Seeking RubisCOs from metagenomic fosmid libraries by a novel activity-based screening technology

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RubisCO (ribulose-1.5-bisphosphate carboxylase/oxygenase) is a key enzyme of the CBB (Calvin-Benson-Bassham) cycle, which is responsible for fixing approximately 200 gigatones of CO2 per year. RubisCO mediates the actual CO2 fixation step in this cycle by attaching CO2 to Rubp (ribulose-1.5-bisphosphate), thereby producing 3-D-phosphoglycerate (3-PGA). Of the four currently known types of RubisCO only type I, II, and III were shown to have the classical Rubp-dependent incorporation abilities. We developed an activity-based screen to detect recombinant Rubp-converting enzymes from metagenomic fosmid libraries. The libraries were constructed with hydrothermal vent material. We screened 12.500 fosmid clones and found forty active RubisCO enzymes encoded on the metagenomic fragments. They resembled type I and type II RubisCO from Alpha-, Beta- or Gammaproteobacteria and even type III RubisCO from Archaea.

Particularly one metagenomic fragment exhibited very interesting properties: the expressed proteins convert Rubp rapidly within seconds and respective DNA sequence does not resemble any known RubisCO types. Work is continued on this fragment and its respective gene products.

 

Previous funding

This project was financed by the German Science Foundation (DFG) (2013-2017). Hamburg’s Excellence Initiative (LEXI), BWF and the Joachim Herz Stiftung financed the C1-Resource and Energy Management Graduate School, which provided funding for this project (2009-2013).

 

Related publications:

Böhnke, S. & M. Perner (2019) Seeking active RubisCOs from the currently uncultured microbial majority colonizing deep-sea hydrothermal vent environments. ISME Journal doi: 10.1038/s41396-019-0439-3

Böhnke, S. & M. Perner (2017) Unraveling RubisCO Form I and Form II Regulation in an Uncultured Organism from a Deep-Sea Hydrothermal Vent via Metagenomic and Mutagenesis studies. Frontiers in Microbiology doi: 10.3389/fmicb.2017.01303

Böhnke, S. & M. Perner (2015) A function-based screen for seeking RubisCO active clones from metagenomes: novel enzymes influencing RubisCO activity. ISME Journal 9 (3) 735-745

 

Contact persons:

Dr. Stefanie Böhnke-Brandt (sboehnke-brandt(at)geomar.de)

Prof. Dr. Mirjam Perner (mperner(at)geomar.de)