Team:Nagahama/Characterization

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Nagahama

Characterization

We have characterized [http://parts.igem.org/Part:BBa_K849000 BBa_K849000].

Fig.1 Overview of the ergosterol biosynthetic pathway in S. cerevisiae and the carotenogenic pathway in X. dendrorhous. The carotenogenic pathway in X. dendrorhous consists of GGPP synthase encoded by crtE, the bifunctional enzyme phytoene synthase and lycopene cyclase encoded by crtYB, and phytoene desaturase encoded by crtI. S. cerevisiae contains a GGPP synthase, encoded by BTS1, which is able to convert FPP into GGPP. HMG1 encodes HMG-CoA reductase, which is the main regulatory point in the ergosterol biosynthetic pathway in many organisms. IPP:isopentenyl diphosphate DMAP:dimethylallyl diphosphate GPP:geranyl diphosphate.

HMG1 encodes HMG-CoA reductase and catalyzes conversion of HMG-CoA to mevalonate, which is a rate-limiting step in sterol biosynthesis. Because mevalonate is synthesized, it is easy to flow to subsequent pathways. Therefore,β-carotene production increase. Color of colony was changed yellow from orange when tHMG1 was inserted into S. cerevisiae synthesizing carotenoid.(Fig.2,3,4) This result showed that tHMG1 was inserted.

tHMG1 has a characteristic of changing color of S. cerevisiae. This result to be due to the fact that as a result of massive synthesis of mevalonate, β-carotene was produced in large amount.

In order to investigate whether tHMG1 increased β-carotene productions, color of S. cerevisiae and β-carotene were compared. It is considered that the amount of β-carotene synthesized increased because the color of yeast introduced tHMG1 was closed the color of 1μg of β-carotene dissolved in 1 ml of hexane.

Fig2.S. cerevisiae synthesizing carotenoid by inserting crtYB, crtI and crtE into genomic DNA of S. cerevisiae.

Fig3.Result of colour of colony by inserting crtYB, crtI,crtE and tHMG1 into genomic DNA of S. cerevisiae.

Fig4.Result of the counterpart control S. cerevisiae which was transformed crtYB, crtI, crtE and empty vector isinto genomic DNA of S. cerevisiae.

Fig5.a:fig1,b:fig2,c:fig3,d:β-carotene

Reference

[http://aem.asm.org/content/73/13/4342.full]Verwaal R, Wang J, Meijnen JP, Visser H, Sandmann G, van den Berg JA, van Ooyen AJ (2007) High-level production of beta-carotene in Saccharomyces cerevisiae by successive transformation with carotenogenic genes from Xanthophyllomyces dendrorhous. Appl Environ Microbiol 73(13):4342–4350