The number of biotechnological applications has skyrocketed and have evolved to be one of the most ubiquitous aspects in our daily life in the past years. From uses in food industry to dietary supplements and science, white biotechnology is everywhere.

That’s why we try to recreate a widespread natural phenomenon called metabolic channelling synthetically to increase the yield biochemical reactions. Metabolic channelling occurs when the enzymes responsible for a specific reaction are in close proximity to each other, and thereby decrease the diffusion time of the substrates (see animation below).

*Animation zur Verdeutlichung von metabolic channelling*

In our first method, we utilize the DNA-binding function of deadCas9 (dCas9) to bind two enzymes of the Indoleacetic acid (IAA) pathway, IAA tryptophan monooxygenase (IAAM) and indoleacetamide hydrolase (IaaH) on a DNA-scaffold which we introduce into E.coli. By binding the enzymes on DNA, we decrease the distance the substrates have to travel in the cytoplasm and hope to achieve a higher output of Indoleacetic acid.

The second method revolves around Liquid-liquid-phase separation, a process naturally occurring in cells^[1]. Through this process, proteins aggregate together to form droplets or membrane-less bodies in the cytoplasm or nucleoplasm. This can be thought of as lipid droplets forming in water, just with proteins. Examples for this process in nature are stress granules or cajal bodies. The formation of droplets has already been studied by attaching YFP to Ddx4 (Nott et al.)^[2], a protein known for aggregation. There are also indicators for this to happen in bacteria3. To use this mechanism for improvement of enzyme efficiency, the YFP-coding region will be replaced by the coding region of the IAA enzymes and expressed in yeast, because of droplet size.

*Video mit der Bildung von droplets in HeLa zellen*

Formation of fluorescing droplets in HeLa cells, courtesy of Tim Nott

IAA concentrations were measured with GC-MS and compared to levels corresponding to free diffusion of the enzymes.

dCas9 approach:

Scaffolds for inducing metabolic channelling with nucleic acids has been tried in several different ways already. For example, by the iGEM ZJU China team of 2012, where RNA cloverleaf structures were used to bind IAA enzymes to RNA.1 They fused the enzymes to RNA-binding proteins from viral coat proteins (MS2 and PP7). These bound to the RNA and thus put the IAA enzymes close to each other (see picture below). With this concept, they were able to record a 1.4-fold increase in IAA output.

NUDT China 2015 mopdified this project and devised a DNA scaffold with the use of TALEs. They fused the IAA enzymes to TALEs and used different lengths between the TALE recognition sites to test is metabolic channelling was dependent on spacer lengths.

With these constructs, they could achieve a up to 10-fold increase in IAA output, with more output being registered at a spacer di8stance of 6 bp.1

Other methods using crisprRNA as a RNA scaffold have also been reported with some success as well as DNA scaffold with ZFNs2.

Our first approach focuses on dCas9. dCas9 is a mutated version of Cas9 where the endonuclease domains were modified so that the protein no longer cuts dsDNA3. This enables us to bind the IAA enzymes to DNA and thereby reduce the diffusion distance for the substrates.

We achieve this by fusing IAAM and IAAH to MS2 and PP7 like ZJU China 2012 did (we created the part BBa_ 2483000 for this, because ZJU China didn’t) and use sgRNA designs from iGEM Warwick 2016. They used these sgRNAs to bring transcription factors closer to promoter regions.4 The sgRNAs contain signal sequences for the RNA-binding proteins MS2 and PP7, the DNA recognition sites were slightly modified and are included in the part BBa_2483004.

When dCas9 binds to the DNA scaffold, the IAA-RBP fusions bind to the signal sequence on the sgRNA. By bringing in specific scaffold DNA, we can control the distance between the IAA enzymes and achieve metabolic channelling.