Type iii polyketide synthase, its encoding gene and use
A technology of polyketide synthase and application, applied in the direction of genetic engineering, plant gene improvement, application, etc., can solve the problem that the research on regulatory genes is still blank and other problems, and achieve the effect of improving the production efficiency or quality
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Embodiment 1
[0095] AsPKS1 prokaryotic expression vector construction and heterologous expression
[0096] The prokaryotic expression plasmid pET28a-AsPKS1 carrying the AsPKS1 gene was constructed and transformed into the expression host Escherichia coli E.coli BL21(DE3) for heterologous expression. The expression of AsPKS1 protein was induced by low temperature of 20℃. According to the fact that the recombinant protein AsPKS1 contains a HIS tag at the N-terminal, the AsPKS1 protein was purified using a nickel ion affinity chromatography column (Ni-NTA His-BindTMResin). The results of protein expression and purification are as follows: Figure 5 As shown, it can be seen that nickel ion affinity chromatography can achieve a good effect on the purification of the target protein, and there are few impurities after purification, and the size of the target protein is about 45kD. AsPKS1 protein with a concentration above 2mg / ml was obtained after concentration and desalination through a filter...
Embodiment 2
[0098] The AsPKS1 protein obtained in Example 1 is used to catalyze p-hydroxycinnamoyl-CoA and malonyl-CoA to produce p-hydroxybenzylidene acetone and bisdemethoxycurcumin with a C6-C4 skeleton. The specific operation steps are as follows:
[0099] The recombinant protein AsPKS1 purified by nickel ion affinity column was used for enzyme reaction experiment. 29nmol of malonyl-CoA, 26nmol of the starting substrate (p-hydroxycinnamoyl-CoA) and 37.5μg of purified AsPKS1 protein were added together to 500μL of 100mM potassium phosphate buffer (KPB buffer) (pH 7.0), in After reacting for 12 hours in a constant temperature water bath shaker (45° C.), 50 μL of 20% by weight HCl was added to terminate the enzyme reaction. The reaction solution was extracted three times with 800 μL of ethyl acetate, the combined extracts were blown dry with nitrogen, and then redissolved in 50 μL of methanol, and detected by a liquid phase mass spectrometer (LCMS-IT-TOF). By analyzing the molecular io...
Embodiment 3
[0102] The AsPKS1 protein obtained in Example 1 is used to catalyze feruloyl-CoA and malonyl-CoA to generate curcumin and 3-methoxy-4-hydroxybenzylideneacetone with a C6-C4 skeleton.
[0103] The operation was carried out according to the specific operation steps of Example 2, and the initial substrate was replaced by feruloyl-CoA, and other conditions and operation methods were the same as in Example 2. According to the measured product and the chemical reaction principle, the reaction formula of the present embodiment is as follows Image 6 As shown, that is, type III polyketide synthase AsPKS1 catalyzes the condensation of feruloyl-CoA and malonyl-CoA to generate curcumin (2b), and produces a small amount of 3-methoxy-4-hydroxyl with a C6-C4 backbone Benzylideneacetone (2a).
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