Application Focus - Genetic Engineering of Brewer's Yeast with Electroporation
By Michelle M. Ng, Ph. D.

Utilizing electroporation to brew a better beerIn honor of National Beer Day, April 7th, the focus of this blog post is how to use electroporation for genetic engineering of brewing yeast. First, we will provide the ideal electroporation parameters for Saccharomyces cerevisiae using either ECM 630 or Gemini X2 electroporators.  Then, we will highlight an example from the primary literature where this electroporation method was used with brewing yeast Saccharomyces cerevisiae ssp. carlsbergensis to modify the amount of ethanol in beer.

 
Below is a summary of the ideal transformation method for S. cerevisiae, using either BTX ECM 630 or Gemini X2 electroporators.  A transformation efficiency of up to 1 x 108 transformants per ug of plasmid vector DNA may be achieved with this protocol.
  1. Grow Saccharomyces cerevisiae up to log phase in YPD media
  2. Cetrifuge and wash pelleted cells several times to remove cell culture media and suspend cells in electroporation buffer at desired density of 1.6 x 109 cells/ml
  3. Electroporate cells in 2 mm gap cuvettes or 2 mm gap High Throughput electroporation plates with the following conditions:
    • Temperature: 4°C
    • Voltage: 2500 V
    • Capacitance: 25 uF
    • Resistance: 100 to 200 Ω
    • Number of Pulses: 1
    • Desired Electrical Field Strength: 12.5 kV/cm
    • Desired Time Constant: 3 to 4.5 ms
  4. Allow cells to incubate at 30C for 1 hour in recovery media made up of 1:1 1M sorbitol:YPD media.
  5. Proceed to desired selection method.
 
 
Researchers Elke Nevoigt, et al. used electroporation to genetically engineer industrial lager brewing yeast to achieve their goal of reducing the content of alcohol in beer.
Previously, it had been shown that overexpression of the GPD1 gene (encoding the glycerol-3-phosphate dehydrogenase enzyme) could shift fermentation of laboratory yeast strains to produce less ethanol and produce more glycerol. Therefore, to see if this method could be applied to ferment a beer with reduced ethanol content, the team created a plasmid expression construct to overexpress the GPD1 gene in industrial lager brewing yeast Saccharomyces cerevisiae spp. carlsbergensis.  The researchers then electroporated the brewing yeast with the GPD1 expression construct and ran an experimental fermentation under the same conditions as are typically used to produce beer, fermenting brewers' wort.  Following the fermentation, the group measured the products of fermentation, such as ethanol and glycerol, as well as other products of fermentation that contribute to the taste of beer, such as acetaldehyde, acetonin, and diacetyl.  With this method, the researchers successfully reduced the ethanol content in the beer by 18%, and increased the amount of glycerol produced by 5.6 times.
 

Reference:
1. Nevoigt, E., et al. Engineering of brewing yeast to reduce the content of ethanol in beer. FEMS Yeast Research 2002; 2: 225–232.
 

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