The global transcriptional response of a raw starch-degrading amylolytic Saccharomyces cerevisiae strain to oxygen limitation and genetic modification

The global transcriptional response of a raw starch-degrading amylolytic Saccharomyces cerevisiae strain to oxygen limitation and genetic modification

This study investigates the global transcriptional response of Saccharomyces cerevisiae strain WTPB-G, which was developed by transforming laboratory strain FY23 with the pPB-G plasmid expressing the Bacillus subtilis α-amylase and the Aspergillus awamori glucoamylase as a fusion protein. Genome-wide analysis of transcript levels revealed the cellular mechanisms and the related pathways that were affected by the genetic modification, which conferred the ability of starch utilization to wild-type cells by the introduction of a plasmid-harboring gene encoding the amylolytic activity. Fermentations were carried out in media containing glucose as the only carbon source under aerated and microaerated conditions, and the effect of aeration was also investigated in terms of fermentation properties and transcriptional response. The genome-wide gene expression analysis highlighted that plasmid replication induced cell wall organization and biogenesis, and repressed approximately 20% of the genes involved in ribosome biogenesis and RNA processing. Although oxygen limitation was found to be less effective in the transcriptional changes, a link between oxygen limitation and genes involved in oxidation-reduction, pH reduction, phosphate-containing compounds, and lipid and fatty acid processes was observed.

___

  • Alfenore S, Cameleyre X, Benbadis L, Bideaux C, Uribelarrea JL, Goma G, Molina-Jouve C, Guillouet SE (2004). Aeration strategy: a need for very high ethanol performance in Saccharomyces cerevisiae fed-batch process. Appl Microbiol Biotechnol 63: 537-542.
  • Bai FW, Anderson WA, Moo-Young M (2008). Ethanol fermentation technologies from sugar and starch feedstocks. Biotechnol Adv 26: 89-105.
  • Balat M, Balat H, Öz C (2008). Progress in bioethanol processing. Prog Energy Combust Sci 34: 551-573.
  • Birol G, Önsan Zİ, Kırdar B, Oliver SG (1998). Ethanol production and fermentation characteristics of Recombinant Saccharomyces cerevisiae strains grown on starch. Enzyme Microb Technol 22: 672-677.
  • Bolstad BM, Irizarry R, Astrand M, Speed TP (2003). A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19: 185-193.
  • Bothast RJ, Schlicher MA (2005). Biotechnological processes for conversion of corn into ethanol. Appl Microbiol Biotechnol 67: 19-25.
  • Boyle EI, Weng S, Gollub J, Jin H, Botstein D, Cherry JM, Sherlock G (2004). GO::TermFinder-open source software for accessing Gene Ontology information and finding significantly enriched Gene Ontology terms associated with a list of genes. Bioinformatics 20: 3710-3715.
  • Cankorur-Cetinkaya A, Eraslan S, Kirdar B (2013). Transcriptional remodelling in response to changing copper levels in the Wilson and Menkes disease model of Saccharomyces cerevisiae. Mol Biosyst 9: 2889-2908.
  • Choi GW, Kang HW, Moon, SK (2009). Repeated-batch fermentation using flocculent hybrid, Saccharomyces cerevisiae CHFY0321 for efficient production of bioethanol. Appl Microbiol Biotechnol 84: 261-269.
  • Costenoble R, Valadi H, Gustafsson L, Niklasson C, Franzén CJ (2000). Microaerobic glycerol formation in Saccharomyces cerevisiae. Yeast 16: 1483-1495.
  • Cvijovic M, Olivares-Hernández R, Agren R, Dahr N, Vongsangnak W, Nookaew I, Patil KR, Nielsen J (2010). BioMet Toolbox: genome-wide analysis of metabolism. Nucleic Acids Res 38: 144-149.
  • De Moraes L, Astolfi-Filho S, Oliver S (1995). Development of yeast strains for the efficient utilisation of starch: evaluation of constructs that express alpha-amylase and glucoamylase separately or as bifunctional fusion proteins. Appl Microbiol Biotechnol 43: 1067-1076.
  • Elkins JG, Raman B, Keller M (2010). Engineered microbial systems for enhanced conversion of lignocellulosic biomass. Curr Opin Biotechnol 21: 657-662.
  • Favaro L, Basaglia M, Saayman M, Rose SH, van Zyl WH, Casello S (2010). Engineering amylolytic yeasts for industrial bioethanol production. Chem Eng Transact 20: 97-102.
  • Gautier L, Cope L, Bolstad BM, Irizarry R (2004). affy-analysis of Affymetrix GeneChip data at the probe level. Bioinformatics 20: 307-315.
  • Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J et al. (2004). Bioconductor: Open software development for computational biology and bioinformatics. Genome Biol 5: R80.
  • Goyal HB, Seal D, Saxena RC (2008). Bio-fuels from thermochemical conversion of renewable resources: a review. Renew Sust Energ Rev 12: 504-517.
  • Gray KA, Zhao L, Emptage M (2006). Bioethanol. Curr Opin Chem Biol 10: 141-146.
  • Guimarães PMR, Teixeira JA, Domingues L (2008). Fermentation of high concentrations of lactose to ethanol by engineered flocculent Saccharomyces cerevisiae. Biotechnol Lett 30: 1953-1958.
  • Heux S, Cachon R, Dequin S (2006). Cofactor engineering in Saccharomyces cerevisiae: expression of a H2O-forming NADH oxidase and impact on redox metabolism. Metab Eng 8: 303-314.
  • Inderwildi OR, King D (2009). Quo vadis biofuels? Energ Environ Sci 2: 343-346.
  • Karim AS, Curran KA, Alper HS (2013). Characterization of plasmid burden and copy number in Saccharomyces cerevisiae for optimization of metabolic engineering applications. FEMS Yeast Res 13: 107-116.
  • Kasavi C, Eraslan S, Arga K, Toksoy Oner E, Kirdar B (2014). A system based network approach to ethanol tolerance in Saccharomyces cerevisiae. BMC Syst Biol 8: 90.
  • Kim JH, Kim HR, Lim MH, Ko HM, Chin JE, Lee HB, Kim C, Bai S (2010). Construction of a direct starch-fermenting industrial strain of Saccharomyces cerevisiae producing glucoamylase, alpha-amylase and debranching enzyme. Biotechnol Lett 32: 713-719.
  • Li Z, Lee I, Moradi E, Hung NJ, Johnson AW, Marcotte EM (2009). Rational extension of the ribosome biogenesis pathway using network-guided genetics. PLoS Biol 7: e1000213
  • Oliveira AP, Patil KR, Nielsen J (2008). Architecture of transcriptional regulatory circuits is knitted over the topology of bio-molecular interaction networks. BMC Syst Biol 2: 17.
  • Ow DSW, Nissom PM, Philp R, Oh SKW, Yap MGS (2006). Global transcriptional analysis of metabolic burden due to plasmid maintenance in Escherichia coli DH5α during batch fermentation. Enzyme Microb Technol 39: 391-398.
  • Patil KR, Nielsen J (2005). Uncovering transcriptional regulation of metabolism by using metabolic network topology. P Natl Acad Sci USA 102: 2685-2689.
  • Saeed AI, Bhagabati NK, Braisted JC, Liang W, Sharov V, Howe EA, Li J, Thiagarajan M, White JA, Quackenbush J (2006). TM4 microarray software suite. Method Enzymol 411: 134-193.
  • Sánchez OJ, Cardona C (2008). Trends in biotechnological production of fuel ethanol from different feedstocks. Bioresour Technol 99: 5270-5295.
  • Sanchez-Gonzalez Y, Cameleyre X, Molina-Jouve C, Goma G, Alfenore S (2009). Dynamic microbial response under ethanol stress to monitor Saccharomyces cerevisiae activity in different initial physiological states. Bioprocess Biosys Eng 32: 459-466.
  • Shen Y, Zhang Y, Ma T, Bao X, Du F, Zhuang G, Qu Y (2008). Simultaneous saccharification and fermentation of acid-pretreated corncobs with a recombinant Saccharomyces cerevisiae expressing beta-glucosidase. Bioresour Technol 99: 5099-5103.
  • Teixeira MC, Monteiro PT, Guerreiro JF, Gonçalves JP, Mira NP, Dos Santos SC, Cabrito TR, Palma M, Costa C, Francisco AP et al. (2013). The YEASTRACT database: an upgraded information system for the analysis of gene and genomic transcription regulation in Saccharomyces cerevisiae. Nucleic Acids Res 42: D161-166.
  • Toksoy Öner E (2006). Optimization of ethanol production from starch by an amylolytic nuclear petite Saccharomyces cerevisiae strain. Yeast 23: 849-856.
  • Toksoy Öner E, Oliver SG, Kırdar B (2005). Production of ethanol from starch by respiration-deficient recombinant Saccharomyces cerevisiae. Appl Environ Microbiol 71: 6443-6445.
  • Wong DWS, Robertson GH, Lee CC, Wagschal K (2007). Synergistic action of recombinant α-amylase and glucoamylase on the hydrolysis of starch granules. Protein J 26: 159-164.
  • Yamada R, Bito Y, Adachi T, Tanaka T, Ogino C, Fukuda H, Kondo A (2009). Efficient production of ethanol from raw starch by a mated diploid Saccharomyces cerevisiae with integrated α-amylase and glucoamylase genes. Enzyme Microb Technol 44: 344-349.
  • Yamada R, Yamakawa SI, Tanaka T, Ogino C, Fukuda H, Kondo A (2011). Direct and efficient ethanol production from high-yielding rice using a Saccharomyces cerevisiae strain that express amylases. Enzyme Microb Technol 48: 393-396.