Difference between revisions of "Team:Tartu TUIT/Human Practices/Gold"

Yes, I think that this is possible as yeast is an excellent system for the expression of recombinant eukaryotic proteins.

You might need to work on some codon modification to optimise the expression process.

I would try the same principle: yeast exposure to higher UV level. See this article: Mycosporine‐glutaminol‐glucoside, a UV‐absorbing compound of two Rhodotorula yeast species. https://febs.onlinelibrary.wiley.com/doi/abs/10.1002/yea.1148

I think that this changes in the ratio of available amino-acids are the possible way to help in manipulation of the final product and to increase the amount of porphyra-334 by addition of threonine.

Regarding the extraction step, I would just start with the protocol explained in the article and then make some adjustments if needed. Identification of MAA should follow the same procedure as described in the article.

Yes, the project is very interesting. The possibility of developing a biotechnological method for obtaining MAAs is promising as an alternative to other natural sources (macroalgae) due to the limitations in the extraction yields, tedious purification protocols, and the inherent variability of the biological material.

Although genetics is beyond my expertise, I suspect that one main difficulty can be found in the yield of the biosynthesis and the cost of UV light sources, if needed to induce de production of the natural sunscreens. Nitrogen is essential for the synthesis of MAAs, thus limitations in this element should be avoided when considering the nutrition conditions of the cultures.

Probably combinations of UV (UVA and /or UVB) and visible light work better. It is not clear which kind of photoreceptors are relevant for MAAs synthesis and the action spectrum may depend on the biological species. This issue should be explored in your system.

The effect of variations in the proportion of amino acids is worth to be tested as a control parameter of the porphyra-334/shinorine biosynthesis ratio. From the chemical point of view, these two MAAs are very similar and there is little to be done in order to promote preferential accumulation of any of them.

MAAs are very photostable in aqueous solution. Even so, the photodecomposition quantum yields are lower in the presence of direct micelles, mainly for cationic ones (see Orallo et al. Photochem. Photobiol. Sci., 2017, 16, 1117–1125). In the case you obtain oxo-MAAs you should prevent them from oxidation; imino-MAAs are more resistant in terms of redox reactions. Under extreme pH values, MAAs tend to decompose via hydrolysis of the amino acid moieties.

It is optimal from the stability point of view. However, combination with other filters may be necessary to extend the protection within the whole UV-B and UV-A ranges. In this case, the stability of the mixture should be verified. On the other side, the poor partition of these MAAs in nonpolar media should be considered in the design of formulations for topical sunscreens containing them.

Expressing heterologous genes in yeast isn't a problem but generating enough MAAs to have photoprotective effects remains to be seen. Can the yeast cell catabolise MAAs? Are MAAs toxic to yeast? If so then it may be difficult to produce enough for what you desire. You'll have to do these experiments to find out

Additional exposure to sunlight will only induce MAAs biosynthesis in yeast if you engineer their expression to be controlled by light inducible promoters (which would be pointless). You may as well engineer yeast to constitutively express the genes.

MAAs stem from amino acid bio-synthetic pathways which are usually negatively regulated by their end product. You could try altering these pathways but it may have detrimental effects on the host organism.

If you can demonstrate they absorb across UV-A/B then I think it is sufficient proof of principle.

The biosynthetic production of yeast would be extremely valuable, especially if the yield of MAA can be increase. Extraction from seaweed is the most viable option currently but the yields are relatively low and this is a restriction on the widespread development of MAA.

Again I am not sure the answer to this. As a guess it will depend on the pathways regulating the synthesis and if they are linked to environmental stress. in cyanobacteria it is not only UV radiation that induces MAA synthesis - other stressors also increase the yield so perhaps one of these will work in yeast if UV doesn’t - these include increasing the nitrate concentration, thermal stress, osmotic stress, increase in salinity. There may be others too.

From our work we have found MAA to be extremely photostable (only a few percent degradation after massive UVR doses), so I would say that nothing would need to be added to increase photostability. The MAAs we have tested are much more photostable than most of the currently available synthetic filters we have tested.

It is a reasonable start as a proof of concept but they both have very similar absorption spectra with the same peak at 334nm. It would be better to have a diverse range of MAA, with peak absorptions across the entire UVR spectrum if possible to create a broad spectrum sunscreen that meets regulatory requirements. For example, covering the shorter wavelengths palythine has a peak at 320nm, gadusol (a precursor of MAA) has a peak at 294nm, and the longer wavelengths usijerene has a peak at 354nm and palythene at 360nm. if it would be possible to create these 6 MAA together it would be almost perfect.

I havent tried these experiments but there is evidence of all of these in the literature. You will find some examples of these in my review on MAA which I have attached. (sent an article)

Photostabilty is very easy to check. Essentially all you need to do is irradiate the MAA dissolved in a solvent (I u sed PBS) with increasing doses of UVR and then measure the absorbance with a spectrophotometer. The percentage degradation can then be assessed using the decrease in absorption or measuring the shift in spectrum. It is important to use a relevant source, either environmentally relevant like a solar simulator or a source relevant to the absorption spectrum (e.g. if it is a UVB absorbing molecule then use a UVB source.

Again I don’t have much experience with the biosynthesis of MAA and I don’t think many of the genes are known. There is a little bit literature on the subject and again in the review the biosynthesis section may put you in the right direction.

Yes.

Codon usage could be the potential problem but you should try first the genes from cyano.

It varies organisms to organisms depending on the nature of the promoter.

You can try changing serine/threonine proportion but I think it will depend on proteins responsible for conversion of MAAs

MAAs are highly stable but its stability will depend on the final composition of sunscreen which needs to be tested.

Efficacy of the final product can be tested against targeted goal.

It would be, assuming you could produce it in large quantities for reasonable price. Main issue with MAAs at the moment is, however, not production of MAA-enriched extracts but getting the pure compounds. MAAs are reasonably easy to get in large amounts as by-products of seaweed production or fish farming, but extracting them from these materials is currently not commercially viable, especially for high purity. Compared to these sources, advantage of yeast-based biotech is that you could have at least some control over which MAAs are produced, higher consistency/replicability, and potential price advantage. Genetically modified organisms, however, have very bad reputation these days (at least in EU), so getting it on the market might be very difficult.

First problem you will run into is cloning and maintaining MAA biosynthesis pathway in yeast - getting multiple genes into yeast and having them expressed is, in general, pretty tough. I can't provide much advice there as I primarily do computational work, but you will need good experimental design with appropriate cloning vectors, good controls and lots of experiments to confirm genes are getting into the yeast. It will almost certainly require plenty of trial and error and quite a bit of work. MAA biosynthesis is not strongly expressed in cyanobacteria, so introducing strong promoters might help with actually getting MAAs. Introducing genes one by one and monitoring if genes are there and what yeasts produce might be a good starting point. Assuming MAA precursors are being produced, they will likely be present and kept inside the cell and in very small amounts, so you might have to grow plenty of yeasts (possibly under UV pressure) and run series of isolation experiments to see what (if anything) is being produced. Assuming you have HPLC/MS system and lab scale bioreactors for growing yeasts, detecting gadusol should be reasonably straightforward. Expect lots of trial and error with cloning. Finally, I would strongly suggest working with experienced molecular biologist (or several) who did this type of experiments before and have well established protocols and lab equipment - if you don't have properly equipped lab with proper expertise in house it will be very hard. Assuming you get MAA biosynthesis pathway cloned into yeast and expressed, next problem will be to get significant amount of MAAs (enough to do structure determination experiments). This will probably require optimisation of grown conditions and upscaling of the growth and isolation process. Growth conditions optimisation comes down to trial and error - try different growth media, temperatures, light... monitor production and repeat. Upscaling is almost entirely up to what equipment and how much resources you have available - assuming yields similar to bacteria, rough estimate is 1-10 L of yeasts to get noticable amount of MAAs, a lot more to produce enough for any practical use. Getting MAAs out of yeasts will probably be tricky - depending on your yeast, they might not lyse readily and they will probably not release MAAs into medium. Purification is currently unsolved problem and one of reasons why it is impossible to buy significant amount of pure MAAs: semi-preparative HPLC is standard practice to get lab-quantities of MAAs, but it hardly works for more then micro-gram amounts; full preparatory HPLC is out of reach of most labs and too expensive to be practical, and "simple" metanol extractions don't produce pure compounds. You will probably not get to isolation optimisation point during the project time-frame, but assuming you do, best advice I can give is to try standard metanol extraction, see what comes out, optimise HPLC conditions and repeat.

Very little work was done on MAA-producing yeasts, so it is unclear whether UV plays a role in MAA producing yeasts - I would assume that UV will activate MAA production in yeasts, but I haven't done any work with MAA-producing yeasts. Simplest approach is to just sistematicaly test lots of different growth conditions - vary the composition of growth medium, exposure to UV and temperature, pick the best and optimise.

There is lots of missing pieces in biosynthesis of individual MAAs, so I don't think it is easy (or possible) to predict individual yields purely on genetics. Trying different substrates is worth a try.

That is a very tricky one as reports about MAA stability are not in agreement at the moment. We did some experiments on it at King's roughly a year ago and our data suggests that MAAs are highly photostable; this is in agreement with early reports from different groups and it makes sense from biological perspective (I would expect selective pressure towards evolution of s table sunscreen because it doesn't do much if it is unstable). Some of our collaborators, however, claim the that MAAs are not photo-stable, so there is convincing evidence for both sides. It might be the effect of formulation, so I think it is impossible to tell without further testing. Personally I tend to be on the 'MAAs are photostable' side, but assuming stability is a problem, playing with solvents (formulation for human application will probably involve some combination of oils and water plus maybe tiny bit of ethanol; most of lab testing is done in methanol + water, so results might be misleading). Adding antioxidants might help as well - ascorbic acid is good one to try. Photostability aside, water solubility of MAAs is a major problem for use of MAAs as sunscreens - MAAs readily dissolve in water and that is major problem for sunscreen. Animals get around the problem by concentrating them inside tissues, but compound which gets inside the skin will almost certainly never get approved for human use (due to safety concerns; this is another problem for practical use of MAAs - they do seem to go into the skin). Clever formulation might be able to solve these problems - perhaps encapsulation of MAAs in liposomes or some other type of carrier or perhaps use of MAAs with better properties. Unfortunately I don't do any formulation work, so can't help much there.

Ideally you would want to mix MAAs with very different absorbance maxima (high wavelength absorbing MAA such as Palythene or Euthalothece with low maxima absorbing such as Palythine); Shinorine and Porphyra have virtually indentical absorbance spectra so mixing them won't do much for UV protection. In reality, practical limit comes down to which MAA is possible to produce in sufficient purity and amount.

Certainly.

Even if the introduction of the 4 genes is successful, one concern is their expression and its efficiency. I am no an expert on Saccharomyces cerevisiae, but my first question is about what is known on the presence of solar radiation receptors that are probably needed to trigger the induction of MAA synthesis. What about the genes Ava_3858 and Ava_3857 that encode demethyl 4-deoxygadusol (DDG) synthase and O-methyltransferase (O-MT), respectively, to form 4-deoxygadusol (4-DG), the core structure of mycosporines?

Well, this is related to my previous answer. Another suggestion is to use a nitrogen-enriched cultivation medium.

If you get both, they have a wide window of UV radiation screening. Myc-gly would made the combination perfect.