Design
Microorganisms used in fermented foods
1) Yeast fungus
Pyruvate circuit → Carbon dioxide ⇒ Bread
2) Acetic acid bacteria
Alcohol → acetic acid ⇒ vinegar
3) Lactic acid bacteria
Lactose → pyruvic acid → L-lactate ⇒ cheese, milk
Let E. coli also have these functions
1) Function of Yeast to E. coli
Since E. coli produces formic acid in the pyruvate circuit, we introduce the antisense chain of Pyruvate formate-lyase, an enzyme that produces formic acid.
2) Function of Acetobacter to E. coli
Acetic acid production can be achieved by growing the bacteria in a carbon-rich medium. When the carbon source is depleted, acetic acid is used, so by introducing the antisense chain of acetyl-CoA synthetase, which promotes acetic acid to Acetyl-coA, acetic acid can be produced.
3) Function of lactic acid bacteria to E. coli
By introducing the antisense chain of histidinoldehydrogenase, which promotes the conversion of lactate to pyruvate under anaerobic conditions, a high amount of L-lactate can be produced.
By cutting at Pst and Xba, the GFP can be removed from the colony, so if the colony does not turn red, you know that the gene of interest is being expressed.（Figure）
The pBad is only expressed when arabinose is added, so by introducing the gene of interest into the SpeI and PstI openings, we know that it is working on the enzyme we want to control.(Figure)
By cutting with SpeI and XbaI, the entire GFP sequence is removed, so if no green colonies are seen when the cells are exposed to UV light, then the gene of interest is being expressed.(Figure)
The expression of the antisense chain of acetyl-CoA synthetase is confirmed by the expression of RFP and the appearance of red colonies when the medium is rich in glucose and IPTG is applied. When the glucose promoter is activated at low concentrations of glucose and GFP is observed, this confirms the expression of the antisense chain which is incorporated to produce more lactate. The absence of formic acid is confirmed by GCMS.
