Dynamics of the cytosol soluble carbohydrates and membrane lipids in response to ambient pH in alkaliphilic and alkalitolerant fungi

Comparative composition of lipids and cytosol soluble carbohydrates at different ambient pH values was studied for two obligately alkaliphilic fungi (Sodiomyces magadii and S. alkalinus) and for two alkalitolerant ones (Acrostalagmus luteoalbus and Chordomyces antarcticus). The differences and common patterns were revealed in responses to pH stress for the fungi with different types of adaptation to ambient pH. While trehalose was one of the major cytosol carbohydrates in alkaliphilic fungi under optimal growth conditions (pH 10.2), pH decrease to 7.0 resulted in doubling its content. In alkalitolerant fungi trehalose was a minor component and its level did not change significantly at different pH. In alkalitolerant fungi, arabitol and mannitol were the major carbohydrate components, with their highest ratio observed under alkaline conditions and the lowest one, under neutral and acidic conditions. In alkaliphiles, significant levels of arabitol were revealed only under alkaline conditions, which indicated importance of trehalose and arabitol for alkaliphily. Decreased pH resulted in the doubling of the proportion of phosphatidic acids among the membrane lipids, which was accompanied by a decrease in the fractions of phosphatidylcholines and sterols. Alkalitolerant fungi also exhibited a decrease in sterol level at decreased pH, but against the background of increased proportion of one of phospholipids. Decreased unsaturation degree in the fatty acids of the major phospholipids was a common response to decreased ambient pH.     

Expression and Characterization of Glucose Oxidase from <Emphasis Type="Italic">Aspergillus niger</Emphasis> in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Glucose oxidase (GOX) is currently used in clinical, pharmaceutical, food and chemical industries. The aim of this study was expression and characterization of Aspergillus niger glucose oxidase gene in the yeast Yarrowia lipolytica. For the first time, the GOX gene of A. niger was successfully expressed in Y. lipolytica using a mono-integrative vector containing strong hybrid promoter and secretion signal. The highest total glucose oxidase activity was 370 U/L after 7 days of cultivation. An innovative method was used to cell wall disruption in current study, and it could be recommended to use for efficiently cell wall disruption of Y. lipolytica. Optimum pH and temperature for recombinant GOX activity were 5.5 and 37 °C, respectively. A single band with a molecular weight of 80 kDa similar to the native and pure form of A. niger GOX was observed for the recombinant GOX in SDS-PAGE analysis. Y. lipolytica is a suitable and efficient eukaryotic expression system to production of recombinant GOX in compered with other yeast expression systems and could be used to production of pure form of GOX for industrial applications.     

Expressing accessory proteins in cellulolytic <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> to improve the conversion yield of recalcitrant cellulose

Background

A recently constructed cellulolytic Yarrowia lipolytica is able to grow efficiently on an industrial organosolv cellulose pulp, but shows limited ability to degrade crystalline cellulose. In this work, we have further engineered this strain, adding accessory proteins xylanase II (XYNII), lytic polysaccharide monooxygenase (LPMO), and swollenin (SWO) from Trichoderma reesei in order to enhance the degradation of recalcitrant substrate.

Results

The production of EG I was enhanced using a promoter engineering strategy. This provided a new cellulolytic Y. lipolytica strain, which compared to the parent strain, exhibited higher hydrolytic activity on different cellulosic substrates. Furthermore, three accessory proteins, TrXYNII, TrLPMOA and TrSWO, were individually expressed in cellulolytic and non-cellulolytic Y. lipolytica. The amount of rhTrXYNII and rhTrLPMOA secreted by non-cellulolytic Y. lipolytica in YTD medium during batch cultivation in flasks was approximately 62 and 52 mg/L, respectively. The purified rhTrXYNII showed a specific activity of 532 U/mg-protein on beechwood xylan, while rhTrLPMOA exhibited a specific activity of 14.4 U/g-protein when using the Amplex Red/horseradish peroxidase assay. Characterization of rhTrLPMOA revealed that this protein displays broad specificity against β-(1,4)-linked glucans, but is inactive on xylan. Further studies showed that the presence of TrLPMOA synergistically enhanced enzymatic hydrolysis of cellulose by cellulases, while TrSWO1 boosted cellulose hydrolysis only when it was applied before the action of cellulases. The presence of rTrXYNII enhanced enzymatic hydrolysis of an industrial cellulose pulp and of wheat straw. Co-expressing TrXYNII and TrLPMOA in cellulolytic Y. lipolytica with enhanced EG I production procured a novel engineered Y. lipolytica strain that displayed enhanced ability to degrade both amorphous (CIMV-cellulose) and recalcitrant crystalline cellulose in complex biomass (wheat straw) by 16 and 90%, respectively.

Conclusions

This study has provided a potent cellulose-degrading Y. lipolytica strain that co-expresses a core set of cellulolytic enzymes and some accessory proteins. Results reveal that the tuning of cellulase production and the production of accessory proteins leads to optimized performance. Accordingly, the beneficial effect of accessory proteins for cellulase-mediated degradation of cellulose is underlined, especially when crystalline cellulose and complex biomass are used as substrates. Findings specifically underline the benefits and specific properties of swollenin. Although in our study swollenin clearly promoted cellulase action, its use requires process redesign to accommodate its specific mode of action.

     

Multiplex gene editing of the <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> genome using the CRISPR-Cas9 system

Yarrowia lipolytica is categorized as a generally recognized as safe (GRAS) organism and is a heavily documented, unconventional yeast that has been widely incorporated into multiple industrial fields to produce valuable biochemicals. This study describes the construction of a CRISPR-Cas9 system for genome editing in Y. lipolytica using a single plasmid (pCAS1yl or pCAS2yl) to transport Cas9 and relevant guide RNA expression cassettes, with or without donor DNA, to target genes. Two Cas9 target genes, TRP1 and PEX10, were repaired by non-homologous end-joining (NHEJ) or homologous recombination, with maximal efficiencies in Y. lipolytica of 85.6 % for the wild-type strain and 94.1 % for the ku70/ku80 double-deficient strain, within 4 days. Simultaneous double and triple multigene editing was achieved with pCAS1yl by NHEJ, with efficiencies of 36.7 or 19.3 %, respectively, and the pCASyl system was successfully expanded to different Y. lipolytica breeding strains. This timesaving method will enable and improve synthetic biology, metabolic engineering and functional genomic studies of Y. lipolytica.     

New recombinant strains of the yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> with overexpression of the aconitate hydratase gene for the obtainment of isocitric acid from rapeseed oil

The yeast Yarrowia lipolytica is capable of high-intensity synthesis (overproduction) of citric (CA) and isocitric (ICA) acids under nitrogen limitation. The ratio of the synthesized acids depends on the producing strains used and the expression level of the aconitate hydratase gene (ACO1). Recombinant variants with overexpression of the multicopy ACO1 gene have been obtained based on the natural ICA-producing strain Y. lipolytica 672. A recombinant strain Y. lipolytica 20, which has an isocitrate-citrate ratio shifted towards ICA (2.3: 1) as compared to the parental strain (1.1: 1), has been selected. Culturing of the 20 variant in a 10 L reactor has resulted in the production of 72.6 g/L of ICA and 29.0 g/L of CA with a ratio of 2.5: 1. This makes it possible to regard Y. lipolytica 20 as a promising producer for the development of an industrial process for isocitrate production.     

Production of <Emphasis Type="Italic">trans</Emphasis>-10,<Emphasis Type="Italic">cis</Emphasis>-12-conjugated linoleic acid using permeabilized whole-cell biocatalyst of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Objective

To improve the production of trans-10,cis-12-conjugated linoleic acid (t10,c12-CLA) from linoleic acid in recombinant Yarrowia lipolytica.

Results

Cells of the yeast were permeabilized by freeze/thawing. The optimal conditions for t10,c12-CLA production by the permeabilized cells were at 28 °C, pH 7, 200 rpm with 1.5 g sodium acetate l^?1, 100 g wet cells l^?1, and 25 g LA l^?1. Under these conditions, the permeabilized cells produced 15.6 g t10,c12-CLA l^?1 after 40 h, with a conversion yield of 62 %. The permeabilized cells could be used repeatedly for three cycles, with the t10,c12-CLA extracellular production remaining above 10 g l^?1.

Conclusion

Synthesis of t10,c12-CLA was achieved using a novel method, and the production reported in this work is the highest value reported to date.

     

Role of Arabidopsis <Emphasis Type="Italic">ABF1</Emphasis>/<Emphasis Type="Italic">3</Emphasis>/<Emphasis Type="Italic">4</Emphasis> during <Emphasis Type="Italic">det1</Emphasis> germination in salt and osmotic stress conditions

Key message

Arabidopsis det1 mutants exhibit salt and osmotic stress resistant germination. This phenotype requires HY5, ABF1, ABF3, and ABF4.

Abstract

While DE-ETIOLATED 1 (DET1) is well known as a negative regulator of light development, here we describe how det1 mutants also exhibit altered responses to salt and osmotic stress, specifically salt and mannitol resistant germination. LONG HYPOCOTYL 5 (HY5) positively regulates both light and abscisic acid (ABA) signalling. We found that hy5 suppressed the det1 salt and mannitol resistant germination phenotype, thus, det1 stress resistant germination requires HY5. We then queried publically available microarray datasets to identify genes downstream of HY5 that were differentially expressed in det1 mutants. Our analysis revealed that ABA regulated genes, including ABA RESPONSIVE ELEMENT BINDING FACTOR 3 (ABF3), are downregulated in det1 seedlings. We found that ABF3 is induced by salt in wildtype seeds, while homologues ABF4 and ABF1 are repressed, and all three genes are underexpressed in det1 seeds. We then investigated the role of ABF3, ABF4, and ABF1 in det1 phenotypes. Double mutant analysis showed that abf3, abf4, and abf1 all suppress the det1 salt/osmotic stress resistant germination phenotype. In addition, abf1 suppressed det1 rapid water loss and open stomata phenotypes. Thus interactions between ABF genes contribute to det1 salt/osmotic stress response phenotypes.

     

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

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

Production of β-carotene by expressing a heterologous multifunctional carotene synthase in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Objectives

To obtain functional expression of a heterologous multifunctional carotene synthase containing phytoene synthase, phytoene dehydrogenase, and lycopene β-cyclase activities encoded by carS from Schizochytrium sp. in order to allow Yarrowia lipolytica to produce β-carotene.

Results

To increase the integration efficiency of a 3.8 kb carS under the control of P _GPD promoter with a 2 kb selection marker, ura3, along with a geranylgeranyl diphosphate synthase (GGS1) expression cassette (~10 kb in total), was inserted into the Y. lipolytica chromosome, and the DNA assembler method was combined with double chromosomal deletions of ku70 and ku80. This method resulted in a 13.4-fold increase in integration efficiency compared with the original method, reaching 63% (10/16). The resulting recombinant Y. lipolytica produced 0.41 mg β-carotene per g dry cell weight, while the wild type did not produce any indicating the functionality of the multifunctional carotene synthase in Y. lipolytica.

Conclusion

Expression of GGS1 and a multifunctional carotene synthase from Schizochytrium sp. in Y. lipolytica led to β-carotene production. DNA assembler efficiency was greatly increased by the deletion of ku70 and ku80, which resulted in decreased in vivo nonhomologous end-joining (NHEJ) in Y. lipolytica.

     

Testing the cost of reproduction with the rotifer <Emphasis Type="Italic">Brachionus</Emphasis><Emphasis Type="Italic">patulus</Emphasis> at different pH levels

In the present work, we tested the hypothesis that the cost of reproduction was evident under stressful conditions with the rotifer Brachionus patulus at different pH levels (5–10 at 1 unit intervals). We used sublethal pH levels (pH 5, 9, and 10) to simulate stressful conditions. We analyzed the correlations between age-specific fecundity (m ₁, m ₂, m ₃, …) versus future survival (l _x + 1, l _x + 2, l _x + 3, … for the entire lifespan) (survival costs) and future expectation of reproduction (\( V_{ 1}^{*} , \, V_{ 2}^{*} , \, V_{ 3}^{*} , \ldots \) for the entire lifespan) (reproductive costs), using the data obtained from life table demographic studies of B. patulus under stressful and favorable (pH 6, 7, and 8) pH levels. The results showed that significant negative correlations were observed between age-specific fecundity and future survival and future expectation of reproduction at all tested pH levels, indicating that costs of reproduction exist in the rotifer B. patulus under stressful and favorable pH conditions. However, the percentage of statistically significant negative correlations from total correlations of survival and reproductive costs differed greatly, depending on the tested pH conditions. The percentage of significant negative correlation of reproductive costs is significantly higher under stressful pH conditions (pH 5, 9, and 10) than favorable pH conditions (pH 6, 7, and 8). For survival costs, the same trends are also observed, suggesting that the costs of reproduction were more obvious under stressful pH than favorable pH.     

Comparative study of chemical composition and antitumor activity of aqueous-ethanol extracts of brown algae <Emphasis Type="Italic">Laminaria cichorioides,Costaria costata</Emphasis>, and <Emphasis Type="Italic">Fucus evanescens</Emphasis>

The chemical composition of water-ethanol extracts of the brown algae Laminaria cichorioides, Costaria costata, and Fucus evanescens were studied. The extracts contained mannitol, iodine, micro elements, free amino acids, glycolipids, polyunsaturated fatty acids, fucosterine, and polyphenols. The extracts were distinguished by high contents of mannitol and iodine (L. cichorioides), lipophilic matter (C. costata), and polyphenol compounds (F. evanescens). All the extracts under study inhibited the growth of DLD-1 and HT-29 human intestine tumor cells. The strongest inhibitory effect was exerted by the extract of F. evanescens at a concentration of 50 μg/ml. The extracts can be recommended for the production of fucosterol, phlorotannins, chlorophyll derivatives, mannitol, and compositions for medical and veterinary application.     

HflX protein protects <Emphasis Type="Italic">Escherichia coli</Emphasis> from manganese stress

The ribosome-binding GTPase HflX is required for manganese homeostasis in E. coli. While under normal conditions ?hflX cells behave like wild type E. coli with respect to growth pattern and morphology, deletion of hflX makes E. coli cells extremely sensitive to manganese, characterized by arrested cell growth and filamentation. Here we demonstrate that upon complementation by hflX, manganese stress is relieved. In phenotypic studies done in a manganese-rich environment, ?hflX cells were highly sensitive to antibiotics that bind the penicillin binding protein 3 (PBP3), suggesting that the manganese stress led to impaired peptidoglycan biosynthesis. An irregular distribution of dark bands of constriction along filaments, delocalization of the dark bands from midcell towards poles and subpoles, lack of septum formation and arrested cell division were observed in ?hflX cells under manganese stress. However, chromosome replication and segregation of nucleoids were unaffected under these conditions, as observed from confocal microscopy imaging and FACS studies. We conclude that absence of HflX leads to manganese accumulation in E. coli cells, affecting cell septum formation, probably by modulating the activity of the cell division protein PBP3 (FtsI), a major component of the divisome apparatus. We propose that HflX acts as a gatekeeper, regulating the influx of manganese into the cell.