Nethaji J. Gallage – University of Copenhagen

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Center for Synthetic Biology > Meet the Researchers > Nethaji J. Gallage

Nethaji J. Gallage

Post Doc (PhD graduate)

Section for Plant Biochemistry

Department of Plant and Environmental Sciences

Faculty for Science

nethaji@plen.ku.dk 

Nethaji J. Gallage’s research focuses on elucidating the properties of biosynthetic pathways of vanillin and capsaicinoids across kingdoms. In addition she tries to biotechnologically optimize sustainable vanillin and capsaicinoids production in different heterologous systems. Nethaji is the first author on the publication that unraveled the biosynthetic pathway of vanillin production in Vanilla planifolia.

Research

I am involved in Plant pathway discovery and metabolic engineering of plant pathways to microbes.
Vanilla and its key flavour component vanillin, is a universally appreciated flavour, a global delicacy and probably the most popular plant natural product. ‘Vanilla’ is the complete extract of the vanilla pod, and is known to include more than 200 different flavour compounds. Vanillin (4-hydroxy-3-methoxybenzaldehyde), the most abundant compound and is the key aroma of the vanilla extract and the cured vanilla pod.

Vanillin and vanilla extracts have an estimated annual total volume of 16,000 metric tonnes, worth some of USD 650 million in total. Natural vanilla extract represents less than 1% by volume, while 99% of all vanillin consumed worldwide is synthetically made primarily from petrochemicals or chemically derived from lignin. This is due to supply chain variability and costly and high labour demanding of growing and processing of natural vanilla. These reasons have promoted intensive research to explore the most suitable metabolic engineering organisms and pathways to establish synthetic biology based routes to vanillin production (Gallage and Møller 2015).

During my PhD I was able to elucidate the pathway for vanillin in the pods of Vanilla planifolia. For decades, the international research community thought the formation of vanillin in the vanilla orchid was a highly specialized and complex process. My discovery showed that a single enzyme is able to catalyse the direct conversion of ferulic acid and ferulic acid glucosides into vanillin and vanillin glucoside respectively. We have now named this enzyme vanillin synthase (VpVAN). Vanillin synthase belongs to the enzyme family of cysteine proteases (Gallage et al., 2014 Nature communications).

The aim of my current project is to replace a part of synthetically made vanillin, which is produced using fossil fuel today (99% of vanillin in the market today is synthetically made), with sustainable production systems such as yeast and other heterologous systems. For this purpose we use metabolic engineering approaches to express vanillin synthase. Our research on investigating the post translation steps of VpVAN and discovering the subcellular site of vanillin synthesis would shed light to a greater understanding of how the vanilla orchid synthesize this well familiar flavour compound in much higher concentrations than any other living cell (Manuscript is on preparation for plant physiology). Furthermore, I have started to develop synthesis of vanillin in yeast by exclusively using genes form the vanilla orchid.

I am also involved in resolving and establishing sustainable routes for capsaicinoids biosynthesis. Capsaicinoids are specialized secondary metabolites found uniquely in Capsicum (pepper) species. Capsaicinoids are bioactive molecules of food and of medicinal importance. These compounds are proven to be useful as a counterirritant, antiarthritic, analgesic, antioxidant, and anticancer agent. Capsaicin is the main capsaicinoids and represents about 60% of the capsaicinoids found in hot pepper fruits and is the main contributor to the pungency of chili. Vanillin is the precursor for capsaicin formation in chili.

Why did you choose to work with this? 
My great desire to involve in applied research made me approaching my PhD advisor, Professor Birger Lindberg Møller, suggesting studying vanillin biosynthesis in the vanilla orchid. I wanted to develop synthesis of vanillin in yeast by exclusively using genes form the vanilla orchid. Such a discovery would set the stage to replace synthetically made vanillin, which is produced using fossil fuel, with sustainable production systems such as yeast and other heterologous systems. I truly believe, a more detailed understanding on the molecular mechanisms behind plant pathways would lead to sustainable solutions for consumer needs without undermining the natural systems and resources that productivity depends on

How would you like your work to be applied?
The identification of vanillin synthase as a hydratase/lyase-type enzyme catalysing conversion of ferulic acid and its glucoside into vanillin and its glucoside offers new opportunities for the Vanilla pod–based industries. The regulation and accumulation of vanillin glucoside in the capsules of cultivated vines in response to abiotic and biotic environmental challenges may now be assessed at the molecular level.

Likewise, in general, each plant species contains about 50 different papain-like cysteine proteinases. They play a role in the turnover of proteins at specific time points of plant growth and development, e.g. in a ripening fruit. In the course of evolution of the vanilla-producing V. planifolia orchid, natural mutations arose in one of these cysteine proteinases, enabling the enzyme to convert ferulic acid into vanillin. Many orchids closely related to the vanilla orchid are present in nature but appear not to be able to synthesize vanillin. Classic mutation breeding to obtain vanillin production in such species may now be initiated. This would offer a more diverse production system less prone to diseases, and maybe the introduction of vanillin synthesizing orchids, which would have natural pollinators in their growth habitats and thus not require hand pollination by humans.

Research on natural flavours, such as biotechnology-derived natural vanillin has been expanded during the last two decades as a result of combinations of market pull and the technical push.  The primary driving force for the biotechnology-derived flavour industry was and still is the fact that flavour compounds produced from natural raw materials by microbial or enzymatic methods can be labelled ‘natural’ in accordance with European and US legislation, thereby satisfying consumer trend towards  ‘bio’ and ‘natural’ products in the food and flavour sectors. In contrast, the involvement of chemical synthesis and advancement of flavours not occurring in nature have led to less consumer appreciated labelling such as ‘nature identical’ and ‘artificial’ (EC flavour Directive 88/ 388/ EEC) (US code of federal regulation 21 CFR 101.22), which have resulted a low demand and reduced the market value of flavours produced by such technologies. 

I am very excited to start on constructing a vanillin glucoside biosynthetic pathway in yeast which is depended exclusively on V. planifolia enzymes. It is likely that engineering a vanillin biosynthesis pathway in microorganisms using genes from the vanilla orchid may govern a more positive attitude from consumers for natural vanillin origins from other sources than the vanilla orchid and would compete with other alternatives for bioengineered natural vanillin and as well as synthetic vanillin. By that I will be able to bring basic science out from the lab to an industrial setup.

Do you collaborate with other researchers?
Currently, I collaborate with investigators in the synthetic biology center, Agnieszka regarding Chloroplast isolation in the vanilla pods and with a proteomic expert Jonas Borch at the University of Southern Denmark. I advise PhD student who is attempting establish a vanillin biosynthesis in cyanobacteria as well. We also started collaboration with Asaph Aharoni and his group at Weizmann Institute of Science who is engineering tomatoes for capsaicin synthesis. In additions, I also work together with Gideon Grogan, York Structural Biology Laboratory, and UK who has recently started working on crystalizing VpVAN. On top of that I have a very respectful and smooth collaboration continuing with vanillin project industrial partner, Evolva A/S.

Do you collaborate with industry?
Yes, during my PhD I have established and continued a very fruitful collaboration with the Danish-Swiss biotech company Evolva on the vanillin project. Esben Halkjær Hansen (senior research scientist/ project manager, Evolva A/S) was my PhD co-supervisor and Jørgen Hansen (Chief scientific officer, Evolva A/S) is still continuing as a research advisor for my postdoctoral research project on vanillin and capsicinoids. Collaboration with Evolva will continue on the basis of technology and information sharing as it has been in my PhD years.

What motivates you in your work?
I am very curious!! For me there are no positive or negative results. It is always useful to be open-minded, because you never know what is hidden behind negative results. Pathway discovery ‘passion’ probably initiated from that. When things do not work as the way it planned and when I am under pressure, I really push my limits and that gives me a thrill. I also have a very practical mindset that would like finding solutions for problems. This may be what drives me as a metabolic engineer. As a scientist it is very important to be able to troubleshoot but what is most difficult is to recognize the trouble. Otherwise all good ideas are useless and wasting a lot of time.

It is my personality having a need for starching boundaries and I am glad that this is what I do at work every day in science. I was born and grew up in Sri Lanka where there was not much freedom for free thinking and as woman there were many limitations for what you can achieve. I am glad that I moved to Denmark and studied here and very grateful for many who have helped along the way.  

What accomplishment are you most proud of?
I am proud that I was able to establish a new pathway discovery project leading to elucidation of biosynthetic pathway for the formation of vanillin - the world’s most popular flavor compound. The goal of the PhD project was reached within the time frame and resulted in a publication in the journal Nature Communications and a subsequent review in Molecular Plant (Review was featured in the cover), and filing of a patent application. Two additional manuscripts on in situ localization of the vanillin pathway in the vanilla pods are in preparation. Now my article on the discovery of VpVAN as well as the review is cited by News and views on Nature Biotechnology on the topic of engineers of scent. I am also proud that during my time with this project I was able to establish many successful collaborations and I have brought my research area out to both scientific e.g. presenting projects on conferences and non-scientific audience e.g. video interviewed by Reuters.

When not doing science; how do you like to spend your time?
I love photography. I have a Canon7D Mark II and a several professional Canon lenses that I play with in my free time. Taking wild type photography, macro as well as portrait photos of my three boys are my favorite categories. I travel to Africa once per year with my husband where we drive long distances across countries and by that we have a unique opportunity to get very close to wild life and where we get to play with our cameras. I also love jogging, playing badminton with my colleges and do Pilates (brain brake). Last but not least, what I enjoy the most is spending time with my husband and three boys at our summerhouse, Bursøe.

CV

Since 2014

Post doctoral researcher, Department of Plant and Environmental Sciences, University of Copenhagen.

2010-2014 PhD in Biochemistry and Biotechnology, Department of Plant and Environmental Sciences, University of Copenhagen. (Supervisor: Birger Lindberg Møller)
2006-2009 MSc Biology and Biotechnology, University of Copenhagen
2006-2009 Student research
assistant– Detergent application II - Novozymes
2006 Student assistant, Department of Plant biology and Plant biotechnology, University of Copenhagen
2003- 2006 BSc Horticulture, University of Copenhagen

Collaborations within the Center for Synthetic Biology
Birger Lindberg Møller

Publications
Gallage,N.J., Hansen,E. H., Kannangara, R., Olsen, C. E., Motawia, M. S., Jørgensen, K., Holme, I., Hebelstrup, K., Grisoni, M., Møller, B.L. Vanillin formation from ferulic acid in Vanilla planifolia is catalysed by a single enzyme. Nature Communications 5, doi:10.1038/ncomms5037(2014). I.F. 10,742

Gallage, N.J. and Møller, B.L. Vanillin – Bioconversion and Bioengineering of the most popular plant flavour and its de novo biosynthesis in the vanilla orchid. Molecular Plant, special edition Synthetic Biology, doi:10.1016/j.molp.2014.11.008 (2014).

Patents

Gallage, N.J., Hansen, E.H., Møller, B.L. and Hansen, J. Microbial organism and methods for producing vanillin, vanillyl alcohol, or vanillin glucoside, by vanillin synthase action on ferulic acid. International Patent Application PCT/DK2013/050357

Presentations

"Pathway discovery of the most popular plant flavour compound vanillin".
Invited speaker at Plants and People 2015: 'Future Plan[t]s' Conference.
Max Planck Institute of Molecular Plant Physiology, Septmeber 9th 2014

Awards

Winner of PhD Brain 2011 - Brain for an evening- University of Copenhagen