1 Introduction

Tea (Camellia sinensis) is one of the most widely consumed beverages globally, second only to water. It holds immense economic, medicinal, and cultural significance. The tea plant is cultivated extensively in various regions, contributing significantly to the agricultural economy (Xia et al., 2017; Wei et al., 2018). The unique flavor, aroma, and health benefits of tea are attributed to its diverse bioactive compounds, making it a valuable crop for both consumption and research (Li et al., 2022; Samanta, 2020).

Tea is rich in bioactive compounds such as catechins, theanine, caffeine, and various polyphenols, which are responsible for its health-promoting properties. These compounds exhibit antioxidant, anti-inflammatory, neuroprotective, and anticancer activities, among other benefits (Shi et al., 2011; Li et al., 2022; Samanta, 2020). For instance, L-theanine, a unique amino acid found in tea, is known for its calming effects and potential applications in functional foods (Li et al., 2022). The presence of these bioactive compounds not only enhances the sensory qualities of tea but also contributes to its therapeutic potential (Cheng et al., 2017; Samanta, 2020).

Despite the known benefits of bioactive compounds in tea, enhancing their production poses several challenges. The complex biosynthetic pathways and the large genome size of the tea plant make genetic manipulation difficult (Shi et al., 2011; Xia et al., 2017; Wei et al., 2018). Additionally, environmental factors, cultivation practices, and microbial interactions in the rhizosphere can significantly influence the levels of these compounds (Bag et al., 2021). Understanding the genetic and biochemical pathways involved in the synthesis of these compounds is crucial for developing strategies to enhance their production (Shi et al., 2011; Xia et al., 2017; Wei et al., 2018).

This study explores the latest advancements in the field of tea metabolic engineering to enhance the production of bioactive compounds; covers the genetic and biochemical pathways involved in the synthesis of key bioactive compounds, the role of environmental and microbial factors, and the potential applications of these findings in improving tea quality and health benefits. By integrating the latest research, this study provides a comprehensive understanding of the challenges and opportunities faced in tea metabolic engineering.

2 Bioactive Compounds in Tea

2.1. Key bioactive compounds: catechins, theaflavins, thearubigins, etc.

Tea, derived from the leaves of Camellia sinensis, is rich in a variety of bioactive compounds, including catechins, theaflavins, and thearubigins. Tea polyphenols are compounds that possess multiple phenolic hydroxyl groups, and catechins are among the most important constituents (Chen, 2024). Catechins, such as (-)-epigallocatechin gallate (EGCG), (-)-epicatechin (EC), and (+)-catechin, are prominent in green tea and are known for their potent antioxidant properties (Punyasiri et al., 2004; Zhang et al., 2019; Samanta, 2020). Theaflavins and thearubigins, on the other hand, are formed during the fermentation process of black tea. These compounds contribute to the color and flavor of black tea and also possess significant health benefits (Zhang et al., 2019; Samanta, 2020). The transformation of catechins into theaflavins and thearubigins during tea processing is a critical aspect of tea chemistry, influencing both the sensory attributes and the bioactive profile of the final product (Zhang et al., 2019; Yu et al., 2020).

In addition to these polyphenolic compounds, tea also contains other bioactive substances such as L-theanine, caffeine, and various flavonols. L-theanine, a unique amino acid found in tea, is known for its calming effects and potential health benefits, including neuroprotection and cardiovascular protection (Jeszka-Skowron et al., 2018; Li et al., 2020). Caffeine and other methylxanthines like theobromine contribute to the stimulating effects of tea and have been shown to regulate intracellular second messenger levels (Jeszka-Skowron et al., 2018; Samanta, 2020). The diverse array of bioactive compounds in tea underscores its multifaceted health benefits and its importance as a functional beverage.

2.2 Health benefits associated with bioactive compounds

The bioactive compounds in tea are associated with a wide range of health benefits. Catechins, particularly EGCG, have been extensively studied for their antioxidant, anti-inflammatory, and anticancer properties. These compounds help in scavenging free radicals, reducing oxidative stress, and inhibiting the growth of cancer cells (Punyasiri et al., 2004; Wei et al., 2018; Samanta, 2020). Theaflavins and thearubigins, found in black tea, also exhibit antioxidant activities and have been linked to cardiovascular health benefits, including the reduction of blood cholesterol levels and improvement of blood vessel function (Zhang et al., 2019; Samanta, 2020).

L-theanine, another significant bioactive compound in tea, has been shown to promote relaxation without drowsiness, enhance cognitive function, and provide neuroprotective effects. It also exhibits potential benefits in regulating blood pressure and supporting immune function (Jeszka-Skowron et al., 2018; Li et al., 2020). Caffeine, while primarily known for its stimulating effects, also contributes to the overall health benefits of tea by enhancing mental alertness and physical performance (Jeszka-Skowron et al., 2018; Samanta, 2020). The combined effects of these bioactive compounds make tea a valuable beverage for promoting overall health and well-being.

2.3 Factors affecting bioactive compound levels in tea

The levels of bioactive compounds in tea can be influenced by various factors, including the type of tea, processing methods, and environmental conditions. For instance, green tea, which undergoes minimal oxidation, retains higher levels of catechins compared to black tea, which is fully fermented and contains higher levels of theaflavins and thearubigins (Zhang et al., 2019; Samanta, 2020). The specific processing steps, such as withering, rolling, fermentation, and drying, play a crucial role in determining the final composition of bioactive compounds in tea (Zhang et al., 2019; Yu et al., 2020).

Environmental factors such as shading, temperature, and soil conditions also significantly impact the levels of bioactive compounds in tea leaves. Shading, for example, has been shown to reduce catechin levels while increasing theaflavin content in preharvest tea leaves by enhancing polyphenol oxidase activity (Liu et al., 2018; Yu et al., 2020). Additionally, the genetic makeup of the tea plant and the specific cultivar can influence the biosynthesis and accumulation of these compounds (Singh et al., 2008; Wei et al., 2018). Understanding these factors is essential for optimizing the production and quality of tea to maximize its health benefits.

3 Metabolic Pathways in Tea Plants

3.1 Overview of metabolic pathways leading to bioactive compound synthesis

Tea plants (Camellia sinensis) are renowned for their rich array of bioactive compounds, primarily catechins, theaflavins, and thearubigins, which are synthesized through complex metabolic pathways. The biosynthesis of these compounds predominantly follows the phenylpropanoid and flavonoid pathways. Catechins, for instance, are synthesized from phenylalanine through a series of enzymatic reactions involving phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), and 4-coumarate-CoA ligase (4CL), leading to the formation of flavan-3-ols such as (-)-epigallocatechin gallate (EGCG) and (-)-epicatechin gallate (ECG) (Punyasiri et al., 2004; Wang et al., 2018; Zhang et al., 2019). The transformation of catechins into theaflavins and thearubigins occurs during the fermentation process, which involves oxidative polymerization catalyzed by polyphenol oxidase (PPO) (Tanaka et al., 2004; Yu et al., 2020).

The biosynthesis of other significant compounds like theanine and caffeine also involves specific pathways. Theanine is synthesized from glutamic acid and ethylamine, catalyzed by theanine synthetase, while caffeine biosynthesis involves the methylation of xanthosine (Wei et al., 2018; Zhang et al., 2021). These pathways are tightly regulated and influenced by various environmental factors, such as shading, which can alter the expression of key enzymes and subsequently the levels of bioactive compounds (Yu et al., 2020; Zeng et al., 2020).

3.2 Key Enzymes and genes involved

Several key enzymes and genes play crucial roles in the metabolic pathways of tea plants. For catechin biosynthesis, enzymes such as anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) are vital, converting anthocyanidins to catechins (Punyasiri et al., 2004; Wang et al., 2018). The gene CsPPO3, encoding polyphenol oxidase, is particularly important for the oxidation of catechins to theaflavins during tea processing. This gene is highly expressed under shading conditions, which enhances PPO activity and theaflavin content in preharvest tea leaves (Yu et al., 2020).

In theanine biosynthesis, the gene encoding theanine synthetase (CsTSI) is regulated by the MYB transcription factor CsMYB6, which binds to the promoter region of CsTSI, enhancing its expression in the roots of tea plants (Zhang et al., 2021). For caffeine biosynthesis, genes involved in the methylation process, such as caffeine synthase, are critical. The draft genome sequence of Camellia sinensis has identified numerous gene families and their duplications, which are essential for the biosynthesis of these key metabolites (Figure 1) (Wei et al., 2018).

Figure 1  Functional analysis of key genes in theanine biosynthesis (Adapted from Wei et al., 2018) Image Caption: A: Theanine biosynthesis pathway, involving key genes such as glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH), arginine decarboxylase (ADC), and theanine synthetase (TS); B: A phylogenetic tree was constructed by comparing glutamine synthetase genes known in other organisms; C: The theanine synthesis activity of CsTSI was detected by overexpressing the tea plant TS gene (CsTSI) in Arabidopsis, with or without feeding ethylamine (Adapted from Wei et al., 2018)

3.3 Regulatory mechanisms of metabolic pathways

The regulation of metabolic pathways in tea plants involves a complex network of genetic and environmental factors. Transcription factors such as MYB play a significant role in regulating the expression of genes involved in the biosynthesis of bioactive compounds. For instance, CsMYB6 regulates theanine synthesis by activating the theanine synthetase gene in the roots (Zhang et al., 2021). Additionally, environmental factors like shading can modulate the expression of genes such as CsPPO3, thereby influencing the levels of catechins and theaflavins in tea leaves (Yu et al., 2020).

Post-transcriptional modifications, such as alternative splicing and the presence of long noncoding RNAs (lncRNAs), also contribute to the regulation of these pathways. These modifications can affect the stability and translation of mRNAs encoding key enzymes, thereby fine-tuning the biosynthesis of bioactive compounds (Zhang et al., 2021). Furthermore, feedback regulation mechanisms, where the accumulation of end products like catechins can down-regulate the expression of biosynthetic genes, ensure a balanced production of these compounds (Singh et al., 2008).

Wei et al. (2018) enhanced the understanding of amino acid metabolism in tea plants by revealing the key pathways and genes involved in the biosynthesis of theanine. The experimental results indicated that the TS candidate gene (CS) in tea plants shows high similarity to known GSI-type genes, and the CsTSI gene plays a crucial role in theanine synthesis. Moreover, treatment with ethylamine significantly increased theanine content. This provides an important molecular foundation for future genetic engineering efforts to improve tea quality.

4.2 CRISPR-Cas9 and genome editing approaches

The advent of CRISPR-Cas9 technology has revolutionized the field of genetic engineering, providing a more precise and efficient method for genome editing. CRISPR-Cas9 allows for targeted modifications at specific genomic loci, enabling the knockout, knock-in, or alteration of genes with high precision. This technology has been successfully applied in various organisms, including plants, to enhance the production of bioactive compounds. For instance, CRISPR-Cas9 has been used to knock out genes involved in the biosynthesis of benzylisoquinoline alkaloids in opium poppy, resulting in altered alkaloid profiles (Alagoz et al., 2016). Additionally, CRISPR-based tools have been employed for multiplex pathway modifications and transcriptional regulations, allowing for comprehensive metabolic engineering (Jakočiūnas et al., 2017; Nishida and Kondo, 2020). The versatility and efficiency of CRISPR-Cas9 make it an ideal tool for metabolic engineering in tea to enhance the production of valuable bioactive compounds.

4.3 Overexpression and silencing of key genes

Overexpression and silencing of key genes are fundamental strategies in metabolic engineering to modulate the biosynthetic pathways of bioactive compounds. Overexpression involves the introduction of additional copies of a gene or the use of strong promoters to increase the expression levels of target genes. Conversely, gene silencing can be achieved through RNA interference (RNAi) or CRISPR interference (CRISPRi), which reduce or eliminate the expression of specific genes. These approaches have been used to enhance the production of various bioactive compounds in plants. For example, the overexpression of genes in the methylerythritol-phosphate (MEP) pathway has been shown to increase the production of β-carotene in Escherichia coli (Li et al., 2015). Similarly, CRISPRi has been employed to temporarily control gene expression without altering the genomic sequence, providing a flexible tool for metabolic pathway regulation (Nishida and Kondo, 2020; Zhao et al., 2020).

4.4 Pathway redirection for enhanced compound production

Pathway redirection involves the reconfiguration of metabolic pathways to channel intermediates towards the desired end products. This can be achieved through the overexpression of key enzymes, the knockout of competing pathways, or the introduction of novel biosynthetic routes. CRISPR-based metabolic pathway engineering has been particularly effective in this regard, enabling the simultaneous modulation of multiple genes to optimize pathway flux. For instance, the CRISPR/Cas9-facilitated multiplex pathway optimization (CFPO) technique has been developed to modulate the expression of multiple genes in the xylose utilization pathway of E. coli, resulting in a significant improvement in xylose utilization (Zhu et al., 2017). Additionally, combinatorial metabolic engineering strategies using CRISPR systems have been employed to enhance the production of β-carotene and other valuable compounds in yeast (Lian et al., 2017). These approaches can be adapted for the metabolic engineering of tea to enhance the production of bioactive compounds by redirecting metabolic fluxes towards the desired pathways.

5 Case Study

5.1 Introduction to the case study

This case study focuses on a metabolic engineering project aimed at enhancing the production of bioactive compounds in tea (Camellia sinensis). Tea is renowned for its health benefits, primarily due to its rich content of bioactive compounds such as catechins, theaflavins, and caffeine. However, the natural levels of these compounds can vary significantly, and there is a growing interest in optimizing their production through metabolic engineering.

5.2 Description of the metabolic engineering project

The project involved the genetic modification of tea plants to either overexpress or silence specific genes involved in the biosynthesis of key bioactive compounds. One approach was to enhance the production of catechins and other flavonoids by targeting genes associated with flavonoid metabolic biosynthesis (Figure 2). This was achieved by leveraging the findings from the tea tree genome, which revealed lineage-specific expansions of these genes (Xia et al., 2017).

Figure 2 Evolutionary differences in key metabolic pathways among 25 Camellia species (Adapted from Xia et al., 2017) Image Caption: A: Phylogenetic relationships among 25 Camellia species based on whole-transcriptome sequencing data, with the percentage content of seven characteristic metabolites in the leaves of each species, detected by HPLC, shown on the right; B: Expression profiles of key functional genes related to the three major metabolic pathways in tea plants across different species, with data presented in log10 values. The boxplot on the right shows expression correlations within the Thea group (green), the non-Thea group (orange), and between the two groups (gray); C: Sequence variations in genes related to flavonoid, theanine, and caffeine metabolic pathways, along with the phylogenetic topology of orthologous genes across species. (Adapted from Xia et al., 2017)

Another strategy focused on reducing or eliminating caffeine content by manipulating the caffeine biosynthesis pathway. This involved either overexpressing caffeine degradative pathway genes or silencing caffeine biosynthesis pathway genes (Yadav and Ahuja, 2007).

5.3 Results and impact on bioactive compound production

The genetic modifications led to significant changes in the levels of bioactive compounds in the engineered tea plants. For instance, the overexpression of flavonoid biosynthesis genes resulted in increased production of catechins, which are crucial for tea flavor and health benefits (Xia et al., 2017). On the other hand, the silencing of caffeine biosynthesis genes successfully reduced caffeine levels, making the tea more suitable for individuals sensitive to caffeine (Yadav and Ahuja, 2007). Additionally, the biotransformation of catechins through enzymatic treatments further enhanced the antioxidant capacity of the tea extracts (Baik et al., 2015). These modifications not only improved the health benefits of the tea but also enhanced its processing suitability and quality (Xia et al., 2017).

Xia et al. (2017) revealed the mechanisms underlying the accumulation of three characteristic secondary metabolites—flavonoids, theanine, and caffeine—in tea leaves. Through a phylogenetic analysis of the transcriptomes of 25 Camellia species, the study demonstrated significant gene expression differences in metabolic pathways between the Thea group (primarily consisting of common tea varieties) and other non-Thea species. These differences are associated with the synthesis of flavonoids, theanine, and caffeine, suggesting that specific gene expression patterns may drive the accumulation of these secondary metabolites in tea plants. This research provides a molecular basis for understanding the evolutionary origins and accumulation of characteristic secondary metabolites in tea plants, which could aid in future tea quality improvement and new cultivar development.

5.4 Lessons learned and future prospects

The case study highlights several important lessons. First, the successful manipulation of specific biosynthesis pathways can lead to significant improvements in the production of desired bioactive compounds. Second, a comprehensive understanding of the tea genome and the metabolic pathways involved is crucial for effective metabolic engineering. Future prospects include further refinement of these genetic modifications to optimize the balance of various bioactive compounds. Additionally, exploring other metabolic pathways and employing advanced biotechnological techniques could lead to the development of new tea varieties with enhanced health benefits and unique flavors (Yadav and Ahuja, 2007; Baik et al., 2015; Xia et al., 2017). The ongoing research in this field promises to make tea an even more valuable commodity in terms of both health benefits and economic value.

6 Challenges and Considerations in Metabolic Engineering of Tea

6.1 Technical challenges in gene editing

Metabolic engineering of tea plants to enhance the production of bioactive compounds faces several technical challenges. One of the primary obstacles is the complexity of plant metabolic pathways, which involves the regulation of multiple genes and intricate protein interactions. This complexity makes it difficult to predict the outcomes of genetic modifications accurately (García et al., 2023). Additionally, the cellular environment in plants is highly complex, which can obscure the effectiveness of metabolic engineering approaches and the predictability of genetic transformations (Leonard et al., 2009). Advanced tools and strategies, such as genome editing and transcriptional regulation, have been developed to reroute metabolic pathways, but their application in tea plants still requires significant optimization (García et al., 2023).

6.2 Regulatory and ethical considerations

The application of metabolic engineering in tea plants also raises several regulatory and ethical considerations. Regulatory frameworks for genetically modified organisms (GMOs) vary significantly across different countries, and obtaining approval for genetically engineered tea plants can be a lengthy and complex process. Ethical concerns related to the consumption of genetically modified tea products also need to be addressed. Public perception and acceptance of GMOs play a crucial role in the commercial success of such products. Therefore, transparent communication about the safety and benefits of genetically engineered tea is essential to gain public trust and regulatory approval (Leonard et al., 2009; García et al., 2023).

6.3 Potential environmental impacts

The environmental impacts of genetically engineered tea plants must be carefully considered. The introduction of genetically modified tea plants into the environment could potentially affect local ecosystems and biodiversity. For instance, there is a risk of gene flow from genetically modified tea plants to wild relatives, which could lead to unintended ecological consequences. Additionally, the large-scale cultivation of genetically engineered tea plants may have implications for soil health and the surrounding flora and fauna. Therefore, comprehensive environmental risk assessments are necessary to evaluate the potential impacts and develop strategies to mitigate any adverse effects (Leonard et al., 2009; García et al., 2023).

7 Future Directions and Research Gaps

7.1 Potential for new bioactive compounds

The exploration of new bioactive compounds in tea is a promising area for future research. Current studies have primarily focused on well-known compounds such as catechins, theanine, and caffeine, which are responsible for many of tea's health benefits and flavors (Xia et al., 2017; Zhang et al., 2018). However, there is potential to discover novel bioactive compounds that could offer additional health benefits or enhance the existing properties of tea. For instance, the study on large yellow tea demonstrated its unique ability to ameliorate metabolic syndrome through mechanisms not fully understood, suggesting the presence of other bioactive compounds that warrant further investigation (Wu et al., 2022). Additionally, the optimization of extraction methods, such as ultrasound-assisted extraction, has shown promise in enhancing the yield of bioactive compounds, indicating that improved extraction techniques could facilitate the discovery of new compounds (Bindes et al., 2019).

7.2 Integration of omics approaches

The integration of omics approaches, including genomics, transcriptomics, and metabolomics, is essential for advancing our understanding of the metabolic pathways involved in the production of bioactive compounds in tea. Recent advancements in these fields have enabled the identification of genes associated with the biosynthesis of key metabolites, such as flavonoids and caffeine (Xia et al., 2017; Zhang et al., 2018). Omics-based network strategies, such as gene co-expression networks and gene-to-metabolite networks, have proven effective in gene discovery and could be further utilized to uncover the complex interactions within tea's metabolic pathways (Zhang et al., 2018). By leveraging these approaches, researchers can identify new targets for metabolic engineering, ultimately enhancing the production of desired bioactive compounds and potentially introducing novel compounds with unique health benefits.

7.3 Need for long-term studies and field trials

While numerous studies have demonstrated the health benefits of tea and its bioactive compounds, there is a significant need for long-term studies and field trials to validate these findings and assess their practical applications. Most current research is limited to short-term clinical trials or in vitro and in vivo studies, which may not fully capture the long-term effects and real-world applicability of tea consumption (Basu et al., 2011; Sánchez et al., 2020). Long-term studies are particularly important for understanding the chronic effects of tea on metabolic and endocrine disorders, as well as its potential role in disease prevention. Additionally, field trials are necessary to evaluate the effectiveness of metabolic engineering approaches in real-world agricultural settings, ensuring that the enhanced production of bioactive compounds can be achieved sustainably and economically (Wilson and Roberts, 2014). These studies will provide critical insights into the long-term health benefits of tea and the feasibility of implementing metabolic engineering strategies on a larger scale.

8 Concluding Remarks

Metabolic engineering has emerged as a powerful tool to enhance the production of bioactive compounds in tea. Various strategies have been explored, including the manipulation of biosynthetic pathways and the use of plant tissue culture techniques. For instance, the production of secondary metabolites, which are crucial for pharmaceutical, cosmetic, and dietary applications, can be significantly increased through metabolic engineering. Additionally, the development of caffeine-free tea through genetic modifications highlights the potential of metabolic engineering to tailor tea's chemical composition for health benefits. The sequencing of the tea tree genome has provided insights into the independent evolution of caffeine biosynthesis and the enhancement of flavonoid metabolic pathways, which are essential for tea flavor and quality.

The advancements in metabolic engineering hold significant implications for the tea industry. By enhancing the production of bioactive compounds, tea can be positioned not only as a beverage but also as a functional food with added health benefits. For example, large yellow tea extract has shown potential in ameliorating metabolic syndrome by modulating lipid metabolism and gut microbiota. This opens up new market opportunities for health-oriented tea products. Furthermore, the ability to produce caffeine-free tea through metabolic engineering can cater to consumers who are sensitive to caffeine, thereby expanding the consumer base. The integration of metabolic engineering with traditional tea cultivation practices can lead to the development of high-value tea varieties with improved flavors and health benefits, thereby boosting the economic value of the tea industry.

The future of metabolic engineering in tea looks promising, with ongoing research likely to yield even more sophisticated techniques for enhancing bioactive compound production. The integration of systems biology and synthetic biology approaches can further unravel the complexities of tea's metabolic pathways, enabling more precise genetic modifications. Additionally, the use of heterologous hosts for the production of plant natural products can provide scalable and sustainable alternatives to traditional extraction methods. As the field progresses, it is essential to address the challenges related to the predictability and efficiency of genetic transformations to fully realize the potential of metabolic engineering in tea. Overall, the continued advancements in this field are expected to revolutionize the tea industry, making it more versatile and health-oriented.

Acknowledgments

Thanks to the peer reviewers for their suggestions on this study.

Conflict of Interest Disclosure

Authors affirm that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

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