedstocks, such as glycerol, into oils, which subsequently can be transesterified to biodiesel. However, the lipogenesis process is a complex phenomenon, and it still remains unknown which genes from the PPP are involved in lipid synthesis. RESULTS: To address this problem we overexpressed five genes from this metabolic pathway: transaldolase (TAL1, YALI0F15587g), transketolase (TKL1, YALI0E06479g), ribulose-phosphate 3-epimerase (RPE1, YALI0C11880g) and two dehydrogenases, NADP+-dependent glucose-6-phosphate dehydrogenase (ZWF1, YALI0E22649g) and NADP+-dependent 6-phosphogluconate dehydrogenase (GND1, YALI0B15598g), simultaneously with diacylglycerol acyltransferase (DGA1, YALI0E32769g) and verified each resulting strain's ability to synthesize fatty acid growing on both glycerol and glucose as a carbon source. Our results showed that co-expression of DGA1 and TKL1 results in higher SCO synthesis, increasing lipid content by 40% over the control strain (DGA1 overexpression). CONCLUSIONS: Simultaneous overexpression of DGA1 and TKL1 genes results in a higher lipid titer independently from the fermentation conditions, such as carbon source, pH and YE supplementation.

Production of tailor-made fatty acids from crude glycerol at low pH by Yarrowia lipolytica.

Nowadays, single cell oil (SCO) can play two distinct roles, first as a supplier of functional oils, and second as a feedstock for the biodiesel industry. These two distinct functions require a different fatty acids (FA) profile in the lipid pool. Moreover, to exploit their potential for industrialization, it is necessary to employ a low-cost substrate. Crude glycerol is the main side-product of biodiesel production. This renewable feedstock is one of Yarrowia lipolytica favorable substrates. In this study we improved polyunsaturated fatty acids (PUFA) synthesis by overexpression of the glycerol phosphate acyltransferase gene (SCT1). Here, we established a method to alter the quantity and FA composition of SCO. The engineered strain showed a 10-fold improvement (>20%) in linoleic acid synthesis (C18:2) in a shake-flask experiment. In a fermenter study co-overexpression of glycerol kinase (GUT1) and SCT1 allowed for 3-fold improvement in C18:2 synthesis from crude glycerol and at low pH.

High-yield expression of extracellular lipase from Yarrowia lipolytica and its interactions with lipopeptide biosurfactants: A biophysical approach.

The unconventional yeast Yarrowia lipolytica is known as a producer of extracellular lipases. Here we overexpressed extracellular lipase (YlLip2) in yeast strain Y. lipolytica AJD ΔXΔA-Lip2 harboring the overexpression cassette of the YALI0A20350 gene under the strong hybrid promoter UAS1B16-TEF. To maintain a high level of YlLip2 production, two extracellular proteases of Y. lipolytica, AEPp and AXPp, were deleted. The purified recombinant YlLip2 presented optimal catalytic activities at 37 °C and pH 8.0. The effect of two lipopeptide biosurfactants, i.e., amphisin produced by Pseudomonas fluorescens DSS73 and viscosinamide secreted by P. fluorescens DR54, on the conformation and activity of YlLip2 was evaluated using spectral methods, surface tension, and the enzyme activity assay. YlLip2 demonstrated high tolerance of the tested biosurfactants and had greater activity retention after incubation with both biosurfactants. Finally, we observed that intrinsic fluorescence intensity of YlLip2 decreased significantly with increasing lipopeptides concentration ranging from 2.5 to 60 µM. Our results showed that both biosurfactants improve enzymatic activity of YlLip2 and might suggest better interaction of the substrate with the active site. These favorable characteristics make YlLip2 a prospective additive in the pharmaceutical, food, cosmetic, and detergent industries.

A comprehensive assessment of microbiome diversity in Tenebrio molitor fed with polystyrene waste.

Recently it was demonstrated that mealworm (Tenebrio molitor) larvae consume and biodegrade polystyrene. Thus, in this study a breeding investigation with various types of polystyrene was performed to follow the changes in the gut microbiome diversity. Polystyrene used for packaging purposes (PSp) and expanded polystyrene (EPS) were perceived as more favorable and attacked more frequently by mealworms compared to raw polystyrene (PS) and material commercially available for parcels (PSp). Although our studies showed that larvae could bite and chew selected materials, they are not able to degrade and use them for consumption purposes. In a next-generation sequencing experiment, among all samples, seven classes, Gammaproteobacteria, Bacilli, Clostridia, Acidobacteria, Actinobacteria, Alphaproteobacteria and Flavobacteria, were indicated as the most abundant, whereas the predominant genera were Enterobacter, Lactococcus and Enterococcus. Additionally, we isolated three bacteria strains able to use diverse types of bioplastic as a sole carbon source. The strains with biodegradable activity against bioplastic were identified as species of the genera Klebsiella, Pseudomonas and Serratia. The presence of a bacterial strain able to degrade bioplastic may suggest a potential niche for further investigations.

Biochemical properties and biotechnological applications of microbial enzymes involved in the degradation of polyester-type plastics.

Application of polyester-degrading enzymes should be considered as an eco-friendly alternative to chemical recycling due to the huge plastic waste disposal nowadays. Many hydrolases from several fungi and bacteria have been discovered and successfully evaluated for their activity towards different aliphatic polyesters (PHA, PBS, PBSA, PCL, PLA), aromatic polyesters (PET, PBT, PMT) as well as their co-polyesters (PBST, PBAT, PBSTIL). This revision gives an up-to-date overview on the main biochemical features and biotechnological applications of those reported enzymes which are able to degrade polyester-based plastics, including different microbial polyester depolymerases, esterases, cutinase-like enzymes and lipases. Summarized information includes available protein sequences with the corresponding accession numbers deposited in NCBI server, 3D resolved structures, and data about optimal conditions for enzymatic activity and stability of many of these microbial enzymes that would be helpful for researchers in this topic. Although screening and identification of new native polyester hydrolases from microbial sources is undeniable according to literature, we briefly highlight the importance of the design of improved enzymes towards recalcitrant aromatic polyesters through different approaches that include site-directed mutagenesis and surface protein engineering.

Heterologous overexpression of bacterial hemoglobin VHb improves erythritol biosynthesis by yeast Yarrowia lipolytica.

BACKGROUND: Yarrowia lipolytica is an unconventional yeast with a huge industrial potential. Despite many advantages for biotechnological applications, it possesses enormous demand for oxygen, which is a bottleneck in large scale production. In this study a codon optimized bacterial hemoglobin from Vitreoscilla stercoraria (VHb) was overexpressed in Y. lipolytica for efficient growth and erythritol synthesis from glycerol in low-oxygen conditions. Erythritol is a natural sweetener produced by Y. lipolytica under high osmotic pressure and at low pH, and this process requires high oxygen demand. RESULTS: Under these conditions the VHb overexpressing strain showed mostly yeast-type cells resulting in 83% higher erythritol titer in shake-flask experiments. During a bioreactor study the engineered strain showed higher erythritol productivity (QERY = 0.38 g/l h) and yield (YERY = 0.37 g/g) in comparison to the control strain (QERY = 0.30 g/l h, YERY = 0.29 g/g). Moreover, low stirring during the fermentation process resulted in modest foam formation. CONCLUSIONS: This study showed that overexpression of VHb in Y. lipolytica allows for dynamic growth and efficient production of a value-added product from a low-value substrate.

Lipid Production From Waste Materials in Seawater-Based Medium by the Yeast Yarrowia lipolytica.

The global limitation of fossil fuels impels scientists to search for new energy sources. A good alternative is biodiesel produced from crop plants. However, its production requires huge quantities of farmland, fertilizers and fresh water, which is in conflict with the human demand for water for consumption and land for food production. Thus, production of single cell oil (SCO) by oleaginous microorganisms remains the best solution for the coming years. Whereas most microorganisms require fresh water for proper cell metabolism, in this study we demonstrate that the unconventional yeast Yarrowia lipolytica is able to produce huge quantities of fatty acid in seawater-based medium. Here we shown that Y. lipolytica is able to produce fatty acids in medium based on seawater and crude glycerol as the main carbon source, which allows for low-cost production of SCO, is beneficial for industrial application and is ecologically friendly.

Aseptic production of citric and isocitric acid from crude glycerol by genetically modified Yarrowia lipolytica.

The unconventional yeast Yarrowia lipolytica is known for its capacity to produce citric or isocitric acid from glycerol. In this study a reduction of production cost was achieved by using cheap crude glycerol and conducting the production at pH 3 to prevent bacterial contamination. In this study a Y. lipolytica strain overexpressing Gut1 and Gut2 was used. For the modified strain, crude glycerol proved to be an excellent substrate for production of citric/isocitric acids in aseptic conditions, as the final concentration of these compounds reached 75.9 ±â€¯1.8 g L-1 after 7 days of batch production. Interestingly, the concentration of isocitric acid was 42.5 ±â€¯2.4 g L-1, which is one of the highest concentrations of isocitric acid obtained from a waste substrate. In summary, these data show that organic acids can be efficiently produced by the yeast Y. lipolytica from crude glycerol without any prior purification in aseptic conditions.