Description

Introduction

Nicotiana benthamiana, a model plant native to Australia, is extensively used in scientific research. Young N.benthamiana plants are highly susceptible to Agrobacterium tumefaciens, which facilitates transient gene expression in leaf tissues through agroinfiltration. Remarkably, N.benthamiana leaves can be agroinfiltrated with a complex mix of Agrobacterium strains, allowing for the transient co-expression of multiple genes without constructing complex genetic circuits[1].

Over the past decade, facilities have been developed to perform agroinfiltration on a large scale, enabling commercial-scale protein production. Medicago, a biopharmaceutical company headquartered in Quebec City, developed COVIFENZ®, the COVID-19 vaccine, by expressing the coronavirus-like particle (CoVLP) of the original strain of SARS-CoV-2 in N.benthamiana[2].

As more natural product pathways are discovered through this method, the facilities designed for industrial protein production are likely to be repurposed for producing high-value plant natural products, such as terpenoids or flavonoids. For example, K.S.et al.[3] synthesized artemisinin sesquiterpenes in tobacco, achieving a yield of 39.5 mg/kg fresh weight of artemisinic acid-12-β-diglucoside.

Problems

While N.benthamiana is a promising chassis for biomanufacturing, it still has inevitable drawbacks.

Like other Nicotiana species, N.benthamiana accumulates high amounts of secondary metabolites, like chlorogenic acid and its derivatives. Its own metabolites will divert too many metabolic streams, reducing the efficiency of heterologous synthesis.

In addition, the current chassis modification logic for N.benthamiana is mostly bottom-up, with chassis designed based on known target products, which is target product specific and not universally applicable [4]. We wondered whether it would be possible to develop a N.benthamiana chassis that could increase the heterologous synthesis yield of different target products.

Our solution: VersaTobacco

VersaTobacco can be mainly divided into two parts: the construction of the low-chlorogenic acid content Nicotiana benthamiana chassis and functional verification of the chassis.

After analyzing the N.benthamiana metabolome, we found that N.benthamiana contains too much chlorogenic acid and its derivatives, which may divert too many metabolic flows and affect the synthesis efficiency(Fig.1).

The chlorogenic acid synthesis pathway of tobacco mainly involves six key enzymes: phenylalnine ammonialyase(PAL)、cinnamic acid 4-hydroxylase(C4H), 4-coumarate-CoA ligase(4CL)、p-coumaroylester 3'-hydroxylases(C3'H), hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferase(HCT) and hydroxycinnamoyl CoA:quinate hydroxycinnamoyl transferase(HQT)[5-8]

At present, it is generally believed that there are three main pathways for the biosynthesis of chlorogenic acid: the first is the synthesis of chlorogenic acid by HQT-catalyzed ester exchange between caffeoyl-CoA and quinic acid (Figure 2, I); the second is the generation of active intermediates of caffeoyl-CoA first, followed by the generation of chlorogenic acid under the catalysis of HQT (Figure 2, II); the third is the generation of p-coumaroylquinic acid under the catalysis of HCT, followed by C3H hydroxylation to generate chlorogenic acid (Figure 2, III); however, when both HCT and C3H are active in Arabidopsis, chlorogenic acid is not accumulated, which raises doubts about the existence of this biosynthetic pathway for chlorogenic acid[9].

We used the COBRApy toolkit to perform flux balance analysis (FBA) on the Genome-scale metabolic network model (GSMM) of Nicotiana benthamiana and found that knocking out the HQT down-regulated the chlorogenic acid production response. Significantly correlated with the upregulation of other metabolic flux reactions.

Part I: Chassis Construction

Constructing NbHQTs knockout N.benthamiana by CRISPR/Cas9

The CRISPR/Cas9 system has been widely used for plant genome editing due to its simplicity, efficiency, and precision. In the study of Nicotiana benthamiana(10), excessive synthesis of chlorogenic acid and its derivatives can divert metabolic streams and hinder the heterologous synthesis of other valuable secondary metabolites. To address this, we planned to precisely knock down four key chlorogenic acid synthesis genes (NbHQT1, NbHQT2, NbHQT3, and NbHQT4) in the genome of the N.benthamiana LAB strain using CRISPR-Cas9 technology. This approach aims to shift the metabolic flow towards other pathways, thereby optimizing N.benthamiana's potential as a chassis for synthetic biology and increasing the yield of medicinal secondary metabolites.

To achieve this goal, we first accessed a database to obtain the gene sequences of NbHQTs. We then designed single-guide RNAs (sgRNAs) using CRISPR-P 2.0 to predict gene editing targets. We constructed single knockout vectors for each of the four genes and double knockout vectors for NbHQT1+3 and NbHQT2+4 through homologous recombination. These vectors were used to transform N. benthamiana, resulting in a stable chassis for synthetic biology. This transformation will reshape the metabolic network of the N. benthamiana chassis, making it more suitable for the production of high-value medicinal natural products.

Part II: Functional validation

Functional validation of N.benthamiana chassis at low chlorogenic acid levels

To further verify the function of the chassis, we planned to reconstruct the synthesis pathways of phaselic acid, resveratrol and crocin, three products from different secondary metabolic pathways of plants, in our modified chassis, hoping to clarify the advantages of our chassis from multiple aspects.

Synthesis of phaselic acid

Phaselic acid(caffeoyl malate)and chlorogenic acid (caffeoylquinic acid) are structurally similar secondary metabolites synthesized by hydroxycinnamoyltransferases. Phaselic acid serves as an important antioxidant and can inhibit cardiovascular disease and breast cancer.It also help with measles. Additionally, Phaselic acid has demonstrated the ability to protect against protein degradation, making it advantageous for preserving nutrient proteins in forage feeds[11].

The hydroxycinnamoyltransferase enzyme found in tobacco has much lower affinity for malate than shikimalte and quinic acid. So in order to test whether increased caffeoyl CoA could be used to synthesize other product after knocking out of HQT gene,we introduce hydroxycinnamoyl transferase:malate transferase from red clover[12], aiming to facilitate the synthesis of phaselic acid in this new tobacco species, thereby assessing the potential of HQT-knockout tobacco as a novel chassis for synthetic biology.

Synthesis of resveratrol

Hydroxycinnamoyl-CoA: quinate hydroxycinnamoyl transferase,HQT catalyzes the conversion of p-coumaroyl-CoA to produce p-coumaroylquinic acid, which serves as an intermediate in the biosynthesis pathway leading to chlorogenic acid. After knocking down HQT, our aim was to investigate whether the accumulated p-coumaroyl coenzyme A could be redirected into alternative metabolic pathways to enhance the production of downstream products.

To further validate the potential of our modified tobacco chassis for synthesizing high-value metabolites, we targeted the production of resveratrol. Resveratrol is a valuable phenolic compound synthesized via the mangiferic acid metabolic pathway, directly downstream from p-coumaroyl-coenzyme A. Previous studies have highlighted resveratrol's diverse pharmacological activities, including antioxidant, anti-inflammatory, anti-aging, and neuroprotective properties. It has shown promise in treating neurodegenerative diseases, cancers, diabetes, and cardiovascular disorders, making it a versatile candidate for pharmaceutical, cosmetic, and nutraceutical applications [13].

Resveratrol (3,5,4'-trihydroxystilbene) presents in more than 70 species of plants, particulaely in grapes. It is mostly present in trans structures and modified products, which are considered as plant antitoxins that help plants to resist different microbial infections and harsh environments [14]. According to the resveratrol metabolic pathway in plants, stilbene synthase (STS) is definitely the key to final resveratrol synthesis [15], while L-phenylalanine competitively binds to DHAP synthase to produce a feedback inhibition, also limiting resveratrol synthesis. Therefore, we chose to introduce a highly efficient stilbene synthase (STS) from Vitis vinifera [15] and overexpress the mutant ARO3 enzyme in 3-Deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase [16], trying to synthesize resveratrol in tobacco.

In addition, modifications such as methylation of resveratrol can produce resveratrol derivatives such as pinostilbene and pterostilbene, which can reduce the toxic effects of resveratrol on plants [14] and further increase the production of resveratrol.

Synthesis of crocin

The methylerythritol 4-phosphate pathway (MEP pathway) is a critical metabolic pathway in Nicotiana benthamiana, and it intersects with the shikimate pathway. Changes in the metabolic flux within this pathway are indicative of the chassis improvement in our study of Nicotiana benthamiana.

Downstream of the MEP pathway, numerous high-value metabolites are produced, among which terpenoids are widely applied in medicine, food, energy and other fields. Crocin, a tetraterpenoid renowned for its distinctive aroma and vibrant color, is usually used as a natural colorant and flavor enhancer. However, its availability is limited, yields are low, and the extraction process is complicated, making industrial production challenging. In a previous study, the synthesis of Crocin in the T1 generation of Nicotiana benthamiana reached as high as 1,058,945 ng/g through the introduction of exogenous genes [17].

In our research, we aim to synthesize Crocin within the modified Nicotiana benthamiana chassis and evaluate whether the yield can be further enhanced using techniques such as HPLC-MS/MS. This assessment aims to confirm the advantages of our modified chassis in terms of metabolic engineering and synthetic biology.