July-2021-Webinar

SynBio FSP monthly seminar date claimer and webex details-202107

 

 

[Image appears of Charlotte Williams talking on the main screen and then the image changes to show the Acknowledgement of Traditional Owners on the screen and participants can be seen in the bar at the top of the screen]

 

Charlotte Williams: I’m just going to wait for maybe one more minute just to give people an opportunity to dial in. I believe we’ve got quite a lot of registered participants, more than we have that are currently logged in. So, I’ll just wait for one more minute and then we shall start, thank you. Hi, welcome everyone. Thank you for joining in on this Synthetic Biology FSP seminar series, monthly seminar series, and today you’ll be hearing from our speakers from the Industrial Biotechnology domain.

 

[Image continues to show the Acknowledgement of Traditional Owners on the main screen and Charlotte can be seen inset in the participant bar talking]

 

So, before I begin I’d just like to remind you that this session is being recorded and it will be made available on the SynBio FSP website after this event. And as you can see on the slide that I’m sharing I’d like to commence by acknowledgement of the traditional owners of the land that we are meeting today. In Melbourne where I live and work it is the lands of the Wurundjeri people. The University of Adelaide, where Joel will be presenting from, recognises the Ghana people as the traditional custodians of the land. And the traditional owners of the land on which UQ is situated, where James is presenting from, are the Turrbal and Jagera people. So, before we begin the talks, I’ll just explain how the Q&A for each speaker will work.

 

[Image changes to show Charlotte talking on the main screen and participants can be seen inset in the participant bar at the top of the screen]

 

I’ll just stop sharing that slide. So, what we’re going to do, if you could please think of questions during each speaker’s session, and could you please post those questions via the Chat function, and make sure you include it marked to everyone. Ask your questions early. Don’t, don’t worry about waiting for other people to ask those questions. So, it’s going to be really good to kind of get that, that communication and feedback for the speakers, and also for internally within the FSP. The questions will be answered at the end of each presentation and I will ask the questions of the speaker. Any unanswered questions that we don’t get to will be directed to the speaker for response after the seminar.

 

As you already are, can I please request that everybody please stay on mute throughout the presentations. Now, before we hop onto the wonderful speakers, for those of you who don’t know me, my name is Charlotte Williams. I am the Application Domain Leader for Industrial Biotechnology within the CSIRO Synthetic Biology FSP. So, this domain bridges synthetic biology and chemical manufacturing, where we look to find new ways to develop and build sustainability in areas of high value added advanced manufacturing, in particular for small molecules. So, what does this mean in reality? It means that we’re using synthetic biology to produce high value chemicals for industry by finding new ways to make or use small molecules of high value for industries such as agriculture, health, and pharmaceuticals, and industrial feedstocks.

 

[Image continues to show Charlotte talking on the main screen and the participant bar can be seen at the top of the screen]

 

The speakers today both will talk about exciting projects, delivering impact and new technologies into the domain. So, the speakers that we have today, first up will be Joel Lee. Joel is a PhD student at the University of Adelaide, and he’s a CSIRO Synthetic Biology Future Science Platform Fellow. So, Joel will talk about “Enzymes and Bleach”. And before Joel gets started, I’ll just also introduce our second speaker, which will be James Heffernan. So, James is also a PhD student. He’s at UQ, at the Australian Institute of Bioengineering and Nanotechnology, and he’s also a CSIRO Synthetic Biology Future Science Platform Fellow. And James is going to be talking about “Heterologous System for Uncovering Transcriptional Architecture and Carbon Dioxide Fermentation”. So, that leaves me just to now introduce Joel and ask Joel to share his screen and kick us off with the first presentation for today’s series. Thank you Joel. Looks good.

 

[Image changes to show Joel talking in the participant bar at the top and a new slide on the main screen showing various diagrams on the left and text on the right: Enzymes and Bleach – Engineering Cytochrome P450 Peroxygenases for Final Chemical Synthesis, Joel Lee, Supervisors – A/Prof Stephen Bell, Dr Charlotte Williams, A/Prof Keith Shearwin]

 

Joel Lee: Thank you Charlotte. Hello everyone. Today, I am going to be talking about using enzymes and hydrogen peroxide or bleach an unlikely combination, in the context of synthesizing chemicals. Now, because I did a Chemistry major, I will lead up with an absolutely terrible joke about hydrogen peroxide or H202.

 

[Image changes to show Joel talking in the participant bar at the top and a new slide on the main screen appears showing four cartoons of two people at a bar ordering a drink, drinking, and then one person falling over and text appears: H₂O₂ is very bad for biological systems but generated in all oxygen breathing organisms, How to get rid of H₂O₂]

 

Namely, two people walk into a bar and one person orders H20, the other person orders H202, the second person “dies”. Now as terrible, terrible as this joke may be, it highlights something really, really important here is that, oh dear, hydrogen peroxide or H202 is really, really bad for biological systems. But it’s generated in all living organisms that breathe oxygen. So, the question is, how does Mother Nature get rid of hydrogen peroxide?

 

[Image changes to show Joel talking in the participant bar at the top and a new slide on the main screen appears showing the process of the biological removal of hydrogen peroxide and text: Biological removal of H₂O_2, Nature uses enzymes, H₂O₂ scavenger enzymes – Found in nearly all aerobic life, E.coli – Alkyl hydroperoxide reductase C, Catalase hydroperoxidase 1/11]

 

Well, nature uses enzymes, more specifically hydrogen peroxide scavenger enzymes that are found in nearly all aerobic life. These enzymes include catalases or hydroperoxide reductases that are able to convert hydrogen peroxide or peroxide species into harmless water.

 

[Image changes to show Joel talking in the participant bar at the top and a new slide on the main screen appears showing a diagram showing the biological removal of hydrogen peroxide and text appears: Biological Removal of H₂O_2,, Peroxygenases – Utilises H₂O_2, to activate C-H bonds]

 

However, there is another class of enzymes that I’m interested in that also uses hydrogen peroxide, are the peroxygenases. Peroxygenases are a heme iron containing enzymes that are able to bind one molecule of hydrogen peroxide, and use this hydrogen peroxide to oxidise carbon-hydrogen bonds into hydroxy species. In conventional chemistry, carbon-hydrogen bonds are very, very hard bonds to oxidise.

 

[Image changes to show Joel talking in the participant bar at the top and a new slide on the main screen appears showing various molecular structure diagrams on the right and text appears on the left: P450 Enzymes, Majority of P450s are heme monooxygenases, Catalyse the insertion of O₂ into C-H bonds, Electrons supplied by NADPH via electron transfer partners, Exist in all domains of life, Diverse substrate range and highly specific]

 

Another class of enzymes that are able to oxidise carbon hydrogen bonds are the P450s. Most P450s exist as heme monooxygenases where they can catalyse the insertion of ambient oxygen into carbon hydrogen bonds. The electrons that are needed by this reaction is supplied by NADPH [6.47] through the use of electron transit partner proteins. P450 enzymes exist in all domains of life, have a very, very diverse substrate range, and are very specific in what sort of substrates they can oxidise, and where they, where they can sub, where they can ox… and where they can oxidise on these substrates. For example camphor can be oxidised by a P450 almost exclusively at this position here to 5-exo-hydroxycamphor.

 

[Image changes to show Joel talking in the participant bar at the top and a new slide on the main screen appears showing a flow diagram of a P450 catalytic cycle and text appears on the right: Two electron transfer events normally, Heterolytic cleavage form active oxidant, Compound 1]

 

This is a normal P450 catalytic cycle. All you really need to pay attention to this is that there are two electron transfer events and that is coupled with the binding of an oxygen molecule to form this hydroperoxo species. Cleavage of this oxygen, oxygen species bond forms Compound 1 which is the active oxidant  in this cycle and Compound 1 is then free to oxidise any bound substrate.

 

[Image shows a circle appearing in the centre of the circular flow diagram and a peroxide shunt appearing across the circle and new text appears: P450 Peroxygenases – Only H₂O₂ needed for Compound 1 formation, Peroxide shunt is inefficient in most P450s]

 

However, some P450s can actually act as peroxygenases where they can bind hydrogen peroxide in this peroxide shunt pathway. Through the binding of one molecule of hydrogen peroxide you can bypass all these steps here to form this peroxo species. The peroxo species form the active oxidant, [8.03].

 

[Image changes to show Joel talking in the participant bar at the top and the circle inside the flow diagram disappears and new text appears on the right: No electron transfer needed, Bypasses the need for Electron transfer partner proteins, Expensive NADH/NADPH Co-factors]

 

Now, because you can bypass all these electron transfer steps, we don’t need any electron transfer when you’re using the peroxide shunt pathway and therefore you do not need to express and purify additional electron transfer partner proteins or use any expensive NADH/NADPH co-factors.

 

[Image changes to show Joel talking in the participant bar at the top and a new slide on the main screen appears showing various diagrams of P450 Peroxygenases and text appears: P450 Peroxygenases, CYP152 family – Natural H₂O₂ driven P450s, Oxidises Fatty-Acids, Capable of hydroxylation at alpha and or beta positions, Also demonstrated fatty acid decarboxylation]

 

Now in the wild, natural P450 peroxygenases mostly come from the CYP152 family. The CYP152 family primarily oxidise only fatty acids and they oxidise these fatty acids at the alpha and beta positions. Some of the CYP152 enzymes have also demonstrated decarboxylation of these fatty acids to alkenes, where alkenes are, have special interest in the generation of biofuels.

 

[Image changes to show Joel talking in the participant bar at the top and a new slide on the main screen appears showing crystal structure diagrams on the right and text heading and text appears: CYP199A4 from Rhodopseudomonas palustris, CYP199A4 binds and rapidly oxidises para-substituted benzoic acids, Different “R” groups readily available, Good model system to study P450 reactions]

 

So, in order, so natural P450 peroxygenases have a very, very narrow substrate range. And we want to expand their capability into other P450 systems and to do this we decide to use a  model system. And this model system is called CYP199A4. It is a bacterial P450 that can bind and rapidly oxidise para-substituted benzoic acids, where these benzoic acids are shown here. Functionality at the R position can be readily changed with, with relative ease, and very cheaply giving it access to a diverse range of substrates that can test, we can test with this with P450. We’ve also managed to generate crystal structures from this P450 and both these traits make it a good model system to study P450 reactions. So we want to study P450 peroxygenase reaction, we first need to convert our model system into a peroxygenase.

 

[Image changes to show Joel talking in the participant bar at the top and a new slide on the main screen appears showing various crystal diagrams and text appears: CYP199A4 from R. palustris, Substrate carboxylic acid near A245 believed critical for peroxygenase activity, T252 was mutated to glutamate residue (T252E)]

 

So, colleagues in Japan have studied natural P450 peroxygenases and they’ve identified when a natural P450 peroxygenase binds to a fatty acid, the carboxylic acid group of the fatty acid is held in a very close position to the heme centre. It is believed that the substrates carboxylic acid group is really, really important for peroxygenase activity where it acts as an acid base catalyst to stabilise interaction to the heme with the hydrogen peroxide molecule. A similar interaction occurs in normal P450s using, that, but it uses an alcohol group instead.

 

[Image shows another diagram appearing on the right showing a glutamate structure]

 

So, we decided to change this alcohol group in CYP199A4 into a glutamic acid residue.

 

[Image changes to show Joel talking in the participant bar at the top and a new slide on the main screen showing text: P450 Peroxygenases, T252 of CYP199AF was mutated to Glu residue (T252E), Comparing WT-CYP199A4 to T252E]

 

Initial protein engineering of this P450 was done by my colleague, Matthew Podgorski, where he converted residue Threonine 252 of CYP199A4 into a glutamic acid residue. He did it to generate mutant T252E. He then decided to test the peroxygenase activity of wild type CYP199A4 to this new T252E by chucking some hydrogen peroxide at it.

 

[Image changes to show Joel talking in the participant bar at the top and a new slide on the main screen appears showing a diagram of a graph on the left, and a flow chart on the right moving from veratric acid via CYP199A4-T252E to vanillic acid, and text appears: P450 Peroxygenases, T252 of CYP199A4 was mutated to Glu residue (T252E), Comparing WT-CYP199A4 to T252E, Both WT and T252D mutant showed peroxygenase activity, T252E mutant showed greater product formation using H₂O₂]

 

And surprisingly both wild type, and T252E did show peroxygenase activity, but the T252E nearly showed sixfold increase in product formation in the presence of hydrogen peroxide when compared to the wild type. This is a good indication that we did generate, or made it more, made it a more efficient peroxygenase through a simple protein engineering step.

 

[Image changes to show Joel talking in the participant bar at the top and a new slide on the main screen appears showing text: Research Aims, Can we turn P450 monooxygenases into more efficient peroxygenases for find chemical synthesis, Aims, Developing in situ methods of H₂O₂ generation for peroxygenase reactions, Immobilisation of P450 peroxygenases for flow chemistry, Understanding P450 peroxygenase activity using crystallography]

 

So the overarching theme today is can we turn P450 monooxygenases into more efficient peroxygenases in the context of synthesising chemicals. We’re going to look at three different things today is, first thing is to develop a method of generating hydrogen peroxides in situ in solution to drive peroxygenase activity. We’re going to immobilise some P450s on to solid surfaces for flow chemistry. And we’re also going to try and understand how P450 peroxygenase activity occurs within crystallography.

 

[Image changes to show Joel talking in the participant bar at the top and a new slide on the main screen appears showing a diagram on the right of the in situ hydrogen peroxide formulation and text appears: In situ H₂O₂ Formation, Chemical oxygen surrogates, urea hydrogen peroxide (UHP), Photocatalytic flavin systems]

 

So, the first thing is generating hydrogen peroxide in solution, or in situ. Then I’m going to talk about two methods today. The first method is the use of chemical oxygen surrogates. And the second method is to use photocatalytic or light-driven flavin systems. Both these approaches emphasize the slow release of hydrogen peroxide to drive P450 peroxygenase activity but also slow enough to mitigate heme damage to avoid deactivating the enzyme.

 

[Image shows a circle appearing around the molecular diagram and a photo of white powdery substance appears in a petri dish and new text appears: Slow release of H₂O₂ drives reactions and mitigates heme damage]

 

So, the first thing is to use an oxygen surrogate such as urea hydrogen peroxide, which is this molecule here. This consists of the urea molecule bound to hydrogen peroxide and exists as a solid where upon dissolution of the solid into water, it will slowly release hydrogen peroxide, and then hydrogen peroxide can then be used by the P450 for reactions.

 

[Image changes to show Joel talking in the participant bar at the top and a new slide on the main screen appears showing new text: Chemical Oxygen Surrogates, UHP as oxygen surrogate, Added as solid to CYP199A4-T252E (3UM), (mM to 64mM tested, Incubation 4 hr]

 

So, I took some solid UHP, added it to our enzyme which is our mutant[?] P450, and tested it over a range of different concentrations of UHP.

 

[Image shows a line graph appearing on the bottom left showing a steep incline and then decline]

 

We found that as the concentration of UHP increases so does the amount of product formation increases. So, this is a good indication that enough hydrogen peroxide was being generated by UHP to drive product formation.

 

[Image shows a new line graph appearing on the right of the screen showing a steep incline and then decline and text appears above the graph: UHP vs H₂O_2, More product formation over 4 h, Likely less damaging to heme centre]

 

I then decided to test the effects of UHP versus hydrogen peroxide. Well we thought that at the same concentration of UHP versus hydrogen peroxide, UHP was able to generate more product and it’s likely that UHP is less damaging to the heme centre.

 

[Image changes to show Joel talking in the participant bar at the top and a new slide on the main screen appears showing a glowing light bulb and below some highlighted flavin and non-highlighted flavin systems and text appears: Photocatalytic Flavin Systems, Visible light excites flavin to a high redox state, EDTA acts as electron/hydrogen donor to generate reduced form, Reduced flavin reacts with oxygen generates H₂O₂]

 

Next, we’re going to look at light driven flavin systems, where flavins are these yellow coloured compounds that are light harvesting molecules where upon exposure to light at 440 nanometres it enters an excited state. This excited state allows it to accept electrons from EDTA to enter a reduced flavin state. This reduced flavin state is highly unstable and will then react with ambient oxygen to form hydrogen peroxide. This hydrogen peroxide is now then free to react with our P450.

 

[Image changes to show Joel talking in the participant bar at the top and a new slide on the main screen appears showing a glowing light bulb, a vial of coloured yellow liquid, and a photo of a table lamp shining on vials of liquid on a  machine]

 

So, essentially I got a clear vial that contains our mutant P450 peroxygenase. It contains, and they added all the basic, all the components of our light driven system and basically shone a light, shine a light, table lamp on our reaction.

 

[Image shows a line graph appearing on the slide in place of the photo and the graph shows a steep incline and then decline]

 

This relatively unsophisticated setup did generate product in the presence of light. This is a really, really good indication that our light driven system was generating enough hydrogen peroxide to drive P450 peroxygenase activity.

 

[Image changes to show Joel talking in the participant bar at the top and a new slide on the main screen appears showing molecular flow diagrams and text appears: Photocatalytic Flavin Systems, CYP152A1 with light-driven system]

 

So, we then decided to test this with a natural P450 peroxygenase namely CYP152A1. CYP152A1, once again primarily oxidises fatty acids and a 14 carbon fatty acid here, at the alpha and beta positions.

 

[Image shows a line graph appearing on the right of the screen and text appears above: CYP152A1 (5uM) + Flavin Mononucleotide (200 uM) +EDTA (1mm) + Myristic acid (C14, 200 uM)]

 

I took this P450, added it to a, our light-driven system along with some 14 carbon Myristic Acid and GCMS analysis revealed that there was product formation occurring using this light driven system.

 

[Image shows diagrams of the process of making shorter chain fatty acids appearing on the left of the graph]

 

But surprisingly short chain, shorter chain fatty acids was also present in increased amounts, or they just also appeared as products alongside their oxidation products. We believe a chain shortening reaction was occurring where Myristic Acid can be oxidised by our P450 through multiple steps to generate this diol product. This diol product is highly reactive and will form this keto acid product. This keto acid product can then be oxidised by hydrogen peroxide, meanwhile to decarboxylate to form a shorter chain fatty acid. This is a really good indication that our light driven system was generating enough hydrogen peroxide to drive these multiple oxidation steps, but also slow enough that it wasn’t damaging the enzyme.

 

[Image changes to show Joel talking in the participant bar at the top and a new slide on the main screen appears showing text: Enzyme Immobilisation, Peroxygenase Enzymes only require H₂O₂ for activity – CYP199A4-T252E (Mutant Peroxygenase), CYP152A1 (Natural Peroxygenase)]

 

Next we’re going to look at immobilising enzymes on to solid surfaces. So, P450 peroxygenases only require hydrogen peroxide for activity.

 

[Image shows a diagram at the bottom showing a P450 peroxygenase and a solid bead side by side and joined by a line]

 

So, if we got some of our peroxygenases, and the idea is we immobilise it onto a solid bead.

 

[Image changes to show Joel talking in the participant bar at the top and a new slide on the main screen appears showing photos of silica, polystyrene and cellulose beads on the right and text appears: Enzyme Immobilisation, Short polypeptide tags, Allow binding to solid beads]

 

So, this work was done in collaboration with Dr Colin Scott at the CSIRO Biocatalysis team where they identified short polypeptide tags that can bind to beads of silica, polystyrene, or cellulose.

 

[Image changes to show a new slide on the main screen showing P450 linked to a bead, and then a chromatography column with beads and P450 inside and text appears: Enzyme immobilisation]

 

The idea is we get some of these beads, stick some P450 on to them, and get a whole bunch of these, and fill an empty chromatography column and from one end we can flow substrate and hydrogen peroxide, and what comes out is our products.

 

[Image changes to show Joel talking in the participant bar at the top and a new slide on the main screen appears showing a photo of test tubes with silica linked to another photo of test tubes with silica and CYP152A1 and then linked to a drawing of P450 joined to a bead, and text appears: Enzyme Immobilisation, Washed with buffer twice, Myristic acid (C14 Substrate) and 5 mM H₂O₂ added] 

 

So, what I’ve done so far with this is I got some silica. I expressed and purified a P450 that is able to bind a silica, mix it with the silica, and incubate it for a while to generate beads that have P450 on them. I washed these beads with buffer twice and added it, and added to these beads some Myristic acid, as substrate, and some hydrogen peroxide to drive reactions.

 

[Image changes to show Joel talking in the participant bar at the top and a new slide on the main screen appears showing a line graph showing the GCS Analysis and text appears: Enzyme Immobilisation, Tested with free silica, Incubated for 24 h at 4 degrees C, Some product formation observed]

 

GCMS analysis revealed luckily that these beads were able to generate some product especially compared to beads that have not been functionalised with P450. This is a really good indication that I’ve managed to functionalise some P450s onto these beads, and we’re planning to optimise this further and hopefully scale it up further to do more flow chemistry stuff.

 

[Image changes to show Joel talking in the participant bar at the top and a new slide on the main screen appears showing a photo of a CYP199A4-T252 co-crystallised with substrate, and a diagram of 4-methoxybenzoic acid bound CYP199A4-T252E and text appears above: In Crystallo Enzymatic Reactions, P450 Peroxygenases, Only require H₂O₂ for catalytic activity]

 

Now, I’m going to look at studying P450 reactions using crystallography. So, once again P450 peroxygenases only require hydrogen peroxide for catalytic activity.

 

[Image shows an arrow showing hydrogen peroxide being added to the photo]

 

So, what happens, if I got a crystal of a P450 peroxygenase that has some substrate in it, and add some hydrogen peroxide on it.

 

[Image changes to show Joel talking in the participant bar at the top and a new slide on the main screen appears showing diagrams of the process of the crystal being soaked and the resulting crystal structure beneath and text appears: In Crystallo Enzymatic Studies, CYP199A4-T252E with 4-methoxybenzoic acid, Soaked in 4mM H₂O₂ for 5 mins]

 

So, I have a crystal of our mutant P450 peroxygenase CYP199A4-T252E that has been crystallised with 4-methoxybenzoic acid. I added it. I soaked this crystal with some hydrogen peroxide for a few minutes and collected diffraction data at the Australian Synchrotron and solved the crystal structure.

 

[Image shows another crystal structure which has not been soaked with hydrogen peroxide appearing on the left]

 

If we were to compare this crystal structure of a crystal that’s been soaked with hydrogen peroxide, and compare it to a crystal that has not been soaked with hydrogen peroxide, you can see a distinct loss of electron density going from here to here. We believe a demethylation reaction has occurred within the crystallised enzyme where essentially I got a solid chunk of crystallised enzyme, chucked some hydrogen peroxide on it, and reaction has occurred within the crystallised enzyme.

 

[Image changes to show Joel talking in the participant bar at the top and a new slide on the main screen appears showing a flow chart of how Rational Design and its processes and Directed Evolution and its processes work together to create an Improved Enzyme and text appears: Screening Systems?, An efficient screening method is essential]

 

So finally, I’m just going to talk about some of the work my lab has been doing recently, is well, so a lot of the proteins we work with were designed rationally to introduce or improve P450 peroxygenase activity. Another approach in protein engineering that can be used to improve peroxygenase activity is the use of directed evolution where we generate a very very large library of mutated, mutated P450 genes, and select for the best P450 peroxygenase activity.

 

[Image shows a red line appearing around the “Screening and selection” on the Directed Evolution side of the flow chart]

 

To do this however, we need a really, really efficient screening method, screening method. And the approach we decided to generate an efficient screening method is to use whole cell systems.

 

[Image changes to show Joel talking in the participant bar at the top and a new slide on the main screen appears showing a photo of Alecia Gee and text appears: E.coli Knockout Strains, Escherichia coli has multiple H₂O₂ scavenger enzymes, 2 H₂O₂ Catalase 2 H₂O + O_2, R-O-OH + H+ + NADH hydroperoxide reductase R-OH + H₂O + NAD+]

 

This work was done by Alecia Gee. She’s an Honours student in my lab, in collaboration with Professor Keith Shearwin, and Dr. Andrew Hao, at the University of Adelaide, where because E.coli has multiple hydrogen peroxide scavenger enzymes, can we get rid of some of them. So, Alecia decided to use λ red recombinase from bacteriophage λ to remove H₂O₂ scavenger enzymes genes from the genome of E.coli, where the recombinase is capable of swapping between two genes of interest that are flanked by regions of homology.

 

[Image changes to show Joel talking in the participant bar at the top and a new slide on the main screen appears showing diagrams of the process of knocking out the H₂O₂ scavengers and transforming into a whole cell peroxygenase, and text appears: E.coli Knockout Strains, Use lambda red recombinase to excise H₂O₂ scavenger genes from E.coli genome, Transform peroxygenase P450s into these E.coli, Potentially increase H₂O₂ levels in E.coli for whole-cell peroxygenase activity]

 

Once the H₂O₂ scavenger enzymes were removed from the E.coli genome, we will transform peroxygenase P450s into these E.coli, where the increased levels of hydrogen peroxide in these E.coli knockout strains, would be able to drive whole-cell peroxygenase activity.

 

[Image shows a diagram appearing on the right to show the process of producing 4-Nitrophenol Yellow, and a photo appears on the right of two beakers of liquid]

 

We can then use these transformed, transformed E.coli for colorimetric assays. For example, our cells with a P450, that can… we have cells that with a P450 that can convert colourless 4-Nitroanisole to yellow coloured Nitrophenol. We are currently trying to apply these whole-cell strains to other P450 systems and generate more colorimetric assays to hopefully do directed evolution studies in the future.

 

[Image changes to show Joel talking in the participant bar at the top and a new slide on the main screen appears showing a photo of a cell at the top and a molecular structure diagram beneath and text appears: Conclusions and Future Work, In crystallo enzymatic reaction, Successfully showed enzymatic reaction in a crystallised enzyme, Testing other substrates currently underway, Oxygen surrogates and light-driven flavin systems, Capable of generating H₂O₂ to allow substrate conversion, Further Optimisation of these systems to increase substrate conversion and scale-up, Test other P450 systems]

 

So in conclusion, I’ve shown that an enzymatic reaction was able to occur within a crystallised enzyme. We’re testing this with other substrates, hopefully. It’s hopefully trapped more product, or more potentially reactions intermediates. I’ve tested two different ways of generating hydrogen peroxide in the solution and we’ll try and further optimise this further to increase substrate conversion, and also test other P450 systems.

 

[Image changes to show Joel talking in the participant bar at the top and a new slide on the main screen appears showing a photo of Jinia Akter inset and a line graph below and text appears: Conclusions and Future Directions, CYP116, Found in a hot spring in Indonesia, Hydroxylates phenaylacetic acids, Highly thermostable enzyme]

 

For example we currently identified a thermostable P450 that is able to convert a fair amount of product, a fair amount of substrate into product with only one micromolar of P450 and some hydrogen peroxide.

 

[Image changes to show Joel talking in the participant bar at the top and a new slide on the main screen appears showing diagrams of enzyme immobilisation and whole cell screening and text appears: Conclusions and Future Work, Enzyme Immobilisation, Peroxygenase enzymes have been designed to have short polypeptide tags to bind silica beads, Peroxygenase activity on beads were present and further optimisation underway, Whole Cell Screening Systems, Knockout strains successfully made, Colorimetric assays are being developed for further whole cell studies]

 

We’ve also successfully immobilised peroxygenase enzymes onto silica beads, and we’ve got to test this further to hopefully scale this up for flow chemistry reactions. We’ve also generated knockout strains that have their H₂O₂ scavenger enzymes removed from their genome and we’re currently developing colorimetric assays to, for further study, and hopefully do directed evolution in the future.

 

[Image changes to show Joel talking in the participant bar at the top and a new slide on the main screen appears showing the University of Adelaide and CSIRO logos and acknowledgements of the FSP scholarship and various personnel involved]

 

This work was supported by the CSIRO, the SynBio Future Science Platform. I’d like to thank Stephen Bell for being an endless source of ideas, Dr Charlotte Williams my co-supervisor for this opportunity to give a talk to the CSIRO community, and also for a lot of input and feedback through the enzyme immobilisation work. Keith Shearwin and Andrew Hao for the whole-cell screening work. Dr Colin Scott and Raquel Rocha from the CSIRO Biocatalysis team for their input in the immobilisation work. Carol Hartley and Annette from CSIRO for, for some input on the whole- cell work. Dr John Bruning’s group at the University of Adelaide for teaching the dark art of crystallography, and all the Bell Group members past and present. Thank you.

 

[Image changes to show Charlotte in the participant bar at the top of the screen and the main screen shows a blank black screen]

 

Charlotte Williams: Wonderful. Thank you Joel. That was, that was great timing, and a great kind of… yeah people are clapping. Thank you. Thank you everyone.

 

[Image changes to show Charlotte talking on the main screen and Joel can be seen listening in the participant bar at the top of the screen and then on the main screen]

 

Joel there’s a couple of questions that, that occurred to me which I’ll just go through while other people are thinking.

 

[Image changes to show Charlotte talking on the main screen and Joel can be seen listening in the participant bar at the top of the screen]

 

And if you, if you think of questions while I’m asking Joel my questions, feel free to pop them in the Chat labelled to everyone. So, Joel the first thing that I was thinking of is in the flavin catalysed reaction, is that dependent on the concentration of flavin that you use, and do you have to optimise that?

 

[Image changes to show Joel talking on the main screen and Charlotte can be seen listening in the participant bar at the top of the screen]

 

Joel Lee: So far, so far yes, it’s dependent on concentration of flavins. So, one of the reactions I’ve had, we only used  40 micromolar of flavin and only got about 40% conversion from substrate to product. But when I upped it to 200 micromolar flavin, it looks like I have full conversion of substrate to products. So, there’s some optimising, so it is probably dependent on the flavin.

 

[Image changes to show Charlotte talking on the main screen and Joel can be seen listening in the participant bar at the top of the screen]

 

Charlotte Williams: Yeah, and so there’s obviously some optimisation that you’ve done in getting the concentration right so there’s enough there but not too much. I don’t know if it does any self-quenching or anything like that.

 

[Image changes to show Joel talking on the main screen and Charlotte can be seen listening in the participant bar at the top of the screen]

 

Joel Lee: Yep, so far we, that’s still on the way.

 

[Image changes to show Charlotte talking on the main screen and Joel can be seen listening in the participant bar at the top of the screen]

 

Charlotte Williams: OK, yeah good. But that’s good. That’s obviously something that, that, that you’re thinking through. The other thing that occurred to me was that we’ve talked about this before with that chain shortening that you see, in those chain shortening events, did you see that happening, or have you had the chance to look at that happening on other fatty acids other than Myristic acid?

 

[Image changes to show Joel talking on the main screen and Charlotte can be seen listening in the participant bar at the top of the screen]

 

Joel Lee: Yeah. I’ve tested fatty acids from C10 to C18. So, 10 carbon fatty acids, 18 carbon fatty acids. We find that from 15 downwards we start seeing chain shortening reactions. Fifteen upward you don’t see any chain shortening reactions.

 

[Image changes to show Charlotte talking on the main screen and Joel can be seen listening in the participant bar at the top of the screen]

 

Charlotte Williams: Right that’s really interesting, and is that because presumably you’re not getting that diol?

 

[Image changes to show Joel talking on the main screen and Charlotte can be seen listening in the participant bar at the top of the screen]

 

Joel Lee: Yes, it’s also presumably the, the larger chain fatty acids probably can’t fit in the active site.

 

[Image changes to show Charlotte talking on the main screen and Joel can be seen listening in the participant bar at the top of the screen]

 

Charlotte Williams: Oh sure, yep, yep, yep. That makes sense. I know… I’ve got another question around those Myristic acid reactions Joel. And when you… how do you characterise the, the different isomer between the alpha and beta?

 

[Image changes to show Joel talking on the main screen and Charlotte can be seen listening in the participant bar at the top of the screen]

 

Joel Lee: At this, at this stage in time it was doing mass, the mass spectrum, the mass spectrum. We have a database on GCMS that has a pretty comprehensive database of the different alpha and beta fatty acids and we compared it to the mass spectrum of the database and it seems to be the alpha and beta variant.

 

[Image changes to show Charlotte talking on the main screen and Joel can be seen listening in the participant bar at the top of the screen]

 

Charlotte Williams: Yep, OK. So, when you do the reaction when you’ve immobilised on beads, do you just take some of the kind of supernatant and put it in the [25:24]?

 

[Image changes to show Joel talking on the main screen and Charlotte can be seen listening in the participant bar at the top of the screen]

 

Joel Lee: We spun the reaction down to the supernatant, and basically, and then analyse the supernatant using GCMS, yes.

 

[Image changes to show Charlotte talking on the main screen and Joel can be seen listening in the participant bar at the top of the screen]

 

Charlotte Williams: Great yep. That sounds fantastic. So, if anyone else comes up with any further questions, oh we’ve just had one drop in. Thank you Ian. So, he’s saying if the P450 is immobilised, can it be reused, or is the hydrogen peroxide too damaging.

 

[Image changes to show Joel talking on the main screen and Charlotte can be seen listening in the participant bar at the top of the screen]

 

Joel Lee: I’m going to make a confession here, that work was done two weeks ago. So, it’s not super optimised yet so we haven’t really tested whether or not it can be used or not but my incubation period was about 24 hours. So, we’re likely next time to do a time course experiment to see if there’s a point where the H₂O₂ will damage the, the bound enzyme.

 

[Image changes to show Charlotte talking on the main screen and Joel can be seen listening in the participant bar at the top of the screen]

 

Charlotte Williams: Yeah, thanks Joel and it would be good to have a look at the lifetime as well of the immobilised enzymes and see if you can… one of the things that, that we looked at was if you could actually put them in the fridge for a while and take them back out and do they still work. But there’s a lot of work involved in looking at stability. Thank you Joel. That was absolutely wonderful. Fantastic presentation and if anyone has any further questions, they’ll be sent through to, to Joel afterwards. Right, so Joel if you want to stop sharing your screen that would be fabulous.

 

[Image changes to show Joel talking on the main screen and Charlotte can be seen listening in the participant bar at the top of the screen]

 

Joel Lee: OK I can do that. Has that stopped sharing?

 

[Image changes to show Charlotte talking on the main screen and Joel and James can be seen listening in the participant bar at the top of the screen]

 

Charlotte Williams: Yep, let me get back to where you are. That’s beautiful. Thank you. You have done. That’s wonderful.

 

[Image flicks to show Joel listening on the main screen and then the image changes to show Charlotte talking on the main screen again]

 

So, I’ll leave it to James. James do you want to… here we go, coming on.

 

[Image changes to show a new slide on the main screen and James can be seen inset in the participant bar listening and text appears on the slide: Developing a heterologous system for uncovering transcriptional architecture in Clostridium autoethanogenum and progressing CO₂ fermentation, James Heffernan]

 

Now, yes that’s in the right mode and you’re good to go. Thank you James, take it away.

 

[Image shows the same slide appearing on the main screen and James can be seen talking in the participant bar at the top of the screen]

 

James Heffernan: Thank you Charlotte. Hi, everyone. So today I’m going to be covering a little bit of the work that we’ve been doing with Clostridium autoethanogenum  the [27.21] layer.

 

[Image changes to show a new slide on the main screen and James can be seen inset in the participant bar talking and text appears on the slide: Gas fermentation background, A heterologous system for a TetR family protein, Development of CO₂ fermentation]

 

So just quickly, I’ll run through a bit of background as I assume gas fermentation is not everyone’s specialty and then we’ll tap into the work with TetR and to CO_2.

 

[Image changes to show a new slide on the main screen showing a diagram of the process of gas fermentation and James can be seen inset in the participant bar talking and text appears on the slide: Industrial C1 recycling – Purpose, Gas fermentation is useful for converting C1 into C2+]

 

So gas fermentation is quite useful for converting C1 carbons into C2 or more carbons. So that typically means we can use waste streams such as here on the left and some of these might require a wee bit of pre-processing to get the composition of the gas stream to a nice specification and then we can send that onto a gas fermentation, or gas fermenter with an autotrophic microbe inside. These are often called acetogens because they can make Acetyl-CoA from C1 compounds and then after the gas fermentation we have some potential downstream processes to either purify or elongate the chains.

 

[Image changes to show a new slide on the main screen showing a bar graph comparing power and ethanol and James can be seen inset in the participant bar talking and text appears on the slide: Industrial C1 recycling – Value, This can have economic and environmental benefits, LanzaTech Business Case – Providing 2x more returns from ethanol than from electricity]

 

And so we worked quite closely with an industrial partner called LanzaTech, who are the main company involved with this sector and their initial value proposition was being able to generate more profit out of converting a high CO or a high carbon monoxide steel mill off gas into ethanol rather than burning that CO to generate electricity.

 

[Image shows a diagram appearing on the right of the slide showing a circular economy type diagram of the industrial C1 recycling]

 

But obviously, as that has continued there’s been more and more of an environmental benefit arising from that with the ability to get those C1 compounds into the bioeconomy.

 

[Image changes to show a new slide on the main screen showing a diagram of LanzaTech’s technology development on the right and James can be seen inset in the participant bar talking and text appears on the left of the slide: Industrial C1 recycling – Technology, LanzaTech’s first commercial scale production facility started operation in 2018, and they recently received their 1,000^th patent, Marcellin group has been working with them since ~2013, focussing on developing systems biology tools]

 

So LanzaTech recently commercialised their first industrial scale operation and along the way, this has led to a lot of technology development, as we can see here. This is just a very brief overview of a lot of their work, but they’ve also had a lot of partners along the way and we have largely been involved with the system’s biology development of the Clostridium autoethanogenum at the Marcellin Group.

 

[Image shows a diagram appearing on the right of the slide labelled “Absolute proteome” and text appears on the right: Still>10% of proteome unknown to us…]

 

But even though there’s been a heap of technology development, there’s still a lot of unknown genes involved in metabolism quite highly expressed, so there’s still quite a lot of interesting things to uncover.

 

[Image shows the diagram on the right changing to show a diagram of a thermodynamic based model and new text appears on the left: With a completed GEM and various multi-omics characterisations we are now working on understanding thermodynamic limitations and developing kinetic models]

 

And now our group is largely focusing on developing thermodynamic-based models to try and explain aspects of the metabolism.

 

[Image changes to show a new slide on the main screen showing diagrams of “Top-3 promoter motifs for syngas” on the right, and James can be seen inset in the participant bar talking, and text appears on the slide: TetR – a sigma factor?, We discovered a novel promotor motif among the most common motifs for autotrophic growth]

 

So into the more synthetic biology end of the work we’re doing. So, recently we’ve discovered a novel promoter motif involved with autotrophic metabolism or involved in the promotors of genes with, that are involved in autotrophic metabolism.

 

[Image shows the diagram on the right changing to show the Wood-Ljungdahl pathway diagram, and new text appears on the left: This motif was also linked to key enzymes of autotrophic growth, And found to be common in other Clostridium species]

 

And as you can see here, this is largely to do with the Wood-Ljungdahl pathway shown here, which is the main autotrophic pathway taking C1 molecules through to Acetyl-CoA but also involved in other aspects of energy metabolism and these are, can be quite distilled from each other throughout the genome. And this motif was also found in other species of Clostridium and other acetogenic species outside of the Clostridium genus.

 

[Image shows a new diagram appearing on the right of the screen, and new text appears on the left: Found TetR family protein using pull-downs and proteomics, But it acted as a promoter, similar to “housekeeping” sigma factor]

 

So through some pull-downs, some proteomics and then add synthetic gene-circuit with those genes of interest, they eventually identified some, an interesting interaction between the promotor motif  and TetR and that interaction was or had a similar effect as with the native housekeeping sigma factor within Clostridium autoethanogenum  and what was interesting was that this TetR family protein was actually acting as a promotor in this situation.

 

[Image changes to show a new slide on the main screen showing two circles showing a genetic circuit – lac inducible system, and a Reporter gene – green fluorescence protein, and James can be seen inset in the participant bar talking, and text appears on the slide: TetR – make it better?, Two-plasmide genetic circuit in E.coli, Easily inducible and detectable]

 

So where, if we had identified TetR as a Sigma factor, we then thought, can we improve the interaction between the sigma factor and the promotor motif. So to do so in E.coli would be quite an arduous undertaking due to a lot of limitations, mainly with growth but also just with a model method metabolic toolbox. So as was done with the previous fluorescence OD graph, we wanted to use a synthetic circuit in E.coli to try and get a better understanding of the interaction. So to do so, we’re just going to use quite a simple lac operon inducible system where we express TetR, that TetR interacts with our novel promotor motif to express GFP.

 

[Image shows a line graph appearing on the slide in place of the diagram, and new text appears on the left: Quantitative and reproducible]

 

And we’d seen that this was a nice and quantifiable interaction, so you can see here the difference between those two blue lines, which is the kind of modification we can obtain when we have an induced and non-induced growth profile.

 

[Image shows a sequence table appearing at the bottom of the slide, and new text appearing on the left: Mutations to the motif showed sensitivity of interaction]

 

And then, after we had that, we then looked at the effect of a few mutations and we could see that, for instance there was an 85 base pair untranslated region that seemed to have quite a limited effect on the fluorescence of OD, compared to our 130 or native promotor sequence upstream of a gene of interest and then when we mutate the, some of those key base pairs involved with the motif, we see quite a rapid reduction or quite a large reduction in the interaction between TetR and that motif.

 

[Image shows the Wood-Ljungdahl pathway appearing in the top right of the same slide]

 

So we could have continued with this and looked at just optimising metabolism based on a sort of gene of interest, mutations to the promotor motif, but we were a bit more interested in seeing if we could engineer TetR itself to try and have more of a global optimisation of the autotrophic metabolism.

 

[Image changes to show a new slide on the main screen showing a flow diagram on the right, and text on the left: TetR – make it better?, To assist high-throughput (HTP) methods, we wanted to integrate the system into the genome of E. coli., First try failed using methods from Jiang et al (2019), but we will try again with new sets of gRNAs, Develop gRNA library based on homology modelling conducted by Dr. Andrew Warden, ClonePix enables HTP screening of colonies, 90-well plate makes further quantification fast]

 

So to do this, we’re aiming to get an… better system integrated into the genome of E.coli so that we have a more robust system or high throughput, for using high throughput methods. We had our first go at trying to integrate that just using a pretty standard CRISPR method but we were unsuccessful initially, so we’ll be trying that again shortly with new sets of gRNAs but following that we’ve then been working with Dr. Andrew Warden to develop some homology models that can help assist us with the development of our gRNA libraries which can then be used for some pooled cloning and editing and we can then use our high throughput methods such as ClonePix for rapid, either selection or identification, of those interesting mutations which can then be quantified again with 96-well growth curves.

 

[Image changes to show a new slide on the main screen showing a flow diagram of the process using CRISPRi technology, and text appears on the right: TetR – what are its metabolic effects?, LanzaTech used CRISPRi on various genes of interest to understand their metabolic role, Small-scale bottle fermentations showed no phenotypic difference when TetR was knocked-down]

 

So just a brief comment on the kind of metabolic effects of TetR. The LanzaTech had developed a TetR knockdown using CRISPRi

 

[Image show a new diagram appearing on the right of the slide showing the knockdown and control results, and text appears on the left: We validated the knock-down with RT-qPCR]

 

and we were then tasked with showing the transcriptional effect of this knockdown using RT-qPCR and what was quite interesting was although we could see quite a clear transcriptional effect, there was no phenotypic difference between the knock-down and the control strains. So as we, as I’ve kind of mentioned we don’t know a lot about the transcriptional architecture of C, Clostridium autoethanogenum  and potentially there’s quite a bit more to understand.

 

[Image changes to show a new slide on the main screen showing the Wood-Ljungdahl pathway diagram in the top right, and James can be seen talking in the participant bar at the top, and text appears: TetR – summary, We aimed to develop a heterologous/synthetic gene circuit to rationally engineer TetR and optimise autotrophic metabolism of C. autoethanogenum]

 

So in summary of that TetR work, so we’ve aimed to develop a heterologous system to enable rapid engineering of TetR and eventually try and optimise the autotrophic metabolism of Clostridium autoethanogenum.

 

[Image shows a diagram appearing below showing the C1 fixation in orange, and new text appears: If TetR doesn’t have a strong phenotypic effect, are there other transcriptional factors that could and can they be engineered?, We aimed to make this tool adaptable for future use]

 

However, there’s a lot of things left to look into, both with the TetR, and also in just the broader transcriptional architecture of Clostridium autoethanogenum. So a key aspect of this was trying to develop the tool in such a way that we could use it in the future for whatever kind of other transcription factors might pop up that are interesting.

 

[Image changes to show a new slide on the main screen showing a diagram of a flux balance, and text appears: Improving CO₂ fermentation by C. autoethanogenum, When fermenting CO₂ with H₂ as energy source, steady growth in CSTRs is difficult, Flux balance analysis suggested a limitation of reduced ferredoxin]

 

So now, moving onto a bit more of the biotech focused, application focused area. So typically, or the standard gas composition for industrial fermentation would normally be a relatively high CO gas so when fermenting CO₂ and hydrogen, CO₂  with hydrogen we come across a similar difficulty. Now I want to stress here, that we are working with continuous bioreactor systems as these are more similar to the style of fermentation at industry scale so there’s a, the technology is a little bit more transferable.

 

[Image shows a graph appearing on the right or the slide, and new text appears on the left: Addition of 2% CO into gas mix improves – Growth, Gas, Production]

 

So with some initial data from our CO₂ hydrogen experiment, we could see that there was quite a limitation of the reduced ferredoxin as, when compared to our normal CO₂ fermentations. So what we wanted to see was if we could help growth or just help the performance of the fermentation by just adding a small amount of CO into the mixture and, quite impressively, we saw greater than double biomass concentration when we did so and we were also able to get to twice as fast growth so here in a continuous system with dilution rate would be equal to the growth rate at steady state.

 

[Image shows a new bar graph appearing on the right of the slide, and James can be seen talking in the participant bar at the top of the screen, and new text appears: Further details in DOI – 10.3389/fbioe.2020.00204]

 

And in that, those same fermentations we also saw interestingly an increase in hydrogen uptake, specific hydrogen uptake and this is quite notable considering CO is a known inhibitor of hydrogenators and we could also see no reduction in the CO₂ uptake which was important as well. And then in the production we also saw an increase in the ethanol product distribution which was quite good as that’s a more valuable product for us. And if you’re interested in a bit more of the read-ups, explanation of all of that, at least check out our paper on that.

 

[Image shows James talking inset in the participant bar at the top of the screen, and a new slide appears showing three line graphs on the right, and text appears: Improving C. autoethanogenum for CO₂ fermentation, Aim – use adaptive laboratory evolution (ALE) to improve the growth of C. autoethanogenum while maintaining ethanol production, We decided to use the gas mix with 2% CO to do so]

 

And then, we’ve also been working on improving Clostridium autoethanogenum for CO₂ fermentation. And to do so we’ve been focusing on using adaptive laboratory evolution primarily because as I’ve mentioned there’s a lot going on in the metabolism that we’re yet to understand, so this is a wee bit more broader purpose method for trying to do so. And a key thing we wanted to try and do was maintain our ethanol production, as well, because that is relatively hard to do and our ethanol concentrations were quite high compared to literature. So we decided to do this with the 2% CO mix as there was some growth profile or the growth profile of the 2% CO mix looked to be a bit more like what we wanted the CO₂ hydrogen to look like. So we can see just the steeper graph profile there, which is good.

 

[Image shows a new data point graph appearing on the right of the screen, and text appears on the left: The culture quickly improved growth in bottles]

 

And when we did so just by routinely passaging over time, we see biode…, decrease in the, or I should say an increase in the growth rate and a decrease in the doubling time. The first couple are probably a little bit exaggerated there, the first couple of data points. We’re probably more likely going from around a 24-hour doubling time to a 12-hour doubling time.

 

[Image shows a new bar graph appearing on the right, and new text appears: Bioreactor growth (CO₂/H₂) has shown a ~40% increase in growth rate, while at the same conditions the product distribution was maintained (initial data)]

 

And then, when we reintroduced this strain back into a bioreactor with CO₂ hydrogen, so without that 2% CO. We found that the new strain was able to grow approximately 40% faster which was pretty great and also that we had closely maintained the production profile.

 

[Image shows two new diagrams appearing on the right of the screen]

 

And this largely agreed with what we were kind of expecting from the theoretical stoichiometry, as typically if you’re fermenting CO₂ hydrogen you’re going to yield more ATP per hydrogen when you’re making acetate compared to ethanol, so we were kind of expecting a little bit of a shift in that production profile. But it was pretty minimal, so that was cool to see.

 

[Image shows James talking inset in the participant bar at the top of the screen, and a new slide appears showing a diagram of the gas fermentation process on the right, and text appears on the left: CO₂ fermentation summary, Fermenting CO₂ with C. autoethanogenum was improved via the gas composition and adaptive laboratory evolution, Each has their own benefits and caveats, CO₂ is a feedstock that should be targeted as an opportunity for the bioeconomy, There are various ways that CO₂ fermentation can be improved and adapted to an application]

 

And just a quick summary of the CO₂ fermentation. So we’ve improved autoethanogenum CO₂ fermentation using two different methods, with gas optimisation and the adaptive laboratory evolution. And each has their own benefits and caveats. Just, I guess, as a personal opinion more so I think, CO₂  is quite an interesting feedstock for the future of the bioeconomy and it should really be targeted. And we’ve kind of shown here that there are various ways that you can kind of tailor the CO₂ fermentation depending on what your application requires.

 

[Image shows James talking inset in the participant bar at the top of the screen, and a new slide appears showing various logos, and text appears: Thank you and questions, Esteban Marcellin, Lars Nielsen, Axa Gonzalez Garcia, Vishnu Mahamkali, Tim McCubbin, Gert Talbo, Manuel Plan, Robin Palfreyman, Kasper Valgepea, Andrew Warden, Sean Simpson, Michael Kopke, Nick Fackler, Ryan Tappel]

 

So yeah. Thanks everyone who’s been involved, quite a large team and thanks specifically to Andrew from CSIRO. Yes.

 

[Image shows the same slide on the main screen, and Charlotte can be seen talking in the participant bar at the top of the screen, and James can be seen listening]

 

Charlotte: Wonderful, thanks James. Thank you very, very much, thank you for sticking to time and thank you for that wonderful presentation and yeah we can see kind of everyone that  you’ve got here on your thank you slide, so yeah, leave that slide open so then we can see all those different folks that have been involved because it’s been a, a fantastic project. And I agree with your final comment about carbon dioxide being a very viable feedstock and it’s something that we should be using as a feedstock and understanding how we can use better for the bioeconomy, absolutely.

 

So I’ve dropped in a couple of questions and these are kind of more around my understanding regarding kind of getting myself up to speed with what you’re doing and as I’m talking if anyone comes up with any other questions, please drop them in that Chat to everyone. So the first thing I was thinking when you were talking about the TetR work, so the mutations that you’re… that you introduced, obviously you were seeing a change in expression which was through the GFP, the intention behind that though was to obviously, was to alter the metabolism or the product formation of the TetR reaction.

 

[Image shows the same slide on the main screen, and James can be seen talking in the participant bar at the top, and Charlotte can be seen listening]

 

James: Ah, yes, in the long term, we’re looking to sort of most likely, we want to increase the interaction between the TetR and the promoter motif but with that, all those mutations we’re seeing a decrease.

 

[Image shows the same slide on the main screen, and Charlotte can be seen talking in the participant bar at the top of the screen, and James can be seen listening]

 

Charlotte: Yeah so, and I’ve just, I was just trying to remember what was the, what were the products of that? Was it ethanol from a single-carbon source, was that the TetR?

 

[Image shows the same slide appearing on the main screen, and James can be seen talking in the participant bar at the top, and Charlotte can be seen listening]

 

James: So the TetR is just more so a sigma factor related to a lot of different genes involved in the Wood-Ljungdahl pathway, which is the main carbon metabolism. So yeah, not the…

 

[Image shows the same slide on the main screen, and Charlotte can be seen talking in the participant bar at the top of the screen, and James can be seen listening]

 

Charlotte: Yep. So what, what metabolytes would you be looking for then if you were trying to see?

 

[Image shows the same slide on the main screen, and James can be seen talking in the participant bar at the top, and Charlotte can be seen listening]

 

James: In terms of metabolytes and the phenotypics acetate, ethanol and growth are the main things we’d be looking for, yep.

 

[Image shows the same slide on the main screen, and Charlotte can be seen talking in the participant bar at the top of the screen, and James can be seen listening]

 

Charlotte: Fantastic, and that’s obviously kind of related across, across the project I can see. So this, this question kind of arises from my lack of knowledge around fermentation. Is additional carbon monoxide something that usually promotes fermentation? You, you mentioned that it can be an inhibitor because presumably it’s got toxicity. Could you just talk a little bit about what’s going on there?

 

[Image shows the same slide on the main screen, and James can be seen talking in the participant bar at the top and Charlotte can be seen listening]

 

James: So in terms of autotrophic growth, that is essentially you only want, or chemolithotrophic is another word, you don’t want any organic carbons present, because those, or it’s never really been seen, where you’ll favour, you’ll see one compound over an organic compound. So typically they just will grow, or the, at an industrial scale level only into C1 compounds because you can’t really get a combinatorial growth so…

 

[Image shows the same slide on the main screen, and Charlotte can be seen talking in the participant bar at the top of the screen, and James can be seen listening]

 

Charlotte: And does it have a balance between promoting fermentation and being toxic or being an inhibitor?

 

[Image shows the same slide on the main screen, and James can be seen talking in the participant bar at the top and Charlotte can be seen listening]

 

James: Yeah, so depending on the application, so steel mill for example is, I think around 50% carbon monoxide and very little hydrogen, so in that situation you’ll have a little bit of CO₂ as a by-product and then CO is essentially your only carbon substrate. So that would then be converting CO to the likes of ethanol, yeah.

 

[Image shows the same slide on the main screen and Charlotte can be seen talking in the participant bar at the top of the screen, and James can be seen listening]

 

Charlotte: Yep, right, fantastic.  Well, thank you very much, I don’t think anything’s come in yet, but I’ll keep checking that stream to see if anyone has any further questions or comments. As we said before anything that comes in can go through to the speakers afterwards but we’re getting close to perfect timing.

 

[Image changes to show Charlotte talking on the main screen, and James can be seen listening in the participant bar at the top of the screen]

 

I just really want to thank Joel and James for agreeing to talk today at this seminar series, both really fantastic projects that have made excellent progress and thank you both for presenting those talks today, I really appreciate it and it’s really great to hear the progress on how you’re going. Thanks to everyone for joining us online today as well. And I’m just going to embarrass him and just say very best wishes and thanks to James, who is actually celebrating his birthday today. So, thank you James for speaking on your birthday and I hope you’re going to have a good relaxing rest of your day now. I’d like to give the speakers a round of, a round of applause.

 

[Image shows Charlotte talking on the main screen and clapping, and then continuing to talk and participants can be seen in the participant bar at the top of the screen]

 

If everyone could please turn their cameras on and please join me in giving the speakers a very big round of applause. Thank you or use that little clapping hand emoji. Thank you, thank you, everyone, thank you for dialling in, thanks again for the speakers, it’s really fantastic and I, what else have I got to tell you about? Yeah, so as I said it was recorded so you should be able to kind of go back in and have a look if you need to or if anyone missed the seminar series and they want to hear these fantastic talks, then it should be available through the FSP platform.

 

Any answered questions we will pass on through. Let me just double back to there and see if there’s anything that’s come through, no just thank yous and thanks again. Invitations will be circulated shortly for the next monthly seminar series which will be held in August. The date is to be confirmed and that will be coming from the Maximising Impact Domain and that just remains to thank everyone and yeah have a really fantastic day. Thanks to the speakers, thanks to the PhD supervisors, Thanks to the FSP committee for helping me get everything organised. Thanks Emily and Alison and yeah, have a great day. Thanks. You’re welcome to stay on for a little bit if you want, Joel and James.

 

[Image shows Charlotte smiling and giving the thumbs up symbol]