K.K Lecture 7&8 Flashcards
Antibiotics
- Sir Alexander Fleming (1928)
- beta-lactams: Penicillins, Cephalosporins
- they are active against gram+ bacteria and target repeating unit of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) in cell wall
- they are natural and semi-synthetic Penicillins
- they are secondary metabolites
What are the factors affecting antibiotic production?
- most antibiotics are produced by fermentation
- antibiotics are mostly secondary metabolites (stationary phase and linked to sporulation)
catabolite repression
- if there is a high amount of glucose, then antibiotic production is decreased
- inhibition of enzyme production
optimise antibiotic production
- add a small amount of glucose or isolate mutant that is not catabolite repressed
The optimal conditions for antibiotic production
(took plates, washed them, isolated them. spores grew in medium containing moss extract)
two-stage fermentation
1. spore - hyphal mass
- submerged culture
- oxygen, optimum temperature, maximum nutrients
- scale up to 101 culture
2. stationary phase - antibiotic production
- slow feed - continuous
- limit glucose which increases antibiotic
- up to 20,000 1 fermentation - 300,000 1 fermentation
10,000 tonnes of penicillin G per annum
Factors affecting Penicillin production
- Nitrogen and phosphorus repression
- high levels of N or PO4 decrease the production of antibiotics - Feed-back inhibition
- antibiotic: can become toxic if there are high levels of intracellular levels
- penicillin: culture of P.chrysogeneum leads to the inhibition of a/b production
- lower intra-cellular a/b levels: add polyene leads to leakage (polyene creates pores in cell membrane and antibiotic which causes leakage but doesn’t kill the fungus. it helps to reduce inhibitory effects of penicillin on fungus)
Optimisation of antibiotic synthesis
- random mutagenesis and selection
- mate a/b producing strains producing hybrids
- target mutagenesis – phage (to disrupt certain genes inside the fungus)
- rational selection - metal chelating agents (EDTA: binds to metal and pulls them out of the solution)
- protoplast fusion (fused cells were stripped of their cell wall to see what grew. aka naked cells)
- cloning (clone gene from one species and introduce it into another)
- produce hybrid antibiotics
- study the genetics of a/b synthesis
Timeline of Penicillin
- Fleming, 1928: P.notatum, 3mg/l
- Florey and Chain, 1938: Penicillin used to treat gram+ bacterial infections
- USA, 1941-1945: P.chrysogenum»_space; P.notatum (30mg/l)
submerged fermentation»_space; surface fermentation - P.chrysogenum mutated strain yield up to 7000mg/l
Production of Penicillin
- Penicillin is the most important antibiotic
- it has low toxicity to humans
- it inhibits peptidoglycan synthesis
- beta-lactams: Penicillin, Cephalosporin, Penems, Carbopenems
Fermentation of Penicillin F,G or V
- today: 20% of V is used, and 80% of g must be modified before using
- they are semi-synthetic penicillins
- why are semi-synthetic penicillins used? to overcome resistance due to bacterial penicillinases
- penicillin G or V use penicillin amylase which modifies ring making it harder for bacteria to breakdown producing 6-Aminopenicillanic acid leading to semi-synthetic penicillin
Other antibiotics
(fungi produce antibiotics to protect themselves to survive, it is not for us)
- Griseofulvin
- chlorine-containing compound (fungus-producing anti-fungal)
- produced by Penicillium griseofulvin
- active against dermatophytes
- fermentation: 24 degrees, pH 5.5-6.5, aerobic, 2-25 days - Fusidic acid (fucidin)
- steroid derived
- produced by Fusidium coccineum (good for bacterial infections on the skin)
Production of gliotoxin by Aspergillus fumigatus
- gliotoxin is a mycotoxin but is also involved in redox balance in fungus (it is good at knocking out aspects of the immune system. it is too powerful, so it is discontinued)
- it displays immunosuppressive properties in vivo - suppresses local and systematic immune response
- gliotoxin inhibits activity of neutrophils
- evaluated as an immunosuppressive agent prior to transplantation
Neutrophil
activated neutrophil
- 1/3 of killing activity is due to superoxide production, and 2/3 is due to degranulation occurring, which is why it’s more important
- gliotoxin blocks these processes, which allows it to kill aspects of the immune system
- amoeba and neutrophils have the same evolutionary origin
- amoeba are natural predators of Aspergillus. Inb an environment, they produce toxins to defend themselves
Genetic manipulation
- improved carbohydrate utilisation
- increased ethanol, temp tolerance (some way to build up resistance to ethanol)
- reduce growth yield
- flocculence changes (make them more likely to stick together, more flocculate)
- altered synthesis of organoleptic (so you don’t have to get an odd-tasting flavour)
- inhibition of contamination (keeps bacteria from colonising and growing)
- novel by-products
- low ethanol beers
- post-fermentation products
Types of genetic manipulation
- mutagenesis
- rare mating (used un brewing industry)
- protoplast fusion
- transformation
Mutagenesis
- creating some sort of mutation
group of mutations: - spontaneous: single nucleotide change (occur naturally)
- chemical: single nucleotide change
- UV: errors/damages in DNA replication
- ionising radiation: deletions, translocations. it’s the most important as it causes gross changes/ huge damages in DNA but also causes mutants of interest
Why do we use mutagenesis?
- to increase resistance to a toxin
- to enhance the production of a substance
Case A: yeast sensitivity to antibiotic
- spread high number of yeast onto agar + antibiotic and look for spontaneous resistance (1 in 10^7)
- UV treats cells prior to spreading which increases chance of selecting (1 in 10^5)
Case B: product formation
- a–>b–>c–>d1 (80%) with d2 (20%), which is a side product being produced)
- a–>b–>c–>d1 (100%) with no d2 being produced
- the problem with this case is that mutagenesis can produce many invisible mutations in a single cell and can alter many unrelated characteristics
Case C: auxotrophic markers
amino acid requiring strains:
- UV
- chemical mutagenesis
heavy metals, MNNG, and antibiotics damage the cells
- you can use these markers in protoplast fusion, complementation of requirements
- A. arg-, thr- + B. met-, his- = (AB)
if we fuse them, the resulting product (AB) will be able to grow without them.
Rare mating (fungi will mate)
- mix high densities (10^8) of non-mating cultures and get some spontaneous mating to give true hybrids
- at high cell densities, fungi will begin to mate to increase the copy number
- cytofusion is also possible
What is single chromosome transfer?
transfer one chromosome from one strain to another
- it is important in brewing strains
Protoplast fusion
- protoplast is a cell totally devoid of cell wall material and it is extremely fragile
- a spheroplast is a membrane covere in cell wall fragments. it is a protoplast with fragments still stuck on walls
Protoplast isolation
- mechanically
- metabolically
- enzymatically (most widely used)
- degrade cell wall in an isotonic buffer, which prevents lysis of the membrane
a. wall may be degraded all around the cell
b. protoplast squeezes out through the aperture (opening/hole) in the cell wall - whether a or b depends on nature of lytic preparation, yeast species and stage of cell cycle
Lytic preparations
- Novozym 234: Trichoderma harzianum – glucanse & chitinase
- Funcelase: Trichoderma virile – glucanase
- Sucd’helix pomatia – chitin’s, glucanase & mannanase
- all of these enzymes chew up the cell wall, and then we have protoplasts formed
Factors affecting protoplast stability
(when protoplast is formed, it is fragile and will easily burst)
- membrane damage
- isotonic buffer: sugar alcohol (sorbitol) or salt (kcl)
- lytic preparation (if kept in too long, enzymes will continue to breakdown and protoplast will burst)
- pH: low pH will lead to lysis (need to keep it at 5/5.5 so it doesn’t burst)
- osmotic concentration
- metabolism (if we add glucose, cells will metabolism and burst. we don’t want this so we don’t add in glucose)
