Tea, the second most popular nonalcoholic beverage consumed worldwide, is favoured by billions of consumers due to its special flavour and numerous health benefits (Higdon and Frei, 2003). Theanine, a unique secondary metabolite in the tea plant (Camellia sinensis L.), confers the umami taste of the tea infusion. Theanine also has many physiological beneficial effects, including promoting relaxation, improving sleep quality and immunity and protecting the cardiovascular system (Kanarek et al., 2011).

Theanine only accumulates at a high level in tea plants (Tadahiro and Shinsuke, 1984). This might be controlled by the specific presence of ethylamine in tea plants (Cheng et al., 2017), as theanine is primarily biosynthesized from ethylamine and glutamate by theanine synthetase in tea roots, with ethylamine being synthesized from alanine, by alanine decarboxylase (AlaDC) (Figure 1a,b), and CsAlaDC being specifically expressed in tea roots (Figure 1c). Although CsAlaDC exhibited AlaDC activity, in vitro (Bai et al., 2019), the in vivo role in tea plants has not been characterized.

Theanine content varies greatly among the different cultivars, and theanine levels appear to be genetically regulated in tea leaves (Fang et al., 2021). To investigate the in vivo role of ethylamine in theanine accumulation, we quantified the key metabolites in the theanine metabolic pathway, in the roots of seven tea plant cultivars (Figure 1d–f). As shown in Figure 1d, theanine contents in the roots of these cultivars varied greatly. Importantly, the ethylamine contents exhibited a similar pattern as the theanine contents (Figure 1F). The correlation coefficient between the contents of ethylamine and theanine was 0.883 (P < 0.01) (Figure 1g). Taken together, these findings provided in vivo evidence for the pivotal role of ethylamine in determining theanine accumulation in tea plants.

Next, we examined CsAlaDC expression in the roots of the seven cultivars (Figure 1h,i). Here, we found that its expression level was highly and positively correlated with the ethylamine contents, and the correlation coefficient reached 0.903 (P < 0.01) (Figure 1j). We also analysed the correlation between CsAlaDC expression and theanine content. Our findings indicated that CsAlaDC expression, in roots, was highly and positively correlated with the theanine contents, and the correlation coefficient was 0.881 (P < 0.01) (Figure 1k). Unexpectedly, the CsTSI expression level was not correlated with the theanine contents (Figure 1k). Taken together, these results suggested that CsAlaDC expression plays a critical role in determining theanine accumulation in roots of tea plants.

We used the Nicotiana benthamiana transient expression system to further characterize the function of CsAlaDC, in planta. To this end, A. tumefaciens strain GV3101 (pSoup-p19), carrying pCAMBIA1305-CsAlaDC-GFP plasmid, was infiltrated into leaves of 5-week-old N. benthamiana plants (Figure 1l); the pCAMBIA1305 empty vector was used as the control (EV). We detected CsAlaDC expression at the mRNA level (Figure 1m) by qRT-PCR, and a high level of ethylamine (Figure 1n; >100 μg/g dry weight) was also identified in the CsAlaDC-expressing tobacco leaves. As anticipated, no ethylamine product was detected in WT tobacco leaves and those infiltrated with EV. Taken together, these findings offer support for the hypothesis that CsAlaDC has the capacity to synthesize ethylamine, in planta.

Theanine is highly demanded, by the market, due to its health effects and medicinal value, as a food constituent, in cosmetics and in other fields (Cheng et al., 2017). The theanine obtained both by direct extraction and chemical synthesis is of very poor quality (Gu et al., 2004). In addition, plant cell culture is disadvantaged by high cost, poor genetic stability and the low content of metabolites, hampering its use for industrial theanine production. An alternative, and intriguing possibility, would be to synthesize theanine in other crops to improve their health-promoting effects of foods. Until now, however, the synthesis of theanine in non-tea plants through synthetic biology has not been achieved. Tobacco (N. benthamiana) is generally used as the model plant for synthetic biology research (Forestier et al., 2021; Li et al., 2019). Thus, we felt it would be important to synthesize theanine in tobacco, as a model for theanine biosynthesis in non-tea plants.

Given the biosynthesis of ethylamine in tobacco, we speculated whether theanine could be produced in the tobacco leaves in the presence of ethylamine. Unexpectedly, theanine production was not detected (Figure 1o), indicating that the presence of ethylamine did not lead to theanine synthesis, at least not in detectable quantities.

We then speculated that the combination of CsAlaDC and CsTSI, in tobacco leaves, could synthesize theanine. Based on this notion, we co-infiltrated A. tumefaciens strains carrying pCAMBIA1305-CsAlaDC and pK7WGF2-CsTSI plasmids (CsAlaDC+CsTSI) into tobacco leaves. A. tumefaciens strains containing pCAMBIA1305 and pK7WGF2-CsTSI (CsTSI) and pK7WGF2 and pCAMBIA1305-CsAlaDC (CsAlaDC) were infiltrated as the controls. Both CsAlaDC and CsTSI were being expressed in the infiltrated tobacco leaves (Figure 1p,q). Ethylamine was produced in CsAlaDC- and CsAlaDC+CsTSI-expressing leaves (Figure 1r). High levels of theanine were produced in leaves co-expressing CsAlaDC and CsTSI, and the theanine content reached to ˜4 mg/g (Figure 1s,t), which is comparable with the theanine contents in tea plant leaves. Thus, theanine biosynthesis requires both the presence of ethylamine and the co-action of CsAlaDC and CsTSI. To our knowledge, this is the first report of theanine biosynthesis, by synthetic biology, in a non-tea plant.

In summary, we established that CsAlaDC works coordinately with CsTSI in determining the unique high theanine accumulation in the roots of tea plants. More importantly, we reported the first biosynthesis of theanine in a model plant, tobacco. Theanine production in this system does not require the provision of additional substrates, thereby greatly reducing costs and avoids causing environmental pollution. As theanine confers the umami taste, but also has various health effects, engineering its synthesis in non-tea plants, including crops, may well contribute to improve the health-promoting quality of the foods derived from such crops, as well as meeting the market demand for theanine.

茶是全球第二大最受欢迎的非酒精饮料，因其独特的风味和众多健康益处而受到数十亿消费者的青睐（Higdon 和 Frei，2003 年）。茶氨酸是茶树 ( Camellia sinensis L.) 中一种独特的次生代谢产物，赋予茶汤的鲜味。茶氨酸还具有许多生理有益作用，包括促进放松、改善睡眠质量和免疫力以及保护心血管系统（Kanarek等人，2011 年）。

茶氨酸仅在茶树中大量积累（Tadahiro 和 Shinsuke，1984 年）。这可能受到茶树中乙胺的特定存在的控制（Cheng等人，2017 年），因为茶氨酸主要由茶根中的茶氨酸合成酶从乙胺和谷氨酸中生物合成，而乙胺是由丙氨酸通过丙氨酸脱羧酶 (AlaDC) 合成的。 )（图 1a、b）和CsAlaDC在茶根中特异性表达（图 1c）。尽管 CsAlaDC在体外表现出 AlaDC 活性（Bai等人，2019 年），但在茶树中的体内作用尚未得到表征。

不同品种的茶氨酸含量差异很大，茶叶中的茶氨酸水平似乎受到基因调控（Fang et al ., 2021）。为了研究乙胺在茶氨酸积累中的体内作用，我们量化了七种茶树品种根中茶氨酸代谢途径中的关键代谢物（图 1d-f）。如图 1d 所示，这些品种根中的茶氨酸含量差异很大。重要的是，乙胺含量表现出与茶氨酸含量相似的模式（图 1F）。乙胺与茶氨酸含量的相关系数为0.883（P  <0.01）（图1g）。总之，这些发现在体内提供乙胺在确定茶树中茶氨酸积累中的关键作用的证据。

接下来，我们检查了七个品种根中的CsAlaDC表达（图 1h，i）。在这里，我们发现其表达水平与乙胺含量呈高度正相关，相关系数达到0.903（P  <0.01）（图1j）。我们还分析了CsAlaDC表达与茶氨酸含量之间的相关性。我们的研究结果表明，根中CsAlaD C的表达与茶氨酸含量呈高度正相关，相关系数为0.881（P  <0.01）（图1k）。出乎意料的是，CsTSI表达水平与茶氨酸含量无关（图 1k）。综上所述，这些结果表明CsAlaDC表达在确定茶树根中茶氨酸积累中起关键作用。

我们使用本氏烟草瞬时表达系统来进一步表征植物中CsAlaDC的功能。为此，将携带 pCAMBIA1305- CsAlaDC - GFP质粒的根癌农杆菌菌株 GV3101 (pSoup-p19)渗入 5 周龄本氏烟草植物的叶子中（图 1l）；pCAMBIA1305 空载体用作对照 (EV)。我们通过 qRT-PCR 在 mRNA 水平（图 1m）检测到CsAlaDC表达，并且在CsAlaDC中也鉴定出高水平的乙胺（图 1n；>100 μg/g 干重）- 表达烟叶。正如预期的那样，在 WT 烟叶和渗入 EV 的烟叶中未检测到乙胺产物。总之，这些发现为 CsAlaDC 有能力在植物中合成乙胺的假设提供了支持。

由于茶氨酸的健康作用和药用价值，作为食品成分，在化妆品和其他领域中，茶氨酸的需求量很大（Cheng等人，2017 年）。通过直接提取和化学合成获得的茶氨酸质量很差（Gu et al ., 2004）。此外，植物细胞培养成本高、遗传稳定性差、代谢物含量低等缺点，阻碍了其在工业茶氨酸生产中的应用。另一种有趣的可能性是在其他作物中合成茶氨酸，以提高食物对健康的促进作用。然而，到目前为止，还没有实现通过合成生物学在非茶树中合成茶氨酸。烟草 (N.benthamiana）通常用作合成生物学研究的模型植物（Forestier et al ., 2021 ; Li et al ., 2019）。因此，我们认为在烟草中合成茶氨酸作为非茶树中茶氨酸生物合成的模型非常重要。

鉴于烟草中乙胺的生物合成，我们推测在乙胺存在下是否可以在烟叶中产生茶氨酸。出乎意料的是，没有检测到茶氨酸的产生（图 1o），这表明乙胺的存在不会导致茶氨酸的合成，至少不能检测到。

然后我们推测CsAlaDC和CsTSI在烟叶中的组合可以合成茶氨酸。基于这个概念，我们将携带 pCAMBIA1305 -CsAlaDC和 pK7WGF2- CsTSI质粒 ( CsAlaDC+CsTSI ) 的根癌农杆菌菌株共同渗透到烟叶中。将含有 pCAMBIA1305 和 pK7WGF2 -CsTSI ( CsTSI ) 和 pK7WGF2 和 pCAMBIA1305 -CsAlaDC ( CsAlaDC ) 的根癌农杆菌菌株作为对照渗入。CsAlaDC和CsTSI都在浸润的烟叶中表达（图 1p，q）。乙胺在CsAlaDC中生产- 和CsAlaDC+CsTSI -表达叶（图 1r）。在共表达CsAlaDC和CsTSI的叶子中产生高水平的茶氨酸，并且茶氨酸含量达到〜4 mg / g（图1s，t），这与茶树叶子中的茶氨酸含量相当。因此，茶氨酸的生物合成需要乙胺的存在以及 CsAlaDC 和 CsTSI 的共同作用。据我们所知，这是合成生物学在非茶树中进行茶氨酸生物合成的第一份报告。

总之，我们确定 CsAlaDC 与 CsTSI 协同工作以确定茶树根中独特的高茶氨酸积累。更重要的是，我们报道了模型植物烟草中茶氨酸的首次生物合成。该系统生产茶氨酸不需要提供额外的底物，从而大大降低了成本，避免了环境污染。由于茶氨酸具有鲜味，而且还具有多种健康作用，因此在包括农作物在内的非茶植物中对其合成进行工程改造，可能有助于提高此类农作物食品的健康促进质量，并满足市场需求对茶氨酸的需求。