Design

Construction of CRISPR-Cas9 Knockout Plants

The high-level accumulation of compounds such as chlorogenic acid in Nicotiana benthamiana diverts a significant amount of metabolic precursors and energy away from the synthesis of secondary metabolites, limiting its application potential in synthetic biology. To overcome this limitation and enhance the synthetic capacity of the N. benthamiana platform, this project has constructed a genome-scale metabolic model (GSMM) for N. benthamiana to devise optimal strategies for metabolic flux regulation.

Under the guidance of the model, we aim to perform targeted knockout of the HQT (hydroxycinnamate CoA transferase) gene in the N. benthamiana genome using CRISPR-Cas9 technology, which will serve as the foundation for subsequent experimental investigations.

Identification of Genes for Knockout

Chlorogenic acid is a phenylpropanoid compound produced by plants via the shikimate pathway during aerobic respiration. Excessive synthesis of chlorogenic acid in tobacco diverts a substantial portion of the metabolic flux, impacting the heterologous synthesis of other pharmaceutically valuable secondary metabolites. We contemplate constructing a Nicotiana benthamiana platform with low chlorogenic acid content to enhance the synthesis throughput of the target product.

During the construction process, we first investigated three synthetic pathways commonly believed to be associated with chlorogenic acid through modeling:

1. Chlorogenic acid is synthesized by HQT-catalyzed transesterification of caffeoyl-CoA, derived from caffeic acid, with quinic acid (Fig.1-1, I);
2. p-Coumaroyl-CoA is first converted to p-coumaroyl shikimate under the catalysis of HCT, then to caffeoyl glycoside as an active intermediate, subsequently to caffeoyl-CoA, and finally to chlorogenic acid under HQT catalysis (Fig.1-1, II);
3. p-Coumaroyl-CoA is converted to p-Coumaroyl quinic acid under HCT catalysis, followed by C3'H-catalyzed hydroxylation to produce chlorogenic acid (Fig.1-1, III).

After conducting metabolic flux analysis based on the refined GSMM model, we ultimately selected the HQT pathway for partial knockout. See details

sgRNA Design for CRISPR-based Gene Knockout

There are four different NbHQT genes in the Nicotiana benthamiana genome, which exist as two pair of homologous genes. We plan to use CRISPR-Cas9 technology for their gene knockout. sgRNA is crucial for guiding Cas9 to specific genomic loci for targeted cleavage, enabling us to achieve our experimental goals. We have designed sgRNA targeting sequences for each gene to construct knockout plasmids that can individually knock out the four genes to generate single mutants. Additionally, we have constructed knockout plasmids capable of simultaneously knocking out two homologous genes to generate double mutants.

Construction of CRISPR-Cas9-knockout vector and plant

We use BG-plant-fast-cas9 vector provided by BIOGROUND company to construct CRISPR-Cas9-knockout plasmids by homologous recombination. We expect to obtain the following vector: NbHQT1-CRISPR, NbHQT2-CRISPR, NbHQT3-CRISPR, NbHQT4-CRISPR, NbHQT1+3-CRISPR, and NbHQT2+4-CRISPR. After constructing the vector, we planned to transform them to the Agrobacterium tumefaciens strain EHA105 and finally infect Nicotiana benthamiana.

Functional Validation of CRISPR-Cas9 Knockout Plants

Synthesis of Phaselic Acid

Phaselic acid is a common acylated acid found in plants with antioxidant and anti-inflammatory properties, which are thought to help prevent cardiovascular diseases, and also protects proteins from degradation, which can be used as feed proteins for ruminants. The synthetic pathway of phaselic acid in plants such as red clover has been elucidated (M.J.P.P. Sullivan, 2009). Therefore, we used HCT-M, a key enzyme in the synthesis pathway, as the main target gene and ligated it into an expression vector using Gateway cloning, and then transformed the expression vector into Agrobacterium tumefaciens strain GV3101 before transfecting the tobacco. In this way, the synthesis of phaselic acid in tobacco was achieved. Finally, phaselic acid was extracted by the corresponding metabolome extraction method and subjected to LC-MS/MS analysis to verify that the synthetic pathway had been successfully translated into tobacco.

Synthesis of Resveratrol

In the upstream pathway of chlorogenic acid synthesis in the shikimic acid pathway, resveratrol is a valuable non-flavonoid polyphenolic organic compound known for its antioxidant, anti-inflammatory, anti-cancer, and cardiovascular protective effects. Therefore, we chose it as a signature product to assess the efficiency of metabolite synthesis in our VersaTobacco chassis.

According to the metabolic pathway of resveratrol (Fig.2-2), STS is the key synthetic enzyme that doesn't exist in the Nicotiana benthamiana chassis, and L-Phe has the feedback inhibition on the upstream enzyme DHAP synthase. Thus, we introduce the efficient enzyme VvSTS gene, and the YIARO3 gene which can release this feedback inhibition (Liu.H, 2023), to synthesize resveratrol efficiently in VersaTobacco. The expression plasmids will be constructed by the Gateway method, transiently transform tobacco with Agrobacterium, and finally detect the target product by LC-MS/MS. Since our VersaTobacco has knocked out the chlorogenic acid synthase, theoretically, it will have a higher resveratrol synthesis efficiency compared to the wild type.

Synthesis of Crocin

Crocins are highly valuable water-soluble carotenoids. The synthetic pathway of crocin in saffron and gardenia has been fully elucidated (Fig.2-3). Therefore, four important enzymes in the synthetic pathway, GjCCD4a, GjALDH2C3, GjUGT74F8 and GjUGT94E13, are used as the main target gene. We tend to use Gateway, Gibson assembly and other methods to attach it to the expression vector, then transform the expression vector into Agrobacterium tumefaciens strain GV3101. And then we infect Nicotiana benthamiana with Agrobacterium. Thus, the purpose of synthesizing crocin in Nicotiana benthamiana is realized. Finally, extract crocin by the corresponding metabolome extraction method and perform the LC-MS/MS analysis to verify that the synthetic pathway is transformed into Nicotiana benthamiana.

Optimization of Function in CRISPR-Cas9 Knockout Plants

Semi-Rational Design of Enzymes

The catalytic activity of HCT2 derived from Trifolium pratense is limited, and its affinity for caffeoyl-CoA is weak. Instead, it will combine with feruloyl-CoA to generate many feruloyl-malic acid by-products. STS uses p-coumaroyl-CoA and malonyl-CoA as substrates to synthesize resveratrol, and its catalytic efficiency will significantly affect the biosynthesis yield of resveratrol. In order to improve the affinity of HCT2 to caffeoyl-CoA and enhance the catalytic efficiency of STS to increase resveratrol production, we conducted a semi-rational design of HCT2 and STS, screened beneficial mutants by computer-aided screening and designed relevant wet experiments for verification.

Directed Evolution of Enzymes

The catalytic activity of HCT-M derived from red clover (Trifolium pratense) is limited, exhibiting weak affinity for caffeoyl-CoA and instead preferentially binding to feruloyl-CoA, leading to the production of substantial amounts of feruloyl malic acid as a by-product (Sullivan, M. L., 2011). To address this issue, we optimized the enzyme activity of HCT-M through directed evolution, aiming to screen for mutants with high substrate affinity for caffeoyl-CoA. We introduced random mutations into the HCT-M gene using error-prone PCR, constructed variants with different HCT-M mutations, purified the mutated HCT-M proteins, and performed enzyme activity assays to screen for mutants with enhanced catalytic activity.