Development of Stable Flocculent Saccharomyces cerevisiae Strain for Continuous Aspergillus niger Ga

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Development of Stable Flocculent Saccharomyces
cerevisiae Strain for Continuous Aspergillus
niger ß-Galactosidase Production

• Carla Oliveira, José A. Teixeira, Nelson Lima,
  Nancy A. Da Silva, and Lucília Domingues

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INTRODUCTION

• ß-Galactosidase
• ß-Galactosidase can cleave ß-linked galactose
  residues from various compounds
• Commonly used to cleave lactose
• Widely used in the food and pharmaceutical
  industries

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INTRO

• Plasmid loss in a nonselective medium led to a
  decrease in productivity during continuous
  culture operation
• A continuous high cell-density system leading to
  increased volumetric productivity
• The utilization of flocculating cells provides an
  important contribution to the improvement of
  downstream processing
• Strain stability is needed so that continuous
  operation does not lead to a progressive loss of
  the product
• Integrating a recombinant DNA into the genome

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INTRO

• To increase the number of the integrated copies
  of the desired gene use repetitive sequences ?
• Target sites for homologous recombination
• The d-sequences flank the regions of the Ty1
  retrotransposon of S. cerevisiae
• Allowing multiple gene copy integration
• BIG PROBLEM ?
• Looped out through excisional recombination

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MATERIALS AND METHODS

• Strains and plasmids
• The lacA expression cassette containing the lacA
  gene flanked by the ADH1 promoter and terminator
  was isolated from plasmid pVK1.1

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MATERIALS AND METHODS

• Plasmid pd-neo was used as carrier vector for the
  integration of the lacA expression cassette into
  the yeast chromosomes
• d-integrating vector containing the bacterial neo
  resistant gene for selection against G418

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MATERIALS AND METHODS

• Plasmid construction

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MATERIALS AND METHODS

• Yeast transformation
• By electroporation
• Selection against G418 antibiotic
• To select for ß-galactosidase producing colonies,
  YPD medium was supplemented with 40 µg/ml (X-gal).

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MATERIALS AND METHODS

• ß-galactosidase activity
• Measuring the release of p-nitrophenol from
  p-nitrophenyl-ß-D-galactopyranoside ( pNPG)
• Absorbance was measured at 405 nm

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MATERIALS AND METHODS

• Southern blot analysis
• Integration copy number and patterns of the
  ß-galactosidase expression cassette were analysed
• Total genomic DNA ? digested with XbaI ? agarose
  gel electrophoresis ?nylon membrane ? Cross
  Linking ? Probes were labelled ? measuring the
  relative ratio of intensities of the multicopy
  band to a single-copy band

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RESULTS

• Chromosomal integration of lacA gene
• A linearized pCO1 plasmid was introduced into S.
  cerevisiae
• The number of transformants obtained was largely
  dependent on the G418 concentration added
• Longer incubation after yeast transformation
  prior to G418 selection was extremely important
  for transformant recovery
• The colonies with a deep blue colour, expressing
  higher ß-galactosidase activity, were screened by
  a standard ß-galactosidase activity assay

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RESULTS

• Chromosomal integration of lacA gene

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RESULTS

• Characterization of integrated gene expression
• Linear relationship can
• be observed up to a
• copy number of eight

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RESULTS

• Stability evaluation of recombinant strains
• Eight sequential batch cultures ? Spread on YPD
  plates ? Analysis of G418 and X-gal ? Southern
  blot analysis
• Copy number maintened !!?

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RESULTS

• Continuous-culture experiments
• During the continuous cultures,the percentage of
  cells expressing ß-galactosidase was higher for
  the integrant strain than for the strain carrying
  the plasmid pVK1.1

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RESULTS

• Continuous-culture experiments
• For the plasmid-carrying strain, 60 white
  colonies were observed in the X-gal plates
• For the integrant strain, all colonies were
  counted as ß-galactosidase producing cells

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DISCUSSION

• The use of the d-integration system enable
  increased production of heterologous proteins
• A linear correlation between ß-galactosidase
  activity and integrated copy number
• It indicates that the integration on different
  d-sites does not alter gene expression
• For the integration of multiple gene copies, it
  is extremely important to check for stability as
  the integrated copy number may decrease by gene
  loop-out events

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Conclusion

• construction of S. cerevisiae strains producing
  extracellular A. niger ß-galactosidase at levels
  comparable to the multicopy Yep plasmid system.
• For the continuous high cell-density culture,
  the integrant strain was more stable and
  presented a higher specific ß-galactosidase
  activity than the plasmid-carrying strain

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