De novo sequencing, assembly and analysis of the genome of the laboratory strain Saccharomyces cerevisiae CEN.PK113-7D, a model for modern industrial biotechnology
Nijkamp, J.F. ; Broek, M. van den; Datema, E. ; Kok, S. de; Bosman, L. ; Luttik, M.A. ; Daran-Lapujade, P. ; Vongsangnak, W. ; Nielsen, J. ; Heijne, W.H.M. ; Klaassen, P. ; Paddon, C.J. ; Platt, D. ; Kotter, P. ; Ham, R.C.H.J. van; Reinders, M.J.T. ; Pronk, J.T. ; Ridder, D. de; Daran, J.M. - \ 2012
Microbial Cell Factories 11 (2012). - ISSN 1475-2859
l-arabinose - alcoholic fermentation - biotin-prototrophy - chemostat cultures - gene prediction - yeast genome - glucose - evolutionary - protein - xylose
Saccharomyces cerevisiae CEN.PK 113-7D is widely used for metabolic engineering and systems biology research in industry and academia. We sequenced, assembled, annotated and analyzed its genome. Single-nucleotide variations (SNV), insertions/deletions (indels) and differences in genome organization compared to the reference strain S. cerevisiae S288C were analyzed. In addition to a few large deletions and duplications, nearly 3000 indels were identified in the CEN.PK113-7D genome relative to S288C. These differences were overrepresented in genes whose functions are related to transcriptional regulation and chromatin remodelling. Some of these variations were caused by unstable tandem repeats, suggesting an innate evolvability of the corresponding genes. Besides a previously characterized mutation in adenylate cyclase, the CEN. PK113-7D genome sequence revealed a significant enrichment of non-synonymous mutations in genes encoding for components of the cAMP signalling pathway. Some phenotypic characteristics of the CEN. PK113-7D strains were explained by the presence of additional specific metabolic genes relative to S288C. In particular, the presence of the BIO1 and BIO6 genes correlated with a biotin prototrophy of CEN. PK113-7D. Furthermore, the copy number, chromosomal location and sequences of the MAL loci were resolved. The assembled sequence reveals that CEN. PK113-7D has a mosaic genome that combines characteristics of laboratory strains and wild-industrial strains.
Toward pectin fermentation by Saccharomyces cerevisiae: Expression of the first two steps of a bacterial pathway for d-galacturonate metabolism.
Huisjes, E.H. ; Luttik, M.A. ; Almering, M.J. ; Bisschops, M.M. ; Dang, D.H. ; Kleerebezem, M. ; Siezen, R.J. ; Maris, A.J. van; Pronk, J.T. - \ 2012
Journal of Biotechnology 162 (2012)2-3. - ISSN 0168-1656 - p. 303 - 310.
uronic acid metabolism - limited chemostat cultures - neighbor-joining method - mold hypocrea-jecorina - xylose isomerase gene - d-altronic acid - escherichia-coli - l-arabinose - alcoholic fermentation - shuttle vectors
Saccharomyces cerevisiae cannot metabolize d-galacturonate, an important monomer of pectin. Use of S. cerevisiae for production of ethanol or other compounds of interest from pectin-rich feedstocks therefore requires introduction of a heterologous pathway for d-galacturonate metabolism. Bacterial d-galacturonate pathways involve d-galacturonate isomerase, d-tagaturonate reductase and three additional enzymes. This study focuses on functional expression of bacterial d-galacturonate isomerases in S. cerevisiae. After demonstrating high-level functional expression of a d-tagaturonate reductase gene (uxaB from Lactococcus lactis), the resulting yeast strain was used to screen for functional expression of six codon-optimized bacterial d-galacturonate isomerase (uxaC) genes. The L. lactis uxaC gene stood out, yielding a tenfold higher enzyme activity than the other uxaC genes. Efficient expression of d-galacturonate isomerase and d-tagaturonate reductase represents an important step toward metabolic engineering of S. cerevisiae for bioethanol production from d-galacturonate. To investigate in vivo activity of the first steps of the d-galacturonate pathway, the L. lactis uxaB and uxaC genes were expressed in a gpd1¿ gpd2¿ S. cerevisiae strain. Although d-tagaturonate reductase could, in principle, provide an alternative means for re-oxidizing cytosolic NADH, addition of d-galacturonate did not restore anaerobic growth, possibly due to absence of a functional d-altronate exporter in S. cerevisiae.
Regulation of pentose catabolic pathway genes of Aspergillus niger
Groot, M.J.L. de; Dool, C. van den; Wosten, H.A.B. ; Levisson, M. ; vanKuyk, P.A. ; Ruijter, G.J.G. ; Vries, R.P. de - \ 2007
Food Technology and Biotechnology 45 (2007)2. - ISSN 1330-9862 - p. 134 - 138.
transcriptional activator xlnr - d-xylose - l-arabinose - alpha-glucuronidase - degrading enzymes - kinase gene - expression - nidulans - polysaccharides - degradation
The aim of this study was to obtain a better understanding of the pentose catabolism in Aspergillus niger and the regulatory systems that affect it. To this end, we have cloned and characterised the genes encoding A. niger L-arabitol dehydrogenase (ladA) and xylitol dehydrogenase (xdhA), and compared the regulation of these genes to other genes of the pentose catabolic pathway. This demonstrated that activation of the pathway depends on two transcriptional regulators, the xylanolytic activator (XlnR) and an unidentified L-arabinose specific regulator (AraR). These two regulators affect those genes of the pentose catabolic pathway that are related to catabolic conversion of their corresponding inducers (D-xylose and L-arabinose, respectively).
Selection and characterisation of a xylitol-derepressed Aspergillus niger mutant that is apparently impaired in xylitol transport
Vondervoort, P.J.I. van de; Groot, M.J.L. de; Ruijter, G.J.G. ; Visser, J. - \ 2006
Applied Microbiology and Biotechnology 73 (2006)4. - ISSN 0175-7598 - p. 881 - 886.
metabolic-control analysis - saccharomyces-cerevisiae - l-arabinose - xylose catabolism - fermentation - xylan - construction - degradation - nidulans - genes
Aspergillus niger is known for its biotechnological applications, such as the use of xylanase enzyme for the degradation of hemicellulose. Depending on culture conditions, several polyols may also be accumulated, such as xylitol during D-xylose oxidation. Also during industrial fermentation of xylose for the production of fuel ethanol by recombinant yeast, xylitol is a by-product. We studied xylitol metabolism by isolating mutants that have impaired xylitol-mediated repression. Genetic and biochemical characterisation revealed that one of these mutants was affected not only in xylitol-mediated carbon repression, but also had impaired xylitol transport
Metabolic control analysis of xylose catabolism in Aspergillus
Prathumpai, W. ; Gabelgaard, J.B. ; Wanchanthuek, P. ; Vondervoort, P.J.I. van de; Groot, M.J.L. de; McIntyre, M. ; Nielsen, J. - \ 2003
Biotechnology Progress 19 (2003). - ISSN 8756-7938 - p. 1136 - 1141.
yeast pichia-stipitis - nad+-xylitol-dehydrogenase - fuel ethanol-production - d-xylulokinase - l-arabinose - purification - niger - fermentation - reductase - polyol
A kinetic model for xylose catabolism in Aspergillus is proposed. From a thermodynamic analysis it was found that the intermediate xylitol will accumulate during xylose catabolism. Use of the kinetic model allowed metabolic control analysis (MCA) of the xylose catabolic pathway to be carried out, and flux control was shown to be dependent on the metabolite levels. Due to thermodynamic constraints, flux control may reside at the first step in the pathway, i.e., at the xylose reductase, even when the intracellular xylitol concentration is high. On the basis of the kinetic analysis, the general dogma specifying that flux control often resides at the step following an intermediate present at high concentrations was, therefore, shown not to hold. The intracellular xylitol concentration was measured in batch cultivations of two different strains of Aspergillus niger and two different strains of Aspergillus nidulans grown on media containing xylose, and a concentration up to 30 mM was found. Applying MCA showed that the first polyol dehydrogenase (XDH) in the catabolic pathway of xylose exerted the main flux control in the two strains of A. nidulans and A. niger NW324, but the flux control was exerted mainly at the first enzyme of the pathway (XR) of A. niger NW 296.