AbstractSteroidal glycoalkaloids (SGAs) are specialized metabolites produced by hundreds of Solanum species including food crops, such as tomato, potato and eggplant. Unlike true alkaloids, nitrogen is introduced at a late stage of SGA biosynthesis through an unknown transamination reaction. Here, we reveal the mechanism by which GLYCOALKALOID METABOLISM12 (GAME12) directs the biosynthesis of nitrogen-containing steroidal alkaloid aglycone in Solanum.

摘要甾体糖生物碱（SGAs）是由数百种茄属植物产生的特殊代谢产物，包括番茄，马铃薯和茄子等粮食作物。与真正的生物碱不同，氮是在SGA生物合成的后期通过未知的转氨反应引入的。在这里，我们揭示了糖生物碱代谢12（GAME12）指导茄中含氮甾体生物碱苷元生物合成的机制。

We report that GAME12, a neofunctionalized γ-aminobutyric acid (GABA) transaminase, undergoes changes in both active site specificity and subcellular localization to switch from its renown and generic activity in core metabolism to function in a specialized metabolic pathway. Moreover, overexpression of GAME12 alone in engineered S. nigrum leaves is sufficient for de novo production of nitrogen-containing SGAs.

我们报道，GAME12是一种新功能化的γ-氨基丁酸（GABA）转氨酶，在活性位点特异性和亚细胞定位方面都发生了变化，从其在核心代谢中的知名度和通用活性转变为在专门的代谢途径中起作用。此外，在工程化的S.nigrum叶片中单独过表达GAME12足以从头生产含氮SGA。

Our results highlight how hijacking a core metabolism GABA shunt enzyme is crucial in numerous Solanum species for incorporating a nitrogen to a steroidal-specialized metabolite backbone and form defensive alkaloids..

我们的研究结果强调了劫持核心代谢GABA分流酶在许多茄属物种中如何将氮结合到甾体特化代谢物骨架并形成防御性生物碱中至关重要。。

MainPlants synthesize a huge repertoire of diverse, lineage-specific steroidal-specialized metabolites. Steroidal glycoalkaloids (SGAs) represent one of the major classes of these metabolites, produced by hundreds of wild and cultivated species of the genus Solanum, including major staple food crops such as tomato (S. lycopersicum), potato (S. tuberosum) and eggplant (S. melongena)1.

主要植物合成了大量不同的谱系特异性甾体特化代谢物。甾体糖生物碱（SGAs）是这些代谢物的主要类别之一，由数百种野生和栽培的茄属物种产生，包括主要的主食作物，如番茄（S.lycopersicum），马铃薯（S.tuberosum）和茄子（S.melongena）1。

SGAs have important roles in plant defense and are classified as antinutrients because of their high toxicity and bitterness2. SGAs are considered to be pseudoalkaloids, which are compounds that contain a basic nitrogen moiety but, in contrast to true alkaloids, are not derived from an amino acid starting precursor3.

SGAs在植物防御中具有重要作用，由于其高毒性和苦味而被归类为抗营养素2。SGA被认为是假生物碱，它是含有碱性氮部分的化合物，但与真正的生物碱相反，它不是来自氨基酸起始前体3。

Instead, SGAs are derived from cholesterol (1) and it is proposed that the nitrogen atom is introduced into the cholesterol backbone at a late stage of biosynthesis4 (Fig. 1a). After introduction of the nitrogen group, the steroidal alkaloid aglycones (for example, dehydrotomatidine (5) in tomato and potato) are subsequently glycosylated by a suite of uridine diphosphate (UDP) glycosyltransferases (UGTs) to generate diverse SGA products (7, 8 and 9)4,5,6,7,8.Fig.

相反，SGA来源于胆固醇（1），并且提出在生物合成的后期将氮原子引入胆固醇骨架4（图1a）。引入氮基团后，甾体生物碱糖苷配基（例如番茄和马铃薯中的脱氢胞苷（5））随后被一套尿苷二磷酸（UDP）糖基转移酶（UGT）糖基化，产生多种SGA产物（7,8和9）4,5,6,7,8.Fig。

1: Predicted biosynthetic pathway of SGAs in Solanum species.a, The predicted biosynthetic pathway of SGAs in potato (S. tuberosum), tomato (S. lycopersicum) and eggplant (S. melongena). Solid arrows represent known biosynthetic steps and dashed arrows show uncharacterized steps. Unconfirmed intermediates in the SGA pathway are placed in brackets.

1： 马铃薯（S.tuberosum），番茄（S.lycopersicum）和茄子（S.melongena）中SGAs的预测生物合成途径。实心箭头表示已知的生物合成步骤，虚线箭头表示未表征的步骤。SGA途径中未经证实的中间体放在括号中。

Colored arrows represent the branches of SGA biosynthesis specific to different Solanum species. The relevant carbon positions modified by the biosynthetic enzymes are numbered in red. b, The primary metabolism GABA-T catalyzes pyruvate (10) transamination, leading to the for.

彩色箭头表示特定于不同茄属物种的SGA生物合成的分支。由生物合成酶修饰的相关碳位置以红色编号。b，初级代谢GABA-T催化丙酮酸（10）转氨，导致for。

nigrum using GAME12Because GAME12 controls the branch point between the amino-containing SGAs and steroidal saponins, we hypothesized that we could use it to introduce nitrogen into a saponin backbone and thereby modify the steroidal metabolite profiles in plants. For a proof of concept, we chose to metabolically engineer S. nigrum plants because the leaves of this plant merely produce steroidal saponins (furostanol-type; for example, uttroside B (16)).

使用GAME12的黑色素由于GAME12控制含氨基的SGA和甾体皂苷之间的分支点，我们假设我们可以使用它将氮引入皂苷骨架，从而改变植物中的甾体代谢物谱。为了证明概念，我们选择代谢工程S.nigrum植物，因为这种植物的叶子仅产生甾体皂苷（呋喃甾醇型；例如，uttroside B（16））。

Moreover, SGAs are only produced in S. nigrum fruits (also known as berries)34. It is still unknown how these two classes of steroidal-specialized metabolites are biosynthesized in S. nigrum plants from their cholesterol precursor.The furostanol product generated from cholesterol by the action of GAME6, GAME8 and GAME11 has been proposed as a key intermediate in Solanum SGA biosynthesis (Fig.

此外，SGA仅在S.nigrum果实（也称为浆果）中产生34。目前尚不清楚这两类甾体特化代谢物是如何从其胆固醇前体在S.nigrum植物中生物合成的。通过GAME6，GAME8和GAME11的作用由胆固醇产生的呋喃甾醇产物已被提议作为茄属SGA生物合成的关键中间体（图）。

1a). Further oxidation and transamination of furostanol by GAME4 and GAME12, respectively, generates steroidal alkaloid aglycone scaffolds (for example, solasodine), which are further glycosylated to produce diverse SGA structures (for example, α-solamargine) (Fig. 1a). Uttroside B is a major steroidal saponin that accumulates in S. nigrum leaves35 (Fig.

1a）。分别通过GAME4和GAME12进一步氧化和转氨呋甾醇，产生甾体生物碱糖苷配基支架（例如，solasodine），其进一步糖基化以产生不同的SGA结构（例如，α-solamargine）（图1a）。Uttroside B是一种主要的甾体皂苷，在S.nigrum叶片中积累35（图）。

5a and Supplementary Fig. 11). Notably, uttroside B shares a common furostanol scaffold with SGAs (Fig. 5a), strongly suggesting that the furostanol scaffold could act as a branching point for the production of the steroidal saponins in the leaves and SGAs in S. nigrum berries. This implies that GAME6, GAME8 and GAME11 generating the furostanol intermediate would likely act as common enzymes in both steroidal saponin and SGA biosynthesis in S. nigrum.

5a和补充图11）。值得注意的是，uttroside B与SGA共享一个共同的呋甾醇支架（图5a），强烈表明呋甾醇支架可以作为叶子中甾体皂苷和S.nigrum浆果中SGA产生的分支点。这意味着产生呋甾醇中间体的GAME6，GAME8和GAME11可能在S.nigrum的甾体皂苷和SGA生物合成中充当常见酶。

Indeed, we explored the S. nigrum transcriptome (generated in-house from young leaves and green fruits) and identified orthologs of G.

事实上，我们探索了S.nigrum转录组（由幼叶和绿色果实内部产生），并鉴定了G的直系同源物。

benthamiana

底栖动物

All four tomato GABA-T homologs C-terminally fused to RFP were coexpressed with free GFP or mitochondrially targeted GFP44, using Agrobacterium-mediated transient expression in N. benthamiana, following the same procedure as in the pathway reconstitution experiments, with the only difference being the lower OD600 of the final inoculum (OD600 = 0.3).

。

Leaf discs (0.5 cm in diameter) were isolated from the leaves 48 h after infiltration. Micrographs of the freshly punched leaf discs mounted in water were acquired on a cLSM 880 (Zeiss) using a Plan-Apochromat ×20/0.8 air or a C-Apochromat ×40/1.20 water immersion objective. Used excitation light sources were a 405-nm laser diode (3% transmission), 488-nm argon laser (4%) and a 543-nm helium–neon laser (30%) for chlorophyll autofluorescence, GFP and RFP respectively.

渗透后48小时，从叶片中分离出叶盘（直径0.5厘米）。使用Plan-Apochromat×20/0.8空气或C-Apochromat×40/1.20水浸物镜在cLSM 880（Zeiss）上获取安装在水中的新冲压叶盘的显微照片。使用的激发光源分别是405 nm激光二极管（3%透射），488 nm氩激光器（4%）和543 nm氦氖激光器（30%），用于叶绿素自发荧光，GFP和RFP。

The transmitted light was captured with a T-PMT added in the GFP channel. Two sequential tracks were acquired to reduce crosstalk between GFP and RFP. The spectral detector was set to an emission detection range of 490–570 nm in combination with a 488 MBS for GFP in the first track and 580–650 nm with a 488/543 MBS for RFP and 670–735 nm for chlorophyll in a second track.

通过在GFP通道中添加的T-PMT捕获透射光。。光谱检测器的发射检测范围为490-570nm，第一轨道中GFP的发射检测范围为488Mbs，第二轨道中GFP的发射检测范围为580-650nm，RFP的发射检测范围为488/543Mbs，叶绿素的发射检测范围为670-735nm。

Acquisition was controlled in ZEN (Zeiss) and executed unidirectionally with 1 Airy unit, frame averaging of 4 and approximately 0.5 µs per pixel dwell time. Contrast improvement, cropping and scale bar insertion were performed in ImageJ45.UHPLC–MS analysis of N..

采集由ZEN（Zeiss）控制，并以1个艾里单位单向执行，帧平均为4，每像素停留时间约为0.5µs。对比度改善，裁剪和比例尺插入在ImageJ45中进行。N的UHPLC-MS分析。。

benthamiana extractsExtracts were analyzed using a Thermo UltiMate 3000 UHPLC system (Thermo Fisher Scientific) coupled with an Impact II ultrahigh-resolution quadrupole time-of-flight (QTOF) MS instrument (Bruker) and the Bruker Compass qToF Control version 6.3 and Bruker Compass Hystar version 6.0.30.0.

使用Thermo UltiMate 3000 UHPLC系统（Thermo Fisher Scientific）结合Impact II超高分辨率四极杆飞行时间（QTOF）MS仪器（Bruker）和Bruker Compass QTOF Control版本6.3和Bruker Compass Hystar版本6.0.30.0分析本塞姆氏烟草提取物。

software. Separation was performed on a Waters Acquity Premier BEH C18 Vanguard FIT column (2.1 × 100 mm, 1.7 μm, 130 Å) at column temperature of 40 °C. The first minute of each run was redirected to waste. Solvent A was 0.1% formic acid in water; solvent B was 100% acetonitrile. The gradient was as follows: 5% B from 0 to 1 min, to 28% B at 9.5 min, to 90% B at 14 min, held at 90% B until 16 min and then 100% B at 18 min.

。分离在Waters Acquity Premier BEH C18 Vanguard FIT柱（2.1×100 mm，1.7μm，130Å）上进行，柱温为40℃。每次跑步的第一分钟都被重定向到浪费。溶剂A是水中的0.1%甲酸；溶剂B是100%乙腈。梯度如下：从0到1分钟的5%B，到9.5分钟的28%B，到14分钟的90%B，保持在90%B直到16分钟，然后在18分钟时保持100%B。

The column was then flushed with 100% B for 2 min and re-equilibrated to 5% B for 2.5 min. The flow was 0.3 ml min−1 and the injection volume was 2 μl. The samples were ionized using a pneumatic-assisted electrospray ionization source in positive mode (ESI+) with a capillary voltage of 3,500 V, plate offset of 500 V, nebulizer gas pressure of 2.0 bar, drying gas flow of 10 L min−1 and drying temperature of 250 °C.

然后将柱用100%B冲洗2分钟，并重新平衡至5%B 2.5分钟。流量为0.3 ml min-1，进样量为2μl。使用气动辅助电喷雾电离源以正模式（ESI+）电离样品，毛细管电压为3500 V，板偏移为500 V，雾化器气体压力为2.0 bar，干燥气体流量为10 L min-1，干燥温度为250℃。

The spectra were acquired at a 12-Hz rate within the 80–1,300 m/z scan range. Fragmentation was triggered at an absolute threshold of 400 counts and limited to a total cycle time of 0.5 s. The stepping mode (20 to 50 eV) was used for the collision energy. Sodium formate solution in isopropanol was injected at the beginning of each run and the m/z values were recalibrated using the expected calibrant ion m/z values.

。碎片在400计数的绝对阈值下触发，总循环时间限制在0.5s。碰撞能量使用步进模式（20至50eV）。在每次运行开始时注入异丙醇中的甲酸钠溶液，并使用预期的校准离子m/z值重新校准m/z值。

Acquired data were analyzed using the Bruker DataAnalysis version 5.3 software and MzMine version 3.4.27 software46. The dehydrotomatidine (5) product was identified on the basis of the presence of previously reported fragment io.

使用Bruker DataAnalysis版本5.3软件和MzMine版本3.4.27软件46分析获取的数据。根据先前报道的片段io的存在，鉴定了脱氢胞苷（5）产物。

GAME4 cloning and microsome preparationThe tomato GAME4 coding sequence was amplified from the previously obtained in planta expression plasmid (3Ω1) using Platinum SuperFi polymerase (Thermo Fisher Scientific). The resulting amplicons were cloned into a SalI-digested and Antarctic phosphatase-treated (New England Biolabs) pESC-Trp vector (Agilent), using the InFusion cloning kit (Clontech Takara).

GAME4克隆和微粒体制备使用Platinum SuperFi聚合酶（Thermo Fisher Scientific）从先前获得的植物表达质粒（3Ω1）中扩增番茄GAME4编码序列。使用输液克隆试剂盒（Clontech Takara）将所得扩增子克隆到SalI消化并经南极磷酸酶处理的（New England Biolabs）pESC-Trp载体（安捷伦）中。

The resulting plasmids were transformed into chemically competent E. coli Top10 cells (Invitrogen). Transformed cells were spread and selected for on LB agar plates supplemented with 100 μg ml−1 carbenicillin. The resulting colonies were used to inoculate liquid LB cultures, further used for plasmid isolation.

将所得质粒转化到具有化学活性的大肠杆菌Top10细胞（Invitrogen）中。将转化的细胞铺展并选择在补充有100μg/ml羧苄青霉素的LB琼脂平板上。所得菌落用于接种液体LB培养物，进一步用于质粒分离。

Plasmids were isolated from the positive colonies using the Wizard Plus SV minipreps DNA purification kit (Promega). The sequence of the introduced fragments was verified using the Sanger sequencing service provided by Genewiz (Azenta Life Sciences). The verified plasmid was transformed into chemically competent WAT11 Saccharomyces cerevisiae cells, according to a previously described protocol48.

使用Wizard Plus SV minipreps DNA纯化试剂盒（Promega）从阳性菌落中分离质粒。使用Genewiz（Azenta Life Sciences）提供的Sanger测序服务验证了引入片段的序列。根据先前描述的方案48，将经验证的质粒转化到具有化学活性的WAT11酿酒酵母细胞中。

Transformed cells were spread and selected for on SD-Trp dropout plates supplemented with 2% (v/v) glucose. The presence of the GAME4 coding sequence in the vector was verified by colony PCR. Briefly, the positive S. cerevisiae colonies were picked and dipped in 100 μl of 20 mM NaOH and incubated at 98 °C for 10 min.

将转化的细胞铺展并选择在补充有2%（v/v）葡萄糖的SD-Trp辍学板上。通过菌落PCR验证了载体中GAME4编码序列的存在。简而言之，挑选阳性酿酒酵母菌落并浸入100μl20mM NaOH中，并在98℃下孵育10分钟。

The suspension was briefly centrifuged and 2 μl was mixed with the Phire II Master Mix (Thermo Fisher Scientific) and appropriate primers. The size of the resulting amplicons was verified using agarose gel electrophoresis. Verified colonies were used to inoculate 30-ml cultures of SD-Trp dropout medium supplemented with 2% (v/v) glucose, which were incubated overni.

将悬浮液短暂离心，并将2μl与Phire II预混液（Thermo Fisher Scientific）和适当的引物混合。使用琼脂糖凝胶电泳验证所得扩增子的大小。使用经验证的菌落接种补充有2%（v/v）葡萄糖的SD-Trp辍学培养基的30 ml培养物，将其在Ni上孵育。

Agrobacterium-mediated stable transformation of S.

农杆菌介导的S的稳定转化。

nigrum

龙葵

S. nigrum (wild type, SN30 bulk) seeds were obtained from seed stocks maintained by the greenhouse team at the Max Planck Institute for Chemical Ecology. Seeds were sterilized using a solution of 0.1 g of sodium dichloroisocyanurate dihydrate and 50 μl of 0.5% Tween 20 in 5 ml of deionized sterile water.

S、  nigrum（野生型，SN30散装）种子是从马克斯·普朗克化学生态学研究所的温室团队保存的种子库中获得的。使用0.1g二氯异氰尿酸钠二水合物和50μl0.5%吐温20在5ml去离子无菌水中的溶液对种子进行灭菌。

Seeds were incubated in the sterilizing solution for 5 min, washed three times with deionized sterile water and then incubated overnight in 5 ml of sterile 1 M KNO3 solution at 4 °C in darkness. Seeds were germinated on Gamborg B5 medium for 7 days under the following day–night cycle: 16 h of light, 26 °C; 8 h of darkness, 24 °C.

将种子在灭菌溶液中孵育5分钟，用去离子无菌水洗涤三次，然后在黑暗中于4℃在5mL无菌1M KNO3溶液中孵育过夜。在第二天的昼夜循环中，种子在Gamborg B5培养基上发芽7天：光照16小时，26°C；黑暗8小时，24℃。

Plasmid-harboring A. tumefaciens (strain GV3101) was used to inoculate 8-ml LB media cultures supplemented with 200 μg ml−1 spectinomycin, 100 μg ml−1 rifampicin and 50 μg ml−1 gentamycin. The cultures were grown until the culture OD600 reached 0.4–0.8. The cells were pelleted by centrifugation at 4,000g at room temperature for 5 min.

含有根癌农杆菌（菌株GV3101）的质粒用于接种补充有200μg/ml大观霉素，100μg/ml利福平和50μg/ml庆大霉素的8ml LB培养基培养物。培养物生长直至培养物OD600达到0.4-0.8。通过在室温下以4000g离心5分钟使细胞沉淀。

The pellet was resuspended in 7 ml of Agrobacterium washing medium (basal medium: 4.41 g L−1 Murashige and Skoog containing vitamins and 30 g L−1 sucrose, supplemented with 0.02 mg L−1 indole-3-acetic acid (IAA) and 1 mg L−1 6-benzylaminopurine (BAP), pH 5.80). The seedling hypocotyls were cut into 3-mm-long pieces using a sterile scalpel dipped in the A. tumefaciens suspension.

将沉淀重悬于7ml农杆菌洗涤培养基（基础培养基：含有维生素和30g L-1蔗糖的4.41g L-1 Murashige和Skoog，补充有0.02mg L-1吲哚-3-乙酸（IAA）和1mg L-16-苄基氨基嘌呤（BAP），pH 5.80）中。使用浸入根癌农杆菌悬浮液中的无菌手术刀将幼苗下胚轴切成3mm长的片。

The explants were transferred onto callus induction medium (basal medium supplemented with 3 g L−1 Phytagel, 0.02 mg L−1 IAA and 1 mg L−1 BAP, pH 5.80) and incubated at 26 °C in darkness for 3 days. The explants were then subcultured onto fresh callus induction medium plates containing antibiotics (25 mg L−1 kanamycin and 125 mg L−1 timentin) and incubated at 30 °C for 16 h and 27 °C for 8 h for a total of 14 days.

将外植体转移到愈伤组织诱导培养基（补充有3g L-1 Phytagel，0.02 mg L-1 IAA和1mg L-1 BAP，pH 5.80的基础培养基）上，并在26℃下在黑暗中孵育3天。然后将外植体传代培养到含有抗生素（25 mg L-1卡那霉素和125 mg L-1 timentin）的新鲜愈伤组织诱导培养基平板上，并在30℃下孵育16小时和27℃，持续8小时，共14天。

The resulting callus was.

产生的愈伤组织是。

nigrum transgenic line leavesThe leaf tissue of the S. nigrum transformant lines was collected, snap-frozen in liquid N2 and pulverized. Metabolites were extracted using 80% methanol with 0.1% formic acid. For 100 mg of weighed-out frozen tissue, 300 μl of the extraction solvent was added. The resulting mixtures were vortexed briefly and sonicated in a sonic bath for 15 min.

黑色素转基因品系叶片收集黑色素转化品系的叶组织，在液氮中速冻并粉碎。使用80%甲醇和0.1%甲酸提取代谢物。对于100毫克称重的冷冻组织，加入300微升提取溶剂。。

The extracts were centrifuged at top speed for 15 min and passed through a 0.22-μm PTFE filter (Thermo Fisher Scientific). Extracts were analyzed using a Waters UHPLC system (Waters Acquity) coupled to a SYANPT-G2QTOF MS instrument (Waters Acquity). Separation was performed on a Waters Acquity Premier BEH C18 Vanguard FIT column (2.1 × 100 mm, 1.7 μm, 130 Å).

将提取物以最高速度离心15分钟，并通过0.22μm PTFE过滤器（Thermo Fisher Scientific）。使用与SYANPT-G2QTOF MS仪器（Waters Acquity）偶联的Waters UHPLC系统（Waters Acquity）分析提取物。在Waters Acquity Premier BEH C18 Vanguard FIT色谱柱（2.1×100 mm，1.7μm，130Å）上进行分离。

Solvent A was 0.1% formic acid in 5% acetonitrile in water; solvent B was 0.1% formic acid in acetonitrile. The gradient was as follows: 0% at 0 min to 28% B at 22 min and 28% to 100% B at 36 min. The column was flushed with 100% B for 2 min, re-equilibrated to 0% B within 0.5 min and conditioned at 0% B for 1.5 min.

溶剂A是在水中的5%乙腈中的0.1%甲酸；溶剂B是乙腈中的0.1%甲酸。梯度如下：0分钟时0%至22分钟时28%B，36分钟时28%至100%B。柱用100%B冲洗2分钟，在0.5分钟内重新平衡至0%B，并在0%B下调节1.5分钟。

The samples were ionized using a pneumatic-assisted ESI+ with a capillary voltage of 3,400 V, cone voltage of 24 V, source temperature of 125 °C, desolvation temperature of 275 °C and desolvation gas flow of 650 L h−1. The spectra were acquired within the 50–1,600 m/z scan range. Data were acquired using the MSE mode with energy ramp.

使用气动辅助ESI+对样品进行电离，毛细管电压为3400V，锥电压为24V，源温度为125℃，去溶剂温度为275℃，去溶剂气流为650L·h-1。光谱在50-1600m/z扫描范围内获得。使用具有能量斜坡的MSE模式获取数据。

The collision energy was set to 4 eV at the low-energy acquisition function and a 10–30 eV ramp for the high-energy function. Sodium formate solution and Leu encephalin were used at the lock mass. Acquired data were analyzed using the MassLynx version 4.2 software. The putative structural assignment of SGAs and steroidal saponins produced by the transgenic S. nigrum plants was based on the identificat.

碰撞能量在低能量采集功能下设置为4 eV，在高能功能下设置为10-30 eV斜坡。在锁定块上使用甲酸钠溶液和亮脑素。使用MassLynx 4.2版软件分析获得的数据。转基因黑曲霉植物产生的SGA和甾体皂苷的推定结构分配基于identificat。

Data availability

数据可用性

The sequences of the gene constructs used in the study are listed in Supplementary Data 1. The sequences used in comparative genomics analyses, the corrected gene models and the sequences used in phylogeny analyses are listed in Extended Data Fig. 1. S. nigrum young leaves and green fruit (berries) transcriptome raw sequence reads were deposited to the NCBI Sequence Read Archive under accession numbers PRJNA1134384 and PRJNA1133681, respectively.

补充数据1列出了研究中使用的基因构建体的序列。扩展数据图1列出了比较基因组学分析中使用的序列，校正后的基因模型和系统发育分析中使用的序列。S、 黑果幼叶和绿色果实（浆果）转录组原始序列读数分别以登录号PRJNA1134384和PRJNA1133681保藏到NCBI序列读取档案中。

The accession numbers of the genomes used for the phylogenomics and synteny analysis of GABA-T homologs are provided in Supplementary Table 6. The crystal structure used in the modeling of GABA-T homologs was retrieved from PDB 4ATQ. Data are available from the corresponding authors upon request. Source data are provided with this paper..

补充表6提供了用于GABA-T同源物的系统基因组学和同线性分析的基因组的登录号。从PDB 4ATQ检索用于GABA-T同源物建模的晶体结构。数据可根据要求从通讯作者处获得。本文提供了源数据。。

ReferencesSchreiber, K. in The Alkaloids: Chemistry and Physiology (ed. Manske, R. H. F.) (Academic Press, 1968).Roddick, J. G. in Saponins Used in Traditional and Modern Medicine (eds Waller, G. R. & Yamasaki, K.) (Springer, 1996).Lichman, B. R. The scaffold-forming steps of plant alkaloid biosynthesis.

《生物碱：化学与生理学》（ed.Manske，R.H.F.）中的参考文献Chreiber，K.（学术出版社，1968年）。Roddick，J.G。在传统和现代医学中使用的皂苷（eds Waller，G.R。＆Yamasaki，K。）（Springer，1996）。Lichman，B.R。植物生物碱生物合成的支架形成步骤。

Nat. Prod. Rep. 38, 103–129 (2021).Article .

《国家生产报告》38103-129（2021）。文章。

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Itkin, M. et al. Biosynthesis of antinutritional alkaloids in solanaceous crops is mediated by clustered genes. Science 341, 175–179 (2013).Article

Itkin，M.等人。茄科作物中抗营养生物碱的生物合成是由聚集基因介导的。科学341175-179（2013）。文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Itkin, M. et al. GLYCOALKALOID METABOLISM1 is required for steroidal alkaloid glycosylation and prevention of phytotoxicity in tomato. Plant Cell 23, 4507–4525 (2011).Article

Itkin，M。等人。糖生物碱代谢M1是甾体生物碱糖基化和预防番茄植物毒性所必需的。。文章

CAS

中科院

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Kohara, A., Nakajima, C., Yoshida, S. & Muranaka, T. Characterization and engineering of glycosyltransferases responsible for steroid saponin biosynthesis in Solanaceous plants. Phytochemistry 68, 478–486 (2007).Article

Kohara，A.，Nakajima，C.，Yoshida，S。＆Muranaka，T。负责茄科植物中甾体皂苷生物合成的糖基转移酶的表征和工程。植物化学68478-486（2007）。文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

McCue, K. F. et al. The primary in vivo steroidal alkaloid glucosyltransferase from potato. Phytochemistry 67, 1590–1597 (2006).Article

McCue，K.F.等人。来自马铃薯的主要体内甾体生物碱葡糖基转移酶。植物化学671590-1597（2006）。文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Moehs, C. P., Allen, P. V., Friedman, M. & Belknap, W. R. Cloning and expression of solanidine UDP-glucose glucosyltransferase from potato. Plant J. 11, 227–236 (1997).Article

Moehs，C.P.，Allen，P.V.，Friedman，M。＆Belknap，W.R。马铃薯中solanidine UDP-葡萄糖基转移酶的克隆和表达。植物J.11227–236（1997）。文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Umemoto, N. et al. Two cytochrome P450 monooxygenases catalyze early hydroxylation steps in thepotato steroid glycoalkaloid biosynthetic pathway. Plant Physiol. 171, 2458–2467 (2016).Article

Umemoto，N。等人。两种细胞色素P450单加氧酶催化磷脂甾体糖生物碱生物合成途径中的早期羟基化步骤。植物生理学。1712458-2467（2016）。文章

CAS

中科院

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Nakayasu, M. et al. A dioxygenase catalyzes steroid 16α-hydroxylation in steroidal glycoalkaloid biosynthesis. Plant Physiol. 175, 120–133 (2017).Article

Nakayasu，M。等人。一种双加氧酶催化甾体糖生物碱生物合成中的甾体16α-羟基化。植物生理学。175120-133（2017）。文章

CAS

中科院

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Nakayasu, M. et al. Characterization of C-26 aminotransferase, indispensable for steroidal glycoalkaloid biosynthesis. Plant J. 108, 81–92 (2021).Article

Nakayasu，M.等人。甾体糖生物碱生物合成必不可少的C-26氨基转移酶的表征。植物J.108，81–92（2021）。文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Li, L., Dou, N., Zhang, H. & Wu, C. The versatile GABA in plants. Plant Signal. Behav. 16, 1862565 (2021).Article

Li，L.，Dou，N.，Zhang，H。＆Wu，C。植物中的多功能GABA。电厂信号。行为。161862565（2021）。文章

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Clark, S. M., Di Leo, R., Van Cauwenberghe, O. R., Mullen, R. T. & Shelp, B. J. Subcellular localization and expression of multiple tomato γ-aminobutyrate transaminases that utilize both pyruvate and glyoxylate. J. Exp. Bot. 60, 3255–3267 (2009).Article

Clark，S.M.，Di Leo，R.，Van Cauwenberghe，O.R.，Mullen，R.T。＆Shelp，B.J。利用丙酮酸和乙醛酸的多种番茄γ-氨基丁酸转氨酶的亚细胞定位和表达。J、 实验Bot 603255–3267（2009）。文章

CAS

中科院

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Weber, N., Ismail, A., Gorwa-Grauslund, M. & Carlquist, M. Biocatalytic potential of vanillin aminotransferase from Capsicum chinense. BMC Biotechnol. 14, 25 (2014).Article

。BMC生物技术。14，25（2014）。文章

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Augustin, M. M. et al. Elucidating steroid alkaloid biosynthesis in Veratrum californicum: production of verazine in Sf9 cells. Plant J. 82, 991–1003 (2015).Article

Augustin，M.M.等人。阐明加利福尼亚藜芦中类固醇生物碱的生物合成：在Sf9细胞中产生藜芦碱。工厂J.82991–1003（2015）。文章

CAS

中科院

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Sonawane, P. D., Jozwiak, A., Panda, S. & Aharoni, A. ‘Hijacking’ core metabolism: a new panache for the evolution of steroidal glycoalkaloids structural diversity. Curr. Opin. Plant Biol. 55, 118–128 (2020).Article

Sonawane，P.D.，Jozwiak，A.，Panda，S。和Aharoni，A.“劫持”核心代谢：甾体糖生物碱结构多样性进化的新招牌。货币。奥平。植物生物学。55118-128（2020）。文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Rao, ΑV. & Gurfinkel, D. M. The bioactivity of saponins: triterpenoid and steroidal glycosides. Drug Metabol. Drug Interact. 17, 211–236 (2000).Article

Rao，ΑV.&Gurfinkel，D.M。皂苷的生物活性：三萜类和甾体糖苷。药物代谢。药物相互作用。17211-236（2000）。文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Cárdenas, P. D. et al. The bitter side of the nightshades: genomics drives discovery in Solanaceae steroidal alkaloid metabolism. Phytochemistry 113, 24–32 (2015).Article

Cárdenas，P.D.等人，《夜影的苦涩面：基因组学推动了茄科甾体生物碱代谢的发现》。植物化学113,24-32（2015）。文章

PubMed

PubMed

Google Scholar

谷歌学者

Roddick, J. G. Subcellular localization of steroidal glycoalkaloids in vegetative organs of Lycopersicon esculentum and Solanum tuberosum. Phytochemistry 16, 805–807 (1977).Article

Roddick，J.G。番茄和马铃薯营养器官中甾体糖生物碱的亚细胞定位。。文章

CAS

中科院

Google Scholar

谷歌学者

Suza, W. P. & Chappell, J. Spatial and temporal regulation of sterol biosynthesis in Nicotiana benthamiana. Physiol. Plant. 157, 120–134 (2016).Article

Suza，W.P。＆Chappell，J。本塞姆氏烟草甾醇生物合成的时空调控。生理学。工厂。157120-134（2016）。文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

De Combarieu, E., Fuzzati, N., Lovati, M. & Mercalli, E. Furostanol saponins from Tribulus terrestris. Fitoterapia 74, 583–591 (2003).Article

De Combarieu，E.，Fuzzati，N.，Lovati，M.和Mercalli，E.蒺藜中的Furostanol皂苷。植物疗法74583-591（2003）。文章

PubMed

PubMed

Google Scholar

谷歌学者

Liu, T. et al. Protodioscin-glycosidase-1 hydrolyzing 26-O-β-d-glucoside and 3-O-(1→4)-α-l-rhamnoside of steroidal saponins from Aspergillus oryzae. Appl. Microbiol. Biotechnol. 97, 10035–10043 (2013).Article

Liu，T。等人。原薯di皂苷-糖苷酶-1水解米曲霉甾体皂苷的26-O-β-d-葡萄糖苷和3-O-（1→4）-α-l-鼠李糖苷。应用。微生物。生物技术。。文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

O’Keeffe, R. et al. Synthesis of novel 24-amino-25,26,27-trinorlanost-8-enes: cytotoxic and apoptotic potential in U937 cells. Bioorg. Med. Chem. 23, 2270–2280 (2015).Article

O'Keeffe，R。等人。新型24-氨基-25,26,27-三聚乳酸-8-烯的合成：U937细胞的细胞毒性和凋亡潜力。生物组织医学化学。232270-2280（2015）。文章

PubMed

PubMed

Google Scholar

谷歌学者

Ronchetti, F. & Russo, G. Stereochemistry of the hydrogen introduction at C-25 in the biosynthesis of tomatidine. J. Chem. Soc., Chem. Commun. 88, 785–786 (1974).Article

Ronchetti，F。＆Russo，G。tomatidine生物合成中C-25处氢引入的立体化学。J、 化学。Soc。，化学。Commun公司。88785-786（1974）。文章

Google Scholar

谷歌学者

Ohyama, K., Okawa, A. & Fujimoto, Y. Biosynthesis of steroidal alkaloids in Solanaceae plants: incorporation of 3β-hydroxycholest-5-en-26-al into tomatine with tomato seedlings. Bioorg. Med. Chem. Lett. 24, 3556–3558 (2014).Article

Ohyama，K.，Okawa，A。＆Fujimoto，Y。茄科植物中甾体生物碱的生物合成：将3β-羟基胆甾-5-en-26-al掺入番茄幼苗的番茄碱中。生物组织医学化学。利特。243556-3558（2014）。文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Ohyama, K., Okawa, A., Moriuchi, Y. & Fujimoto, Y. Biosynthesis of steroidal alkaloids in Solanaceae plants: involvement of an aldehyde intermediate during C-26 amination. Phytochemistry 89, 26–31 (2013).Article

Ohyama，K.，Okawa，A.，Moriuchi，Y。和Fujimoto，Y。茄科植物中甾体生物碱的生物合成：在C-26胺化过程中醛中间体的参与。植物化学89,26-31（2013）。文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Tschesche, R. & Spindler, M. Zur biogenese des aza-oxa-spiran-systems der steroidalkaloide vom spirosolan-typ in Solanaceen. Phytochemistry 17, 251–255 (1978).Article

Česche，R.和Spindler，M.茄科植物螺茄型甾体生物碱氮杂-氧-螺环系统的生物发生。植物化学17251-255（1978）。文章

CAS

中科院

Google Scholar

谷歌学者

Tschesche, R. & Brennecke, H. R. Side chain functionalization of cholesterol in the biosynthesis of solasodine in Solanum laciniatum. Phytochemistry 19, 1449–1451 (1980).Article

Tschesche，R。＆Brennecke，H。R。Solanum laciniatum中solasodine生物合成中胆固醇的侧链官能化。植物化学191449-1451（1980）。文章

CAS

中科院

Google Scholar

谷歌学者

Sonawane, P. D. et al. Short-chain dehydrogenase/reductase governs steroidal specialized metabolites structural diversity and toxicity in the genus Solanum. Proc. Natl Acad. Sci. USA 115, E5419–E5428 (2018).Article

Sonawane，P.D。等人。短链脱氢酶/还原酶控制茄属中甾体特化代谢物的结构多样性和毒性。程序。国家科学院。科学。美国115，E5419–E5428（2018）。文章

CAS

中科院

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Huang, J. et al. Nuclear phylogeny and insights into whole-genome duplications and reproductive development of Solanaceae plants. Plant Commun. 4, 100595 (2023).Article

Huang，J.等人。茄科植物的核系统发育和全基因组重复和生殖发育的见解。植物群落。4100595（2023）。文章

CAS

中科院

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Smith, M. D. et al. Less is more: an adaptive branch-site random effects model for efficient detection of episodic diversifying selection. Mol. Biol. Evol. 32, 1342–1353 (2015).Article

Smith，M.D.等人，《少即是多：一种自适应分支位点随机效应模型，用于有效检测情景多样化选择。分子生物学。进化。321342-1353（2015）。文章

CAS

中科院

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Gururaj, H. B., Padma, M. N., Giridhar, P. & Ravishankar, G. A. Functional validation of Capsicum frutescens aminotransferase gene involved in vanillylamine biosynthesis using Agrobacterium mediated genetic transformation studies in Nicotiana tabacum and Capsicum frutescens calli cultures.

Gururaj，H.B.，Padma，M.N.，Giridhar，P。＆Ravishankar，G.A。使用农杆菌介导的遗传转化研究在烟草和辣椒愈伤组织培养物中对参与香草胺生物合成的辣椒转氨酶基因进行功能验证。

Plant Sci. 195, 96–105 (2012).Article .

植物科学。195,96-105（2012）。文章。

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Murrell, B. et al. Detecting individual sites subject to episodic diversifying selection. PLoS Genet. 8, e1002764 (2012).Article

Murrell，B。等人。检测受情景多样化选择影响的单个站点。PLoS Genet。8，e1002764（2012）。文章

CAS

中科院

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Zhao, W., Yan, T., Huang, X. & Zhang, Y. Analysis of steroidal glycoalkaloids and their metabolites in Solanum nigrum fruits based on liquid chromatography–tandem mass spectrometry and molecular networking. J. Sep. Sci. 46, 2200804 (2023).Article

Zhao，W.，Yan，T.，Huang，X.＆Zhang，Y.基于液相色谱-串联质谱和分子网络分析龙葵果实中的甾体糖生物碱及其代谢产物。J、 9月Sci。462200804（2023）。文章

CAS

中科院

Google Scholar

谷歌学者

Nath, L. R. et al. Evaluation of uttroside B, a saponin from Solanum nigrum Linn, as a promising chemotherapeutic agent against hepatocellular carcinoma. Sci. Rep. 6, 36318 (2016).Article

Nath，L.R.等人对龙葵皂苷B作为一种有前途的抗肝细胞癌化疗药物的评价。科学。代表636318（2016）。文章

CAS

中科院

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Jozwiak, A. et al. Plant terpenoid metabolism co-opts a component of the cell wall biosynthesis machinery. Nat. Chem. Biol. 16, 1–9 (2020).Article

Jozwiak，A。等人。植物萜类代谢共同选择细胞壁生物合成机制的一个组成部分。自然化学。生物学16，1-9（2020）。文章

Google Scholar

谷歌学者

Leong, B. J. et al. Evolution of metabolic novelty: a trichome-expressed invertase creates specialized metabolic diversity in wild tomato. Sci. Adv. 5, eaaw3754 (2019).Article

Leong，B.J.等人，《代谢新颖性的进化：毛状体表达的转化酶在野生番茄中产生了特殊的代谢多样性》。科学。。文章

CAS

中科院

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Flagel, L. E. & Wendel, J. F. Gene duplication and evolutionary novelty in plants. New Phytol. 183, 557–564 (2009).Article

Flagel，L.E。＆Wendel，J.F。植物中的基因复制和进化新颖性。新植物醇。183557-564（2009）。文章

PubMed

PubMed

Google Scholar

谷歌学者

Kusaka, H. et al. An evolutionary view of vanillylamine synthase pAMT, a key enzyme of capsaicinoid biosynthesis pathway in chili pepper. Plant J. 117, 1453–1465 (2024).Article

Kusaka，H.等人。辣椒中辣椒素生物合成途径的关键酶香草胺合酶pAMT的进化观点。工厂J.1171453–1465（2024）。文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Bruce, H. et al. Structures of a γ-aminobutyrate (GABA) transaminase from the s-triazine-degrading organism Arthrobacter aurescens TC1 in complex with PLP and with its external aldimine PLP-GABA adduct. Acta Crystallogr. F 68, 1175–1180 (2012).Article

Bruce，H.等人。来自s-三嗪降解生物金黄色节杆菌TC1的γ-氨基丁酸（GABA）转氨酶的结构与PLP及其外部醛亚胺PLP-GABA加合物复合。晶体学报。F 681175-1180（2012）。文章

CAS

中科院

Google Scholar

谷歌学者

Kochnev, Y., Hellemann, E., Cassidy, K. C. & Durrant, J. D. Webina: an open-source library and web app that runs AutoDock Vina entirely in the web browser. Bioinformatics 36, 4513–4515 (2020).Article

Kochnev，Y.，Hellemann，E.，Cassidy，K.C.＆Durrant，J.D.Webina：一个开源库和web应用程序，完全在web浏览器中运行AutoDock Vina。生物信息学364513-4515（2020）。文章

CAS

中科院

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Fernandez-Pozo, N. et al. The Sol Genomics Network (SGN)—from genotype to phenotype to breeding. Nucleic Acids Res. 43, D1036–D1041 (2015).Article

Fernandez-Pozo，N。等人。Sol基因组学网络（SGN）-从基因型到表型再到育种。核酸研究43，D1036–D1041（2015）。文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Cárdenas, P. D. et al. Pathways to defense metabolites and evading fruit bitterness in genus Solanum evolved through 2-oxoglutarate-dependent dioxygenases. Nat. Commun. 10, 5169 (2019).Article

Cárdenas，P.D.等人。茄属植物通过2-氧戊二酸依赖性双加氧酶进化出防御代谢物和逃避果实苦味的途径。国家公社。105169（2019）。文章

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Nelson, B. K., Cai, X. & Nebenführ, A. A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J. 51, 1126–1136 (2007).Article

Nelson，B.K.，Cai，X。＆Nebenführ，A。一组多色的体内细胞器标记，用于拟南芥和其他植物的共定位研究。植物J.511126–1136（2007）。文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).Article

Schindelin，J。等人。斐济：生物图像分析的开源平台。《自然方法》9676-682（2012）。文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Schmid, R. et al. Integrative analysis of multimodal mass spectrometry data in MZmine 3. Nat. Biotechnol. 41, 447–449 (2023).Article

Schmid，R.等人。MZmine 3中多峰质谱数据的综合分析。美国国家生物技术公司。41447-449（2023）。文章

CAS

中科院

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Heinig, U. & Aharoni, A. in Plant Isoprenoids: Methods and Protocols (ed. Rodríguez-Concepción, M.) (Springer, 2014).Gietz, R. D. & Schiestl, R. H. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat. Protoc. 2, 31–34 (2007).Article

Heinig，U。和Aharoni，A。植物类异戊二烯：方法和方案（编辑Rodríguez-Concepción，M.）（Springer，2014）。Gietz，R.D。＆Schiestl，R.H。使用LiAc/SS载体DNA/PEG方法进行高效酵母转化。。2，31-34（2007）。文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Berrow, N. S. et al. A versatile ligation-independent cloning method suitable for high-throughput expression screening applications. Nucleic Acids Res. 35, e45 (2007).Article

Berrow，N.S.等人。一种适用于高通量表达筛选应用的多功能连接非依赖性克隆方法。核酸研究35，e45（2007）。文章

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Agrawal, P. K. Dependence of 1H NMR chemical shifts of geminal protons of glycosyloxy methylene (H2-26) on the orientation of the 27-methyl group of furostane-type steroidal saponins. Magn. Reson. Chem. 42, 990–993 (2004).Article

Agrawal，P.K。糖基氧基亚甲基（H2-26）孪生质子的1H NMR化学位移对呋喃甾烷型甾体皂苷27甲基取向的依赖性。马格纳。雷森。化学。42990–993（2004）。文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Agrawal, P. K. Assigning stereodiversity of the 27-Me group of furostane-type steroidal saponins via NMR chemical shifts. Steroids 70, 715–724 (2005).Article

Agrawal，P.K。通过NMR化学位移分配呋甾烷型甾体皂苷27-Me基团的立体多样性。类固醇70715-724（2005）。文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Acharya, D. et al. Furostane-type steroidal saponins from the roots of Chlorophytum borivilianum. Helv. Chim. Acta 91, 2262–2269 (2008).Article

Acharya，D。等人。来自绿藻borivilianum根的呋喃甾烷型甾体皂苷。你好。奇姆。Acta 912262–2269（2008）。文章

CAS

中科院

Google Scholar

谷歌学者

Hulse-Kemp, A. M. et al. Reference quality assembly of the 3.5-Gb genome of Capsicum annuum from a single linked-read library. Hortic. Res. 5, 4 (2018).Article

Hulse Kemp，A.M.等人从单链阅读文库中对辣椒3.5-Gb基因组进行参考质量组装。霍特语。第5、4（2018）号决议。文章

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Wu, S. et al. Genome sequences of two diploid wild relatives of cultivated sweetpotato reveal targets for genetic improvement. Nat. Commun. 9, 4580 (2018).Article

Wu，S。等人。栽培甘薯的两个二倍体野生近缘种的基因组序列揭示了遗传改良的目标。国家公社。94580（2018）。文章

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Ranawaka, B. et al. A multi-omic Nicotiana benthamiana resource for fundamental research and biotechnology. Nat. Plants 9, 1558–1571 (2023).Article

Ranawaka，B.等人。用于基础研究和生物技术的多组分本塞姆氏烟草资源。《自然植物》91558-1571（2023）。文章

CAS

中科院

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Lu, J. et al. The Physalis floridana genome provides insights into the biochemical and morphological evolution of Physalis fruits. Hortic. Res. 8, 1–19 (2021).Article

Lu，J。等人。Physalis floridana基因组提供了对Physalis果实的生化和形态进化的见解。霍特语。第8、1-19号决议（2021年）。文章

Google Scholar

谷歌学者

Hosmani, P. S. et al. An improved de novo assembly and annotation of the tomato reference genome using single-molecule sequencing, Hi-C proximity ligation and optical maps. Preprint at bioRxiv https://doi.org/10.1101/767764 (2019).Pham, G. M. et al. Construction of a chromosome-scale long-read reference genome assembly for potato.

Hosmani，P.S。等人。使用单分子测序，Hi-C邻近连接和光学图谱改进的番茄参考基因组的从头组装和注释。bioRxiv预印本https://doi.org/10.1101/767764。Pham，G.M.等人。马铃薯染色体规模长读参考基因组装配的构建。

GigaScience 9, giaa100 (2020).Article .

GigaScience 9，giaa100（2020）。文章。

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Dainat, J. NBISweden/AGAT: AGAT-v1.2.0. Zenodo https://doi.org/10.5281/zenodo.3549546 (2023).Emms, D. M. & Kelly, S. OrthoFinder: phylogenetic orthology inference for comparative genomics. Genome Biol. 20, 238 (2019).Article

Dainat，J。NBISweden/AGAT:AGAT-v1.2.0。泽诺多https://doi.org/10.5281/zenodo.3549546（2023年）。Emms，D。M。和Kelly，S。OrthoFinder：比较基因组学的系统发育正交推断。基因组生物学。20238（2019）。文章

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Minh, B. Q. et al. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Mol. Biol. Evol. 37, 1530–1534 (2020).Article

Minh，B.Q.等人，《IQ-TREE 2：基因组时代系统发育推断的新模型和有效方法》。分子生物学。进化。371530-1534（2020）。文章

CAS

中科院

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Kalyaanamoorthy, S., Minh, B. Q., Wong, T. K. F., von Haeseler, A. & Jermiin, L. S. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat. Methods 14, 587–589 (2017).Article

Kalyanamoorthy，S.，Minh，B.Q.，Wong，T.K.F.，von Haeseler，A.＆Jermiin，L.S.ModelFinder：快速模型选择以进行准确的系统发育估计。自然方法14587-589（2017）。文章

CAS

中科院

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Hoang, D. T., Chernomor, O., von Haeseler, A., Minh, B. Q. & Vinh, L. S. UFBoot2: improving the ultrafast bootstrap approximation. Mol. Biol. Evol. 35, 518–522 (2018).Article

Hoang，D.T.，Chernomor，O.，von Haeseler，A.，Minh，B.Q。＆Vinh，L.S。UFBoot2：改进超快自举近似。分子生物学。进化。35518-522（2018）。文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Guindon, S. et al. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst. Biol. 59, 307–321 (2010).Article

Guindon，S.等人。估计最大似然系统发育的新算法和方法：评估PhyML 3.0的性能。系统。生物学59307-321（2010）。文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Tang, H., Krishnakumar, V. & Li, J. JCVI utility libraries. Zenodo https://doi.org/10.5281/zenodo.594205 (2015).Wang, Y. et al. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res 40, e49 (2012).Article

Tang，H.，Krishnakumar，V。＆Li，J。JCVI实用程序库。泽诺多https://doi.org/10.5281/zenodo.594205（2015年）。Wang，Y。et al。MCScanX：用于基因同线性和共线性检测和进化分析的工具包。核酸Res 40，e49（2012）。文章

CAS

中科院

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Li, H. Protein-to-genome alignment with miniprot. Bioinformatics 39, btad014 (2023).Article

Li，H。蛋白质与miniprot的基因组比对。生物信息学39，btad014（2023）。文章

CAS

中科院

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Bombarely, A. et al. Insight into the evolution of the Solanaceae from the parental genomes of Petunia hybrida. Nat. Plants 2, 1–9 (2016).Article

Bombarely，A.等人，《从矮牵牛（Petunia hybrida）亲本基因组中洞察茄科的进化》，《自然植物》2，1-9（2016）。文章

Google Scholar

谷歌学者

Lin, X. et al. Solanum americanum genome-assisted discovery of immune receptors that detect potato late blight pathogen effectors. Nat. Genet. 55, 1579–1588 (2023).Article

Lin，X。等人。Solanum americanum基因组协助发现检测马铃薯晚疫病病原体效应物的免疫受体。纳特·吉内特。551579-1588（2023）。文章

CAS

中科院

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Leebens-Mack, J. H. et al. One thousand plant transcriptomes and the phylogenomics of green plants. Nature 574, 679–685 (2019).Article

Leebens Mack，J.H.等人，《1000种植物转录组和绿色植物的系统发育基因组学》。自然574679-685（2019）。文章

Google Scholar

谷歌学者

Price, M. N., Dehal, P. S. & Arkin, A. P. FastTree 2—approximately maximum-likelihood trees for large alignments. PLoS ONE 5, e9490 (2010).Article

Price，M.N.，Dehal，P.S。＆Arkin，A.P.FastTree 2-用于大型比对的近似最大似然树。PLoS ONE 5，e9490（2010）。文章

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Weaver, S. et al. Datamonkey 2.0: a modern web application for characterizing selective and other evolutionary processes. Mol. Biol. Evol. 35, 773–777 (2018).Article

Weaver，S.等人，《Datamonkey 2.0：用于表征选择性和其他进化过程的现代web应用程序》。分子生物学。进化。35773-777（2018）。文章

CAS

中科院

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Wisotsky, S. R., Kosakovsky Pond, S. L., Shank, S. D. & Muse, S. V. Synonymous site-to-site substitution rate variation dramatically inflates false positive rates of selection analyses: ignore at your own peril. Mol. Biol. Evol. 37, 2430–2439 (2020).Article

Wisotsky，S.R.，Kosakovsky Pond，S.L.，Shank，S.D。＆Muse，S.V。同义位点替代率变化极大地增加了选择分析的假阳性率：忽略不计，风险自负。Mol。Biol。进化。372430-2439（2020）。文章

CAS

中科院

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Van Cauwenberghe, O. R., Makhmoudova, A., McLean, M. D., Clark, S. M. & Shelp, B. J. Plant pyruvate-dependent gamma-aminobutyrate transaminase: identification of an Arabidopsis cDNA and its expression in Escherichia coli. Can. J. Bot. 80, 933–941 (2002).Article

Van Cauwenberghe，O.R.，Makhmoudova，A.，McLean，M.D.，Clark，S.M。＆Shelp，B.J。植物丙酮酸依赖性γ-氨基丁酸转氨酶：拟南芥cDNA的鉴定及其在大肠杆菌中的表达。可以。J、 Bot.80933–941（2002年）。文章

Google Scholar

谷歌学者

Mirdita, M. et al. ColabFold: making protein folding accessible to all. Nat. Methods 19, 679–682 (2022).Article

Mirdita，M。等人，ColabFold：使所有人都可以进行蛋白质折叠。自然方法19679-682（2022）。文章

CAS

中科院

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).Article

Pettersen，E.F.等人，《UCSF Chimera——探索性研究和分析的可视化系统》。J、 计算机。化学。251605-1612（2004）。文章

CAS

中科院

PubMed

PubMed

Google Scholar

谷歌学者

Meng, E. C. et al. UCSF ChimeraX: tools for structure building and analysis. Protein Sci. 32, e4792 (2023).Article

Meng，E.C.等人，《UCSF ChimeraX：结构构建和分析工具》。蛋白质科学。32，e4792（2023）。文章

CAS

中科院

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Wang, S. et al. Identification of anthocyanin composition and functional analysis of an anthocyanin activator in Solanum nigrum fruits. Molecules 22, 876 (2017).Article

Wang，S.等人。龙葵果实中花青素组成的鉴定和花青素激活剂的功能分析。分子22876（2017）。文章

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Download referencesAcknowledgementsWe thank K. Eljounaidi and A. Higginson for initial work on alkaloid aminotransferase phylogenetics. We thank J. Wurlitzer for help with molecular cloning and imaging and the Max Planck Institute for Chemical Ecology greenhouse staff for plant husbandry.

。我们感谢J.Wurlitzer在分子克隆和成像方面的帮助，以及马克斯·普朗克化学生态研究所温室工作人员在植物饲养方面的帮助。

We thank M. Pliner at the Weizmann Institute of Science, Israel, for kind help in S. nigrum transformation experiments. D.G. thanks M. Florean for helpful discussions. This work was supported by the European Research Council (788301) and the Max Planck Society. S.J.S. is supported by the Biotechnology and Biological Sciences Research Council (BB/V006452/1) and B.R.L.

我们感谢以色列魏茨曼科学研究所的M.Pliner在S.nigrum转化实验中的善意帮助。D、 G.感谢Florean先生的有益讨论。这项工作得到了欧洲研究理事会（788301）和马克斯·普朗克学会的支持。S、 J.S.得到了生物技术和生物科学研究委员会（BB/V006452/1）和B.R.L.的支持。

is supported by the UK Research and Innovation funding service (MR/S01862X/1). N. benthamiana, mitochondria and chloroplast icons used in the figures was generated in BioRender. A.A. is the incumbent of the Peter J. Cohn Professorial Chair. We thank the Adelis Foundation, Leona M. and Harry B. Helmsley Charitable Trust, Jeanne and Joseph Nissim Foundation for Life Sciences, Tom and Sondra Rykoff Family Foundation Research and the Raymond Burton Plant Genome Research Fund for supporting the A.A.

由英国研究与创新资助服务（MR/S01862X/1）支持。N、 。A、 是Peter J.Cohn教授主席的现任者。我们感谢阿德利斯基金会、莱昂娜·M.和哈里·B·赫尔姆斯利慈善信托基金会、珍妮和约瑟夫·尼西姆生命科学基金会、汤姆和桑德拉·瑞科夫家庭基金会研究基金会以及雷蒙德·伯顿植物基因组研究基金会对A.A.的支持。

lab activity.FundingOpen access funding provided by Max Planck Society.Author informationAuthors and AffiliationsDepartment of Natural Product Biosynthesis, Max Planck Institute for Chemical Ecology, Jena, GermanyDagny Grzech, Ryan M. Alam, Marianna Boccia, Yoko Nakamura, Benke Hong, Sarah Heinicke, Maritta Kunert, Lorenzo Caputi, Sarah E.

实验室活动。基金马克斯·普朗克协会提供的开放获取资金。作者信息作者和附属机构马克斯·普朗克化学生态研究所天然产物生物合成系，耶拿，德国达尼·格泽奇，瑞安·M·阿拉姆，玛丽安娜·博西亚，中村洋子，本克·洪，莎拉·海尼克，玛丽塔·库内特，洛伦佐·卡普蒂，莎拉·E。

O’Connor & Prashant D. SonawaneCentre for Novel Agricultural Products, Department of Biology, University of York, York, UKSamuel J. Smit & Benjamin R. LichmanDepartment of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot, IsraelRanjit Barbole & Asaph Aharoni.

英国约克约克大学生物学系新型农产品研究中心O'Connor＆Prashant D.SonawaneCentre Samuel J.Smit＆Benjamin R.LichmandDepartment of Plant and Environmental Sciences，Weizmann Institute of Science，Rehovot，IsraelRanjit Barbole＆Asaph Aharoni。

PubMed Google ScholarSamuel J. SmitView author publicationsYou can also search for this author in

PubMed Google ScholarSamuel J.SmitView作者出版物您也可以在

PubMed Google ScholarRyan M. AlamView author publicationsYou can also search for this author in

PubMed Google ScholarRyan M.AlamView作者出版物您也可以在

PubMed Google ScholarMarianna BocciaView author publicationsYou can also search for this author in

PubMed Google ScholarMarianna BocciaView作者出版物您也可以在

PubMed Google ScholarYoko NakamuraView author publicationsYou can also search for this author in

PubMed Google ScholarYoko NakamuraView作者出版物您也可以在

PubMed Google ScholarBenke HongView author publicationsYou can also search for this author in

PubMed Google ScholarBenke HongView作者出版物您也可以在

PubMed Google ScholarRanjit BarboleView author publicationsYou can also search for this author in

PubMed Google ScholarRanjit BarboleView作者出版物您也可以在

PubMed Google ScholarSarah HeinickeView author publicationsYou can also search for this author in

PubMed Google ScholarSarah HeinickeView作者出版物您也可以在

PubMed Google ScholarMaritta KunertView author publicationsYou can also search for this author in

PubMed Google ScholarMaritta KunertView作者出版物您也可以在

PubMed Google ScholarWibke SeibtView author publicationsYou can also search for this author in

PubMed Google ScholarWibke SeibtView作者出版物您也可以在

PubMed Google ScholarVeit GrabeView author publicationsYou can also search for this author in

PubMed Google ScholarVeit GrabeView作者出版物您也可以在

PubMed Google ScholarLorenzo CaputiView author publicationsYou can also search for this author in

PubMed Google ScholarBenjamin R. LichmanView author publicationsYou can also search for this author in

PubMed Google ScholarBenjamin R.LichmanView作者出版物您也可以在

PubMed Google ScholarSarah E. O’ConnorView author publicationsYou can also search for this author in

PubMed Google ScholarSarah E.O'ConnorView作者出版物您也可以在

PubMed Google ScholarAsaph AharoniView author publicationsYou can also search for this author in

PubMed Google ScholarAsaph AharoniView作者出版物您也可以在

PubMed Google ScholarPrashant D. SonawaneView author publicationsYou can also search for this author in

PubMed Google ScholarPrashant D.SonawaneView作者出版物您也可以在

PubMed Google ScholarContributionsD.G. and P.D.S. designed and performed the research. S.J.S. and B.R.L. performed comparative genomic and phylogeny analyses. R.M.A. performed 26-furostanol aldehyde intermediate trapping. M.B. and S.H. purified uttroside B. Y.N. performed the structural characterization of uttroside B by NMR.

PubMed谷歌学术贡献SD。G、 和P.D.S.设计并进行了这项研究。S、 J.S.和B.R.L.进行了比较基因组和系统发育分析。R、 M.A.进行了26次呋甾醇醛中间体捕获。M、 B.和S.H.纯化的uttroside B.Y.N.通过NMR对uttroside B进行了结构表征。

B.H. assisted with in vitro assay design. R.B. assisted with cloning of the GABA-T homologs. S.H. and M.K. assisted in UHPLC–MS method development and operated the instruments. V.G. performed the confocal microscopy. W.S. generated S. nigrum transgenic lines. L.C. assisted with in vitro assay design, mutagenesis studies and data analysis.

B、 H.协助体外测定设计。R、 。S、 H.和M.K.协助UHPLC-MS方法开发并操作仪器。五、 G.进行共聚焦显微镜检查。W、 S.产生了S.nigrum转基因品系。五十、 C.协助体外测定设计，诱变研究和数据分析。

A.A., S.E.O’C. and P.D.S. conceptualized the study. D.G., S.J.S., B.R.L., P.D.S., S.E.O’C. and A.A. wrote the paper.Corresponding authorsCorrespondence to.

A、 A.，S.E.O'C。P.D.S.将这项研究概念化。D、 G.，S.J.S.，B.R.L.，P.D.S.，S.E.O'C。A.A.写了这篇论文。通讯作者通讯。

Sarah E. O’Connor, Asaph Aharoni or Prashant D. Sonawane.Ethics declarations

Sarah E.O'Connor、Asaph Aharoni或Prashant D.Sonawane。道德宣言

Competing interests

相互竞争的利益

The authors declare no competing interests.

作者声明没有利益冲突。

Peer review

同行评审

Peer review information

同行评审信息

Nature Chemical Biology thanks Zhang Fangyuan, Amelia Palermo, Junlan Zeng and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

《自然化学生物学》感谢张方元，阿米莉亚·巴勒莫，曾俊兰和另一位匿名审稿人对这项工作的同行评审做出的贡献。

Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Extended dataExtended Data Fig. 1 Phylogenomic analysis of GABA-Ts.a. Maximum likelihood gene tree of GABA-T homologues. Thick branches show those selected for diversifying selection tests.

Additional informationPublisher的注释Springer Nature在已发布的地图和机构隶属关系中的管辖权主张方面保持中立。扩展数据扩展数据图1 GABA-Ts.a.GABA-T同源物的最大似然基因树的系统基因组分析。。

Branches are coloured if significant selection was detected, with color showing ω1 rate class value in model with one rate class per branch. Grey circles show branches with >80% and >95% support as judged by 1000X SH-aLRT and UltraFast Boostrapping replicates, respectively. An extended phylogentic tree can be found in Supplementary Fig.

如果检测到显着选择，则将分支着色，颜色显示模型中的ω1速率类值，每个分支具有一个速率类。灰色圆圈显示分别通过1000X SH-aLRT和超快bootrapping复制判断的支持率>80%和>95%的分支。扩展的系统发生树可以在Supplementary Fig.中找到。

25. b. Microsynteny analysis of GABA-T homologues. Curved lines connecting genomes represent homologous genes, with colors added manually to track genes of interest. Gaps (10-70 Mb) in synteny represented with double slash. Pseudogenes (genes that have premature stop codons in exons, highly divergent sequences or missing expected coding regions.

25.b.GABA-T同源物的微同步分析。连接基因组的曲线代表同源基因，手动添加颜色以跟踪感兴趣的基因。用双斜杠表示的同步间隙（10-70Mb）。假基因（外显子中具有过早终止密码子，高度不同序列或缺少预期编码区的基因）。

are marked with the Ψ symbol. Larger versions of the results of each syntenic block analysis in panel (b) can be found in Supplementary Fig. 26.Source dataSupplementary informationSupplementary InformationSupplementary Figs. 1–36 and Tables 1–7.Reporting SummarySupplementary Data 1Sequences of constructs used in the study.Supplementary Data 2Source data for Supplementary Figs.

用Ψ符号标记。图（b）中每个同线性块分析结果的较大版本可以在补充图26中找到。源数据补充信息补充信息补充图1-36和表1-7。报告摘要补充数据1研究中使用的构建体序列。补充数据2补充图的源数据。

10, 31, 32b and 35.Source dataSource Data Fig. 5Peak areas for uttroside B and product E.Source Data Extended Data Fig. 1Gene sequences used for construction of the phylogenetic tree and sequences of corrected gene models.Rights and permissions.

10,31,32b和35。源数据源数据图5 uttroside B和产物E的峰值区域。源数据扩展数据图1用于构建系统发育树的基因序列和校正基因模型的序列。权限和权限。

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.

开放获取本文是根据知识共享署名4.0国际许可证授权的，该许可证允许以任何媒体或格式使用，共享，改编，分发和复制，只要您对原始作者和来源给予适当的信任，提供知识共享许可证的链接，并指出是否进行了更改。

The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

本文中的图像或其他第三方材料包含在文章的知识共享许可中，除非在材料的信用额度中另有说明。如果材料未包含在文章的知识共享许可中，并且您的预期用途不受法律法规的许可或超出许可用途，则您需要直接获得版权所有者的许可。

To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/..

要查看此许可证的副本，请访问http://creativecommons.org/licenses/by/4.0/..

Reprints and permissionsAbout this articleCite this articleGrzech, D., Smit, S.J., Alam, R.M. et al. Incorporation of nitrogen in antinutritional Solanum alkaloid biosynthesis.

转载和许可本文引用本文Grzech，D.，Smit，S.J.，Alam，R.M.等人在抗营养茄生物碱生物合成中掺入氮。

Nat Chem Biol (2024). https://doi.org/10.1038/s41589-024-01735-wDownload citationReceived: 15 November 2023Accepted: 19 August 2024Published: 13 September 2024DOI: https://doi.org/10.1038/s41589-024-01735-wShare this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard.

Nat Chem Biol（2024）。https://doi.org/10.1038/s41589-024-01735-wDownload引文接收日期：2023年11月15日接收日期：2024年8月19日发布日期：2024年9月13日OI：https://doi.org/10.1038/s41589-024-01735-wShare本文与您共享以下链接的任何人都可以阅读此内容：获取可共享链接对不起，本文目前没有可共享的链接。复制到剪贴板。

Provided by the Springer Nature SharedIt content-sharing initiative

由Springer Nature SharedIt内容共享计划提供

Subjects

主题

BiosynthesisMetabolic engineeringMetabolic pathwaysNatural productsPlant sciences

生物合成代谢工程代谢途径天然产物植物科学