Agnieszka Zygadlo Nielsen – University of Copenhagen

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Center for Synthetic Biology > Meet the Researchers > Agnieszka Zygadlo Nielsen

Agnieszka Zygadlo Nielsen

Assistant professor

Photosystem I Group
Section for Molecular Plant Biology
Department of Plant and Environmental Sciences
Faculty of Science

az@plen.ku.dk

Agnieszka Zygadlo Nielsen’s research focuses in on specific P450 enzymes involved in the Light Driven Biosynthesis of natural compounds.

She is first author of the article that solidly established the concept in the scientific community: " Redirecting Photosynthetic Reducing Power toward Bioactive Natural Product Synthesis " leading  to both a podcast interview and profile the June 2013 issue.


What is your research focus?
Currently, I am working on P450 enzymes with a synthetic biology approach with the goal of producing bioactive compounds of commercial value. The work elaborates on our approach to drive enzymatic reactions shunting the NADPH regeneration system using the reducing power of photosynthesis instead. Light-driven synthesis requires a creative approach, creating new enzymatic activity assays, channeling metabolic pathways, optimizing heterologous expression of the enzymes in cyanobacterial and eukaryotic hosts (tobacco plants and moss). Future work will be directed towards establishing stable transformants with multi enzyme pathways expressed in the chloroplast and thereby producing interesting bioactive compounds directly driven by light.

How could your work be applied outside the world of research?
Light-driven biosynthesis constitutes a promising approach for sustainable production of high value natural compounds of complex chemical structures using photosynthesis as the energy source.  We focus on diterpenoids which are highly complex molecules that are almost impossible to synthetize chemically. They have high market value as they are used as active compounds in cancer treatments or other disease treatments, as well as in fragrances.

How do you collaborate with the researchers from the other scientific disciplines of Center for Synthetic Biology?
We have extensive collaborations through-out the center. We collaborate closely with Björn Hamberger’s group. They make the diterpenoids biosynthesis pathway discovery work. We also work with Birger Lindberg Møller and Tomas Laursen for their expertise in cytochrome P450 enzymes. Furthermore we have initiated collaboration with Kenneth Lindegaard Madsen and Ulrik Gether to try a scaffolding strategy in order to concentrate enzymes involved in a pathway together. Finally, I collaborate with Søren Roi Midtgaard to by SAXS-analyse photosystem I reconstituted into nanodiscs.

Why did you choose to work with synthetic biology?
Synthetic biology is an emerging field where everything seems possible. It gives space for creative ideas, to think out of the box and meet scientists from different fields. The latter has given rise to great ideas and collaborations while doing good science.

What accomplishment are you most proud of?
I am proud of that we established the proof-of-concept of Light Driven Biosynthesis which was a wild concept five years ago. Now, with our work, it is considered to be a possible approach to produce high value compounds in photosynthetic hosts.

Research

Our research group aims to use light as the main resource for the production of bioactive molecules in plants, which are of medicinal or agricultural significance. This development will be enabled by the sustainable and efficient production of valuable natural compounds in photosynthetic organisms.

Figure: Light driven dhurrin metabolon

Photosynthetic protein complexes use sunlight to oxidize water and transfer the released electrons to stable electron carriers and produce NADPH. Photosystem I (PSI) produces the most negative redox potential in nature and is perfectly suited to drive a number of redox reactions. In this work, we have introduced a new biosynthetic pathway consisting of cytochrome P450 enzymes (P450s) into the chloroplast. Plant P450s are naturally localized in the endoplasmatic reticulum where they catalyse hydroxylations of bioactive compounds. Normally, electrons necessary for their reactions are delivered by a cytochrome P450 reductase using NADPH.

We used the enzymes involved in the biosynthesis of the plant defence compound Dhurrin. In this pathway, the first enzyme is the membrane-bound CYP79A1 which converts the amino acid tyrosine into an oxime. This oxime is converted by another membrane bound P450, CYP71E1, into a nitrile and finally the soluble glucosyltransferase UGT85B1 completes the formation of Dhurrin by adding a sugar moiety.

The three genes encoding CYP79A1, CYP71E1 and UGT85B1 were cloned and transiently expressed, both individually and in combination, in leaves of N. benthamiana. A transit-peptide derived from another chloroplast localized protein was engineered in front of each protein ensuring targeting of the three enzymes to the chloroplast. Activity assays on isolated thylakoids and intact chloroplast showed that all three enzymes are active in the chloroplast. More interestingly, it was shown that the activities of CYP79A1 and CYP71E1 – individually and combined – can be supported by reducing power from PSI in a light-dependent manner. This demonstrates that it indeed is possible to express all the enzymes of the pathway in an active state in the chloroplast."

CV

April 2010 Post-doc in UNIK synthetic biology program, Faculty of Life Sciences, Plant biochemistry laboratory, University of Copenhagen in Prof. Poul Erik Jensen’s group
2009-2010 Post-doc in RENEWALL EU program, AarhusUniversity, Frederiksberg in Prof. Peter Ulvskov’s group
2007-2008 Research scientist at Aresa A/S
2006 Post-docFaculty of Life Sciences, Plant biochemistry laboratory, University of Copenhagen
2005 PhD degree in plant molecular biology Faculty of Life Sciences, University of Copenhagen, Denmark
2001 Postgraduate degree in cellular biology and physiology, obtained with Honours University D. Diderot, Paris VII, France
2000 MSc in oncogenesis and additional research engineer degree (Magistère) in Genetics University D. Diderot, Paris VII, France

 

Collaboration partners within UNIK

Søren Roi Midtgaard
Tomas Laursen
Birger Lindberg Møller
All from Poul Erik Jensen's group
All from Björn Hamberger’s group
Kenneth Lindegaard

Selected Scientific Publications

Lassen, L.M., Nielsen, A.Z., Olsen, C.E., Bialek, W., Jensen, K., Møller, B.L., Jensen, P.E. Anchoring a plant cytochrome P450 via PsaM to the thylakoids in Synechococcus sp. PCC 7002: evidence for light-driven biosynthesis. PLoS One 15, 9(7):e102184, doi:10.1371/journal.pone.0102184 (2014).

Lassen, L.M., Nielsen, A.Z., Ziersen, B., Gnanasekaran, T., Møller, B.L., Jensen, P.E. Redirecting photosynthetic electron flow into light-driven synthesis of alternative products including high-value bioactive natural compounds. ACS Synthetic Biology 17, 3(1):1-12, doi:10.1021/sb400136f (2014).

Nielsen, A.Z., Ziersen, B., Jensen, K., Lassen, L.M., Olsen, C.E., Møller, B.L., Jensen, P.E. Redirecting photosynthetic reducing power toward bioactive natural product synthesis. ACS Synthetic Biology 21, 2(6):308-15, doi:10.1021/sb300128r(2013).

Zygadlo, A., Robinson, C., Scheller, H.V., Mant, A., Jensen, P.E. The properties of the positively charged loop region in PSI-G are essential for its "spontaneous" insertion into thylakoids and rapid assembly into the photosystem I complex. Journal of Biological Chemistry 14, 281(15):10548-54, doi:10.1074/jbc.M512687200(2006).

Kouril, R., Zygadlo, A., Arteni, A.A., de Wit, C.D., Dekker, J.P., Jensen, P.E., Scheller, H.V., Boekema, E.J. Structural characterization of a complex of photosystem I and light-harvesting complex II of Arabidopsis thaliana. Biochemistry 23, 44(33):10935-10940, doi: 10.1021/bi051097a (2005).

Rosgaard, L., Zygadlo, A., Scheller, H.V., Mant, A., Jensen, P.E. Insertion of the plant photosystem I subunit G into the thylakoid membrane. FEBS Journal 272, (15):4002-10, doi:10.1111/j.1742-4658.2005.04824.x (2005).

Zygadlo, A., Jensen, P.E., Leister, D., Scheller, H.V. Photosystem I lacking the PSI-G subunit has a higher affinity for plastocyanin and is sensitive to photodamage. Biochimica et Biophysica Acta 30, 1708(2):154-63, doi:10.1016/j.bbabio.2005.02.003 (2005).