Papers by Bjorn Johansson
Scientific Reports, 2021
Carbohydrate rich substrates such as lignocellulosic hydrolysates remain one of the primary sourc... more Carbohydrate rich substrates such as lignocellulosic hydrolysates remain one of the primary sources of potentially renewable fuel and bulk chemicals. The pentose sugard-xylose is often present in significant amounts along with hexoses.Saccharomyces cerevisiaecan acquire the ability to metabolized-xylose through expression of heterologousd-xylose isomerase (XI). This enzyme is notoriously difficult to express inS. cerevisiaeand only fourteen XIs have been reported to be active so far. We cloned a newd-xylose isomerase derived from microorganisms in the gut of the wood-feeding beetleOdontotaenius disjunctus. Although somewhat homologous to the XI fromPiromycessp. E2, the new gene was identified as bacterial in origin and the host as aParabacteroidessp. Expression of the new XI inS. cerevisiaeresulted in faster aerobic growth than the XI fromPiromycesond-xylose media. Thed-xylose isomerization rate conferred by the new XI was also 72% higher, while absolute xylitol production was ident...
FEMS yeast research, 2018
Lignocellulosic hydrolysates remain one of the most abundantly used substrates for the sustainabl... more Lignocellulosic hydrolysates remain one of the most abundantly used substrates for the sustainable production of second generation fuels and chemicals with Saccharomyces cerevisiae. Nevertheless, fermentation inhibitors such as acetic acid, furfural and hydroxymethylfurfural are formed during the process and can lead to slow or stuck fermentations and/or act as genotoxic agents leading to production strain genetic instability. We have developed a novel dominant deletion (DEL) cassette assay for quantification of DNA damage in both wild-type and industrial yeast strains. Using this assay, the ethanol production strain S. cerevisiae PE-2 was shown to be more resistant to hydrogen peroxide and furfural than the laboratory DEL strain RS112. Indeed, the PE-2 strain also showed a lower tendency for recombination, consistent with a more efficient DNA protection. The dominant DEL assay presented herein should prove to be a useful tool in the selection of robust yeast strains and process con...
Enzyme and microbial technology, 2017
A prerequisite to the use of any enzyme in any industrial process is an understanding of its acti... more A prerequisite to the use of any enzyme in any industrial process is an understanding of its activity and stability under process conditions. Glycoside hydrolase family 8 enzymes include many important biotechnological biocatalysts yet little is known of the performance of these with respect to pH. A better understanding of this parameter and its relationship to structure and function in these enzymes will allow for an improved use of these in industry as well as an enhanced ability in their engineering and optimisation for a particular application. An in-depth analysis of the pH induced changes in activity, irreversible inactivation, conformation, stability and solubility of a commercial glycoside hydrolase family 8 xylanase was carried out with the aim of identifying the factors determining the pH dependence of this enzyme. Our study showed that different phenomena play different roles at the various pHs examined. Both reversible and irreversible processes are involved at acidic p...
Food Technology and Biotechnology, 2016
The eff ect of xanthohumol, a prenylfl avonoid isolated from the hop plant (Humulus lupulus L.), ... more The eff ect of xanthohumol, a prenylfl avonoid isolated from the hop plant (Humulus lupulus L.), on Saccharomyces cerevisiae DNA oxidative damage and viability was evaluated. Yeast cultures under oxidative stress, induced by H 2 O 2 , displayed stronger growth in the presence of 5 mg/L of xanthohumol than cultures with only H 2 O 2. Likewise, DNA damage assessed by the comet assay was signifi cantly lower in cells co-incubated with xanthohumol and H 2 O 2. Accordingly, fl uorescence of dichlorofl uorescein in cells treated with H 2 O 2 and xanthohumol was considerably lower than in cells exclusively treated with H 2 O 2 , indicative of a reactive oxygen species scavenging mechanism and consequent formation of oxidation products, as detected by mass spectrometry. However, at concentrations above 5 mg/L, xanthohumol elicited an opposite eff ect, leading to a slower growth rate and significant increase in DNA damage. A yeast yap1 deletion mutant strain sensitive to oxidative stress grew more slowly in the presence of at least 5 mg/L of xanthohumol than cultures of the wild type, suggesting that xanthohumol toxicity is mediated by oxidative stress. This evidence provides further insight into the impact of xanthohumol on yeast cells, supporting dose-dependent antioxidant/antigenotoxic and prooxidant/genotoxic eff ects.
FEMS Yeast Research, 2015
With the widespread use of blue LED for the treatment of human infections such as oropharyngeal c... more With the widespread use of blue LED for the treatment of human infections such as oropharyngeal candidiasis, concerns about the safety of this technology in photodynamic therapy have been raised.
Methods in molecular biology (Clifton, N.J.), 2004
Multiple gene expression can be introduced in a yeast strain with using only two markers by means... more Multiple gene expression can be introduced in a yeast strain with using only two markers by means of the two new vectors described, the expression vector pB3 PGK and the CRE recombinase vector pCRE3. The pB3 PGK has a zeocin-selectable marker flanked by loxP sequences and an expression cassette consisting of the strong PGK1 promoter and the GCY1 terminator. The gene of interest (YFG1) is cloned between the promoter and terminator of pB3 PGK. The pB3 PGK-YFG1 is integrated into the genome by a single restriction cut within the YFG1 gene and integrated in the YFG1 locus. The strain is further transformed with the pCRE3 vector. The CRE recombinase expressed from this vector removes the zeocin marker and makes it possible to use the pB3 PGK vector over again in the same strain after curing of the pCRE3 vector. The 2 micro -based pCRE3 carries the aureobasidin A, zeocin and URA3 markers. pCRE3 is easily cured by growth in nonselective medium without active counterselection. The screening...
Molecular Microbiology, 2005
During the last years, several reports described an apoptosis-like programmed cell death process ... more During the last years, several reports described an apoptosis-like programmed cell death process in yeast in response to different environmental aggressions. Here, evidence is presented that hyperosmotic stress induces in Saccharomyces cerevisiae a cell death process accompanied by morphological and biochemical indicators of apoptotic programmed cell death, namely chromatin condensation along the nuclear envelope, mitochondrial swelling and reduction of cristae number, production of reactive oxygen species and DNA strand breaks, with maintenance of plasma membrane integrity. Disruption of AIF1 had no effect on cell survival, but lack of Yca1p drastically reduced metacaspase activation and decreased cell death indicating that this death process was associated to activation of this protease. Supporting the involvement of mitochondria and cytochrome c in caspase activation, the mutant strains cyc1∆ cyc7∆ and cyc3∆, both lacking mature cytochrome c, displayed a decrease in caspase activation associated to increased cell survival when exposed to hyperosmotic stress. These findings indicate that hyperosmotic stress triggers S. cerevisiae into an apoptosis-like programmed cell death that is mediated by a caspase-dependent mitochondrial pathway partially dependent on cytochrome c.
Yeast, 2003
Disruption of the ZWF1 gene encoding glucose-6-phosphate dehydrogenase (G6PDH) has been shown to ... more Disruption of the ZWF1 gene encoding glucose-6-phosphate dehydrogenase (G6PDH) has been shown to reduce the xylitol yield and the xylose consumption in the xylose-utilizing recombinant Saccharomyces cerevisiae strain TMB3255. In the present investigation we have studied the influence of different production levels of G6PDH on xylose fermentation. We used a synthetic promoter library and the copper-regulated CUP1 promoter to generate G6PDH-activities between 0% and 179% of the wildtype level. G6PDH-activities of 1% and 6% of the wild-type level resulted in 2.8and 5.1-fold increase in specific xylose consumption, respectively, compared with the ZWF1-disrupted strain. Both strains exhibited decreased xylitol yields (0.13 and 0.19 g/g xylose) and enhanced ethanol yields (0.36 and 0.34 g/g xylose) compared with the control strain TMB3001 (0.29 g xylitol/g xylose, 0.31 g ethanol/g xylose). Cytoplasmic transhydrogenase (TH) from Azotobacter vinelandii has previously been shown to transfer NADPH and NAD + into NADP + and NADH, and THoverproduction resulted in lower xylitol yield and enhanced glycerol yield during xylose utilization. Strains with low G6PDH-activity grew slower in a lignocellulose hydrolysate than the strain with wild-type G6PDH-activity, which suggested that the availability of intracellular NADPH correlated with tolerance towards lignocellulosederived inhibitors. Low G6PDH-activity strains were also more sensitive to H2 O2 than the control strain TMB3001.
Yeast, 2010
Chromosomal DNA damage can be a result of several processes and agents of endogenous or exogenous... more Chromosomal DNA damage can be a result of several processes and agents of endogenous or exogenous origin. These cause strand breaks or oxidized bases that lead to strand breaks, which relax the normally supercoiled genomic DNA and increase its electrophoretic mobility. The extent of DNA damage can be assessed by single cell gel electrophoresis, where the chromosomal DNA migration distance correlates with the extent of DNA damage. This technique has been used for a variety of applications with several organisms, but only a few studies have been reported for Saccharomyces cerevisiae. A possible reason for this absence is that low cellular DNA content could hamper visualization. Here we report an optimization of the comet assay protocol for yeast cells that is robust and sensitive enough to reproducibly detect background DNA damage and oxidative damage caused by hydrogen peroxide. DNA repair was observed and quantified as diminishing comet tail length with time after oxidative stress removal in a process well described by first order kinetics with a tail length half life of 11 minutes at 37ºC. This is to our knowledge the first quantitative measurement of DNA repair kinetics in S. cerevisiae by this method. We also show that diet antioxidants protect from DNA damage as shown by a threefold decrease in comet tail length. The possibility of assessment of DNA damage and repair in individual cells applied to the model organism S. cerevisiae creates new perspectives for studying genotoxicity and DNA repair.
Molecular Biology of the Cell, 2006
Saccharomyces cerevisiae has been reported to die, under certain conditions, from programmed cell... more Saccharomyces cerevisiae has been reported to die, under certain conditions, from programmed cell death with apoptotic markers. One of the most important markers is chromosomal DNA fragmentation as indicated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. We found TUNEL staining in S. cerevisiae to be a consequence of both single- and double-strand DNA breaks, whereas in situ ligation specifically stained double-strand DNA breaks. Cells treated with hydrogen peroxide or acetic acid staining positively for TUNEL assay stained negatively for in situ ligation, indicating that DNA damage in both cases mainly consists of single-strand DNA breaks. Pulsed field gel electrophoresis of chromosomal DNA from cells dying from hydrogen peroxide, acetic acid, or hyperosmotic shock revealed DNA breakdown into fragments of several hundred kilobases, consistent with the higher order chromatin degradation preceding DNA laddering in apoptotic mammalian cells. DNA frag...
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2008
Mitochondrial involvement in yeast apoptosis is probably the most unifying feature in the field. ... more Mitochondrial involvement in yeast apoptosis is probably the most unifying feature in the field. Reports proposing a role for mitochondria in yeast apoptosis present evidence ranging from the simple observation of ROS accumulation in the cell to the identification of mitochondrial proteins mediating cell death. Although yeast is unarguably a simple model it reveals an elaborate regulation of the death process involving distinct proteins and most likely different pathways, depending on the insult, growth conditions and cell metabolism. This complexity may be due to the interplay between the death pathways and the major signalling routes in the cell, contributing to a whole integrated response. The elucidation of these pathways in yeast has been a valuable help in understanding the intricate mechanisms of cell death in higher eukaryotes, and of severe human diseases associated with mitochondria-dependent apoptosis. In addition, the absence of obvious orthologues of mammalian apoptotic regulators, namely of the Bcl-2 family, favours the use of yeast to assess the function of such proteins. In conclusion, yeast with its distinctive ability to survive without respirationcompetent mitochondria is a powerful model to study the involvement of mitochondria and mitochondria interacting proteins in cell death.
Generation of gene disruption cassettes with long flanking regions via fusion PCR. Andreas Karoly... more Generation of gene disruption cassettes with long flanking regions via fusion PCR. Andreas Karoly Gombert (1), Björn Johansson (2), Nelson Lima (3), José Teixeira (3), Lucília Domingues (3)
Pre-incubation with GBE protects yeast cells from H O-induced DNA damage Yeast spheroplasts (BY47... more Pre-incubation with GBE protects yeast cells from H O-induced DNA damage Yeast spheroplasts (BY4741 strain) were incubated with GBE (diluted 2, 4, 20 or 100 fold) for 20min, washed, and subsequently incubated with 5mM H O for 20min. Spheroplasts were washed and analyzed by the comet assay.
Production of renewable liquid biofuels that can substitute fossil fuel, has emerged as a major c... more Production of renewable liquid biofuels that can substitute fossil fuel, has emerged as a major challenge for applied biology. Biodiesel, in the form of fatty acid esters, produced by oleaginous microorganisms could be an attractive alternative, since the utilization of diesel fuel is more efficient than for example ethanol. Oleaginous yeasts may accumulate very high (60%) levels of intracellular lipids but two drawbacks are the relatively complicated extraction process and the subsequent transesterification with the accompanying glycerol by-product formation. The objective of this work is to apply metabolic engineering of fatty acid synthesis and secretion in the model yeast S. cerevisiae in order to create a microorganism able to produce and secrete free fatty acids or fatty acid esters. S. cerevisiae is a proper model, since lipid metabolism has been studied extensively and all genes encoding enzymes directly involved in lipid synthesis are known. This organism has also been reported to acquire oleaginous properties by no more than three genetic modifications(1). In the yeast S. cerevisiae, activation of exogenous long-chain fatty acids to coenzyme A derivatives, prior to metabolic utilization, is mediated by the fatty acyl-CoA synthetases Faa1p and Faa4p. It has been shown that free fatty acids are secreted from a FAA1,4 double mutant (2). This modification will be combined with modifications of core fatty acid elongation, such as overexpression of acetyl-CoA synthetase (Acs1p), in an attempt to improve fatty acid production rate. Essential for this work is to facilitate a biological platform for efficient fatty acid or lipid production. In this work, the "delitto perfetto" method (3) was applied to delete these two fatty acyl-CoA synthetases generating genetically clean strains without markers or bacterial DNA. Results show that this technique can be used to generate multiple knockouts by recycling the marker gene.
FEMS Yeast Research, 2003
Recombinant Saccharomyces cerevisiae TMB3001, harboring the Pichia stipitis genes XYL1 and XYL2 (... more Recombinant Saccharomyces cerevisiae TMB3001, harboring the Pichia stipitis genes XYL1 and XYL2 (xylose reductase and xylitol dehydrogenase, respectively) and the endogenous XKS1(xylulokinase), can convert xylose to ethanol. About 30% of the consumed xylose, however, is excreted as xylitol. Enhanced ethanol yield has previously been achieved by disrupting the ZWF1 gene, encoding glucose-6phosphate dehydrogenase, but at the expense of the xylose consumption. This is probably the result of reduced NADPH-mediated xylose reduction. In the present study, we increased the xylose reductase (XR) activity 4^19 times in both TMB3001 and the ZWF1-disrupted strain TMB3255. The xylose consumption rate increased by 70% in TMB3001 under oxygen-limited conditions. In the ZWF1-disrupted background, the increase in XR activity fully restored the xylose consumption rate. Maximal specific growth rates on glucose were lower in the ZWF1-disrupted strains, and the increased XR activity also negatively affected the growth rate in these strains. Addition of methionine resulted in 70% and 50% enhanced maximal specific growth rates for TMB3255 (zwf1v) and TMB3261 (PGK1-XYL1, zwf1v), respectively. Enhanced XR activity did not have any negative effect on the maximal specific growth rate in the control strain. Enhanced glycerol yields were observed in the high-XR-activity strains. These are suggested to result from the observed reductase activity of the purified XR for dihydroxyacetone phosphate.
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Papers by Bjorn Johansson