Team:BNDS China/Design
Carminic Production
The pathway of the biosynthesis from malonyl-CoA and acetyl-CoA to CA (carminic acid) comprises at least four enzymatic reactions—octaketide synthase, ZhuI or ZhuJ, Monooxygenase, and DcUGT2. However, because the biosynthesis of CA in yeast has not been reported yet , we planned to construct the pathway reaction by reaction to verify whether each enzyme remain its activity when it is expressed in yeast (S. cerevisiae).
The Choice of Backbone
As for the backbone of our plasmids, we use a high copy vector pYES, which can ensure enough copy of our genes. To balance between expression level and cellular burden, inducible promoter or medium-strong promoter is used to control gene expression, for which we can not only guarantee a certain amount of enzyme expression, but also avoid too heavy cellular burden . Details of chosen promoters will be discussed in following parts.
Backbone formation
First, OKS(octaketide synthase) is cloned to pYES downstream a galactose inducible promoter pGAL1. After transformation to yeast, the production of SEK4 and SEK4b, which are folded by the non-reduced octaketides generated through the catalysis of OKS, can be detected by HPLC (High Performance Liquid Chromatograph) analysis.
The Catalysis from OKS
Next, we introduced the aromatase (ZhuI) and/or cyclase (ZhuJ) to S. cerevisize. Since it is not clear whether these two enzymes are both essential for the conversion from non-reduced octaketides to FKA (flavokermesic acid anthrone), we designed three plasmids to test the expression and the function of our enzymes. Plasmid 1: heterologous coexpression of OKS and ZhuI Plasmid 2: heterologous coexpression of OKS and ZhuJ Plasmid 3: heterologous coexpression of OKS, ZhuI, and ZhuJ On each plasmid, moderate promoters pTEF1 and pHHF2 are added before ZhuI and ZhuJ respectively. As FKA is an unstable intermediate which can spontaneously convert into FK(flavokermesic acid ), we planned to test the FK production rate via HPLC analysis.
The Catalysis from FA
After the verification of the enzyme activity of OKS, ZhuI, and ZhuJ, we introduce last two enzymes, Monooxygenase and DcUGT2, to the previous plasmids that already contain OKS ZhuI and/or ZhuJ gene codons. The expressions of these two enzymes are controlled by two moderate promoters—pTEF2 and pPGK1 respectively. After the construction of the whole pathway, we planned to use HPLC to demonstrate the production of CA. We also planned to model our pathway by measuring the expression level of each enzyme, and the concentration of each intermediate product, in which way we could finely adjust the expression of each enzyme to achieve an optimum production of CA by yeast. If achieve so, Carminic acid could be produced in yeast in factories by fermentation instead of cochineal insect farming.