Team:Uppsala/Design

The saffron biosynthetic pathway is an extension of the β-carotene pathway, with zeaxanthin being a key intermediate(1). The pathway from farnesyl pyrophospate (FPP) to zeaxanthin is a BioBrick since before. We wanted to extend the β-carotene pathway to continue from zeaxanthin to crocin.



Plan integrating FPP to Zeaxanthin in Chromosome

The BioBrick includes a set of five genes thus making it a very large operon. As we wanted to extend the strain by adding three more genes we realised this would make the stain very unstable. To solve this our team created a zeaxanthin producing strain (link to their page) with chromosome integration using lambda red.

Plan going from Zeaxanthin to Crocin

To extend the pathway for the conversion of zeaxanthin to crocin, we needed to add the three step pathway, catalyzed by three different enzyme classes:
  1. Carotenoid cleavage dioxygenases (CCD) are responsible for the symmetric cleavage of zeaxanthin at the 7,8/7′,8′ positions to form crocetin dialdehyde from zeaxanthin.(2)
  2. Aldehyde Dehydrogenases (ADH) converts the 20 carbon cleavage product, crocetin dialdehyde to crocetin.(3)
  3. UDP-glucuronosyltransferase (UGT) catalyzes the glucuronidation reaction of forming crocin from crocetin.(3)

    4. Next we researched for the most appropriate enzymes under the above enzyme classes to execute the successful conversion of all the intermediates to crocin. We found CaCCD2(4), CsADH2946(3)and UGTCs2(5) to be the most promising for our project.
    The genes of the three individual enzymes were identified and synthesized as a gBlocks by IDT. Prior to synthesis, the genes were also codon optimized for E. coli, to get the desired overexpression of our enzymes. An N-terminal His-tag was added to aid the downstream purification process. This was done after drawing conclusions from the modelling results(link homology modelling) that predicted the N-terminal to be outside of the enzymes and far from the active sites after folding. The promoter used was an inducible promoter BBa_J04500 from the iGEM kit. The enzymes were created into BioBricks by using plasmid pSB1C3-J04500 with IPTG-inducible expression. The plasmid was linearised with Phusion PCR(link protocol) and the constructs were inserted into the iGEM plasmid using Gibson assembly(link protocol). We transformed the plasmid into E. coli (TOP10 competent cells), screened the colonies using colony PCR(link protocol) and run gel electrophoresis(link protocol) to validate that the insert had been successfully assembled into the plasmid. We also created BioBricks with a part for RFP expression. Our next course of action was then to overexpress the individual enzymes in E. coli strain BL21(DE3*) and purify them. Immobilized metal ion affinity chromatography (IMAC) was used as the preferred purification step (explaining the presence of the His-tag in all our enzyme)s. Thereafter we wanted to check the in vitro enzyme activity and for this we used commercially available zeaxanthin, crocetin dialdehyde and crocetin respectively as substrates for the three enzymes. Crocin was also obtained pure, to compare with the results of the last enzymatic step.
References

  1. Ángela, L. G., and Ahrazem, R. O. 2010. Understanding Carotenoid Metabolism in Saffron Stigmas : Unravelling Aroma and Colour Formation. Functional Plant Science and Biotechnology 4, 56–63.
  2. Frusciante S, Diretto G, Bruno M, Ferrante P, Pietrella M, Prado-Cabrero A, et al. Novel carotenoid cleavage dioxygenase catalyzes the first dedicated step in saffron crocin biosynthesis. Proceedings of the National Academy of Sciences. 2014 Aug 19;111(33):12246–51.
  3. Gómez-Gómez L, Parra-Vega V, Rivas-Sendra A, Seguí-Simarro JM, Molina RV, Pallotti C, et al. Unraveling Massive Crocins Transport and Accumulation through Proteome and Microscopy Tools during the Development of Saffron Stigma. Int J Mol Sci [Internet]. 2017 Jan 1 [cited 2017 Oct 29];18(1).
  4. Ahrazem O, Rubio-Moraga A, Berman J, Capell T, Christou P, Zhu C, et al. The carotenoid cleavage dioxygenase CCD2 catalysing the synthesis of crocetin in spring crocuses and saffron is a plastidial enzyme. New Phytol. 2016 Jan 1;209(2):650–63
  5. Moraga AR, Nohales PF, Pérez JAF, Gómez-Gómez L. Glucosylation of the saffron apocarotenoid crocetin by a glucosyltransferase isolated from Crocus sativus stigmas. Planta. 2004 Oct;219(6):955–66.