Centre of Exellence DNA guided assembly line Rok

Centre of Exellence DNA guided assembly line Rok Gaber Laboratory of Biotechnology The 2 nd International Symposium Vera Johanides Biotechnology in Croatia by 2020 11. 5. 2013

Many useful compounds are produced by natural microorganisms Biomaterials. Biologically active substances. Pigments, Antioxidants, Food supplements. Biofuels. … More. 2

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Inovative approach - scaffolding of biosynthetic pathway enzymes Enzyme 3 4 Enzyme 2 1 4

Inovative approach - scaffolding of biosynthetic pathway enzymes Enzyme 3 4 1 2 5

Inovative approach - scaffolding of biosynthetic pathway enzymes • Direct covalent linkeage of succesive enzymes • Introduction of protein interacting domains • Protein scaffolding • RNA scaffolding • DNA scaffolding 6

Selection of DNA binding domains Zinc finger • • Leucine zipper Helix-turn-helix Sequence specific High affinity Similar characteristics Available in large number 7

Testing of selected Zinc Fingers Zinc finger expression is not induced. X p. BAD Zinc finger expression is induced. p. BAD Zinc Finger RNA polymerase X RNA polymerase -35 ZFBS -10 Reporter gene -35 ZF ZFBS X -10 Reporter gene 8

Testing of Zinc Fingers 9

Proof of principle • Can Zinc Fingers simultaneously bind to adjacent binding sites? 10

Proof of principle • Assay 1: – DNA enabled split fluorescent protein reconstitution. N-YFP C-YFP 2 bp 11

Proof of principle • Assay 1: – DNA enabled split fluorescent protein reconstitution. 12

Model biosynthetic pathway – resveratrol biosynthesis TAL OMT • Enzymes from two different organisms • Direct covalent linkeage of 4 CL and STS increases production of resveratrol (Zhang et al. 2006). • Extendable biosynthetic pathway 13

Design of Zinc Finger – enzyme fusions TAL ( ) 16 X ( OMT 2 bp )( 4 bp )( 8 bp ) 14

pterostilbene trans-resveratrol Final results p-coumaric acid Standards No program 2 bp spacer 3 -fold increase of resveratrol production in presence of DNA scaffold. 15

Acknowledgements and contributions Roman Jerala i. GEM 2010 team Slovenia Jernej Turnšek Nejc Tomšič Tjaša Stošicki Tina Ilc Tina Lebar Matej Žnidarič Mattia Petroni Jure Bordon Rok Pustoslemšek Rok Črešnovar Miha Mraz Miha Moškon Monika Avbelj Tomaž Koprivnjak Jerneja Mori Rok Gaber Jernej Turnšek Tina Lebar Mojca Benčina Gregor Anderluh Irena Vovk Vesna Hodnik Vesna Glavnik Breda Simonovsk a