Lecture 3 - redone

Question 1

What is lignocellulosic biomass?

Answer 1

Strong, fibre composite material. Durable and rich in polysaccharides.

Question 2

What is the composition of lignocellulosic biomass?

Answer 2

• 65-75% polysaccharides (50-70% cellulose)
• 10-35% lignin
• Variable sugar content depending on the biomass type
  
  • High: Bark on harvest and green plant tissue
  • Low: Seneseced material e.g. micanthus stems
• 2-3% protein
• Hydrophobics (sterols, fatty acids, waxes)
• Inorganics (Si (up to 10% in rice), K, Na, Cl, P)

Question 3

What are the considerations of linocellulose for sustainable bioethanol production?

Answer 3

• Lignocelulose is the greatest untapped biological raaw material
• Strong and durable material built to last, hard to digest because of lignin and cellulose indigestibility. This is a challenge for sustainable bioethanol production
• Composed of polysaccharide that can be converted to fermentable sugars

Question 4

What are the two types of cell walls?

Answer 4

Primary

Secondary

Question 5

What are the features of primary cell walls?

Answer 5

• Strong but extensible
• Polymer networks highly hydrated
• Polymers held together by non-covelent cross links
• Thin
• contains pectin, cellulose and hemicellulose

Question 6

What are the features of secondary cell walls?

Answer 6

• Strong and rigid
• Contain less water
• Polymers are highly packed
• Sealed and crosslinked by the hydrophobic polyphenol lignin

Question 7

What are the features of cellulose?

Answer 7

• Most abundant biological polymer
• 30% of cell wall dry weight, 25-40% of total lignocellulosic biomass

Question 8

What is the composition of cellulose?

Answer 8

• b-1-4-linked glucan chain (hard to digest)
• eachy glycosyl unit is inverted 180 to its neighbour
• b(1-4)-linked glucan chains formed into microfibrils, 36 glucan chains form a microfibril
• Cellulose microfibrils form a crystalline structure (stable, hard to digest, need lots of enzymes)
• Decorated with more complex polysacharides with lots of side chains
  
  • don’t for crystalline structure
  • form links between cellulose microfibrils (flexible)
  • stop cellulose microfibrils sticking to one another, plasticising the cell wall

Question 9

What is the mechanism of action of the bacterial cellulase sythase?

Answer 9

• Membrane bound

1. Takes UDP glucose from the cytoplasm, polymerises it to generate a chain of b(1-4)-linked glucan which is extended out of the bacteria (into the cell wall space in plants)
2. After glucosyl transfer, newly added glc rotates around the acetyl lineage (180 degrees) into the plane of the polymer
3. The rotation direction is determined by steric interactions and the formation of the b(1-4)-glucan hydrogen bond
4. During this relaxation, glucan is translocated into the enzyme channel and the process is repeated with a second UDP-glc
5. The rotation direction after glycosyl transfer is in the opposite direction due to steric constraints

Question 10

What enzyme complex synthesises the cellulose microfibrils?

Answer 10

• Rosettes
• Travel in the membrane whilst undergoing synthesis, producing microtubules in a coordinated manner, pushing the complex along
• Six lobed rosette comprised of hexameric cellulase synthase complexes
• 6 b(1-4)-linked glucan chains are synchronosly produced and stick together as they are highly hydrophobic
• 20-100b glucan chains in a cellulose microbfibril

Question 11

What are the features of hemicelluloses?

Answer 11

• complex polysaccharides
• bind to the surface of crystalline fibre by hydrogen bonding
  
  • this forms the main structural network
  • sugars in the hemicelluloses have the same spacing as glucose units in the cellulose microfibrils to allow H bonding
  • where they are not bound to the microfibrils they form tethers between microbfibrils
• side chains prevent them from being part of the crystalline structure
  
  • network has more branching, less regular structure

Question 12

Outline secondary cell wall hemicellulases

Answer 12

• 25-35% cell wall dry mass
• form links to lignin
• ferulic acid linkages on arabinoxylans form crosslinks between polymers so can dimerise/trimerise; forming hemicellulose-hemicellulose and hemicellulose-lignin bonds

Question 13

What is the structure and composition of the hemicelluloses?

Answer 13

• long polysaccharides based on (1,4)b-D-glucans or (1,4)b-D-xyans or glucomannans
• substatial side chains
• rich in 5C sugars except for gymnosperms (galacomannans)

Question 14

What are the features of hemicelluloses in connifers?

Answer 14

• Main hemicellulose is galactoglucomannans (hexose polymer)
• Alternating glucan/mannose residues made by a single polysaccharide synthase
• Directed with a(1-6)-galactose residues to prevent the formation of crystalline polymers

Question 15

What are the features of hemicelluloses in angiosperms?

Answer 15

• Main hemicelulose are xylans and arbinoxylans
• b(1-4)-D-xylanopyranase polymers
• 6 ring structure which can hydrogen bond to cellulose microfibrils and form non-hairy crosslinks on cellulose microfibrils
• provide linkage between polysaccharides and lignin (ferulic acid linkages)

Question 16

Outline Xylan biosynthesis

Answer 16

• complex
• in the golgi
• need lots of different proteins putting all the units onto the polymers
• backbone is made by three different proteins
  
  1. adds glucuronic acid
  2. adds methyl groups to glucuronic acid
  3. adds arabinose onto xylose backbond and xylose onto arabinose
• Hypothetical how most of the substrates get into the cell. Some (e.g. ferryl units) as UDP sugars and then modified

Question 17

What is the structure of xylans in dicots?

Answer 17

• End primer where the chain is extended
• Rhamnose/galacturonic acid at the reducing ends
• linear b(1-4)-xylan backbone substituted with:
  
  • GlcA
  • Arabinose (but frequently not found)
  • (Me-)GlcA
  • Acetate
• Minor domain: (Me-)GlcA closely spaced
• Major domain: (Me-)GlcA on evenly spaced residues (8 xylose units apart)

Question 18

What is the structure of xylans in grasses?

Answer 18

• Xylan backbone substituted with:
  
  • Ferulic acid (form a link between the arabinoxylan and lignin)
  • Coumeric acid (makes up lignin)
  • Arabinose
  • Xylose
  • Galactose

Question 19

What are the features of lignin?

Answer 19

• In secondary cell walls
• second most abundant biological polymer
• attached to the polysaccharide netweork in situ by linking to hemicellulose
• Gives secondary cell walls:
  
  • rigidity
  • renders network insoluble
  • seal and crosslink polymers so they are more tightly packed
  • network resistant to degradation
• Formed of monolignols (change composition in different organisms)

Question 20

Outline the formation of lignin of monolignols

Answer 20

• 3 monolignols produced in a biosynthetic pathway in the cytoplasm, moved out and secreted as monomers ino the cell wall space by a particular transporter
  
  • H lignin (p-coumaryl)
  • G lignin (conniferyl)
  • S-lignin (sinaptyl alcohols)
• polymerised in the cell wall to form a complex structure by free radical coupling ‘oxidative chemistry’
  
  1. Lacases/peroxidases form free radicals
  2. Radialised monomers added onto the existing chain in 1 of 3 positions, or polymers are generated than condense to one another
  3. Form different linkages to help build up the lignin complexity; the heterogeneity and lack of repeat pattern make it hard to degrade
     *

Question 21

How does the composition of lignin monolignols changes in different organisms?

Answer 21

• Connifers: coniferyl and coumaryl units only (G, H)
• Monocots: rougly 3 in equal quantities (G, S, H)
• Dicots: mostly conniferyl and sinaptyl units (G, S)

Question 22

What is pectin?

Answer 22

• 30% promary cell wall dry weight
• acidic polysaccharide
• For wall adhesion and elasticity
• forms a coextensive network with cellose and hemicellulose

Question 23

What are the features of woody cell walls?

Answer 23

• Polysaccharides and lignin matrix
• Cellulose and hemicellulose

Question 24

What is Radially swollen walls (RWS) and how was it identified?

Answer 24

• Radially swollen walls is a conditional mutant in a cellulose synthase gene
• Identified following a screen for the swelling of plant cells in mutants (primary cell walls)
• Normal at 20 degress, at 25 degrees cells become swollen and the cell dies
  
  • Mutants in primary cell wall synthase in the catalytic subunit holding the rosette together
  • Rosette falls apart at elevated temperature
• RSW has homology to bacterial synthase

Question 25

What are the Irx mutants and how were they identified?

Answer 25

• Mutation in secondary cell wall cellulose synthase
• Not lethal
• Loss of structure, dwarf plants, doesn’t make secondary cell wall properly
• Some collapse of the xylem due to the negative pressure during transpiration
• Identified by a mutated population of arabidopsis thaliana
  
  1. Screened for changes in xylem cells
  2. Mapped collapsed xylem cells
  3. identified 3 different cellulase synthase genes that are required for making microfibre

Question 26

Why is xylan biosynthesis (the hemicellulose of angiosperms) an important research target?

Answer 26

• Most available form of plant biomas
• Makes up 30% of lignocellulistic biomass
• Likely provides a link to lignin

Question 27

Describe the coexpression analysis in Brown to identify novel genes in arabidopsis involved in cell wall formation

Answer 27

Used expression profiling and reverse genetics to identify novel genes in arabidopsis involved in cell wall formation

Background knowledge: Secondary cell wall generated by cellulase synthase

1. Looked for genes coexpressed together with secondary cell wall synthase using a microarray analysis
2. Prepared RNA from stems of different arabidopsis plants
3. Used transcriptome studies to identify genes showing significan expression with IRX1/2
4. Found 150 genes with the same pattern of expression as the secondary cell wall synthase
5. Ordered tDNA insertion lines

Phenotype analysis of insertion lines generated from transcriptome studies

1. Took cross section of stem vascular bundles in WT and lrx mutant plants
2. Stained transverse sections with toludine blue to look for collapsed xylem phenotypes
3. Identified genes that play an important role in secondary cell walls
4. Looked at non-cellulosic carbohydrate composition of the cell wall material from stems of the WT/mutant linkes. Looked at individual sugars at % of the total wall sugar.
5. Three mutants had low xylase compared to other mutants
6. Genes likely to be involved in xylan biosynthesis (lrx 7,8,9)