Lecture 7 - Consolidated bioprocessing and synthetic biology Flashcards
What are the previous uses of enzymes and why is it such a big market?
- washing powders
- brewing
- cheese making
- flour processing
- major cost
- can only be used once
When are enzymes required in a working cellulosic biomass biofuel plant producing ethanol?
- 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
How do companies like Nonzymes and DSM make enzymes for biofuels?
- 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
What is consolidated bioprocessing?
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
What are the ways by which consolidated bioprocessing can become a reality?
Need to take a bug that is:
- Good at making a biofuel and add the ability to secrete large amounts of cellulolytic enzymes and degrade cellulosic sugars
- Good at secreting celluloytic enzymes and add biofuel production pathways
- 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
What problems need to be overcome to produce a consolicdated bioprocessing cell?
- Growing on multiple sugars
- Protein secretion
- The problem of the outer membrane
How can the problem of ‘growing on multiple sugars’ be overcome?
- 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
How can the problem of preferential metabolism be overcome?
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
What are the sugar hierachies in bacteria and how did Chris Rao’s group demonstrate why and how this is?
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)
How can the problem of protein secretion be overcome?
- 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
How can empirical screening be used to improve secretion?
- 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
How can secretory apparatus machinery be optimised?
- increase # of copies of secretory apparatus
- use stronger promoters
- more gene copies
Works well in G+ as there is only one barrier
In what organisms is the outer membrane problematic?
Gram negatives
What are two options for overcoming the outer membrane in gram - bacteria?
- use a secretion system from a pathogen e.g. Type I-VII
- make a fusion to a protein that is secreted
What are the features of type I secretion? How can this be used to make E.coli secrete proteases?
- 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
What are the features of type II secretion?
- 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
What is the possibility of using a secretion sytem with no signal?
- 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
What are four different techiniques to overcoming the outer membrane in gram - bacteria?
- Type I secretion
- Type II secretion
- Secretion system with no signal
- Protein fusions for secretion
How can protein fusions for secretion help to overcome the outer membrane in gram - bacteria?
- 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
What was the title of Jay Keaslings paper on biofuels in E.coli
Synthesis of three advanced biofuels from ionic liquid-pretreated switchgrassusing engineered E.coli
What was the pitch of Jay Keaslings paper/
- 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:
- Pinene synthesis
- Butanol synthesis
- Fatty acid ethyl ester synthesis (as a biodiesel precursor)
What was the process of Jay Keaslings paper?
- Pitch
- Getting secreted cellulases and hemicellulases
- making short oligosaccharides
- getting e.coli to grow on short oligosaccharides
- switching to a native promoter
- Using synthetic clusters for cellulose and hemicellulose utilisation
- Testing on real substrate
- Connecting to biobutanol
How did Jay Keasling get secreted cellulases and hemicellulases?
- 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
How did Jay keasling ensure that (3) short oligosacharides were made?
- 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
How did Jay Keasling get E.coli to grow on oligosaccharides? (4)
- 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
How and why did Jay keasling switch to a native promoter (5)?
- 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
How did Jay Keasley generate synthetic clusters for cellulose and hemicellulose utilisation?
- 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
What was the result when Jay Keasley tried their synthetic bugs on real substrates?
- 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
How did Jay keasley connect the growth of their synthetic bug to biobutanol?
- 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
How can you get over the problems with butanol being problematic to cell membranes?
Use fed/drained culture
