| 1. |
- Bao, Jichen, 1988, et al.
(författare)
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Balanced trafficking between the ER and the Golgi apparatus increases protein secretion in yeast
- 2018
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 8:1
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Tidskriftsartikel (refereegranskat)abstract
- The yeast Saccharomyces cerevisiae is widely used as a cell factory to produce recombinant proteins. However, S. cerevisiae naturally secretes only a few proteins, such as invertase and the mating alpha factor, and its secretory capacity is limited. It has been reported that engineering protein anterograde trafficking from the endoplasmic reticulum to the Golgi apparatus by the moderate overexpression of SEC16 could increase recombinant protein secretion in S. cerevisiae. In this study, the retrograde trafficking in a strain with moderate overexpression of SEC16 was engineered by overexpression of ADP-ribosylation factor GTP activating proteins, Gcs1p and Glo3p, which are involved in the process of COPI-coated vesicle formation. Engineering the retrograde trafficking increased the secretion of α-amylase but did not induce production of reactive oxygen species. An expanded ER membrane was detected in both the GCS1 and GLO3 overexpressio n strains. Physiological characterizations during batch fermentation showed that GLO3 overexpression had better effect on recombinant protein secretion than GCS1 overexpression. Additionally, the GLO3 overexpression strain had higher secretion of two other recombinant proteins, endoglucanase I from Trichoderma reesei and glucan-1,4-α-glucosidase from Rhizopus oryzae, indicating overexpression of GLO3 in a SEC16 moderate overexpression strain might be a general strategy for improving production of secreted proteins by yeast.
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| 2. |
- Bergman, Alexandra Linda, 1985, et al.
(författare)
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Functional expression and evaluation of heterologous phosphoketolases in Saccharomyces cerevisiae
- 2016
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 6:1
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Tidskriftsartikel (refereegranskat)abstract
- Phosphoketolases catalyze an energy-and redox-independent cleavage of certain sugar phosphates. Hereby, the two-carbon (C2) compound acetyl-phosphate is formed, which enzymatically can be converted into acetyl-CoA-a key precursor in central carbon metabolism. Saccharomyces cerevisiae does not demonstrate efficient phosphoketolase activity naturally. In this study, we aimed to compare and identify efficient heterologous phosphoketolase enzyme candidates that in yeast have the potential to reduce carbon loss compared to the native acetyl-CoA producing pathway by redirecting carbon flux directly from C5 and C6 sugars towards C2-synthesis. Nine phosphoketolase candidates were expressed in S. cerevisiae of which seven produced significant amounts of acetyl-phosphate after provision of sugar phosphate substrates in vitro. The candidates showed differing substrate specificities, and some demonstrated activity levels significantly exceeding those of candidates previously expressed in yeast. The conducted studies also revealed that S. cerevisiae contains endogenous enzymes capable of breaking down acetyl-phosphate, likely into acetate, and that removal of the phosphatases Gpp1 and Gpp2 could largely prevent this breakdown. An evaluation of in vivo function of a subset of phosphoketolases was conducted by monitoring acetate levels during growth, confirming that candidates showing high activity in vitro indeed showed increased acetate accumulation, but expression also decreased cellular fitness. The study shows that expression of several bacterial phosphoketolase candidates in S. cerevisiae can efficiently divert intracellular carbon flux toward C2-synthesis, thus showing potential to be used in metabolic engineering strategies aimed to increase yields of acetyl-CoA derived compounds.
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| 3. |
- Bonzom, Cyrielle, 1987, et al.
(författare)
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Glycosylation influences activity, stability and immobilization of the feruloyl esterase 1a from Myceliophthora thermophila
- 2019
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- Heterologous protein production is widely used in industrial biotechnology. However, using non-native production hosts can lead to enzymes with altered post-translational modifications, such as glycosylation. We have investigated how production in a non-native host affects the physicochemical properties and enzymatic activity of a feruloyl esterase from Myceliophthora thermophila, MtFae1a. The enzyme was produced in two microorganisms that introduce glycosylation (M. thermophila and Pichia pastoris) and in Escherichia coli (non-glycosylated). Mass spectrometric analysis confirmed the presence of glycosylation and revealed differences in the lengths of glycan chains between the enzymes produced in M. thermophila and P. pastoris. The melting temperature and the optimal temperature for activity of the non-glycosylated enzyme were considerably lower than those of the glycosylated enzymes. The three MtFae1a versions also exhibited differences in specific activity and specificity. The catalytic efficiency of the glycosylated enzymes were more than 10 times higher than that of the non-glycosylated one. In biotechnology, immobilization is often used to allow reusing enzyme and was investigated on mesoporous silica particles. We found the binding kinetics and immobilization yield differed between the enzyme versions. The largest differences were observed when comparing enzymes with and without glycosylation, but significant variations were also observed between the two differently glycosylated enzymes. We conclude that the biotechnological value of an enzyme can be optimized for a specific application by carefully selecting the production host.
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| 4. |
- Fellahi, Soltana, et al.
(författare)
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Identification of two new keratinolytic proteases from a Bacillus pumilus strain using protein analysis and gene sequencing
- 2016
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 6:1
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Tidskriftsartikel (refereegranskat)abstract
- The Bacillus strain (CCUG 66887) has a high capacity to excrete keratinase with the ability to degrade both alpha- and beta keratin. In this study we aimed to show the characteristics of the keratinolytic protease and to identify its gene by using liquid chromatography-electrospray ionization tandem mass spectrometry methods (nanoHPLC-ESI-MS/MS) followed by Mascot data base search. The results showed that the enzyme in fact consists of two different keratinases, both with a molecular mass of 38 kDa. Further, DNA sequencing generated the open reading frame (ORF) of one of the genes (Ker1), and de novo genome sequencing identified the ORF of the second gene (Ker2). The two keratinase genes contain 1153 base pairs each and have a gene similarity of 67 %. In addition, the Bacillus strain was classified as Bacillus pumilus and its genes were annotated in the GeneBank at NCBI (accession: CP011109.1). Amino acid sequences alignment with known B. pumilus proteases indicated that the two keratinases of B. pumilus strain C4 are subtilisin-like serine proteases belonging to the Protease S8 family. Taken together, these result suggest the two keratinases as promising candidates for enzymatic processing of keratinous wastes in waste refinery.[on SciFinder (R)]
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| 5. |
- Fernando, Dinesh, et al.
(författare)
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Comparison of traditional field retting and Phlebia radiata Cel 26 retting of hemp fibres for fibre-reinforced composites
- 2017
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Classical field retting and controlled fungal retting of hemp using Phlebia radiata Cel 26 (a mutant with low cellulose degrading ability) were compared with pure pectinase treatment with regard to mechanical properties of the produced fibre/epoxy composites. For field retting a classification of the microbial evolution (by gene sequencing) and enzyme profiles were conducted. By phylogenetic frequency mapping, different types of fungi, many belonging to the Ascomycota phylum were found on the fibres during the first 2 weeks of field retting, and thereafter, different types of bacteria, notably Proteobacteria, also proliferated on the field retted fibres. Extracts from field retted fibres exhibited high glucanase activities, while extracts from P. radiata Cel 26 retted fibres showed high polygalacturonase and laccase activities. As a result, fungal retting gave a significantly higher glucan content in the fibres than field retting (77 vs. 67%) and caused a higher removal of pectin as indicated by lower galacturonan content of fibres (1.6%) after fibres were retted for 20 days with P. radiata Cel 26 compared to a galacturonan content of 3.6% for field retted fibres. Effective fibre stiffness increased slightly after retting with P. radiata Cel 26 from 65 to 67 GPa, while it decreased after field retting to 52 GPa. Effective fibre strength could not be determined similarly due to variations in fibre fracture strain and fibre-matrix adhesion. A maximum composite strength with 50 vol% fibres of 307 MPa was obtained using P. radiata Cel 26 compared to 248 MPa with field retting.
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| 6. |
- García-Hidalgo, Javier, et al.
(författare)
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Identification of the two-component guaiacol demethylase system from Rhodococcus rhodochrous and expression in Pseudomonas putida EM42 for guaiacol assimilation
- 2019
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- A diversity of softwood lignin depolymerization processes yield guaiacol as the main low molecular weight product. This key aromatic compound can be utilized as a carbon source by several microbial species, most of which are Gram positive bacteria. Microbial degradation of guaiacol is known to proceed initially via demethylation to catechol, and this reaction is catalyzed by cytochrome P450 monooxygenases. These enzymes typically require a set of redox partner proteins, whose number and identities were not described until very recently in the case of guaiacol. In this work we identified two proteins involved in guaiacol demethylation by the actinomycete Rhodococcus rhodochrous. Additionally, we constructed four different polycistronic operons carrying combinations of putative redox partners of this guaiacol demethylation system in an inducible expression plasmid that was introduced into the Gram negative host Pseudomonas putida EM42, and the guaiacol consumption dynamics of each resulting strain were analyzed. All the polycistronic operons, expressing a cytochrome P450 together with a putative ferredoxin reductase from R. rhodochrous and putative ferredoxins from R. rhodochrous or Amycolatopsis ATCC 39116 enabled P. putida EM42 to metabolize and grow on guaiacol as the sole carbon source.
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| 7. |
- Hernández, Armando, et al.
(författare)
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Impact of the fermentation parameters pH and temperature on stress resilience of Lactobacillus reuteri DSM 17938
- 2019
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- This study was undertaken to investigate the impact of culture pH (4.5–6.5) and temperature (32–37 °C) on the stress resilience of Lactobacillus reuteri DSM 17938 during freeze-drying and post freeze-drying exposure to low pH (pH 2)and bile salts. Response-surface methodology analysis revealed that freeze-drying survival rates Ncells after drying/Ncells before drying*100 were linearly related to pH with the highest survival rate of 80% when cells were cultured at pH 6.5 and the lowest was 40% when cells were cultured at pH 4.5. The analysis further revealed that within the chosen temperature range the culture temperature did not significantly affect the freeze-drying survival rate. However, fermentation at pH 4.5 led to better survival rates when rehydrated cells were exposed to low pH shock or bile salts. Thus, the effect of pH onfreeze-drying survival was in contrast to effects on low pH and bile salts stress tolerance. The rationale behind this irreconcilability is based on the responses being dissimilar and are not tuned to each other. Culturing strain DSM17938 at pH values higher than 5.5 could be a useful option to improve the survivability and increase viable cell numbers in the final freeze-dried product. However, the dissimilar responses for the process- and application parameterstested here suggest that an optimal compromise has to be found in order to obtain the most functional probiotic product possible.
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| 8. |
- Hernandez-Cortes, G., et al.
(författare)
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Improvement on the productivity of continuous tequila fermentation by Saccharomyces cerevisiae of Agave tequilana juice with supplementation of yeast extract and aeration
- 2016
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 6:1
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Tidskriftsartikel (refereegranskat)abstract
- Agave (Agave tequilana Weber var. azul) fermentations are traditionally carried out employing batch systems in the process of tequila manufacturing; nevertheless, continuous cultures could be an attractive technological alternative to increase productivity and efficiency of sugar to ethanol conversion. However, agave juice (used as a culture medium) has nutritional deficiencies that limit the implementation of yeast continuous fermentations, resulting in high residual sugars and low fermentative rates. In this work, fermentations of agave juice using Saccharomyces cerevisiae were put into operation to prove the necessity of supplementing yeast extract, in order to alleviate nutritional deficiencies of agave juice. Furthermore, continuous fermentations were performed at two different aeration flow rates, and feeding sterilized and non-sterilized media. The obtained fermented musts were subsequently distilled to obtain tequila and the preference level was compared against two commercial tequilas, according to a sensorial analysis. The supplementation of agave juice with air and yeast extract augmented the fermentative capacity of S. cerevisiae S1 and the ethanol productivities, compared to those continuous fermentations non supplemented. In fact, aeration improved ethanol production from 37 to 40 g L-1, reducing sugars consumption from 73 to 88 g L-1 and ethanol productivity from 3.0 to 3.2 g (Lh)(-1), for non-aerated and aerated (at 0.02 vvm) cultures, respectively. Supplementation of yeast extract allowed an increase in specific growth rate and dilution rates (0.12 h(-1), compared to 0.08 h(-1) of non-supplemented cultures), ethanol production (47 g L-1), reducing sugars consumption (93 g L-1) and ethanol productivity [5.6 g (Lh)(-1)] were reached. Additionally, the effect of feeding sterilized or non-sterilized medium to the continuous cultures was compared, finding no significant differences between both types of cultures. The overall effect of adding yeast extract and air to the continuous fermentations resulted in 88 % increase in ethanol productivity. For all cultures, pH was not controlled, reaching low pH values (from 2.6 to 3). This feature suggested a reduced probability of contamination for prolonged continuous cultures and explained why no significant differences were found between continuous cultures fed with sterilized or non-sterilized media. Concentrations of volatile compounds quantified in the distillates (tequila) were in the allowed ranges established by the Mexican regulation of tequila (NOM-006-SCFI-2012, Norma Oficial Mexicana: Bebidas alcoholicas-Tequila-specificaciones, 2012). The preference level of the distillates was similar to that of two well-known commercial tequilas. The results suggested the possibility of implementing this innovative technology on an industrial scale, attaining high productivities and using non-sterilized agave juice.
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| 9. |
- Hidayat, B.J., et al.
(författare)
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The binding of cellulase variants to dislocations: a semi-quantitative analysis based on CLSM (confocal laser scanning microscopy) images
- 2015
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 5:1, s. 1-14
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Tidskriftsartikel (refereegranskat)abstract
- © 2015, Hidayat et al. Binding of enzymes to the substrate is the first step in enzymatic hydrolysis of lignocellulose, a key process within biorefining. During this process elongated plant cells such as fibers and tracheids have been found to break into segments at irregular cell wall regions known as dislocations or slip planes. Here we study whether cellulases bind to dislocations to a higher extent than to the surrounding cell wall. The binding of fluorescently labelled cellobiohydrolases and endoglucanases to filter paper fibers was investigated using confocal laser scanning microscopy and a ratiometric method was developed to assess and quantify the abundance of the binding of cellulases to dislocations as compared to the surrounding cell wall. Only Humicola insolens EGV was found to have stronger binding preference to dislocations than to the surrounding cell wall, while no difference in binding affinity was seen for any of the other cellulose variants included in the study (H. insolens EGV variants, Trichoderma reesei CBHI, CBHII and EGII). This result favours the hypothesis that fibers break at dislocations during the initial phase of hydrolysis mostly due to mechanical failure rather than as a result of faster degradation at these locations.
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| 10. |
- Kaczmarzyk, Danuta, et al.
(författare)
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Arabidopsis acyl-acyl carrier protein synthetase AAE15 with medium chain fatty acid specificity is functional in cyanobacteria
- 2016
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Ingår i: AMB Express. - : BioMed Central (BMC). - 2191-0855. ; 6:1, s. 1-9
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Tidskriftsartikel (refereegranskat)abstract
- Cyanobacteria are potential hosts for the biosynthesis of oleochemical compounds. The metabolic precursors for such compounds are fatty acids and their derivatives, which require chemical activation to become substrates in further conversion steps. We characterized the acyl activating enzyme AAE15 of Arabidopsis encoded by At4g14070, which is a homologue of a cyanobacterial acyl-ACP synthetase (AAS). We expressed AAE15 in insect cells and demonstrated its AAS activity with medium chain fatty acid (C10-C14) substrates in vitro. Furthermore, we used AAE15 to complement a Synechocystis aas deletion mutant and showed that the new strain preferentially incorporates supplied medium chain fatty acids into internal lipid molecules. Based on this data we propose that AAE15 can be utilized in metabolic engineering strategies for cyanobacteria that aim to produce compounds based on medium chain fatty acids.
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