Index
Overview
In order to utilize the MVA pathway inherent in Saccharomyces cerevisiae for the production of medicinally active compounds in the traditional Chinese medicine Danshen (Salvia miltiorrhiza), such as tanshinone, as well as a diterpene carnosic acid with a similar structure, we integrated the genes of the constructed enzymes related to the phenolic diterpenes through three consecutive Golden Gate assembly reactions and ligated them into the plasmid pYTK096, which was expressed in Saccharomyces cerevisiae cells for the production of tanshinone and carnosic acid capable of treating myocarditis.
1 Source of the target genes
We obtained all CDS sequences of interest from Salvia plants.
1.1 Synthesis pathways of tanshinones and carnosic acid in Salvia miltiorrhiza
Our project generates a molecule of IPP through six enzymatic reactions via the MVA pathway that occurs naturally in the chassis organism, Saccharomyces cerevisiae. After formation of IPP, it can be reversibly isomerized to DMAPP by isomerase (IDI). After DMAPP and IPP start to condense head to tail, we integrate the genes of key enzymes of the diterpene pathway by Golden Gate molecular cloning to integrate the key enzyme genes of the diterpene pathway, and then follow the pathway shown in Figure 2 to obtain tanshinones and carnosic acid.
1.2 Key enzymes in the synthesis pathway of tanshinones and carnosic acid
Tanshinones and carnosic acid are representative components of Salvia plants. The biosynthesis of these two diterpenoids requires the catalytic completion of key enzymes of the multistep diterpene pathway, and the full names and functions of the various synthases are shown in the table below.
| Abietane-Type Diterpenoids Synthase Gene | Full Name | Function in Synthesizing Tanshinone |
|---|---|---|
| SmGGPPS | Geranylgeranyl diphosphate (GGPP) | Geranylgeranyl pyrophosphate synthase in the downstream MEP pathway of Salvia miltiorrhiza |
| SmCPS1 | Copalyl diphosphate (CP) | A key enzyme in tanshinone synthesis downstream of the MEP pathway that catalyzes the production of tanshinones in this species |
| SmKSL | Kaurene synthase like (KSL) | A key enzyme in tanshinone synthesis downstream of the MEP pathway that catalyzes the production of tanshinones |
| SmCPR | NADPH genes of Danshen | Cytochrome P450 reductases |
| CYP76AHs | The CYP76AH subfamily of cytochrome P450 oxygenases |
SmCYP76AH1 is a ferredoxin synthase that catalyzes the C12 hydroxylation of miltiradiene. SmCYP76AH3 is a hybrid catalytic P450 that catalyzes the hydroxylation of ferredoxin at the C11 position and the carbonylation of tanshinone at the C7 position, generating 11-hydroxyferredoxin, lupulin, and 11-hydroxylupulin. The carbonylation function at the C7 position allows for the extension of the tanshinone biosynthesis pathway into an additional branched chain, resulting in the formation of a complex catalytic network. SmCYP76AH22 is homologous to CYP76AH3 and also produces 11-hydroxyferriol. Next, three successive C20 oxidations catalyzed by CYP76AK6-8 converted 11-hydroxyferrocenol to carnosic acid, which in turn spontaneously oxidized to carnosol. |
| CYP76AKs | The CYP76AK subfamily of cytochrome P450 oxygenases (oxygenases) | CYP76AKs catalyze the oxidation of the C20 position. CYP76AK6 catalyzes three consecutive C20 oxidations, converting 11-hydroxyferrocenol to carnosic acid, which in turn spontaneously oxidizes to carnosol. |
2 Chassis Selection
The advantages of choosing yeast cells as chassis organisms are their clear genetic background, ease of genetic manipulation, short growth cycle, good environmental tolerance, large-scale fermentation, few by-products, simple product purification and high safety. Saccharomyces cerevisiae has subcellular structures such as endoplasmic reticulum, Golgi apparatus, peroxisome and post-translational modification mechanisms, which can well express proteins of plant and animal origin, such as cytochrome P450-like proteins [4]. The naturally occurring MVA pathway in Saccharomyces cerevisiae provides precursors for terpene synthesis compared to E. coli, algae, molds, etc. and is considered by researchers to be more suitable for use as a chassis organism for terpene product synthesis. In addition to this, yeast cells can be fermented at high density[5], which provides a good platform for us to extract tanshinone derivatives by fermentation.
3 Golden Gate Core Component Build
We applied the Golden Gate method to integrate the genes of enzymes controlling the synthesis of tanshinones, and ligated them into the plasmid pYTK096 to construct the gene pathways TY9 and TY10, and then integrated them into the genomes of S. cerevisiae BY4742 by transformation. Then the engineered yeasts obtained from the constructs were fermented and cultured on a large scale to synthesize and accumulate large amounts of tanshinones and other diterpenoids. The fermentation products were extracted and purified from the fermentation broth, and the composition of the products was analyzed qualitatively and quantitatively.
We constructed plasmid vectors for TY9 and TY10 by three steps:
(1) Level-0 Vector Construction
The sequences containing the target genes (SmCPS1, SmKSL, SmCPR, CYP76AH1, CYP76AH3, CYP76AH22, CYP76AK6) were cloned by PCR reaction and inserted into vector pYTK001, respectively. The plasmids were constructed as follows: pYTK001-SmGGPPS (or SmCPS1, SmKSL, SmCPR, CYP76AH1, CYP76AH3, CYP76AH22, CYP76AK6).
In Saccharomyces cerevisiae, promoters can be categorized into inducible and constitutive types [6]. Inducible promoters are represented by galactose metabolism-related promoters, which are used to control the dynamic regulation of bacterial growth and gene expression. In contrast, the most studied and widely used constitutive promoters are used in our project (Table 2). This type of promoter is simple to operate and initiates the regulation of gene expression together with bacterial growth.
In Saccharomyces cerevisiae, terminator sequences not only terminate transcription, but also affect the expression level of proteins encoded upstream of the terminator, for example, the SSA1 gene has been speculated to act as an RNA editor to promote efficient expression of upstream genes [7]. By reviewing the literature we decided to select the corresponding promoters and terminators used for transcription in Saccharomyces cerevisiae to control the expression of downstream products according to the roles of the enzymes expressed by different genes.
The plasmid was constructed as follows:
a) pYTK001-PTDH3 (or PCCW12, PPGK1, PTEF1, PTEF2)
b) pYTK001-TENO1 (or TSSA1, TADH1, TTDH1, TENO2)
The constitutive promoters and terminators we used are shown in the table below:
| Constitutive Promoters | Full Name | Pathways |
|---|---|---|
| PTDH3 | Glyceraldehyde-3-phosphate dehydrogenase | Glycolytic Metabolic Pathway [4, 8] |
| PCCW12 | Cell wall glycoprotein | Protein folding [9] |
| PPGK1 | Phosphoglycerate kinase 1 | Glycolytic Metabolic Pathway [8, 10] |
| PTEF1 | Translation Elongation Factor | RNA transport [8, 10] |
| PTEF2 | Translation Elongation Factor | RNA transport [8, 10] |
| Terminators | Full Name | Pathways |
|---|---|---|
| TENO1 | Phosphopyruvate hydratase | Glycolytic Metabolic Pathway [11, 12] |
| TSSA1 | Hsp70 family ATPase | RNA transcription [7] |
| TADH1 | Alcohol Dehydrogenase | Glycolytic Metabolic Pathway [5] |
| TTDH1 | Glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) 1 | Glycolytic Metabolic Pathway |
| TENO2 | Phosphopyruvate hydratase | Glycolytic Metabolic Pathway [11, 12] |
(2) Level-1 Vector Construction
The modules of each Level 0 were combined and constructed into vector pYTK095 by BsaI digestion and ligation, and the plasmids were constructed as follows:
- pYTK095-PTDH3-SmGGPPS-TENO1
- pYTK095-PCCW12-SmCPS1-TSSA1
- pYTK095-PPGK1-SmKSL-TADH1
- pYTK095-PTEF2-SmCPR-TTDH1
- pYTK095-PTEF1-CYP76AH1-TENO2
- pYTK095-PTEF2-CYP76AH3-TTDH1
- pYTK095-PTEF1-CYP76AH22-TENO2
- pYTK095-PTEF2-CYP76AK6-TTDH1
CDS: SmGGPPS, SmCPS1, SmKSL, SmCPR, CYP76AH1, CYP76AH3, CYP76AH22, CYP76AK6
Promoters: PTDH3, PCCW12, PPGK1, PTEF1, PTEF2;
Terminators: TENO1, TSSA1, TSSA1, TADH1, TTDH1, TENO2
(3) Level-2 Vector Construction
Each vector of Level 1 was assembled and constructed into pYTK096 vector by BsmBI digestion and ligation.
The plasmids were constructed as follows:
In these two vectors, GFP and Kana genes were used as genetic selection markers.
4 Transformation of Saccharomyces cerevisiae
The constructed vector was linearized by NotI endonuclease and transferred into BY4742 yeast receptor cells, which were screened by -ura-deficient medium, and the yeast genomic DNA was extracted for PCR identification to determine the positive single clones.
5 Extraction and Detection of Fermentation Products
5.1 Fermentation culture and product extraction and isolation of engineered yeasts
The successful monoclonal plasmid was expanded in culture, shaken overnight and preserved. The strain was further fermented and cultured using liquid YPDA medium for 5 days before centrifugation to collect the fungal cells. Yeast cells were extracted by ultrasonic extraction with methanol for 1 h. After centrifugation at low temperature, the supernatant extract was filtered through a filter membrane, and the filtered sample was transferred to a liquid phase vial with a liner tube for Q-Exactive analysis.
5.2 Detection and analysis of fermentation products
The Q-Exactive assay was performed using a Thermo Fisher analytical instrument, and the mass spectrometry data was analyzed using Xcalibur software, which was used to detect our synthesized products by comparing them with standards and graphing them using Excel.
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