Lecture 6 redone Flashcards
Outline aerobic respiration in E.coli
- Glucose is completely oxidised to CO2, generating NADH from NAD (producing 1ATP)
- NADH is reoxidised in the aerobic respiritory chain which terminates in oxygen reduced to H2O
- During this process, protons are pumped across the membrane by NADH dehydrogenase and cytochrome C and O oxidase to generate a membrane potential
- Membrane potential is used by F1F0 ATPase to conert ADP + Pi to ATP. This is where most of the ATP is made.
Uses the pyruvate dehyndrogenase enzyme to convert pyruvate to Acetyl-CoA and Co2
What are the different types of microbial fermentation and what organisms perform these reactions?
- E.coli
- Engineered E.coli ethanol fermentation
- Manipulation of the E.coli mixed acid fermentation to make H
- Saccharomyces cereviase
- Ethanol fermentation
- Clostridium species
- ABE fermentation for ethanol, butanol
- Zymonas mobilis
- Ethanol fermentation
Describe the loss of the extra carbon molecule in glucose conversion to pyruvate
By Embden-Meyerhoff pathway or the Entero-deuderoff pathway
In both, when pyruvate is concerted to acetyl-CoA the extar C molecule is lost
- either directly to CO2
- Pyruvate dehydrogenase - aerobic respiration (PHD)
- Pyruvate fromate lyase - bacterial fermentation (PFL)
- Pyruvate decarboxylase - yeast fermentation (PDC)
- Or indirectly, later, from the breakdown of formate
How can loss of the extrac carbon molcule in glucose conversion to pyruvate be overcome?
- James Lios
- Replacing emdben-meyerhoff pathway with an alternative route to get all the carbon from glucose to acetyl-CoA
- Enzymes from the pentose phosphate system rearrange sugars to get all the C from glucose to acetyl-CoA
- 4C and 2C from 6C, not 3C and 3C
- 4C molecules can be rearranged with the other 4C molecules to make 6C again and cycle round
Outline hydrogen production in E.coli
- Hydrogen produced by mixed acid fermentation by the formate-hydrogen lyase (FHL) enzymes
- E.coli can be engineered to increase FHL activity and decrease competing pathways by knocking out other pathways and enzymes
- No succinate/lactose
Describe the FHL (formate hydrogen lyase) enzyme complex
- Membrane bound enzyme complex of different subunits
- Requires range of metal cofactors
- Fe-S clusters
- Molybdenum cofactors
- 21st amino ascid selenocysteine
- Insertion requires dedicated apparatus to get it in and is rate limiting
- hycA and fhlA mutants can increase levels of hydrogen over the wildtype but limit other pathways/products at higher levels
What type of fermentation does E.coli use and what does this produce?
E.coli uses mixed acid fermentation to produce acetate succinate, ethanol, H2 and CO2
Outline yeast fermentation of ethanol as done by saccharomyces cerevisae
- Saccharomyces cerevisae anaerobically ferments glucose to pyruvate by the embden meyerhoff pathway. Pyruvate is then converted to ethanol and CO2.
- 1 mol of glucose is converted to 2mol pyruvate
- Pyruvate is decarboxylated by pyruvate decarboxylase to aldehyde
- 2 mol ethanol is generated in a redox balanced pathways generating 2 ATP per glucose
How can yeast fermentation of ethanol be pushed towards glycerol production?
- Adding sodium sulfite
- complexes with acetayldehyde so it cannot act as an alternative electron accepted
- Flux is redirect from DHAP to glycerol
Describe bacterial fermentation of ethanol
- Anarobic - where there is no alternative electron acceptor (oxygen)
- Glucose (c-source) is converted to pyruvate
- Pyruvate formate lyase catalyses the non-oxidative cleavage of pyrvuate into CoA and formate
- 2ATPs and 2NADHs are produced through the embden-meyerhoff pathway
- Cells try to produce as much ATP as possible by using substrate level phosphorylation whilst reoxidising NADH (redox balancing)
What was the impact of Lonnie Ingram?
Engineered ssmall 2Mb genome of Z.mobilis to generate high yields of ethanol from a range of feedstocks
Describe Zymonas mobilis
- Waterborne bacterium
- 2mb genome
- Produces acetalydehyde, hydrogen sulfite
- Produces more thanol than yeast in the Entero-Deuderoff pathway for glycolysis
- 1ATP produced from glucose instead of 2 from EMP
- Need to convert more glucose to ethanol go get the same amount of ATP
Compare bacterial and yeast fermentation
- Z. mobilis produces more ethanol than yeast, easier to GM than yeast, uses Entero-douderoff pathway
- Saccharomyces produces more biomass than Z.mobilis, Embden-meyerhoff pathway
- Both tolerant to 12% ethanol
- Both limited to growth on lignocellulose as cannot ferment pentose sugars (D-xylose and L-arabinose)
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What was the aim of the Woodruff paper?
Open strain of E.coli to produce bioethanol: free from patents and usable fo academicand commercial processes
What is the process used by the Woodruff paper to generate an open strain of E.coli to produce bioethanol?
- Used ingrams technology to create and open-access strain (genes involved in ethanol production)
- More stable integrated onto the chromosome than on a plasmid. Used recombineering; generated a long dsDNA containing cloned PDC and adhB from Z. mobilis downsteam of the lac promoter, and a selectable marker bLac (b-lactamase) which confers resistance to ampicillin
- Used plate based assay to give proxy for ethanol production by looking for a red colour upon production of acetalydehyde when IPTG is added to reprepress plac.
–> Poor expression from this promoter when only a single copy
- Switched to a stronger promoter (PLlac0-1 ) from bacteriophage lambda using an engineered version. Used a engineered version of this promoter controlled by LacI.
- Evidence for ethanol production on the plate assay. Colour change same as seen with pLO1297 - also seen without IPTG addition. Suggests this is a mutant promoter with constituent expression.
–> LW02 strain: increased ethanol production
- Grew LW02 in batch fermentation in a medium good for ethanol production, saw yeilds of 16g/L ethanol (50% of theoretical yield)
- Measured others fermentation products - Lactate, succinate, formate, acetate
- Removed major competing pathways in mixed acid fermentation. Made mutants in lactate dehydrogenase, acetate kinase, endogenous aldehyde dehydrogenase (not redox balanced) and succinate oxidase
–> Saw a higher total yield of 28g/L of ethanol, and lower levels of the other fermation products. Not at 0 because enzymes just mutated, still productive as otehr genes driven by other promoters may still be neccessary.
Further optimisation: codon usage, promoter optimisation
