Part:BBa_K3711048
AOX1-α factor-CUS-AOX1 Terminator
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1187
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1289
Description
This is a composite component for expressing CUS outside the cell. CUS is transcribed and translated into curcuminoid synthase，which is the key enzyme in the synthesis of lycopene. It participates in the transformation from p-Coumaroyl-CoA and malonyl coenzyme to curcumin. AOX1 promoter is a strong promoter induced by methanol. Under the condition of methanol induction, with the help of α factor, CUS is translated and excreted from the cell.
Usage and Biology
Curcumin is synthesized sequentially by two different type III polyketone synthase (PKS) in curcumin rhizome, which is named dipeptide CoA synthase (DCS) and curcumin synthase (CURS). In addition to the DCS/CURS biosynthesis system in curcuma rhizome, we also found and characterized another type III PKS in rice plant Oryza sativa, curcumin synthase (CUS). The synthesis of curcumin catalyzed by CUS is as follows: firstly, p-gumaryl-CoA and malonyl-CoA are condensed to form dipeptide-CoA intermediate. The synthesized dipeptide CoA condensed with another p-coumaryl CoA to synthesize didemethoxycurcumin. CUS itself catalyzes both reactions of DCS/CUS, so the CUS system is simpler than the DCS/CURS system. In this respect, CUS is a better enzyme than DCS/CURS and is used in the metabolic engineering of curcumin in microorganisms. Besides. CUS can produce cinnamyl methane and curcumin from cinnamyl CoAn and ferulyl CoA.
Molecular cloning
Plasmid with target gene is transformed into E.coli. From them, we acquire large amount of target gene using as raw material for further operation.
The bands of AOX1-α factor-CUS-AOX1 Terminator (3000bp) , AOX1-α factor-ACC-AOX1 Terminator (3000+bp), AOX1-α factor-4CL-AOX1 Terminator (3000+bp) and AOX1-α factor-LOX2-AOX1 Terminator (almost 5000bp) from colony PCR are identical to the theoretical lengths of 3046bp, 3619bp, 3523bp and 4528bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these target plasmid had successfully transformed into E.coli.
Using E.coli for amplification, we extract and digest them with Bgl I or Sal I to get linear plasmid, which could be integrated into yeast genome to avoid getting lost while being frozen. Then, concentration of linear plasmid is also applied to achieve higher copy number and higher expression level. Several rounds of electroporation later, we successfully get all the plasmid with AOX1 as promoter into yeast.
The bright bands are identical to the theoretical lengths, which could demonstrate that this target plasmid had successfully transformed into yeast.
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