Summary
Important information we found out:
- Prof. R. Sommaruga and Prof. S. Churio proposed to use nitrogen-enriched media.
- Our first intention was to test our strains under the UV light exposure to prove the presence of UV-absorbing compounds and to choose the strains with the highest yield.
Prof. S. Churio proposed us to use not only UV but also visible light to favor MAAs biosynthesis.
- Dr. K. P. Lawrence suggested us in the interview to apply some other types of environmental stress to induce shinorine and porphyra-334, such as osmotic stress, thermal stress or enhanced salinity.
- In order to increase the effectiveness of the sunscreen, the possibility of incorporation of more diverse MAAs with distinct spectral characteristics can be considered. It was also recommended by the researchers in the field (Dr. K. P. Lawrence, Prof. R. Sommaruga, Dr. R. Garcesa). Although, this will require a thorough investigation of the information available since the genes of the enzymes for the biosynthesis of many MAAs are not yet identified.
All the proposals mentioned above were taken into consideration while developing our project.
Nedeljka Rosic
Adjunct Fellow
School of Biological Sciences
Faculty of science
The University of Queensland
Brisbane, Australia
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Do you think that production of yeast extract enriched with MAA would be a valuable combination?
Yes, I think that this is possible as yeast is an excellent system for the expression of recombinant eukaryotic proteins.
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What can be the possible difficulties in producing MAA in yeast? What do you think how we can overcome them?
You might need to work on some codon modification to optimise the expression process.
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Whether the additional exposure to sunlight have the same effect in yeast as in Cyanobacteria in terms of increasing concentration of MAA? In your opinion, what can be done to increase the yield of MAAs in yeast?
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Would you please suggest anything to increase the yield of porphyra-334 compared to shinorine? Do you think whether the change in serine/threonine proportion would have an effect on the ratio porphyra-334/shinorine?
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.
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Do you think that MAA extraction and analysis protocols described in your article are suitable for yeast culture? Do you have any advice about the identification of MAAs obtained in yeast?
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.
Sandra Churio
Tenured Professor at Departamento de Química
Facultad de Ciencias Exactas y Naturales
Universidad Nacional de Mar del Plata
Mar del Plata, Argentina
Independent Researcher, National Research Council (CONICET), Instituto de Física de Mar del Plata (IFIMAR), Argentina
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Do you think that production of yeast extract enriched with MAA would be a valuable combination?
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.
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What can be the possible difficulties in producing MAA in yeast? What do you think how we can overcome them?
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.
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Whether the additional exposure to sunlight have the same effect in yeast as in Cyanobacteria in terms of increasing concentration of MAA? In your opinion, what can be done to increase the yield of MAAs in yeast?
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.
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Would you please suggest anything to increase the yield of porphyra-334 compared to shinorine? Do you think whether the change in serine/threonine proportion would have an effect on the ratio porphyra-334/shinorine?
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.
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In your opinion, do we need to add some additional compounds to our sunscreens to increase photostability of MAAs. If yes, what compounds we can use?
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.
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How do you think, is the combination of Shinorine and Porphyra-334 reasonable for sunscreens?
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.
Matthew Pope
PhD Candidate
Department of Life Sciences
Imperial College London
London, United Kingdom
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What can be the possible difficulties in producing MAA in yeast? What do you think how we can overcome 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
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Whether the additional exposure to sunlight have the same effect in yeast as in Cyanobacteria in terms of increasing concentration of MAA? In your opinion, what can be done to increase the yield of MAAs in yeast?
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.
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Would you please suggest anything to increase the yield of porphyra-334 compared to shinorine? Do you think whether the change in serine/threonine proportion would have an effect on the ratio porphyra-334/shinorine?
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.
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How do you think, is the combination of Shinorine and Porphyra-334 reasonable for sunscreens?
If you can demonstrate they absorb across UV-A/B then I think it is sufficient proof of principle.
Karl Lawrence
Photobiology Group
St John's Institute of Dermatology
School of Basic and Medical Biosciences
King's College London (KCL)
London, United Kingdom
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Do you think that production of yeast extract enriched with MAA would be a valuable combination?
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.
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Whether the additional exposure to sunlight have the same effect in yeast as in Cyanobacteria in terms of increasing concentration of MAA? In your opinion, what can be done to increase the yield of MAAs in yeast?
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.
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In your opinion, do we need to add some additional compounds to our sunscreens to increase photostability of MAAs. If yes, what compounds we can use?
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.
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How do you think, is the combination of Shinorine and Porphyra-334 reasonable for sunscreens?
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.
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I would like to ask you if you already tried similar experiments and maybe you can suggest more precise values or methods?
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)
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Could you please advise if there are simple basic tests that can be done to check photostability?
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.
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I would like to ask you if you have the information about the genes coding for these MAAs?
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.
Shailendra P Singh
Assistant professor
Institute of Science
Banaras Hindu University
Varanasi, India
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Do you think that production of yeast extract enriched with MAA would be a valuable combination?
Yes.
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What can be the possible difficulties in producing MAA in yeast? What do you think how we can overcome them?
Codon usage could be the potential problem but you should try first the genes from cyano.
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Whether the additional exposure to sunlight have the same effect in yeast as in Cyanobacteria in terms of increasing concentration of MAA? In your opinion, what can be done to increase the yield of MAAs in yeast?
It varies organisms to organisms depending on the nature of the promoter.
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Would you please suggest anything to increase the yield of porphyra-334 compared to shinorine? Do you think whether the change in serine/threonine proportion would have an effect on the ratio porphyra-334/shinorine?
You can try changing serine/threonine proportion but I think
it will depend on proteins responsible for conversion of MAAs
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In your opinion, do we need to add some additional compounds to our sunscreens to increase photostability of MAAs. If yes, what compounds we can use?
MAAs are highly stable but its stability will depend on the final composition of sunscreen which needs to be tested.
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How do you think, is the combination of Shinorine and Porphyra-334 reasonable for sunscreens?
Efficacy of the final product can be tested against targeted goal.
Ranko Gacesa
PostDoctoral Researcher
Weersma Group,
Department of Gastroenterology,
University Medical Center Groningen,
Groningen, The Netherlands
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Do you think that production of yeast extract enriched with MAA would be a valuable combination?
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.
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What can be the possible difficulties in producing MAA in yeast? What do you think how we can overcome them?
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.
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Whether the additional exposure to sunlight have the same effect in yeast as in Cyanobacteria in terms of increasing concentration of MAA? In your opinion, what can be done to increase the yield of MAAs in yeast?
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.
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Would you please suggest anything to increase the yield of porphyra-334 compared to shinorine? Do you think whether the change in serine/threonine proportion would have an effect on the ratio porphyra-334/shinorine?
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.
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In your opinion, do we need to add some additional compounds to our sunscreens to increase photostability of MAAs. If yes, what compounds we can use?
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.
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How do you think, is the combination of Shinorine and Porphyra-334 reasonable for sunscreens?
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.
Ruben Sommaruga
Full Professor of Limnology
Head of the Research Group:
Lake and Glacier Ecology
Director Institute of Ecology University of Innsbruck
Innsbruck, Austria
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Do you think that production of yeast extract enriched with MAA would be a valuable combination?
Certainly.
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What can be the possible difficulties in producing MAA in yeast? What do you think how we can overcome them?
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?
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Whether the additional exposure to sunlight have the same effect in yeast as in Cyanobacteria in terms of increasing concentration of MAA? In your opinion, what can be done to increase the yield of MAAs in yeast?
Well, this is related to my previous answer. Another suggestion is to use a nitrogen-enriched cultivation medium.
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How do you think, is the combination of Shinorine and Porphyra-334 reasonable for sunscreens?
If you get both, they have a wide window of UV radiation screening. Myc-gly would made the combination perfect.
