Biofuels and Biotech 2

Question 1

Describe a lignocellulosic feedstock

Answer 1

Bagasse (remains of sugar cane once sugar is removed - 50% celloluse, 30% hemicellulose, 20% lignin)

Question 2

Describe a cellulolytic bacteria and the basic mechnism of action

Answer 2

Clostridium thermocellum.
Lives on cellulose by direct binding to the crystalline surface
breaks down cellulose and hemicellulose to sugars
anaerobic
Sticks to cellulose via cellulose binding factor (CBF)

Question 3

Describe the structure of the cellulosome

Answer 3

Main protein CipA - a scaffoldin subunit. Has three main features: Type 1 cohesion domains (x9), CMBs (cellulose binding module), Type 2 dockerin domain

Scaffoldin held to the cell surface by anchoring subunit on plant cell via a range of carbohydrate active enzymes attached to it’s type 1 cohesin domain

Question 4

Describe the cohesin/dockerin interaction

Answer 4

• Conserved interaction between surface of the cohesin and helices of the dockerin
• Any protein with a type 1 dockerin domain can bind to a cohesin on the scaffolding domains
• Binding site includes CA2+ ion and is extremely high affinity

Question 5

Describe the cellulose binding module (CBM)

Answer 5

• Sticks the complex to the cellulose
• Many types (67 families) grouped into three groups:
  A: binds crystalline structures
  B: Binds internal regions of long glycanchains
  C: binds short oligosaccharides or the ends of long chains

Question 6

How do ABE fermentation microbes take up their sugars and why is this special?

Answer 6

Phosphotransferase transporters
.ThisdoesnotuseATPasasourceofenergy,butrather
canrelyonthesmalleramountoffreeenergyreleasedintheconversionof phosphoenolpyruvatetopyruvateasthesourceofphosphate

Question 7

Where are monolignols made in the cell

Answer 7

cytoplasm

Question 8

How is lignin biodegraded and which oragnisms are capable of this

Answer 8

Aerobic conditions
Oxidative free radical attack
White rot fungi, Limnoria quadripunctata (marine animal),

Question 9

Describe the process of recombineering

Answer 9

Genetic modifications in vivo
Mediated by bacterial Red recombinase (Exo, Beta and Gam proteins)
Short section of DNA containing H1 and H2 homologous crossover regions and modified gene in middle. Exo binds to linear DNA at each end (H1 and H2) and exonuclease activity generate overhangs. Beta bings to 3’ ssDNA and through homologous recombination DNA is inserted into target DNA