Lecture 5 - Biologicaly degradation of lignocellulose

Question 1

What is the struture of lignocellulose?

Question 2

What is the cellulosome?

Question 3

What is the function of the cellulosome?

Answer 3

A nanomachine for cellulose breakdown

Question 4

What are some routes to break down hemicellulose and lignin?

Question 5

What are some of the strategies microbes use to break down lignocelluose?

Question 6

What are the three main components of lignocellulose?

Answer 6

Cellulose

Hemicellulose

Lignin

Question 7

What is cellulose?

Answer 7

linear polymer of β(1-4)-linked glucose organized in a regular crystalline arrangement and forming insoluble linear microfibrils

Question 8

What is hemicellulose?

Answer 8

complex polysaccharides often rich in pentose sugars such as xylose and arabinose with a variety of other modifications that allow them to attach to both the cellulose and the lignin

Question 9

What is lignin?

Answer 9

highly irregular network of cross-linked phenylpropanoid type molecules

Question 10

How does lignocellulose differ in different plants?

Answer 10

The relative composition of the lignocellulose differs e.g. hardwood v. grasses

Question 11

What is bagasse?

Answer 11

fibrous lignocellulosic material left after the sucrose has been removed from sugar cane and the remainder has been milled.

Approx.

• 50% cellulose
• 30% hemicellulose
• 20% lignin

Question 12

Give an example of a cellulolytic bacteria?

Answer 12

Clostridium thermocellum

• thermophilic bacterium
• lives on cellulose by direct binding to the crystalline surface of cellulose
• forms a dense monolayer on the surface
• breaks down cellulose and hemicellulose to sugars for fermentation
• strict anaerobe
• on Avicel (model crystalilne cellulose substrate) can grow at a rate of 0.1h^-1

Question 13

What was the cellulose binding factor of C.thermocellum as discovered by Edward Bayer and Raphael Lamed?

Answer 13

Cellulosomes: A large (>3MDa) surface located protein complex which produces a range of different proteins

Found in a number of anaerobic bacteria

Question 14

What is the modular structure of the C/thermocellum cellulosome?

Answer 14

• Main protein: CipA which is a scaffoldin subunit with three main features (different organisms have different numbers of the sacffoldin subunits)
  
  • Type-1 cohesin domains (has 9 - acts as the docking complex)
  • CBMs
  • type-II dockerin domain (sticks tightly to anchoring subunit)
• Scaffoldin is held to the outside of the cell surface by an anchoring subunit
• range of carbohydrate active enzymes (CAZys) are attached to its type-I domains
• releassed in small subsunits, excereted and then quickly caught

Question 15

Describe the cohesin-dockerin interaction of the cellulosome

Answer 15

• conserved interaction between the surface of the cohesin and the helices of the dockerin (type-I to type-II)
• any proteins (enzyme subunits) with a type-I dockerin domain can bind to a cohesin (type-I cohesins) on the scaffolding proteins
• not an ordered process and the scaffolding cannot control what binds where (enzymes in any random order)
• the binding site includes a Ca²⁺ ion and is extremely high affinity (sub nM) intereaction
• type II dockerin/cohesins have a larger binding interface and bind even tighter

Question 16

What complex sticks the cellulosome to the cellulose?

Answer 16

• The carbohydrate binding molecule CMB3a (specific to cellulose)
  
  • linear strip of residues interacts with glucose rings
  • planar face allows stacking interactions between cellulose and the linear strip

Question 17

What are the different types of CBMs? (carbohydrage binding molecule)

Answer 17

types of CBMs (67 families 2013)

• A - binds crystalline surfaces
• B- binds short internal regions of long glycan chains
• C- bind short oligosaccharides or the ends of long glycans

Question 18

What enzymes are in the cellulosome?

Answer 18

• C. thermocellum contains 72 cellulosomal enzymes
  
  • many have CBMs targetting them to the cellulose
• To break down crystalline cellulose, must first nick the surface with an endo-cellulase (endo-glucanase) to produce new chain ends
  
  • could be at the region of amorphous cellulose
• Then an exo-cellulase (exo-glucanase) degrages this back
• Bacteria express a repetoire of these enzymes (different enzymes have different functions)
  
  • enzymes are from many glycoside hydrolast (GH) families which synergistically break down the cellulose
• Products are short glucosides (glucose oligomers) which are either taken up directly or first broken down by beta-glucosidases on the cell surface

Question 19

What are the advantages of having a cellulosome?

Answer 19

Sticking to the substrate is (theoretically) useful as:

1. bring armoury of enzymes closer to their targets
2. the released products won’t diffuse away and can be transported quickly into the cell
3. rapid removal of the enzyme products by uptake into the cell, decreading feedback inhibition of the cellulose breakdown

Question 20

give examples of other bacteria and how they break down cellulose

Answer 20

• Clostridium cellulolyticum have a cellulose but secrete other extracellular enzymes
• Anaerocellum thermophilim is efficient at growing on cellose
  
  • producs similar enzymes to Clostriduim thermocellulm but without a cellolosome-like strucutre
  • however some of these enzymes are large proteins which contrain mutiple enzymatic domauns fused together
• Trichoderma has a 50 fold less cellulose breakdown rate than C. thermocellum
  
  • cellulolytic fungi
  • just secretes enzymes (although in contest)

Question 21

What alternative enzyme can be used to break cellulose?

Answer 21

Lytic polysaccharide monooxidases (LPMOs)

• a class of metal-dependent monooxygenases that can cleave cellulose
• AA9 protein from the aerobic fungus Thermaoascus aurantiacus
  
  • ​has a wide flat surface to bind to the crystalline substrate
  • with a central copper containing active site
  • Cu ion is coordinated by two histadines and uses oxygen in the catalytic mechanism to break the glycosidic bonds in the polysaccharide

Question 22

What effect does the digestibility of lignin have of digestibility and how is this overcome?

Answer 22

• llignin component most difficult to break down
• ignin content of a feedstock has the biggest influence on total digestibility
• linked by ether and C-C bonds (make it difficult to get the enzyme in and break down the molecule)
• many ways by which it can be cross linked
• current pretreatment methods include steam explosion
  
  • blast it to bits to expose the cellulose

Question 23

What fungi grow on dead trees and what is the significance of this?

Answer 23

• Brown rot fungi
• White rot fungi
  
  • Xylaria hypoxylon ‘candle snuff fungus’ seen on rotting stumps
  • Xylaria polymorphs ‘Dead man’s fingers’

Live on dead tree and break down cellulose (slow growth) it is possible to break down lignin!

Question 24

How do white rot basidomycetes break down lignin? And brown rot?

Answer 24

White rot basidomycetes

• Secrete a range of oxidative enzymes
  
  • heme-dependent lignin peroxidases
  • manganese peroxidases
  • copper dependent laccases

Brown rot fungi

• partially degrade lignin mainly to get at the cellulose and hemicellulose

Question 25

What bacteria can degrade lignin and how do they do this?

Answer 25

• Sphinobium sp. SYK-6
• certain actinobacteria, a-proteobacteria and y-proteobacteria
• needs large number of enzymes to trim down the aromatics and catalyse the ring opening
• evidence for anaerobic bacteria that can degrade and grown on lignin

Question 26

What are the main hemicelluloses in hardwood and softwood?

Answer 26

Hardwood

• hemicellulose mainly xylan
• backbone of b-1,4-linked xylose with decorations

Softwood

• hemicellulose main galactoglucan
• b-1,4- linked D-glucose and D-galactose

Question 27

What is the targeted hemicellulose source of the funded project looking at engineering E.coli to ‘eat grass’?

Answer 27

The gemicellulose targeted is that of the biofuel grass miscanthus

Question 28

What bacterium did the Yejun Han group isolate to look at the biochemical and strucutral insights into xylan utilisation? Where from? What did they do?

Answer 28

Caldanaerobius polysaccharolyticus

• gram +ve thermophillium
• from garbag dump in illinois
• characterised a range of its hemicellulose degrading enzymes following a partial genome sequence
• take a biochemical and structural approach to characterise components of a xylan utilisation cluster

Question 29

How did the Yejun Han group investigating the Caldanerobius polysaccharolyticus first look for a xylanase?

Answer 29

1. used simple BlastP with known endo-xylanases
2. found one in caldanerobius polysaccharolyticus
3. took sequence and examined it in software (InterPro, Pfam) revealing it had mutliple domains
4. Found that it contains:
   
   1. a GH10 flanked by 2 family 22 CBMs on the N-term
   2. 2 family 9 CMBs on the C-term
   3. 3 surface later homology (SLH) domains also at the c-terminus (attach to the surface of the cell)
   4. Clear N-terminal signal peptide
5. Cloned by PCR into an E.coli expression vecetor to make recombinant protein

Question 30

Following the identification of a xylanase in Caldanerobius polysaccharolyticus, how did the Yejun Han group measure xylanase activity?

Answer 30

• Ran protein on gel, identified a soluble protein at 170 kDa as expected
• First functional assay: (prior to: had to clone protein and his-tag for affinity chromotography. Cleaned up protein by breaking open E.coli heat cell lystae to ~50degrees C, E.coli proteins denature but not protein from the thermophile) -> incubate the protein with birchwood xylan (BWX) overnight and measure the increase in reducing sugar ends. This was shown, demonstrating xylanase activity
• Also followed the reaction using TLC (thin layer chromotography plate) and can resolve the different forms of the xylan, although bands don’t match to the standards exactly
  
  • spot products of the reaction onto thin layer chromotography plates and this separates the molecules
  • products stop at X3 - are they modified in some way?
• Identified that their protein (Xy10A) produced mainly xylo-oligomers

Question 31

Following the ietification of xylase activity in Caldanerobius polysacharolyticus, how did Yejun Han group identify a hemicellulose cluster?

Answer 31

1. Xy10A produced mainly xlo-oligomers
2. must be a region on the genome that encodes genes for further breakdown and use of the xylo-oligomers
3. found a large 18kb cluser with:
   
   1. ABC transporter
   2. genes for pentose phosphate pathway
   3. xylanase was not in this cluster
4. Looked further at the xylosidase (Xyl3A), the glucuronidase (Agu67A) and the ABC transporter

Question 32

How did the Yejun Han group further investigate the Caldanerobius polysaccharolyticus Xyl3a?

Answer 32

• expressed and purified the Xyl3A (xylosidase) in e.coli in the same way as the xylanase
• protein at the expected size of 86kDa (SDS-PAGE)
• size exclusion chromatography: protein eluted at a position that was consistent with it being a dimer in solution (157kDa)

Tested for b-xylosidase activity of the Xyl3A

• screened for activity using a range of pNP-linked substrates
• most active on pNP-D-xylopyranoside (pNPX)
• then tested against a range of real xylo-oligomers and saw efficient cleavage down to xylose (monomer)
• therefore clearly a xylosidase
• has weak activity against cellulo-oligosaccharides (has a weak glucosidase activity)

Question 33

How did the Yejun Han group further investigate the Caldanerobius polysaccharolyticus Agu67A (glucuronidase)?

Answer 33

Expression of Agu67A

• expressed and purified the Agu67A in E.coli the same was as the xylanase
• See a protein of ~ expected size (below 75kDa when should be 79kDa)
• size exclusion chromotography: protein is a dimer
• Identified some GH domains (N, M, C)
• Expected to be a debranching enzyme to remove the 4-O-methylglucuronic acid groups attached to the xylose backbone

Activity of Agu67A

• looked at whether it produced undecorated xylo-oligomers from branched substrate (aldobiouronic, aldotriouronic and aldotetrauronic)
• then look at the release of glucuronic acid equivelents in the reaction
• used HPLC - to look at the substrates that were put in with and without the enzyme. With the enzyme other molecular weights begin to appear
• Works best on alduronic acids

Question 34

How did the Yejun Han group investigate the synergism of Xyn10A (xylanase), Xyl3A (xylosidase) and Agu67A (glucuronidase)?

Answer 34

• mixed three enzymes together to show that the combination of the 3 could convert more of the BWX xylan into free xylose
• the addition of the xylosidase to the xylanase gave the largest increase
• the extra addition of the a-glucuronisdase made a small further improvement (helping the xylanase)

Odd experiment to do as would expect all these enzymes to be in diff places in the cell.

With all 3 proteins get more single sugar products than separate

Question 35

What did Yejun Han group investigate abnout the ABC transporter, why and how?

Answer 35

• whether the ABC-T is specific for xylo-oligosaccharides
• as the xylanase is extracellular and the xylosidase and glucuronidase are intracellular substrates must be transported in as branched xylo-oligomers
• Expressed the SBP from the candidate ABC transporters in teh cluster and examined ligand binding by isothermal titration chromotrography
• found that it doesn’t bind xylose but binds tightly to xylo -biose, -triose and -tetraose with the highest affinity for xylotriose, then xylobiose, then xylotetraose.

Question 36

What is the process of isothermal titration chromatography used to examine ligand binding by the ABC transporter?

Answer 36

1. looks at enzyme-substrate interactions
2. when proteins bind to a substrate, small amounts of energy are released
3. compare to a reference cell (kept at constant temperature) and measure small temperature changes
4. measure the amount of energy need to be added/taken out in order to match test cell to reference cell
5. allows the calculation of the affinity and how well substrate binds

Steepest = best binding

Question 37

How did the Yejun Han group learn more about the binding of the ABC SBP to the xylo-oligomers?

Answer 37

• solved the structure of the SBP
• to learn about how it bound the xylo-oligomers
• structure resembles other monosaccharide transporters from other thermophiles
• clearly binds the xylo-oligosaccharide
• analysis of the binding site suggests it is suited to binding the triose and nothing longer (confirming ITC data)

Question 38

What was the final model from the Yejun Han group about Caldanerobious polysaccharolyticus xylan utilisation cluster?

Answer 38

1. enzyme binds to cell surface and breads down hemicellulose to alduronic acid (Xylanase - Xyn10A)
2. This is transported into the cell (ABC transporter)
3. Processes inside the cell to xylase monomers which are ran through the cells metabolism to generate energy (Xylosidase - Xyl3A, glucuronidase - Agu67A)