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It is a part composed of trGPPS and limonene synthase whose expression is regulated by Ptrc, and LacI to enable IPTG induction of expression of those enzymes. This part was created and used in a paper reported by Alonso-Gutierrez, Jorge et al. in 2013. Limonene can be synthesized by combining this part with an upstream pathway that can supply the products of the mevalonate pathway (IPP and DMAPP).

Sequence and Features Status: 500 Content-type: text/html

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Experiment

Fig. 1 Parts and metabolic pathways in the experiment of monoterpene synthesis and a schematic diagram of the experiment

In this cell-free monoterpene synthesis, we mixed two E. coli extracts each of which has either first 7 or last 2 enzymes of a pathway from Ac-CoA, which is a major intermediate of cell central metabolism. Through mevalonate pathway, the former extract one (derived from E. coli into which pBbA5c-MevT-MBI has been introduced) can provide IPP and DMAPP, which can also be used as intermediates for other important biosynthesis. Here we indeed supplemented only glucose and acetate as carbon sources. We obtained expression system for those seven genes from addgene and have converted this into Biobrick RFC 1000 format by synonymous replacement (BBa_K3580103). In order to take advantage of an engineering principle of synthetic biology we provided two biobrick parts (BBa_K3580101, BBa_K3580102) for the source for the latter extract. BBa_K3580101 has GPP synthase (GPPS) and limonene synthase. Although GPP synthase is shared with BBa_K3580101, BBa_K3580102 has sabinene synthase, which has one point mutation in limonene synthase (Srividya Narayanan et al 2015) and a new coding sequence for Parts registry of iGEM (See here for more details on this experiments).

Results

Similar to a very recent study from Jwett lab (Dudly et al 2019) who mixed 7 extracts, we synthesized limonene using only two extracts. Although fine-tuning can be possible when a larger number of extracts is prepared, we are sure that entry projects in iGEM should be simple but has engineering principle in order to expand iGEM sucess to an educational tool. This is why we selected the division into two extracts.

Fig. 2 GC/MS analysis results of cell-free limonene synthesis system