Team:IISER-Pune-India/TeamNotebook/Team Notebook 11e9906b405543a29361e38a50c81024/Meeting with Shams Yazdani 17438cb52b604d97a8552b5193fb5d2f

Meeting with Shams Yazdani

Meeting with Shams Yazdani

DescriptionPresented Idea and got feedback
HP sub-branch
Property 1
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Main stuff: 1. anaerobic pathway/compounds (why?: NADH balance, energy intensive(aerobic), more yield in anaerobic because in aerobic, carbon flux will end up increasing biomass more than the end product)

Why sucrose over glucose? cuz sucrose naturally produced as an osmo-protectant

Important to analyze our current reaction from glucose to succinate and check for the balance of electrons, ATP, NADH, carbon. Microbe will favour product formation when these are balanced.

Aerobic processes: better growth, ATP production but more carbon will go towards growth/biomass

Anaerobic processes: more NADH available/conserved(?), less energy intensive, higher efficiency, more carbon available for desired metabolite

Advantage of e coli: microaerobic conditions(in between aerobic and anaerobic) can be used where oxygen availability increases growth but doesn't hinder anaerobic pathways so it can be used for the production of metabolites. Anaerobic provides more yields of metabolites, oxygen provided by cyano will promote growth of E coli but will not hamper anaerobic pathways.

So we should look at literature for anaerobic succinate pathways and other compounds from anaerobic pathways.

Some options he suggested: lactic acid, PHB, butanol

He said he would send papers where he worked on butanol and has 2 strains of E.coli he could send us:

1. MG1655 based with complete butanol pathway 'in genome' but wasnt balanced so produces much more pyruvate (20 g/L) than butanol (3-4 g/L). Said it was marker/plasmid free?

2. E.coli B based strain where the electrons were balanced in the pathway and n-butanol was the main product.

Said max 2 interventions for cyano and 4/5 for E coli (3 knockouts and 2 plasmids)

E. coli is a good option - cuz robust and it wont die easily, even if its not growing, the cells will persist until it can grow again.

Don't worry about encapsulation for now as you will lose out on the benefits E coli might have on cyano growth due to the relieving of oxidative stress. You can control e coli growth with temp and he also said he's done a study where he stretched the doubling time of e coli to 8-10 hrs just by limiting glucose in the medium. So basically they are robust and can live as long as there is some continous sugar there, even if its less.

One option is to have cyano and e coli in separate tanks and take supernatant of one to the other. Here you can optimize by calculating the best possible flow rates, which could be different for both species. The advantage is you'll get high cell density (the cells will continue to grow in their separate tanks and the density will keep on rising) so high efficiency. He will send us models for modeling the flow rate. (we don't know if we really want to do this idea cuz we don't get the benefits of co-culture)

In independent cultures it is easier to control the volumes in order to control dilution, productivities, cell density, growth rates to maximise yields. We can balance flow rate to optimise transfer of sucrose from one tank to the other??

We don't need to immobilise cells if we are transferring the supernatant, you can make use of 0.22 micron tangential flow filtration?? by which you can recycle cells and continually increase cell density.

Continuous culture for co-culture might be difficult and we need to explore batch cultures. We need to look into the life cycles of both bacteria when it's in exponential growth phase/stationary phase etc. We need to decide at what stage of cyanobacteria growth would we have to add E coli for the co-culture.

Organic compounds can be easily extracted with solvent from supernatant? Mentioned dodecane for some reason? Said that you could continually preferentially extract metabolite over sucrose from the supernatant.

E coli is fine as a heterotroph, dont have to switch to yeast. Yeast only provides the added benefit that along with the metabolites it produces, its biomass is also useful in industry.

He sent 3 papers.

Team is now re-evaluating aerobic succinate production and will look into other final products