Lecture 7 - Consolidated bioprocessing and synthetic biology

Question 1

What are the previous uses of enzymes and why is it such a big market?

Answer 1

• washing powders
• brewing
• cheese making
• flour processing
• major cost
• can only be used once

Question 2

When are enzymes required in a working cellulosic biomass biofuel plant producing ethanol?

Answer 2

• Imbicon based in denmark
• Have a large opperational cellulosic biomass biofuell plant producing bioethanol
• Genencor provide enzymes for:
• after a chemical or physical pretreatment to break open the lignin, a battery of cellulases and hemicellulases are needed to release the sugars
• significant components of the overall costs of the process as they are added at high concentrations, nearly 100g/L

Question 3

How do companies like Nonzymes and DSM make enzymes for biofuels?

Answer 3

• use defined growth media
• standard batch and fed-batch fermentation
• generally use fungi (Aspergillus oryzae) or Bascillus subtilis to make primary enzymes as these are good at secreting certain proteins naturally
• mainly not recombinant protein production
• certain strains of bascillus can secrete large quantites 20-25g/L of extracellular enzymes

Question 4

What is consolidated bioprocessing?

Answer 4

To overcome the problem of the size of cellulase. Needs to be broken down in chunks to use.

An idea to integrate into a single organism the ability to:

• degrade cellulosic material directly in the timescale of an industrial fermentation and use the released sugars (including pentoses) to convert these various chamicals into a biofuel

Question 5

What are the ways by which consolidated bioprocessing can become a reality?

Answer 5

Need to take a bug that is:

1. Good at making a biofuel and add the ability to secrete large amounts of cellulolytic enzymes and degrade cellulosic sugars
2. Good at secreting celluloytic enzymes and add biofuel production pathways
3. take a model organism which is easy to engineer and add whatever phenotypes needed to add in both of these charactersitics (biofuels and cellulose degradation)

Problems with 1 and 2: these organisms are generally not genetically tractable. Need to develop them into genetically tractable organisms initially which takes a lot of time and input. Need to be aware of:

• strong promoters
• selectable markers
• methods of transformation

Question 6

What problems need to be overcome to produce a consolicdated bioprocessing cell?

Answer 6

1. Growing on multiple sugars
2. Protein secretion
3. The problem of the outer membrane

Question 7

How can the problem of ‘growing on multiple sugars’ be overcome?

Answer 7

• Add the ability to use pentose metabolism to Z.mobilis
• engineered plasmid into a cell using a shuttle vector with 2 groups of genes (xylose metabolism/pentose metabolism) run from different promoters
• will comentabolise D-xylose with glucose
• however didn’t add an efficent means of getting the sugars into the cell, need the correct transporters

bad:

• get the preferential use of one sugar
• Z. mobilis not robust enough bug for large scale fermentation

Question 8

How can the problem of preferential metabolism be overcome?

Answer 8

Not ideal

• BP developed a process using Lonnie Ingrams Z.mobilis homoethanol pathway engineered into E.coli
• problem with simaeltaeonously using hexose (glucose) and pentose (D-xylose, L-arabinose) sugars
• do 2 fermentations with 2 strains of e.coli

Question 9

What are the sugar hierachies in bacteria and how did Chris Rao’s group demonstrate why and how this is?

Answer 9

Sugar hierachies

• Glucose mediated cataboite repression
• also other additional hierachies
• Given the two pentose sugars xylose and arabinose, E.coli prefers to use L-arabinose before D-xylose

How?

• Arabinose bound AraC protein (activates the expression of the arabinose utilisation genes) binds and represses xylose utilisation genes

Why?

• The transporter of L-arabinose is via a secondary carrier (doesn’t require energy) whilst that for D-xylose is an ABC transporter (requires ATP)

Question 10

How can the problem of protein secretion be overcome?

Answer 10

• Need to get cellulases/hemicellulases out of the cell
• use sec/tat pathways (pathways by which proteins are secreted across the cytoplasmic membrane)
• a signal peptide can be added to any soluble protein for secretion
• Tat: secretes folded proteins
• Sec: unfold/secretes unfolded proteins

Question 11

How can empirical screening be used to improve secretion?

Answer 11

• Assume the signal peptide not as efficent as it could be
• signal peptide can be mutated to increase the level of secretion
• EXAMPLE: gene encoding a protease (cellulase or hemicellulase) was put into a vector system whereb ythe signal peptide sequence can be easily changed
• used nearly 400 diff natural signal peptides
• screen with an activity assay to identify an increase in secretion activity
• found one that gave nearly 700% increased efficiency

Question 12

How can secretory apparatus machinery be optimised?

Answer 12

• increase # of copies of secretory apparatus
• use stronger promoters
• more gene copies

Works well in G+ as there is only one barrier

Question 13

In what organisms is the outer membrane problematic?

Answer 13

Gram negatives

Question 14

What are two options for overcoming the outer membrane in gram - bacteria?

Answer 14

1. use a secretion system from a pathogen e.g. Type I-VII
2. make a fusion to a protein that is secreted

Question 15

What are the features of type I secretion? How can this be used to make E.coli secrete proteases?

Answer 15

• protein is moved through a complex that spans both membranes with no periplamic stage
• Tolc: OM protein that can reach into the periplasm to couple with other proteins
• HylBD: ATP-dependeent inner membrane components that bind the substrate protein and catalyse its export
• HlyA is the natural substrate
  
  • C-terminal secretion signal
  • exported as an unfolded preotein to the extracellular environment
  • on the outside the GGxGxD motif uses a free calcium ion to help the protein fold
• E.coli will secrete a range of recombinant proteins by adding the secretion signal
  
  • yields (best 0.1g/L) are not sufficent for consolidated bioprocessing

Question 16

What are the features of type II secretion?

Answer 16

• used primarily for producing pilli
• two-step metabolism
  
  • protein first secreted via Sec to the periplasm where the signal peptide is removed and the exoprotein folds and waits for secretion
  • the exoprotein binds to the parts of the T2SS apparatus which stimulates the ATPAse activity of GspE so that the pseudopilin subunits are added to the pseudopilis
  • the growing pseudopilus physically ejects the exoprotein across the OM
• Still inefficient

Question 17

What is the possibility of using a secretion sytem with no signal?

Answer 17

• a number of gram - bacteria use these systems to secrete degradative enzymes into the environment
  
  • sialidase from Vibrio cholerae
  • Chitinase from V. harveyi
  • Cellulase from Dickeya dadantii
  • mostly slow growing when doing this
• T2SS signal has yet to be idetified
  
  • many substrates have a beta-sheet components, not all
  • difficult to engineer
  • limited yeild

Question 18

What are four different techiniques to overcoming the outer membrane in gram - bacteria?

Answer 18

1. Type I secretion
2. Type II secretion
3. Secretion system with no signal
4. Protein fusions for secretion

Question 19

How can protein fusions for secretion help to overcome the outer membrane in gram - bacteria?

Answer 19

• gene fusions between a recombinant protein and a carrier protein (e.g. malose binding proteins) results in high levels of protein in the periplasm
• Sup Yan Lees: Identified the secreted proteins from E.coli using proteonomics
  
  • overexpressed each in turn to see which secreted to high levels
  • chose OsmY as best protein
    
    • could lead to the secreteion of a number of proteins, including alpha-aylase
    • fuse OsmY to the target protein

Question 20

What was the title of Jay Keaslings paper on biofuels in E.coli

Answer 20

Synthesis of three advanced biofuels from ionic liquid-pretreated switchgrassusing engineered E.coli

Question 21

What was the pitch of Jay Keaslings paper/

Answer 21

• E.coli great as a host
• high costs of enzymes in using cellulosic feedstocks
• vision of consolidated bioprocessing
• a cellulolytic strain of E.coli would be desirable but doesn’t exist due to poor protein secretion
• need to be secreted about 1000-fold better to get enough enzyme

Tested the production of 3 different molecules:

1. Pinene synthesis
2. Butanol synthesis
3. Fatty acid ethyl ester synthesis (as a biodiesel precursor)

Question 22

What was the process of Jay Keaslings paper?

Answer 22

1. Pitch
2. Getting secreted cellulases and hemicellulases
3. making short oligosaccharides
4. getting e.coli to grow on short oligosaccharides
5. switching to a native promoter
6. Using synthetic clusters for cellulose and hemicellulose utilisation
7. Testing on real substrate
8. Connecting to biobutanol

Question 23

How did Jay Keasling get secreted cellulases and hemicellulases?

Answer 23

• Had previously shown that the clostridium stercorarim endoxylase Xyn10B can be produced by E.coli when fused with the protein OsmY
• Screened 10 GH9-family cellulases with OsmY fusions and measured the amount of activity in the supernatent
• used a spectrophotometric assay with azo-CMC assay (colour liberated as cellulase degrades cellulose)
• enzyme chops the CMC into small pieces which stay in solution after a chemical precipitation step at the end to remove the remaining substrate
• small pieces with the azo-dye can be detected by measuring A590

Found: the Cel enzyme from Bascillus sp DO4 (cellulase #7) was the best

Question 24

How did Jay keasling ensure that (3) short oligosacharides were made?

Answer 24

• using their purified enzymes Cel and Xyn10B, demonstrated that IL (ionic liquid) treated switchgrass can be partially degraded
• took a small amount of cell extract from cells making either enzyme
• HPLC analysis of sugars being released
• OsmYCel released glucose levels equivelent to 5% of the cellulobiose producing Cellotriose and biose, whilst OsmY-Xyn10B hydrolysed 11% of the xylan, mostly into xylotriose and biose
• Only release 8% of the sugars available

Demonstrates principle: switchgrass into ionic liquid, enzymes still function

Question 25

How did Jay Keasling get E.coli to grow on oligosaccharides? (4)

Answer 25

• E.coli doesn’t grow on cellotroise, cellobiose, xylanotriose or xylanobiose
• Need to be broken down into monosaccharides
• screened four b-glucosidases cloned from Cellvibrio japonicus (gram - cellulolytic bacterium) looked to see whether this enable growth on glucose
• Look at the OD of cultures to determine growth
• Cel3B works in e.coli much better than the others
  
  • however not clear where these enzymes are located
  • may work better because secreted more efficiently
  • how are the substrates getting in
• screened 12 xylobiosidase genes from C. japonicus
• Gly43F enabled growth on enzymatically hydrolysed beechwood xylan

Question 26

How and why did Jay keasling switch to a native promoter (5)?

Answer 26

• determined which enzymes worked well, now looked at promoters
  
  • didn’t look at transporters for cellubiose even though this is a major limiting step
• screened a number of e.coli promoters to work upstream of cel3A
• wrbA gives growth rates nearly the same on cellubiose as on glucose
  
  • no cellulobiose transporter so must be using glucose transporter fine
• Repeated with xylosidase gly43F, other promoters were better but none had as good growth on free xylose

Problems: synthetic bilogy but highly empirical and shows we don’t understand the relationship between promoter strength and final activity

Question 27

How did Jay Keasley generate synthetic clusters for cellulose and hemicellulose utilisation?

Answer 27

• assembled their respective enzyme/promoter combination on a plasmid
• transcriptional terminators added on to insulate each gene and stop transcription from ongoing which could create prolems
• saw growth on phosphoric acid swollen cellulose with pCellulose
• See much faster growth with pXylan in beechwood xylan (growth better on xylan than cellulose) - may be due to how substrates getting in to the cell

Question 28

What was the result when Jay Keasley tried their synthetic bugs on real substrates?

Answer 28

• tested on real substrates, IL treated switchgrass, eucalyptus and yard waste (grasses)
• used either E.coli with cellulose/xylan or both plasmids
• when co-culture (both plasmids) see twice the yield, suggesting both are accessing different carbon sources (hexoses and pentoses)
• But not actually efficient
• but room for impreovemnt in transport

Question 29

How did Jay keasley connect the growth of their synthetic bug to biobutanol?

Answer 29

• made a synthetic gene cluster for butanol production in E.coli mutant(triangle)adhE
• grew on a cellulosic feedstock
• when carrying eith pXylan or pCellulose, E.coli DH1 pButanol produced butanol from either xylan or cellulbiose respectively
• Coculture of both strains yeilded 30 mg/L biobutanol from a defined rich medium containing 2.2% w/col IL-treated switchgrass as the main carbon source
• WT clostridium can get over 20g/L but now matter of optimisation

Question 30

How can you get over the problems with butanol being problematic to cell membranes?

Answer 30

Use fed/drained culture