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.
Malic acid production by Saccharomyces cerevisiae: engineering of pyruvate carbosylation, oxaloacetate reduction and malate export
Zelle, R.M. ; Hulster, E. de; Winden, W.A. van; Waard, P. de; Dijkema, C. ; Winkler, A.A. ; Geertman, J.M.A. - \ 2008
Applied and Environmental Microbiology 74 (2008)9. - ISSN 0099-2240 - p. 2766 - 2777.
metabolic-flux analysis - aspergillus-flavus - chemostat cultures - alcoholic fermentation - carbon metabolism - escherichia-coli - organic-acids - mdh2 isozyme - yeast - glucose
Malic acid is a potential biomass-derivable "building block" for chemical synthesis. Since wild-type Saccharomyces cerevisiae strains produce only low levels of malate, metabolic engineering is required to achieve efficient malate production with this yeast. A promising pathway for malate production from glucose proceeds via carboxylation of pyruvate, followed by reduction of oxaloacetate to malate. This redox- and ATP-neutral, CO2-fixing pathway has a theoretical maximum yield of 2 mol malate (mol glucose)¿1. A previously engineered glucose-tolerant, C2-independent pyruvate decarboxylase-negative S. cerevisiae strain was used as the platform to evaluate the impact of individual and combined introduction of three genetic modifications: (i) overexpression of the native pyruvate carboxylase encoded by PYC2, (ii) high-level expression of an allele of the MDH3 gene, of which the encoded malate dehydrogenase was retargeted to the cytosol by deletion of the C-terminal peroxisomal targeting sequence, and (iii) functional expression of the Schizosaccharomyces pombe malate transporter gene SpMAE1. While single or double modifications improved malate production, the highest malate yields and titers were obtained with the simultaneous introduction of all three modifications. In glucose-grown batch cultures, the resulting engineered strain produced malate at titers of up to 59 g liter¿1 at a malate yield of 0.42 mol (mol glucose)¿1. Metabolic flux analysis showed that metabolite labeling patterns observed upon nuclear magnetic resonance analyses of cultures grown on 13C-labeled glucose were consistent with the envisaged nonoxidative, fermentative pathway for malate production. The engineered strains still produced substantial amounts of pyruvate, indicating that the pathway efficiency can be further improved