6. Yogurt mission extra lecture

Which bacteria are used as starter cutures

Lactic acid bacteria

Role of lactic acid bacteria in starter cultures for milk/yogurt

Ferment milk into product

Role of lactic acid in food preservation for starter cultures

acid production during fermentation helps preserve food by lowering the pH, inhibiting pathogens

How would you genetically engineer a new L. lactis strain for better starter cultures

Use transduction to transfer your GOI from a different GRAS L. lactis strain

Why is it best to use transduction for this project

Using a natural strategy with a GRAS strain MAY result in the generated strain also qualifying for GRAS status!

Natural strategies for genetic engineering

HGT (within-species only)
 Random mutagenesis
 Directed evolution
 Dominant selection

Natural engineering: Random mutagenesis

Introduce random mutations and hope that the desired property is easily screenable

Natural engineering: directed evolution

a strain is slowly adapted to certain growth conditions but off-site mutations can occur

Natural engineering: dominant selection

designing a selection scheme in which only strains
with the desired property can grow

One of the most important challeges in dairy fermentations

Preventing phage attack on starter cultures

Given the huge burden of phage, why aren’t all bacteria naturally phage-receptor mutants?

there is likely a fitness cost to loss of the receptor(s) (i.e., outcompeted when no phage present)

Phage receptor

can use several cell surface
moieties as receptors, including glycolipids, integral
membrane proteins, or flagella proteins

What would be the best way to determine the identity of a phage receptor?

A dominant selection approach using a transposon mutant library

Bacteriocins: Nisin

positively-charged factors that have antibacterial activity that is added as a food preservative

Which of the answers is a property you would want in a bacteriocin used as a preservative
in the food industry?
(1) alters the taste, smell, and texture of the food
(2) has no lytic activity against foodborne pathogens
(3) has high cost to manufacture
(4) has activity against, and toxicity for, eukaryotic cells
(5) is sensitive to digestive proteases

is sensitive to digestive proteases

How is nisin produced

synthesized as an inactive precursor peptide that contains an N-terminal leader peptide attached to the C-terminal propeptide. These then undergo a
“maturation” process via several enzymes

Q10: Which of the below is NOT a reason why nisin is produced as an inactive precurser?
(1) The leader peptide enhances bacteriocin production as the codons of the leader peptide are rare
(2) The leader peptide keeps the bacteriocin in an inactive form inside the producing cell
(3) The leader peptide directs the bacteriocin for secretion
(4) The leader peptide serves as a binding site for
processing molecules

False: The leader peptide enhances bacteriocin production as the codons of the leader peptide are rare

Nisin biosynthesis gene cluster: NisFEG

provide protection to the producing cell by expelling nisin out of the membrane (which is where killing occurs),

Nisin biosynthesis gene cluster: NisI

binds to nisin for additional protection for the producing cell

Nisin biosynthesis gene cluster: NisI/NisFEG

Genes that give the producing cell immunity and protection as it produces nisin

nisin-controlled gene expression (NICE)

the nisRK genes need to be introduced into a nisin-negative strain, the gene of interest needs to be
placed downstream of the nisA promoter in the same strain, and nisin needs to be added to induce

How could you modify your strain for use during soy fermentation or as a
probiotic?

Induce production of α-galactosidase to break down raffinose and decrease the gas produced from indigestion