Publications

The identification of genetic determinants of methanol tolerance in yeast suggests differences in methanol and ethanol toxicity mechanisms and candidates for improved methanol tolerance engineering

Mota, M.N.; Martins, L.C.; Sá-Correia, I., J. Fungi 2021, 7(2), 90.

Methanol is a promising feedstock for metabolically competent yeast strains-based biorefineries. However, methanol toxicity can limit the productivity of these bioprocesses. Therefore, the identification of genes whose expression is required for maximum methanol tolerance is important for mechanistic insights and rational genomic manipulation to obtain more robust methylotrophic yeast strains. The present chemogenomic analysis was performed with this objective based on the screening of the Euroscarf Saccharomyces cerevisiae haploid deletion mutant collection to search for susceptibility phenotypes in YPD medium supplemented with 8% (v/v) methanol, at 35 °C, compared with an equivalent ethanol concentration (5.5% (v/v)). Around 400 methanol tolerance determinants were identified, 81 showing a marked phenotype. The clustering of the identified tolerance genes indicates an enrichment of functional categories in the methanol dataset not enriched in the ethanol dataset, such as chromatin remodeling, DNA repair and fatty acid biosynthesis. Several genes involved in DNA repair (eight RAD genes), identified as specific for methanol toxicity, were previously reported as tolerance determinants for formaldehyde, a methanol detoxification pathway intermediate. This study provides new valuable information on genes and potential regulatory networks involved in overcoming methanol toxicity. This knowledge is an important starting point for the improvement of methanol tolerance in yeasts capable of catabolizing and copying with methanol concentrations present in promising bioeconomy feedstocks, including industrial residues.

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Complete utilization of the major carbon sources present in sugar beet pulp hydrolysates by the oleaginous red yeasts Rhodotorula toruloides and R. mucilaginosa

Martins, L.C.; Palma, M.; Angelov, A.; Nevoigt, E.; Liebl, W.; Sá-Correia, J. Fungi 2021, 7(3), 215.

Agro-industrial residues are low-cost carbon sources (C-sources) for microbial growth and production of value-added bioproducts. Among the agro-industrial residues available, those rich in pectin are generated in high amounts worldwide from the sugar industry or the industrial processing of fruits and vegetables. Sugar beet pulp (SBP) hydrolysates contain predominantly the neutral sugars d-glucose, l-arabinose and d-galactose, and the acidic sugar d-galacturonic acid. Acetic acid is also present at significant concentrations since the d-galacturonic acid residues are acetylated. In this study, we have examined and optimized the performance of a Rhodotorula mucilaginosa strain, isolated from SBP and identified at the molecular level during this work. This study was extended to another oleaginous red yeast species, R. toruloides, envisaging the full utilization of the C-sources from SBP hydrolysate (at pH 5.0). The dual role of acetic acid as a carbon and energy source and as a growth and metabolism inhibitor was examined. Acetic acid prevented the catabolism of d-galacturonic acid and l-arabinose after the complete use of the other C-sources. However, d-glucose and acetic acid were simultaneously and efficiently metabolized, followed by d-galactose. SBP hydrolysate supplementation with amino acids was crucial to allow d-galacturonic acid and l-arabinose catabolism. SBP valorization through the production of lipids and carotenoids by Rhodotorula strains, supported by complete catabolism of the major C-sources present, looks promising for industrial implementation.

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Xanthophyllomyces dendrorhous, a Versatile Platform for the Production of Carotenoids and Other Acetyl-CoA-Derived Compounds

Sandmann G., Pollmann H., Gassel S., Breitenbach J., In: Misawa N. (eds) Carotenoids: Biosynthetic and Biofunctional Approaches. Advances in Experimental Medicine and Biology 2021, 1261. Springer, Singapore.

Xanthophyllomyces dendrorhous (with Phaffia rhodozyma as its anamorphic state) is a basidiomycetous, moderately psychrophilic, red yeast belonging to the Cystofilobasidiales. Its red pigmentation is caused by the accumulation of astaxanthin, which is a unique feature among fungi. The present chapter reviews astaxanthin biosynthesis and acetyl-CoA metabolism in X. dendrorhous and describes the construction of a versatile platform for the production of carotenoids, such as astaxanthin, and other acetyl-CoA-derived compounds including fatty acids by using this fungus.

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Utilization of biomass derived from cyanobacteria-based agro-industrial wastewater treatment and raisin residue extract for bioethanol production

Tsolcha O.N., Patrinou V., Economou C.N., Dourou M., Aggelis G., Tekerlekopoulou A.G., Water (Switzerland) 2021, 13(4), 486.

Biofuels produced from photosynthetic microorganisms such as microalgae and cyanobacteria could potentially replace fossil fuels as they offer several advantages over fuels produced from lignocellulosic biomass. In this study, energy production potential in the form of bioethanol was examined using different biomasses derived from the growth of a cyanobacteria-based microbial consortium on a chemical medium and on agro-industrial wastewaters (i.e., dairy wastewater, winery wastewater and mixed winery–raisin effluent) supplemented with a raisin residue extract. The possibility of recovering fermentable sugars from a microbial biomass dominated by the filamentous cyanobacterium Leptolynbgya sp. was demonstrated. Of the different acid hydrolysis conditions tested, the best results were obtained with sulfuric acid 2.5 N for 120 min using dried biomass from dairy wastewater and mixed winery–raisin wastewaters. After optimizing sugar release from the microbial biomass by applying acid hydrolysis, alcoholic fermentation was performed using the yeast Saccharomyces cerevisiae. Raisin residue extract was added to the treated biomass broth in all experiments to enhance ethanol production. Results showed that up to 85.9% of the theoretical ethanol yield was achieved, indicating the potential use of cyanobacteria-based biomass in combination with a raisin residue extract as feedstock for bioethanol production.

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Use of Hanseniaspora guilliermondii and Hanseniaspora opuntiae to enhance the aromatic profile of beer in mixed-culture fermentation with Saccharomyces cerevisiae

Bourbon-Melo, N.; Palma, M.; Rocha, M.P.; Ferreira, A.; Bronze, M.R.; Elias, H.; Sá-Correia, I., Food Microbiology 2021, 95, 103678.

Beer production is predominantly carried out by Saccharomyces species, such as S. cerevisiae and S. pastorianus. However, the introduction of non-Saccharomyces yeasts in the brewing process is now seen as a promising strategy to improve and differentiate the organoleptic profile of beer. In this study, 17 non-Saccharomyces strains of 12 distinct species were isolated and submitted to a preliminary sensory evaluation to determine their potential for beer bioflavouring. Hanseniaspora guilliermondii IST315 and H. opuntiae IST408 aroma profiles presented the highest acceptability and were described as having ‘fruity’ and ‘toffee’ notes, respectively. Their presence in mixed-culture fermentations with S. cerevisiae US-05 did not influence attenuation and ethanol concentration of beer but had a significant impact in its volatile composition. Notably, while both strains reduced the total amount of ethyl esters, H. guilliermondii IST315 greatly increased the concentration of acetate esters, especially when sequentially inoculated, leading to an 8.2-fold increase in phenylethyl acetate (‘rose’, ‘honey’ aroma) in the final beverage. These findings highlight the importance of non-Saccharomyces yeasts in shaping the aroma profile of beer and suggest a role for Hanseniaspora spp. in improving it.

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Toward green flotation: Interaction of a sophorolipid biosurfactant with a copper sulfide

Dhar, P.; Havskjold, H.; Thornhill, M.; Roelants, S.; Soetaert, W.; Kota, H.R.; Chernyshova, I., Journal of Colloid and Interface Science 2021, 585, pp. 386-399.

The United Nations’ Sustainable Development Goals have sparked growing interest in biosurfactants from many surfactant-loaded industries including those utilizing froth flotation for mineral separation. However, the interaction of biosurfactants with mineral surfaces is currently poorly understood. We bridge this gap by studying adsorption of a yeast-derived bola acidic sophorolipid (ASL) biosurfactant on djurleite (Cu1.94S). The methods used include Hallimond flotation, contact angle, adsorption isotherm, zeta potential, leaching measurements, and X-ray photoelectron spectroscopy (XPS). To facilitate the interpretation of the adsorption results, we characterize the activity of ASL at the air-water interface and measure its critical micelle concentration (CMC) at different pH using static surface tension. We find ASL to be a multifunctional surfactant with an unusual, pH-sensitive interfacial behavior. At the air-water interface, ASL is most active at pH 8, while its CMC goes through minimum as low as 40 μM at pH 7. The surfactant adsorption at the djurleite-water interface makes the sulfide surface hydrophilic at acidic pH and hydrophobic at neutral and basic pH. In addition, ASL has strong affinity to copper sulfide and demonstrates metal leaching properties. Finally, ASL demonstrates detergency properties. We offer a mechanistic interpretation of these findings. Our results provide a basis for the application of acidic glycolipids in froth flotation and have implications for their application in ion separation using hydrometallurgical routes, as well as for the chemical stability of metal sulfides in environmental systems.

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The role of transport proteins in the production of microbial glycolipid biosurfactants

Claus S., Jenkins Sánchez L., Van Bogaert I.N.A., Applied Microbiology and Biotechnology 2021, 105(5), 1779-1793.

Several microorganisms are currently being used as production platform for glycolipid biosurfactants, providing a greener alternative to chemical biosurfactants. One of the reasons why these processes are commercially competitive is the fact that microbial producers can efficiently export their product to the extracellular environment, reaching high product titers. Glycolipid biosynthetic genes are often found in a dedicated cluster, amidst which genes encoding a dedicated transporter committed to shuttle the glycolipid to the extracellular environment are often found, as is the case for many other secondary metabolites. Knowing this, one can rely on gene clustering features to screen for novel putative transporters, as described and performed in this review. The above strategy proves to be very powerful to identify glycolipid transporters in fungi but is less valid for bacterial systems. Indeed, the genetics of these export systems are currently largely unknown, but some hints are given. Apart from the direct export of the glycolipid, several other transport systems have an indirect effect on glycolipid production. Specific importers dictate which hydrophilic and hydrophobic substrates can be used for production and influence the final yields. In eukaryotes, cellular compartmentalization allows the assembly of glycolipid building blocks in a highly specialized and efficient way. Yet, this requires controlled transport across intracellular membranes. Next to the direct export of glycolipids, the current state of the art regarding this indirect involvement of transporter systems in microbial glycolipid synthesis is summarized in this review.

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The potassium transporter Hak1 in Candida albicans, regulation and physiological effects at limiting potassium and under acidic conditions.

Ruiz-Castilla, F.J.; Rodríguez-Castro, E.; Michán, C.; Ramos, J., J. Fungi 2021, 7, 362.

The three families of yeast plasma membrane potassium influx transporters are represented in Candida albicans: Trk, Acu, and Hak proteins. Hak transporters work as K+-H+ symporters, and the genes coding for Hak proteins are transcriptionally activated under potassium limitation. This work shows that C. albicans mutant cells lacking CaHAK1 display a severe growth impairment at limiting potassium concentrations under acidic conditions. This is the consequence of a defective capacity to transport K+, as indicated by potassium absorption experiments and by the kinetics parameters of Rb+ (K+) transport. Moreover, hak1− cells are more sensitive to the toxic cation lithium. All these phenotypes became much less robust or even disappeared at alkaline growth conditions. Finally, transcriptional studies demonstrate that the hak1− mutant, in comparison with HAK1+ cells, activates the expression of the K+/Na+ ATPase coded by CaACU1 in the presence of Na+ or in the absence of K+.

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Single Cell Oil (SCO)–Based Bioactive Compounds: I—Enzymatic Synthesis of Fatty Acid Amides Using SCOs as Acyl Group Donors and Their Biological Activities

El-Baz H.A., Elazzazy A.M., Saleh T.S., Dritsas P., Mahyoub J.A., Baeshen M.N., Madian H.R., Alkhaled M., Aggelis G., Applied Biochemistry and Biotechnology 2021, 193(3), 822-845.

Fatty acid amides (FAAs) are of great interest due to their broad industrial applications. They can be synthesized enzymatically with many advantages over chemical synthesis. In this study, the fatty acid moieties of lipids of Cunninghamella echinulata ATHUM 4411, Umbelopsis isabellina ATHUM 2935, Nannochloropsis gaditana CCAP 849/5, olive oil, and an eicosapentaenoic acid (EPA) concentrate were converted into their fatty acid methyl esters and used in the FAA (i.e., ethylene diamine amides) enzymatic synthesis, using lipases as biocatalysts. The FAA synthesis, monitored using in situ NMR, FT-IR, and thin-layer chromatography, was catalyzed efficiently by the immobilized Candida rugosa lipase. The synthesized FAAs exhibited a significant antimicrobial activity, especially those containing oleic acid in high proportions (i.e., derived from olive oil and U. isabellina oil), against several human pathogenic microorganisms, insecticidal activity against yellow fever mosquito, especially those of C. echinulata containing gamma-linolenic acid, and anticancer properties against SKOV-3 ovarian cancer cell line, especially those containing EPA in their structures (i.e., EPA concentrate and N. gaditana oil). We conclude that FAAs can be efficiently synthesized using microbial oils of different fatty acid composition and used in specific biological applications.

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Regulation and activity of CaTrk1, CaAcu1 and CaHak1, the three plasma membrane potassium transporters in Candida albicans

Ruiz-Castilla F.J., Bieber J., Caro G., Michán C., Sychrova H., Ramos J., Biochimica et Biophysica Acta – Biomembranes 2021, 1863(1), 183486.

Wild-type cells of Candida albicans, the most common human fungal pathogen, are able to grow at very low micromolar concentrations of potassium in the external milieu. One of the reasons behind that behaviour is the existence of three different types of K+ transporters in their plasma membrane: Trk1, Acu1 and Hak1. This work shows that the transporters are very differently regulated at the transcriptional level upon exposure to saline stress, pH alterations or K+ starvation. We propose that different transporters take the lead in the diverse environmental conditions, Trk1 being the “house-keeping” one, and Acu1/Hak1 dominating upon K+ limiting conditions. Heterologous expression of the genes coding for the three transporters in a Saccharomyces cerevisiae strain lacking its endogenous potassium transporters showed that all of them mediated cation transport but with very different efficiencies. Moreover, expression of the transporters in Scerevisiae also affected other physiological characteristics such as sodium and lithium tolerance, membrane potential or intracellular pH, being, in general, CaTrk1 the most effective in keeping these parameters close to the usual wild-type physiological levels.

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Production of Industrial Enzymes via Pichia pastoris as a Cell Factory in Bioreactor: Current Status and Future Aspects

Duman-Özdamar, Z.E.; Binay, B., Protein Journal 2021, E-pub Ahead of Print.

Industrial enzymes have been widely preferred in various industries such as chemical production, food & beverage, pharmaceutical, textile, cosmetics, etc. due to the advancements in recent years. They are considered more economic than using whole cells and more environmental-friendly than chemical alternatives. Since the demand for industrial enzymes has been rising, the development of production strategies has been gathered speed. In this respect, the efficiency of Pichia pastoris (P. pastoris) as a host for heterologous protein expression has proved and gained attention due to its great potential for large-scale studies. Especially high-cell density fermentation of P. pastoris is a well-studied and efficient method. Moreover, the improvements in the state of art gene-editing tools have broadened the possibilities of strain improvement for P. pastoris. This review summarized the role of P. pastoris as a cell factory by accentuating the accomplishments in biocatalyst production. Moreover, the benefits and challenges of the most relevant expression systems named Escherichia coli (E. coli), Saccharomyces cerevisiae (S. cerevisiae), P. pastoris and recent evolvements and future directions were revealed in detail. Subsequently, offers for prospects and the latest evolvements to enhance the recombinant protein production were discussed.

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Optimization of synthetic media composition for Kluyveromyces marxianus fed-batch cultivation

Dubencovs, K.; Liepins, J.; Suleiko, A.; Suleiko, A.; Vangravs, R.; Kassaliete, J.; Scerbaka, R.; Grigs, O., Fermentation 2021, 7(2), 62.

The Kluyveromyces marxianus yeast recently has gained considerable attention due to its applicability in high-value-added product manufacturing. In order to intensify the biosynthesis rate of a target product, reaching high biomass concentrations in the reaction medium is mandatory. Fed-batch processes are an attractive and efficient way how to achieve high cell densities. However, depending on the physiology of the particular microbial strain, an optimal media composition should be used to avoid by-product synthesis and, subsequently, a decrease in overall process effi-ciency. Thus, the aim of the present study was to optimise the synthetic growth medium and feeding solution compositions (in terms of carbon, nitrogen, phosphorous, magnesium, and calcium concentrations) for high cell density K. marxianus fed‑batch cultivations. Additionally, the biomass yields from the vitamin mixture and other macro/microelements were identified. A model predictive control algorithm was successfully applied for a fed-batch cultivation control. Biomass growth and substrate consumption kinetics were compared with the mathematical model predictions. Finally, 2‑phenylethanol biosynthesis was induced and its productivity was estimated. The determined optimal macronutrient ratio for K. marxianus biomass growth was identified as C:N:P = 1:0.07:0.011. The maximal attained yeast biomass concentration was close to 70 g·L-1 and the 2-PE biosynthesis rate was 0.372 g·L−1·h−1, with a yield of 74% from 2-phenylalanine.

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Optimisation of the Production and Bleaching Process for a New Laccase from Madurella mycetomatis, Expressed in Pichia pastoris: from Secretion to Yielding Prominent

Tülek, A.; Karataş, E.; Çakar, M.M.; Aydın, D.; Yılmazcan, Ö.; Binay, B., Molecular Biotechnology 2021, 63(1), 24-39.

Laccases are polyphenol oxidoreductases used in a number of industrial applications. Due to the increasing demand for these “green catalysis” enzymes, the identification and biochemical characterisation of their novel properties is essential. In our study, cloned Madurella mycetomatis laccase (mmlac) genes were heterologously expressed in the methylotrophic yeast host Pichia pastoris. The high yield of the active recombinant protein in P. pastoris demonstrates the efficiency of a reliably constructed plasmid to express the laccase gene. The optimal biochemical conditions for the successfully expressed MmLac enzyme were identified. Detailed structural properties of the recombinant laccase were determined, and its utility in decolourisation and textile bleaching applications was examined. MmLac demonstrates good activity in an acidic pH range (4.0–6.0); is stable in the presence of cationic metals, organic solvents and under high temperatures (50–60 °C); and is stable for long-term storage at − 20 °C and − 80 °C for up to eight weeks. The structural analysis revealed that the catalytic residues are partially similar to other laccases. MmLac resulted in an increase in whiteness, whilst demonstrating high efficiency and stability and requiring the input of fewer chemicals. The performance of this enzyme makes it worthy of investigation for use in textile biotechnology applications, as well as within environmental and food technologies.

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Optimisation of recombinant myrosinase production in pichia pastoris

Rosenbergová Z., Kántorová K., Šimkovič M., Breier A., Rebroš M., International Journal of Molecular Sciences 2021, 22(7), 3677.

Myrosinase is a plant defence enzyme catalysing the hydrolysis of glucosinolates, a group of plant secondary metabolites, to a range of volatile compounds. One of the products, isothiocyanates, proved to have neuroprotective and chemo-preventive properties, making myrosinase a pharmaceutically interesting enzyme. In this work, extracellular expression of TGG1 myrosinase from Arabidopsis thaliana in the Pichia pastoris KM71H (MutS) strain was upscaled to a 3 L laboratory fermenter for the first time. Fermentation conditions (temperature and pH) were optimised, which resulted in a threefold increase in myrosinase productivity compared to unoptimised fermentation conditions. Dry cell weight increased 1.5-fold, reaching 100.5 g/L without additional glycerol feeding. Overall, a specific productivity of 4.1 U/Lmedium/h was achieved, which was 102.5-fold higher compared to flask cultivations.

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Microbial biosurfactant research: time to improve the rigour in the reporting of synthesis, functional characterization and process development

Twigg M.S., Baccile N., Banat I.M., Déziel E., Marchant R., Roelants S., Van Bogaert I.N.A., Microbial Biotechnology 2021, 14(1), 147-170.

The demand for microbially produced surface-active compounds for use in industrial processes and products is increasing. As such, there has been a comparable increase in the number of publications relating to the characterization of novel surface-active compounds: novel producers of already characterized surface-active compounds and production processes for the generation of these compounds. Leading researchers in the field have identified that many of these studies utilize techniques are not precise and accurate enough, so some published conclusions might not be justified. Such studies lacking robust experimental evidence generated by validated techniques and standard operating procedures are detrimental to the field of microbially produced surface-active compound research. In this publication, we have critically reviewed a wide range of techniques utilized in the characterization of surface-active compounds from microbial sources: identification of surface-active compound producing microorganisms and functional testing of resultant surface-active compounds. We have also reviewed the experimental evidence required for process development to take these compounds out of the laboratory and into industrial application. We devised this review as a guide to both researchers and the peer-reviewed process to improve the stringency of future studies and publications within this field of science.

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Metabolite transport and its impact on metabolic engineering approaches

Agrimi, G.; Steiger, M.G.,  FEMS Microbiology Letters 2021, 368(1), fnaa211.

Cells are the structural units of life and are separated from the environment by at least one cellular membrane consisting of a lipid bilayer. Thus, metabolite transport across cellular membranes is a key feature of living organisms. Specialized proteins or protein complexes mediate transport processes and are accessible to metabolic engineering approaches. Genetic modifications in metabolic engineering has mostly involved the deletion or overexpression of genes encoding for enzymes. The role of transporters has received much less attention, but as this special issue shows, it is a key factor to consider when rationally designing microbial cell factories. Transporters have been employed in metabolic engineering endeavors to target three fundamental aspects: 1) Import of substrates; 2) Export of products; 3) Modification of intracellular fluxes.

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Kinetic modelling of the biochemical 9-octadecenedioic acid production and lipid accumulation using differently functionalised C18:1 substrates

Bauwelinck J., Wittner N., Broos W., Wijnants M., Tavernier S., Cornet I., Biochemical Engineering Journal 2021, 166.

The potential use of different hydrophobic substrates (oleic acid, oleyl alcohol, methyl oleate and rapeseed oil) for the production of high concentrations of 9-octadecenedioic acid by the genetically engineered C. tropicalis ATCC-20962 yeast was researched. For comparison purposes, data for growth, intracellular triacylglyceride (TAG) accumulation and dicarboxylic acid (DCA) production were obtained for mathematical description using both known and newly adapted models. Fermentations were carried out in an aerated, stirred 2L-fermenter in which temperature (30°C) and pH (8.2) were controlled automatically. Using oleic acid resulted in the highest DCA production, i.e. 31.3 g.L−1 in 144 h, as well as the highest intracellular TAG concentration (60.5 m%). Mass balance calculations proved that 35 % of the consumed oleic acid was used for TAG accumulation. The oleyl alcohol and rapeseed oil substrates showed high initial DCA production rates as well, however, after 80 h, DCA production stopped to reach a maximum. In contrast, with methyl oleate substrate, a very slow production was observed without approaching a maximum within the experimental time.

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Highly-stable Madurella mycetomatis laccase immobilized in silica-coated ZIF-8 nanocomposites for environmentally friendly cotton bleaching process

Tülek, A.; Yıldırım, D.; Aydın, D.; Binay B., Colloids and Surfaces B: Biointerfaces 2021, 202, 111672.

In this study, a laccase from Madurella mycetomatis (MmLac) was produced heterologously in Pichia pastoris; the initial immobilization in a metal-organic framework (MOF) (MmLac/ZIF-8) was achieved using zinc nitrate and 2-methylimidazole. Due to the instability of MmLac/ZIF-8 in an acidic medium, a silica layer was created on the surface of MmLac/MOF-8. The immobilized laccase composite (silica@MmLac/ZIF-8) obtained was further treated with glutaraldehyde (silica@Glu-MmLac/ZIF-8) to increase stability of composite. Fourier-transform infrared spectroscopy, X-ray diffraction and scanning electron microscopy techniques were used to confirm the immobilization of MmLac and to investigate the morphology of the immobilized laccase samples. The MmLac samples were also characterised in terms of optimum pH, temperature and thermal stability. The optimum pH of all the MmLac samples was determined to be 4.0. The free MmLac showed maximum activity at 55 °C, whereas both silica@MmLac/ZIF-8 and silica@Glu-MmLac/ZIF-8 were maximumly active at 65 °C. The silica@MmLac/ZIF-8 and silica@Glu-MmLac/ZIF-8 were 9.3- and 11.8-fold higher in stability, respectively, than the free MmLac at 65 °C. Furthermore, both silica@MmLac/ZIF-8 and silica@Glu-MmLac/ZIF-8 showed a higher bleaching performance than free MmLac on cotton woven fabric. According to these results, silica@MmLac/ZIF-8 and silica@Glu-MmLac/ZIF-8 may be promising candidates for biocatalysts in laccase-based biotechnological applications.

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From secretion in Pichia pastoris to application in apple juice processing: Exo-polygalacturonase from Sporothrix schenckii 1099-18

Karataş, E.; Tülek, A.; Çakar, M.M; Tamtürk, F.; Aktaş, F.; Binay, B., Protein and Peptide Letters 2021, E-pub Ahead of Print.

Background: Polygalacturonases are a group of enzymes under pectinolytic enzymes related to enzymes that hydrolyse pectic substances. Polygalacturonases have been used in various industrial applications such as fruit juice clarification, retting of plant fibers, wastewater treatment drinks fermentation, and oil extraction.
Objectives: The study was evaluated at the heterologous expression, purification, biochemical characterization, computational modeling, and performance in apple juice clarification of a new exo-polygalacturonase from Sporothrix schenckii 1099-18 (SsExo-PG) in Pichia pastoris.
Methods: Recombinant DNA technology was used in this study. Two different pPIC9K plasmids were constructed with native signal sequence-ssexo-pg and alpha signal sequence-ssexo-pg separately. Protein expression and purification performed after plasmids transformed into the Pichia pastoris. Biochemical and structural analyses were performed by using pure SsExo-PG.
Results: The purification of SsExo-PG was achieved using a Ni-NTA chromatography system. The enzyme was found to have a molecular mass of approximately 52 kDa. SsExo-PG presented as stable at a wide range of temperature and pH values, and to be more storage stable than other commercial pectinolytic enzyme mixtures. Structural analysis revealed that the catalytic residues of SsExo-PG are somewhat similar to other Exo-PGs. The KM and kcat values for the degradation of polygalacturonic acid (PGA) by the purified enzyme were found to be 0.5868 µM and 179 s-1, respectively. Cu2+ was found to enhance SsExo-PG activity while Ag2+ and Fe2+ almost completely inhibited enzyme activity. The enzyme reduced turbidity up to 80% thus enhanced the clarification of apple juice. SsExo-PG showed promising performance when compared with other commercial pectinolytic enzyme mixtures.
Conclusion: The clarification potential of SsExo-PG was revealed by comparing it with commercial pectinolytic enzymes. The following parameters of the process of apple juice clarification processes showed that SsExo-PG is highly stable and has a novel performance.

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Ethanol production from wheat straw hydrolysate by issatchenkia orientalis isolated from waste cooking oil

Zwirzitz A., Alteio L., Sulzenbacher D., Atanasoff M., Selg M., Journal of Fungi 2021, 7(2), 121.

The interest in using non-conventional yeasts to produce value-added compounds from low cost substrates, such as lignocellulosic materials, has increased in recent years. Setting out to discover novel microbial strains that can be used in biorefineries, an Issatchenkia orientalis strain was isolated from waste cooking oil (WCO) and its capability to produce ethanol from wheat straw hydrolysate (WSHL) was analyzed. As with previously isolated I. orientalis strains, WCO-isolated I. orientalis KJ27-7 is thermotolerant. It grows well at elevated temperatures up to 42 °C. Furthermore, spot drop tests showed that it is tolerant to various chemical fermentation inhibitors that are derived from the pre-treatment of lignocellulosic materials. I. orientalis KJ27-7 is particularly tolerant to acetic acid (up to 75 mM) and tolerates 10 mM formic acid, 5 mM furfural and 10 mM hydroxymethylfurfural. Important for biotechnological cellulosic ethanol production, I. orientalis KJ27-7 grows well on plates containing up to 10% ethanol and media containing up to 90% WSHL. As observed in shake flask fermentations, the specific ethanol productivity correlates with WSHL concentrations. In 90% WSHL media, I. orientalis KJ27-7 produced 10.3 g L−1 ethanol within 24 h. This corresponds to a product yield of 0.50 g g−1 glucose (97% of the theoretical maximum) and a volumetric productivity of 0.43 g L−1 h−1. Therefore, I. orientalis KJ27-7 is an efficient producer of lignocellulosic ethanol from WSHL.

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Enzymatic synthesis of glucose fatty acid esters using scos as acyl group-donors and their biological activities

El-Baz H.A., Elazzazy A.M., Saleh T.S., Dourou M., Mahyoub J.A., Baeshen M.N., Madian H.R., Aggelis G., Applied Sciences (Switzerland), 2021, 11(6), 2700.

Sugar fatty acid esters, especially glucose fatty acid esters (GEs), have broad applications in food, cosmetic and pharmaceutical industries. In this research, the fatty acid moieties derived from polyunsaturated fatty acids containing single-cell oils (SCOs) (i.e., those produced from Cunninghamella echinulataUmbelopsis isabellina and Nannochloropsis gaditana, as well as from olive oil and an eicosapentaenoic acid (EPA) concentrate) were converted into GEs by enzymatic synthesis, using lipases as biocatalysts. The GE synthesis was monitored using thin-layer chromatography, FTIR and in situ NMR. It was found that GE synthesis carried out using immobilized Candida antarctica B lipase was very effective, reaching total conversion of reactants. It was shown that EPA-GEs were very effective against several pathogenic bacteria and their activity can be attributed to their high EPA content. Furthermore, C. echinulata-GEs were more effective against pathogens compared with U. isabellina-GEs, probably due to the presence of gamma linolenic acid (GLA) in the lipids of C. echinulata, which is known for its antimicrobial activity, in higher concentrations. C. echinulata-GEs also showed strong insecticidal activity against Aedes aegypti larvae, followed by EPA-GEs, olive oil-GEs and N. gaditana-GEs. All synthesized GEs induced apoptosis of the SKOV-3 ovarian cancer cell line, with the apoptotic rate increasing significantly after 48 h. A higher percentage of apoptosis was observed in the cells treated with EPA-GEs, followed by C. echinulata-GEs, U. isabellina-GEs and olive oil-GEs. We conclude that SCOs can be used in the synthesis of GEs with interesting biological properties.

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Engineering Yarrowia lipolytica for the selective and high-level production of isocitric acid through manipulation of mitochondrial dicarboxylate–tricarboxylate carriers

Yuzbasheva, E.Y.; Scarcia, P.; Yuzbashev, T.V.; Messina, E.; Kosikhina, I.M.; Palmieri, L.; Shutov, A.V.; Taratynova, M.O.; Amaro, R.L.; Palmieri, F.; Sineoky, S.P.; Agrimi, G., Metabolic Engineering 2021, 65, 156-166.

During cultivation under nitrogen starvation, Yarrowia lipolytica produces a mixture of citric acid and isocitric acid whose ratio is mainly determined by the carbon source used. We report that mitochondrial succinate–fumarate carrier YlSfc1 controls isocitric acid efflux from mitochondria. YlSfc1 purified and reconstituted into liposomes transports succinate, fumarate, oxaloacetate, isocitrate and α-ketoglutarate. YlSFC1 overexpression determined the inversion of isocitric acid/citric acid ratio towards isocitric acid, resulting in 33.4 ± 1.9 g/L and 43.3 ± 2.8 g/L of ICA production in test-tube cultivation with glucose and glycerol, respectively. These titers represent a 4.0 and 6.3-fold increase compared to the wild type. YlSFC1 gene expression was repressed in the wild type strain grown in glucose-based medium compared to olive oil medium explaining the reason for the preferred citric acid production during Y. lipolytica growth on carbohydrates. Coexpression of YlSFC1 and adenosine monophosphate deaminase YlAMPD genes together with inactivation of citrate mitochondrial carrier YlYHM2 gene enhanced isocitric acid accumulation up to 41.4 ± 4.1 g/L with an isocitric acid/citric acid ratio of 14.3 in a small-scale cultivation with glucose as a carbon source. During large-scale cultivation with glucose pulse-feeding, the engineered strain produced 136.7 ± 2.5 g/L of ICA with a process selectivity of 88.1%, the highest reported titer and selectivity to date. These results represent the first reported isocitric acid secretion by Y. lipolytica as a main organic acid during cultivation on carbohydrate. Moreover, we demonstrate for the first time that the replacement of one mitochondrial transport system for another can be an efficient tool for switching product accumulation.

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Chocolate industry side streams as a valuable feedstock for microbial long-chain dicarboxylic acid production

Bauwelinck J., Caluwé M., Wijnants M., Wittner N., Broos W., Dries J., Akkermans V., Tavernier S., Cornet I., Biochemical Engineering Journal 2021, 167.

Two high-fat containing side streams from the chocolate industry were evaluated as an alternative renewable substrate for biochemical long-chain dicarboxylic acid (DCA) production by Candida tropicalis. Cleaning water from liquid chocolate transport and dissolved air flotation (DAF) sludge from the wastewater treatment plant contained 29 and 18 m% fat respectively. The addition of up to 100 g.L−1 of the ‘chocolate water’ and 80 % (v/v) of DAF sludge had no negative effects on both growth and DCA production. In a fed-batch reactor, a DCA concentration of 5.8 g.L−1 was obtained after 120 h and 47.5 % of the consumed fats were converted to DCA upon glucose addition when using chocolate water. Using DAF sludge, a DCA concentration of 4.9 g.L−1 was obtained after 72 h, equalling 37.5 % of the consumed fats. This research shows the potential of the researched side streams as a substrate for DCA production.

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Xylose Metabolism and the Effect of Oxidative Stress on Lipid and Carotenoid Production in Rhodotorula toruloides: Insights for Future Biorefinery

Pinheiro MJ, Bonturi N, Belouah I, Miranda EA, Lahtvee PJ, Front. Bioeng. Biotechnol, 2021.

The use of cell factories to convert sugars from lignocellulosic biomass into chemicals in which oleochemicals and food additives, such as carotenoids, is essential for the shift toward sustainable processes. Rhodotorula toruloides is a yeast that naturally metabolises a wide range of substrates, including lignocellulosic hydrolysates, and converts them into lipids and carotenoids. In this study, xylose, the main component of hemicellulose, was used as the sole substrate for R. toruloides, and a detailed physiology characterisation combined with absolute proteomics and genome-scale metabolic models was carried out to understand the regulation of lipid and carotenoid production. To improve these productions, oxidative stress was induced by hydrogen peroxide and light irradiation and further enhanced by adaptive laboratory evolution. Based on the online measurements of growth and CO2 excretion, three distinct growth phases were identified during batch cultivations. Majority of the intracellular flux estimations showed similar trends with the measured protein levels and demonstrated improved NADPH regeneration, phosphoketolase activity and reduced β-oxidation, correlating with increasing lipid yields. Light irradiation resulted in 70% higher carotenoid and 40% higher lipid content compared to the optimal growth conditions. The presence of hydrogen peroxide did not affect the carotenoid production but culminated in the highest lipid content of 0.65 g/gDCW. The adapted strain showed improved fitness and 2.3-fold higher carotenoid content than the parental strain. This work presents a holistic view of xylose conversion into microbial oil and carotenoids by R. toruloides, in a process toward renewable and cost-effective production of these molecules.

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Screening and growth characterization of non-conventional yeasts in a hemicellulosic hydrolysate

Monteiro De Oliveira, P., Aborneva, D., Bonturi N., Lahtvee, PJ., Front. Bioeng. Biotechnol2021.

Lignocellulosic biomass is an attractive raw material for the sustainable production of chemicals and materials using microbial cell factories. Most of the existing bioprocesses focus on second-generation ethanol production using genetically modified Saccharomyces cerevisiae, however, this microorganism is naturally unable to consume xylose. Moreover, extensive metabolic engineering has to be carried out to achieve high production levels of industrially relevant building blocks. Hence, the use of non-Saccharomyces species, or non-conventional yeasts, bearing native metabolic routes, allows conversion of a wide range of substrates into different products, and higher tolerance to inhibitors improves the efficiency of biorefineries. In this study, nine non-conventional yeast strains were selected and screened on a diluted hemicellulosic hydrolysate from Birch. Kluyveromyces marxianus CBS 6556, Scheffersomyces stipitis CBS 5773, Lipomyces starkeyi DSM 70295, and Rhodotorula toruloides CCT 7815 were selected for further characterization, where their growth and substrate consumption patterns were analyzed under industrially relevant substrate concentrations and controlled environmental conditions in bioreactors. K. marxianus CBS 6556 performed poorly under higher hydrolysate concentrations, although this yeast was determined among the fastest-growing yeasts on diluted hydrolysate. S. stipitis CBS 5773 demonstrated a low growth and biomass production while consuming glucose, while during the xylose-phase, the specific growth and sugar co-consumption rates were among the highest of this study (0.17 h–1 and 0.37 g/gdw*h, respectively). L. starkeyi DSM 70295 and R. toruloides CCT 7815 were the fastest to consume the provided sugars at high hydrolysate conditions, finishing them within 54 and 30 h, respectively. R. toruloides CCT 7815 performed the best of all four studied strains and tested conditions, showing the highest specific growth (0.23 h–1), substrate co-consumption (0.73 ± 0.02 g/gdw*h), and xylose consumption (0.22 g/gdw*h) rates. Furthermore, R. toruloides CCT 7815 was able to produce 10.95 ± 1.37 gL–1 and 1.72 ± 0.04 mgL–1 of lipids and carotenoids, respectively, under non-optimized cultivation conditions. The study provides novel information on selecting suitable host strains for biorefinery processes, provides detailed information on substrate consumption patterns, and pinpoints to bottlenecks possible to address using metabolic engineering or adaptive evolution experiments.

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Optimised Fractionation of Brewer’s Spent Grain for a Biorefinery Producing Sugars, Oligosaccharides, and Bioethanol

Soma Bedő, Margaréta Rozbach, Leonóra Nagy, Anikó Fehér and Csaba Fehér, Processes 20219(2), 366.

Brewer’s spent grain (BSG) is the main by-product of the beer brewing process. It has a huge potential as a feedstock for bio-based manufacturing processes to produce high-value bioproducts, biofuels, and platform chemicals. For the valorisation of BSG in a biorefinery process, efficient fractionation and bio-conversion processes are required. The aim of our study was to develop a novel fractionation of BSG for the production of arabinose, arabino-xylooligomers, xylose, and bioethanol. A fractionation process including two-step acidic and enzymatic hydrolysis steps was investigated and optimised by a response surface methodology and a desirability function approach to fractionate the carbohydrate content of BSG. In the first acidic hydrolysis, high arabinose yield (76%) was achieved under the optimised conditions (90 °C, 1.85 w/w% sulphuric acid, 19.5 min) and an arabinose- and arabino- xylooligomer-rich supernatant was obtained. In the second acidic hydrolysis, the remaining xylan was solubilised (90% xylose yield) resulting in a xylose-rich hydrolysate. The last, enzymatic hydrolysis step resulted in a glucose-rich supernatant (46 g/L) under optimised conditions (15 w/w% solids loading, 0.04 g/g enzyme dosage). The glucose-rich fraction was successfully used for bioethanol production (72% ethanol yield by commercial baker’s yeast). The developed and optimised process offers an efficient way for the value-added utilisation of BSG. Based on the validated models, the amounts of the produced sugars, the composition of the sugar streams and solubilised oligo-saccharides are predictable and variable by changing the reaction conditions of the process.

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Optimised bioconversion of xylose derived from pre-treated crop residues into xylitol by using Candida boidinii

Bedő, S.; Fehér, A.; Khunnonkwao, P.; Jantama, K.; Fehér, C. Agronomy 2021, 11, 79.

Crop residues can serve as low-cost feedstocks for microbial production of xylitol which offers many advantages over the commonly used chemical process. However, enhancing the efficiency of xylitol fermentation is still a barrier to industrial implementation. In this study, the effects of oxygen transfer rate (OTR) (1.1, 2.1, 3.1 mmol O2/(L*h)) and initial xylose concentration (30, 55, 80 g/L) on xylitol production of Candida boidinii NCAIM Y.01308 on xylose medium were investigated and optimized by response surface methodology, and xylitol fermentations were performed on xylose-rich hydrolysates of wheat bran and rice straw. High values of maximum xylitol yields (58%-63%) were achieved at low initial xylose concentration (20-30 g/L) and OTR values (1.1-1.5 mmol O2/(L*h)). The highest value for maximum xylitol productivity (0.96 g/(L*h)) was predicted at 71 g/L initial xylose and 2.7 mmol O2/(L*h) OTR. Maximum xylitol yield and productivity obtained on wheat bran hydrolysate were 60% and 0.58 g/(L*h), respectively. On detoxified and supplemented hydrolysate of rice straw, maximum xylitol yield and productivity of 30% and 0.19 g/(L*h) were achieved. This study revealed the terms affecting the xylitol production by C. boidinii and provided validated models to predict the achievable xylitol yields and productivities under different conditions. Efficient pre-treatments for xylose-rich hydrolysates from rice straw and wheat bran were selected. Fermentation using wheat bran hydrolysate and C. boidinii under optimised condition is proved as a promising method for biotechnological xylitol production.

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Microbial lipids from organic wastes: Outlook and challenges

E. Tomás-Pejó, S. Morales-Palomo, C. González-Fernández. Bioresource Technology Volume 323, March 2021, 124612

Microbial lipids have recently drawn a lot of attention as renewable sources for biochemicals production. Strong research efforts have been addressed to efficiently use organic wastes as carbon source for microbial lipids, which would definitively increase the profitability of the production process and boost a bio-based economy. This review compiles interesting traits of oleaginous microorganisms and highlights current trends on microbial- and process-oriented approaches to maximize microbial oil production from inexpensive substrates like lignocellulosic sugars, volatile fatty acids and glycerol. Furthermore, downstream processes such as cell harvesting or lipid extraction, that are decisive for the cost-effectiveness of the process, are discussed. To underpin microbial oils within the so demanded circular economy, associated challenges, recent advances and possible industrial applications that are also identified in this review.

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Characterization of a Maltase from an Early-Diverged Non-Conventional Yeast Blastobotrys adeninivorans

Visnapuu, T.; Meldre, A.; Põšnograjeva, K.; Viigand, K.; Ernits, K.; Alamäe, T., Int. J. Mol. Sci. 2020, 21, 297.

Genome of an early-diverged yeast Blastobotrys (Arxulaadeninivorans (Ba) encodes 88 glycoside hydrolases (GHs) including two α-glucosidases of GH13 family. One of those, the rna_ARAD1D20130g-encoded protein (BaAG2; 581 aa) was overexpressed in Escherichia coli, purified and characterized. We showed that maltose, other maltose-like substrates (maltulose, turanose, maltotriose, melezitose, malto-oligosaccharides of DP 4‒7) and sucrose were hydrolyzed by BaAG2, whereas isomaltose and isomaltose-like substrates (palatinose, α-methylglucoside) were not, confirming that BaAG2 is a maltase. BaAG2 was competitively inhibited by a diabetes drug acarbose (Ki = 0.8 µM) and Tris (Ki = 70.5 µM). BaAG2 was competitively inhibited also by isomaltose-like sugars and a hydrolysis product—glucose. At high maltose concentrations, BaAG2 exhibited transglycosylating ability producing potentially prebiotic di- and trisaccharides. Atypically for yeast maltases, a low but clearly recordable exo-hydrolytic activity on amylose, amylopectin and glycogen was detected. Saccharomyces cerevisiae maltase MAL62, studied for comparison, had only minimal ability to hydrolyze these polymers, and its transglycosylating activity was about three times lower compared to BaAG2. Sequence identity of BaAG2 with other maltases was only moderate being the highest (51%) with the maltase MalT of Aspergillus oryzae.

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YEASTRACT+: A portal for cross-species comparative genomics of transcription regulation in yeasts

Monteiro, P.T.; Oliveira, J.; Pais, P.; Antunes, M.; Palma, M.; Cavalheiro, M.; Galocha, M.; Godinho, C.P.; Martins, L.C.; Bourbon, N.; Mota, M.N.; Ribeiro, R.A.; Viana, R.; Sá-Correia, I.; Teixeira, M.C.,Nucleic Acids Research 2020, 48(D1), D642-D649.

The YEASTRACT+ information system (http://YEASTRACT-PLUS.org/) is a wide-scope tool for the analysis and prediction of transcription regulatory associations at the gene and genomic levels in yeasts of biotechnological or human health relevance. YEASTRACT+ is a new portal that integrates the previously existing YEASTRACT (http://www.yeastract.com/) and PathoYeastract (http://pathoyeastract.org/) databases and introduces the NCYeastract (Non-Conventional Yeastract) database (http://ncyeastract.org/), focused on the so-called non-conventional yeasts. The information in the YEASTRACT database, focused on Saccharomyces cerevisiae, was updated. PathoYeastract was extended to include two additional pathogenic yeast species: Candida parapsilosis and Candida tropicalis. Furthermore, the NCYeastract database was created, including five biotechnologically relevant yeast species: Zygosaccharomyces bailliiKluyveromyces lactisKluyveromyces marxianusYarrowia lipolytica and Komagataella phaffii. The YEASTRACT+ portal gathers 289 706 unique documented regulatory associations between transcription factors (TF) and target genes and 420 DNA binding sites, considering 247 TFs from 10 yeast species. YEASTRACT+ continues to make available tools for the prediction of the TFs involved in the regulation of gene/genomic expression. In this release, these tools were upgraded to enable predictions based on orthologous regulatory associations described for other yeast species, including two new tools for cross-species transcription regulation comparison, based on multi-species promoter and TF regulatory network analyses.

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Valorisation of pectin-rich agro-industrial residues by yeasts: potential and challenges

Martins, L.C.; Monteiro, C.C.; Semedo, P.M.; Sá-Correia, I., Applied Microbiology and Biotechnology 2020, 104(15), 6527-6547.

Pectin-rich agro-industrial residues are feedstocks with potential for sustainable biorefineries. They are generated in high amounts worldwide from the industrial processing of fruits and vegetables. The challenges posed to the industrial implementation of efficient bioprocesses are however manyfold and thoroughly discussed in this review paper, mainly at the biological level. The most important yeast cell factory platform for advanced biorefineries is currently Saccharomyces cerevisiae, but this yeast species cannot naturally catabolise the main sugars present in pectin-rich agro-industrial residues hydrolysates, in particular D-galacturonic acid and L-arabinose. However, there are non-Saccharomyces species (non-conventional yeasts) considered advantageous alternatives whenever they can express highly interesting metabolic pathways, natively assimilate a wider range of carbon sources or exhibit higher tolerance to relevant bioprocess-related stresses. For this reason, the interest in non-conventional yeasts for biomass-based biorefineries is gaining momentum. This review paper focuses on the valorisation of pectin-rich residues by exploring the potential of yeasts that exhibit vast metabolic versatility for the efficient use of the carbon substrates present in their hydrolysates and high robustness to cope with the multiple stresses encountered. The major challenges and the progresses made related with the isolation, selection, sugar catabolism, metabolic engineering and use of non-conventional yeasts and S. cerevisiae-derived strains for the bioconversion of pectin-rich residue hydrolysates are discussed. The reported examples of value-added products synthesised by different yeasts using pectin-rich residues are reviewed.

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The evaluation of oleic acid alternatives for the biochemical production of 9-octadecenedioic acid

Bauwelinck J., Wijnants M., Tavernier S., Cornet I., Biochemical Engineering Journal 2020, 161.

Despite considerable foaming problems, oleic acid is still the most commonly used substrate for the biochemical production of 9-octadecenedioic acid (DCA). In this paper, the suitability of alternative substrates such as oleyl alcohol, methyl-, ethyl- and butyl oleate, was investigated. First, the toxicity of the alternative substrates to the fermenting yeast C. tropicalis ATCC20962 was determined for different substrate concentrations. It was found that the addition of up to 100 g L−1 of the ester and alcohol substrates showed no growth influence. Since alcohols are expected side products when using ester substrates, the toxicity of the corresponding released alcohols was also evaluated. Methanol and ethanol showed no effect on the growth up to 36 g L−1 and 55 gL−1 respectively, whereas a half maximum inhibitory concentration (IC50) of 1.4 g L−1 for butanol was found. Using 20 g L−1 oleic acid, oleyl alcohol, methyl oleate and butyl oleate as substrate showed a yield of 14 g L−1, 7 g L−1, 5 gL-1 and 0.9 g L-1 9-octadecenedioic acid respectively. A qualitative study showed that all four substrates were converted to 9-octadecenedioic acid. Oleic acid resulted in the highest yield whereas oleyl alcohol is the closest candidate for alternative DCA production.

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Screening of oleaginous yeasts for lipid production using volatile fatty acids as substrate

Llamas M., Dourou M., González-Fernández C., Aggelis G., Tomás-Pejó E., Biomass and Bioenergy 2020, 138, 105553.

Using residual material instead of sugars as substrate for oleaginous microorganisms is a promising approach that may reduce the production costs of microbial lipid. In this study, five oleaginous yeasts were screened for their ability to grow and produce lipid utilizing volatile fatty acids (VFAs), generated from anaerobic fermentation of microalgal biomass, as the only carbon and energy source. Yeasts growth and lipid accumulation capacity at three VFAs concentrations (i.e. 5, 10 and 15 g L−1) were evaluated. Regardless of VFAs concentration four of the five strains were able to grow in digestates reaching biomass yields from VFAs between 0.22 and 0.37 g g−1. The highest lipid content in dry biomass was observed in Cutaneotrichosporon curvatum and Cyberlindnera saturnus (36.9 and 33.9% on dry biomass, respectively) corresponding to lipid yields from VFAs of 0.11 and 0.13 g g−1, respectively. Oleic, palmitic and linoleic acids were the major fatty acids, accounting for more than 70% of the fatty acids contained in total yeast lipids, profile similar to that of common vegetable oils. The above findings suggest that microalgal biomass derived VFAs could be converted into yeast lipid suitable as feedstock in the chemical (including biofuel) industry.

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Revealing the potential of lipid and β-coproduction in basidiomycetes yeast

Byrtusová, D.; Shapaval, V.; Holub, J.; Šimanský, S.; Rapta, M.; Szotkowski, M.; Kohler, A.; Márová, I., Microorganisms 2020, 8(7), 1034.

Beta (β)–glucans are polysaccharides composed of D-glucose monomers. Nowadays, β-glucans are gaining attention due to their attractive immunomodulatory biological activities, which can be utilized in pharmaceutical or food supplementation industries. Some carotenogenic Basidiomycetes yeasts, previously explored for lipid and carotenoid coproduction, could potentially coproduce a significant amount of β–glucans. In the present study, we screened eleven Basidiomycetes for the coproduction of lipids and β–glucans. We examined the effect of four different C/N ratios and eight different osmolarity conditions on the coproduction of lipids and β–glucans. A high-throughput screening approach employing microcultivation in microtiter plates, Fourier Transform Infrared (FTIR) spectroscopy and reference analysis was utilized in the study. Yeast strains C. infirmominiatum CCY 17-18-4 and R. kratochvilovae CCY 20-2-26 were identified as the best coproducers of lipids and β-glucans. In addition, C. infirmominiatum CCY 17-18-4, R. kratochvilovae CCY 20-2-26 and P. rhodozyma CCY 77-1-1 were identified as the best alternative producers of β-glucans. Increased C/N ratio led to increased biomass, lipid and β-glucans production for several yeast strains. Increased osmolarity had a negative effect on biomass and lipid production while the β-glucan production was positively affected.

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Patterns of Lignocellulosic Sugar Assimilation and Lipid Production by Newly Isolated Yeast Strains From Chilean Valdivian Forest

Valdés G., Mendonça R.T., Parra C., Aggelis G., Applied Biochemistry and Biotechnology 2020, 192(4), 1124-1146.

Three yeast strains were isolated from decaying wood of Chilean Valdivian forest and identified as Meyerozyma guilliermondiiScheffersomyces coipomensis, and Sugiyamaella paludigena. These strains were able to efficiently grow on the major monomers contained in Pinus spp. and Eucalyptus spp. wood that includes glucose (Glc), xylose (Xyl), and mannose (Man), showing at 28 °C higher uptake rates for Man, and in some cases for Glc, than for Xyl, used as single carbon sources. Nevertheless, in cultures performed on sugar mixtures, the strains displayed a notable preference for Glc. Additionally, in sugar mixtures, the absence of regulatory mechanisms in sugar assimilation (e.g., catabolic repression) was observed and documented when the activities of several enzymes involved in sugar assimilation (i.e., phosphoglucose isomerase, phosphomannose isomerase, and xylulokinase) were determined. The activity of the key enzymes involved in the onset of lipid accumulation (i.e., NAD+ICDH) and in fatty acid (FA) biosynthesis (i.e., ATP:CL) indicated a significant accumulation of storage lipids (i.e., up to 24%, w/w) containing oleic and palmitic acids as the major components. The present paper is the first report on the potential of M. guilliermondiiS. coipomensis, and S. paludigena as oleaginous yeasts. We conclude that the new isolates, being able to simultaneously assimilate the major lignocellulosic sugars and efficiently convert them into oily biomass, present a biotechnological potential which deserve further investigation.

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Microbial sources of polyunsaturated fatty acids (PUFAs) and the prospect of organic residues and wastes as growth media for PUFA-producing microorganisms

Kothri M., Mavrommati M., Elazzazy A.M., Baeshen M.N., Moussa T.A.A., Aggelis G., FEMS Microbiology Letters 2020, 367(5), fnaa028.

The discovery of non-fish sources of polyunsaturated fatty acids (PUFAs) is of great biotechnological importance. Although various oleaginous microalgae and fungi are able of accumulating storage lipids (single cell oils – SCOs) containing PUFAs, the industrial applications utilizing these organisms are rather limited due to the high-fermentation cost. However, combining SCO production with other biotechnological applications, including waste and by-product valorization, can overcome this difficulty. In the current review, we present the major sources of fungi (i.e. members of Mucoromycota, fungoid-like Thraustochytrids and genetically modified strains of Yarrowia lipolytica) and microalgae (e.g. Isochrysis, NannochloropsisandTetraselmis) that have come recently to the forefront due to their ability to produce PUFAs. Approaches adopted in order to increase PUFA productivity and the potential of using various residues, such as agro-industrial, food and aquaculture wastes as fermentation substrates for SCO production have been considered and discussed. We concluded that several organic residues can be utilized as feedstock in the SCO production increasing the competitiveness of oleaginous organisms against conventional PUFA producers.

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Lignocellulosic biomass as a substrate for oleaginous microorganisms: A review

Valdés G., Mendonça R.T., Aggelis G., Applied Sciences (Switzerland) 2020, 10(21), 7698.

Microorganisms capable of accumulating lipids in high percentages, known as oleaginous microorganisms, have been widely studied as an alternative for producing oleochemicals and biofuels. Microbial lipid, so-called Single Cell Oil (SCO), production depends on several growth parameters, including the nature of the carbon substrate, which must be efficiently taken up and converted into storage lipid. On the other hand, substrates considered for large scale applications must be abundant and of low acquisition cost. Among others, lignocellulosic biomass is a promising renewable substrate containing high percentages of assimilable sugars (hexoses and pentoses). However, it is also highly recalcitrant, and therefore it requires specific pretreatments in order to release its assimilable components. The main drawback of lignocellulose pretreatment is the generation of several by-products that can inhibit the microbial metabolism. In this review, we discuss the main aspects related to the cultivation of oleaginous microorganisms using lignocellulosic biomass as substrate, hoping to contribute to the development of a sustainable process for SCO production in the near future.

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Identification of genes involved in xylose metabolism of Meyerozyma guilliermondii and their genetic engineering for increased xylitol production

Atzmüller D., Ullmann N., Zwirzitz A., AMB Express 2020, 10(1), 78.

Meyerozyma guilliermondii, a non-conventional yeast that naturally assimilates xylose, is considered as a candidate for biotechnological production of the sugar alternative xylitol. Because the genes of the xylose metabolism were yet unknown, all efforts published so far to increase the xylitol yield of this yeast are limited to fermentation optimization. Hence, this study aimed to genetically engineer this organism for the first time with the objective to increase xylitol production. Therefore, the previously uncharacterized genes of M. guilliermondii ATCC 6260 encoding for xylose reductase (XR) and xylitol dehydrogenase (XDH) were identified by pathway investigations and sequence similarity analysis. Cloning and overexpression of the putative XR as well as knockout of the putative XDH genes generated strains with about threefold increased xylitol yield. Strains that combined both genetic modifications displayed fivefold increase in overall xylitol yield. Enzymatic activity assays with lysates of XR overexpressing and XDH knockout strains underlined the presumed functions of the respective genes. Furthermore, growth evaluation of the engineered strains on xylose as sole carbon source provides insights into xylose metabolism and its utilization for cell growth.

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Identification and importance of mitochondrial citrate carriers and ATP citrate lyase for glycolipid production in Starmerella bombicola

Jezierska S., Claus S., Van Bogaert I.N.A., Applied Microbiology and Biotechnology 2020, 104(14), 6235-6248.

Starmerella bombicola is a non-conventional yeast commercially used as a microbial cell factory for sophorolipid production. Sophorolipids are glycolipid biosurfactants composed of a glucose disaccharide sophorose and a fatty acid. In de novo sophorolipid synthesis, the fatty acid moiety is derived from the fatty acid synthesis (FAS) complex; therefore, the yeast’s lipid metabolism plays a crucial role in sophorolipid biosynthesis. As a fatty acid precursor, citric acid is a key primary metabolite that connects carbohydrate and lipid metabolism, and in S. bombicola, it also has a regulatory effect on sophorolipid composition and productivity. We aimed to identify the mitochondrial transporters involved in citrate shuttling and the ATP citrate lyase (Acl), the enzyme that converts citric acid into acetyl-CoA. Subsequently, we studied their role in the citric acid shuttle and glycolipid synthesis and the potential of citrate metabolism as a genetic manipulation target for increased glycolipid synthesis. Bioinformatics analyses predicted 32 mitochondrial carriers of which two were identified as citrate transporters, named SbCtp1 and SbYhm2. Deletion of these mitochondrial carriers led to a lesser sophorolipid yield and a shift in the lactonic/acidic sophorolipid ratio. However, only the knockout of SbYhm2 caused a decrease of citric and an increase of malic acid extracellular concentrations. Additionally, deletion of SbAcl1 had a negative effect on S. bombicola’s specific growth rate and sophorolipid synthesis and contributed to extra- and intracellular citric acid accumulation. Unexpectedly, SbAcl1 overexpression also decreased glycolipid production.

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High-level heterologous expression of active Chaetomium thermophilum FDH in Pichia pastoris

Duman, Z.E.; Duraksoy, B.B.; Aktaş, F.; Woodley, J.M.; Binay, B., Enzyme and Microbial Technology 2020, 137, 109552.

Nowadays, the use of formate dehydrogenase (FDH, EC 1.17.1.9) is well established as a means of NADH regeneration from NAD+ via the coupled conversion of formate into carbon dioxide. Recent studies have been reported that specifically Chaetomium thermophilum FDH (CtFDH) is the most efficient FDH catalyzing this reaction in reverse (i.e. using CO2 as a substrate to produce formate, and thereby regenerating NAD+). However, to date the production of active CtFDH at high protein expression levels has received relatively little attention. In this study, we have tested the effect of batch and high cell density fermentation (HCDF) strategies in a small stirred fermenter, as well as the effect of supplementing the medium with casamino acids, on the expressed level of secreted CtFDH using P. pastoris. We have established that the amount of expressed CtFDH was indeed enhanced via a HCDF strategy and that extracellular protease activity was eliminated via the addition of casamino acids into the fermentation medium. On this basis, secreted CtFDH in an active form can be easily separated from the fermentation and can be used for subsequent biotechnological applications.

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Fine-Tuning of Transcription in Pichia pastoris Using dCas9 and RNA Scaffolds

Baumschabl M., Prielhofer R., Mattanovich D., Steiger M.G., ACS Synthetic Biology 2020, 9(12), 3202-3209.

For metabolic engineering approaches, fast and reliable tools are required to precisely manipulate the expression of target genes. dCas9 can be fused via RNA scaffolds to trans-activator domains and thus regulate the gene expression when targeted to the promoter region of a gene. In this work we show that this strategy can be successfully implemented for the methylotrophic yeast Pichia pastoris. It is shown that the thiamine repressible promoter of THI11 can be activated under repression conditions using a scgRNA/dCas9 construct. Furthermore, the RIB1 gene required for riboflavin production was activated, leading to increased riboflavin production exceeding the riboflavin titers of a conventional RIB1 overexpression with a pGAP promoter.

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Factors influencing adhesion of bacteria Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and yeast Pichia membranifaciens to wooden surfaces

Tomičić R., Tomičić Z., Thaler N., Humar M., Raspor P., Wood Science and Technology 2020, 54(6), 1663-1676.

The aim of this study was to assess the potential of bacteria Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923 and yeast Pichia membranifaciens ZIM 2417 to adhere to wooden surfaces such as poplar (Populus sp.), Norway spruce (Picea abies), European beech (Fagus sylvatica), beech coated with the commercial Belinka oil food contact and disinfectant P3-oxonia active 150, and investigate their survival on the beech wood surface under different relative humidities (RH; 65%, 75%, 85%, 98%) and temperatures (10 °C, 20 °C, 27 °C/37 °C). To extend the research goals, the scanning electron microscopy (SEM) analysis was also performed. The adhesion was determined by the number of colony-forming units per mm2 of sample (CFU/mm2). Results showed that all tested bacteria and yeast were able to adhere to the wooden surfaces, although differences were observed according to strains and type of wood. It was evident that number of adhered cells of S. aureus was lower on spruce (3.62 × 103 CFU/mm2) compared to poplar and beech (1.09 × 105 and 2.11 × 104 CFU/mm2, respectively). Furthermore, oil and disinfectant promoted the adhesion of P. aeruginosa (155.93 and 130.50%, respectively) on the beech surfaces, while they had a strong inhibitory effect on the other tested microorganisms E. coli (87.44 and 88.44%, respectively), S. aureus (91.24 and 96.80%, respectively) and P. membranifaciens (92.45 and 87.24%, respectively). These findings are consistent with SEM micrographs. The current data also indicated that relative humidity and temperature significantly affected the adhesion of tested bacteria and yeast. The highest degree of adhesion was observed at a relative humidity of 98% and temperature of 20 and 37 °C for bacteria, or 20 and 27 °C for yeast. Thus, the knowledge of how these microorganisms adhere to wooden surfaces and which factors affect this phenomenon proves to be of great importance in order to avoid their colonization.

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Candida intermedia CBS 141442: A novel glucose/xylose co-fermenting isolate for lignocellulosic bioethanol productionon link

Moreno, A.D.; Tomás-Pejó, E.; Olsson, L.; Geijer, C. Energies 2020, 13(20):5363.

The present study describes the isolation of the novel strain Candida intermedia CBS 141442 and investigates the potential of this microorganism for the conversion of lignocellulosic streams. Different C. intermedia clones were isolated during an adaptive laboratory evolution experiment under the selection pressure of lignocellulosic hydrolysate and in strong competition with industrial, xylose-fermenting Saccharomyces cerevisiae cells. Isolates showed different but stable colony and cell morphologies when growing in a solid agar medium (smooth, intermediate and complex morphology) and liquid medium (unicellular, aggregates and pseudohyphal morphology). Clones of the same morphology showed similar fermentation patterns, and the C. intermedia clone I5 (CBS 141442) was selected for further testing due to its superior capacity for xylose consumption (90% of the initial xylose concentration within 72 h) and the highest ethanol yields (0.25 ± 0.02 g ethanol/g sugars consumed). Compared to the well-known yeast Scheffersomyces stipitis, the selected strain showed slightly higher tolerance to the lignocellulosic-derived inhibitors when fermenting a wheat straw hydrolysate. Furthermore, its higher glucose consumption rates (compared to S. stipitis) and its capacity for glucose and xylose co-fermentation makes C. intermedia CBS 141442 an attractive microorganism for the conversion of lignocellulosic substrates, as demonstrated in simultaneous saccharification and fermentation processes.

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Sources of microbial oils with emphasis to Mortierella (Umbelopsis) isabellina fungus

Papanikolaou S., Aggelis G., World Journal of Microbiology and Biotechnology 2019, 35(4), 63.

The last years a constantly rising number of publications have appeared in the literature in relation to the production of oils and fats deriving from microbial sources (the “single cell oils”—SCOs). SCOs can be used as precursors for the synthesis of lipid-based biofuels or employed as substitutes of expensive oils rarely found in the plant or animal kingdom. In the present review-article, aspects concerning SCOs (economics, biochemistry, substrates, technology, scale-up), with emphasis on the potential of Mortierella isabellina were presented. Fats and hydrophilic substrates have been used as carbon sources for cultivating Zygomycetes. Among them, wild-type M. isabellina strains have been reported as excellent SCO-producers, with conversion yields on sugar consumed and lipid in DCW values reported comparable to the maximum ones achieved for genetically engineered SCO-producing strains. Lipids produced on glucose contain γ-linolenic acid (GLA), a polyunsaturated fatty acid (PUFA) of high dietary and pharmaceutical importance, though in low concentrations. Nevertheless, due to their abundance in oleic acid, these lipids are perfect precursors for the synthesis of 2nd generation biodiesel, while GLA can be recovered and directed to other usages. Genetic engineering focusing on over-expression of Δ6 and Δ12 desaturases and of C16 elongase may improve the fatty acid composition (viz. increasing the concentration of GLA or other nutritionally important PUFAs) of these lipids.

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Physiological Genomics of the Highly Weak-Acid-Tolerant Food Spoilage Yeasts of Zygosaccharomyces bailii sensu lato

Palma, M.; Sá-Correia, I., In: Sá-Correia I. (ed) Yeasts in Biotechnology and Human Health. Progress in Molecular and Subcellular Biology 2019, vol 58, pp 85-109. Springer, Cham.

Zygosaccharomyces bailii and two closely related species, Z. parabailii and Z. pseudobailii (“Z. bailii species complex”, “Z. bailii sensu lato” or simply “Z. bailii (s.l.)”), are frequently implicated in the spoilage of acidified preserved foods and beverages due to their tolerance to very high concentrations of weak acids used as food preservatives. The recent sequencing and annotation of these species’ genomes have clarified their genomic organization and phylogenetic relationship, which includes events of interspecies hybridization. Mechanistic insights into their adaptation and tolerance to weak acids (e.g., acetic and lactic acids) are also being revealed. Moreover, the potential of Z. bailii (s.l.) to be used in industrial biotechnological processes as interesting cell factories for the production of organic acids, reduction of the ethanol content, increase of alcoholic beverages aroma complexity, as well as of genetic source for increasing weak acid resistance in yeast, is currently being considered. This chapter includes taxonomical, ecological, physiological, and biochemical aspects of Z. bailii (s.l.). The focus is on the exploitation of physiological genomics approaches that are providing the indispensable holistic knowledge to guide the effective design of strategies to overcome food spoilage or the rational exploitation of these yeasts as promising cell factories.

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Influence of various factors on adhesion of yeast Candida spp. and Pichia spp. to abiotic surfaces

Tomičić, R., Tomičić, Z., Dodić, S., Raspor, P., Acta Microbiologica Bulgarica, 2019, 35(1), 19-28.

The aim of this study was to assess the potential of Candida species and Pichia species to adhere to stainless steel (AISI 304) material with different degrees of surface roughness and polystyrene as most frequently used contact materials. Cell surface hydrophobicity (CSH) of Candida and Pichia strains was determined in order to assess correlation between the cell surface hydrophobicity and yeast adhesion to polystyrene. Candida albicans showed a higher ability to adhere to both surfaces compared with non-albicans Candida species. Regarding Pichia species, P. membranifaciens strains were less adherent to stainless steel than P. pijperi. Surface roughness of stainless steel was found to affect the adhesion of Candida and Pichia strains, whereas cell surface hydrophobicity was not correlated with adhesion. We also investigated the antimicrobial and antibiofilm activity of plant extracts such as Humulus lupulus, Alpinia katsumadai and Evodia rutaecarpa against C. albicans, C. glabrata and P. membranifaciens. According to the MIC values, all plant extracts were effective in inhibiting yeast strains. It was observed that biofilms of C. glabrata were more resistance to plant extracts as compared to C. albicans. However, extracts of A. katsumadai and E. rutaecarpa promoted the growth and development of a preformed biofilm of P. membranifaciens.

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Laboratory evolution strategies for improving lipid accumulation in Yarrowia lipolytica

Daskalaki A., Perdikouli N., Aggeli D., Aggelis G., Applied Microbiology and Biotechnology 2019, 103(20), 8585-8596.

Oleaginous microorganisms are of high biotechnological interest being considered as alternative sources of oil (single cell oil—SCO). Current research for increasing productivity of oleaginous microorganisms is focused on the overexpression of genes implicated in lipid synthesis, the inactivation of genes implicated in storage lipid turnover, and on the suppression of competitive to lipid biosynthesis pathways. An alternative strategy, described here, relies on evolution of Yarrowia lipolytica under alternating environments that promote growth, encourage storage lipid synthesis, and reward high energy-containing cells. Derived populations were characterized biochemically, especially on their ability to accumulate lipids, and compared with the starting strain. Interestingly, lipid-accumulating ability early in the evolution was decreased compared with the starting strain. Subsequently, oleaginous lineages dominated, leading to populations able to accumulate lipids in high amounts. A population obtained after 77 generations was able to accumulate 44% w/w of lipid, which was 30% higher than that of the starting strain. We conclude that evolution-based strategies can be utilized as a robust tool for improving lipid accumulation capacity in oleaginous microorganisms.

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