胡椒碱生物合成机理研究进展

2016-05-30 07:04胡丽松邬华松范睿伍宝朵郝朝运
热带作物学报 2016年5期

胡丽松 邬华松 范睿 伍宝朵 郝朝运

摘 要 以胡椒碱生物合成的前体为主线,结合模式植物生物碱生物合成的研究成果,从胡椒碱合成前体鉴定、莽草酸代谢途径、赖氨酸代谢途径3个方面概述了胡椒碱生物合成机理的研究进展,并对胡椒中胡椒碱生物合成调控的研究进行了讨论,提出了提高胡椒碱合成效率及含量的初步设想。

关键词 胡椒碱;生物合成;赖氨酸代谢;莽草酸途径

中图分类号 TQ464;TS201.2 文献标识码 A

Abstract Piperine is the main quality trait and functional substance in Piper spp. Due to its activity in promoting digestion, anti-inflammatory, decreasing blood-lipid and anti-cancer etc., more and more research was focused on the biosynthesis mechanism of piperine. In the present paper, based on the recent research progress in model plants, the biosynthesis of piperine was reviewed, mainly including the precursor identification, lysine metabolism and shikimic acid pathway. Furthermore, some suggestions for piperine biosynthesis control were discussed.

Key words Piperine; Biosynthesis; Lysine metabolism; Shikimic acid pathway

doi 10.3969/j.issn.1000-2561.2016.05.031

胡椒(Piper nigrum L.)是世界上古老而著名的香料作物,素有“香料之王”的美誉,用途广泛,经济价值高。在医学领域,胡椒被用作健胃剂、镇痛剂、解毒剂等[1-4]。在食品工业中,胡椒常被用作防腐剂、保鲜剂等[5-6]。胡椒中化学成分包括胡椒碱、挥发油、酚类化合物和微量元素等,胡椒碱是其中含量最大、活性最高的生物碱[7]。胡椒碱既承载着胡椒口感热辣和促进食欲的两大传统功能,同时还有抗炎症、抗抑郁、提高药效、降血脂、抑制肿瘤等多种功能[2,7-12]。因此,胡椒碱被公认为是胡椒的主要功能物质以及衡量胡椒品质高低的主要因子[7]。

研究结果表明,胡椒碱是赖氨酸源的生物碱,同时属于苯丙素类衍生物。苯丙素类化合物是广泛存在植物体内的一类苯环与3个直链碳连接(C6-C3基团)构成的天然化合物,一般具有苯环结构,可根据C6-C3基团的数目,分为苯丙素、木脂素、香豆素三大类,而每一大类又可分出多个衍生物[13-16]。在生物合成上,苯丙素类化合物多数由莽草酸通过苯丙氨酸和酪氨酸等芳香氨基酸,经脱氨、羟基化等一系列反应形成[17-20]。植物体内赖氨酸源的生物碱主要包括吲哚里西啶类、喹嗪烷类、哌啶类三大类生物碱。这三类生物碱因其氮原子来源于同一个氨基酸,碳原子骨架构成比较保守,而被称为“True alkaloids”,在合成的初期所经历的赖氨酸代谢也较为保守,在模式植物中赖氨酸代谢的中间产物、参与催化反应的关键基因等相关研究比较清楚[21-23]。

不同作物的生物碱种类及构成各不相同。胡椒碱属于哌啶类生物碱,主要存在于胡椒科植物中,目前,关于胡椒中胡椒碱生物合成的机理研究仍然滞后。本研究将参考模式生物中苯丙素和赖氨酸代谢的研究成果,概述胡椒碱生物合成机制,为胡椒育种、胡椒碱生物工程利用提供参考。

1 胡椒碱的发现及一般性质

胡椒碱为胡椒科植物特有的一种生物碱,存在于多种胡椒属植物中,如胡椒(Piper nigrum)、荜茇(Piper longum)、几内亚胡椒(Piper guineense)等植物的果实和根都发现含胡椒碱[21,24]。1918年,Oersted首次从胡椒果实中分离出一种具有辛辣味的黄色结晶物-胡椒碱(Piperine),分子式为C17H19NO3,结构式见图1[25]。

胡椒碱是一种不溶于水的淡黄色结晶物质,熔点128~133 ℃,溶于乙酸、苯、乙醇和氯仿。在酸性介质下胡椒碱可稳定存在,但在碱性环境中,胡椒碱极不稳定会水解为六氢嘧啶和胡椒碱酸。另外,胡椒碱对光敏感,在自然光照射条件下,胡椒碱易发生异构化反应,生成3种同分异构体: chavicine(异胡椒碱)、piperanine(哌嗪)和piperettine(胡椒亭)(图1)。与胡椒碱相比,其异构体的辛辣味相对比较微弱[26-28]。

2 胡椒碱生物合成途径

2.1 胡椒碱合成前体

为鉴定胡椒碱生物合成的前体物质,Georg G等开展了大量的工作[12,27,29-32]。1987年,胡椒酰胺辅酶A(Piperoyl-coenzyme A)通过体外化学方法成功合成,使得通过体外生物化学的方法研究胡椒碱合成机理成为可能[30]。1990年,Geisler等[29]从胡椒的根尖发现了能催化胡椒碱合成的胡椒碱合成酶(piperidine piperoyltransferase; EC 2.3.1.145),通过比较催化不同底物反应生成胡椒碱的效率,确定了胡椒酰胺辅酶A与六氢吡啶(piperidine)是胡椒碱生物合成的直接前体(图2)。因此,根据胡椒碱生物合成2个前体物质的特征,胡椒碱又名1-胡椒酰哌啶(1-Piperyl piperidine)。胡椒碱合成前体的鉴定为胡椒碱生物合成研究奠定了重要基础,后续的工作主要围绕胡椒酰胺辅酶A和六氢吡啶两个直接前体的代谢及调控等研究展开。

2.2 胡椒酰胺辅酶A生物合成的莽草酸途径

胡椒酰胺辅酶A合成起源于莽草酸途径[30,32]。莽草酸途径的苯丙素类(phenylpropanoids)中间产物是大多数植物芳香类物质的合成前体,如反肉桂酸(trans-cinnamic acid)、咖啡酸(caffeic acid)、阿魏酸(ferulic acid)等,不同的苯丙素所衍生的芳香物质各不相同[18-20,33]。莽草酸途径起始于糖代谢生成的磷酸烯醇丙酮酸(phosphoenolpyruvic acid, PEP)与D-磷酸赤藓糖(D-erythrose-4-phosphate, E4P),两者在合成酶催化下反应生成3-脱氧-阿拉伯庚酮糖酸-7-磷酸(3-deoxy-D-arabino-heptulosonic acid 7-phosphate, DAHP)[34-37]。DAHP通过经去磷酸化、环化、脱水一些列酶促反应生成脱氢奎尼酸(3-dehydroquinic acid, DHQ)、脱氢莽草酸(3-dehydroshikimic acid)、莽草酸(shikimic acid)[20,38-42]。随后,莽草酸在激酶的催化下发生磷酸化反应,生成3-磷酸-莽草酸,3-磷酸-莽草酸与磷酸烯醇丙酮酸反应生成5-烯醇丙酮酰莽草酸-3-磷酸(5-enlpyruvylshikimic acid 3-phosphate, EPSP),EPSP通过去磷酸反应生成分支酸(chorismic acid)(图3)[18-19,33,43-46]。

分支酸的合成是莽草酸途径的一个重要枢纽节点,在这个节点上莽草酸途径产生了两条不同的分支。(1)分支酸经邻氨基苯甲酸合成酶催化反应生成对羟基苯丙酮酸,从而进入色氨酸合成途径[47-48];(2)分支酸在变位酶的作用下生成预苯酸(Prephenic acid),进入苯基丙氨酸、酪氨酸合成途径,胡椒酰胺辅酶A合成的前体香豆酸来自这一支路[49]。预苯酸在转氨酶的作用下生成前酪氨酸(arogenic acid),前酪氨酸可在脱氢酶的作用下生成酪氨酸,也可再经脱水反应生成苯丙氨酸(phenylalanine)[50-52]。苯基丙氨酸在苯丙氨酸氨基裂解酶的作用下脱掉氨基生成肉桂酸(cinnamic acid),肉桂酸在羟化酶的作用下生成香豆酸,此外,酪氨酸也可以通过脱氨基反应生成香豆酸,最终进入胡椒酰胺辅酶A合成途径(图4)[53-57]。

胡椒酰胺辅酶A的合成以香豆酸(coumaric acid)为起始底物。香豆酸通过3-羟化反应生成咖啡酸,咖啡酸被甲基化后生成阿魏酸,阿魏酸的羟基再经氧化反应生成松柏醇(coniferyl alcohol)[58-60]。松柏醇通过辅酶A连接、基团修饰、环化作用后生成基本的胡椒酰胺辅酶A骨架[61-64]。胡椒酰胺辅酶A骨架再通过Claisen延伸(克莱森缩合反应)、脱水反应后最终形成胡椒碱合成的直接前体:胡椒酰胺辅酶A(图5)。

2.3 六氢吡啶生物合成的赖氨酸代谢途径

六氢吡啶的合成起源于赖氨酸,其生物活性、代谢路径已在模式生物中开展大量研究。首先,赖氨酸在脱羧酶的作用下生成戊撑二胺,戊撑二胺经氧化、环化、脱水反应生成四氢吡啶,四氢吡啶经还原反应最终生成六氢吡啶[21]。六氢吡啶是很多生物碱合成的前体物质,如与烟酸反应则生成尼古丁,与乙酰乙酰辅酶A反应则生成石榴碱,与胡椒酰胺辅酶A反应则生成胡椒碱。这类物质统称为哌啶类生物碱[65]。

模式植物的研究结果表明,哌啶类生物碱的合成并不是赖氨酸代谢的唯一途径,其代谢主要有3条路径:第一,赖氨酸在氧化酶的作用下生成ε-醛赖氨酸,然后进入吲哚里西啶生物碱合成途径,如苦马豆素和流涎素的生物合成都是通过这一反应实现,此反应广泛存在于植物、动物、微生物中[66-68]。第二,赖氨酸在脱羧酶的作用下生成戊撑二胺。戊撑二胺可以和丙酮酸盐反应生成氧代鹰爪豆碱,为喹嗪烷类生物碱的合成提供底物。羽扇豆宁、金雀花碱都经由这一支路合成[69-72]。第三,戊撑二胺经氧化、环化、脱水反应生成四氢吡啶,四氢吡啶经还原反应最终生成六氢吡啶,进入哌啶类生物碱合成途径(图6)[23,73-77]。

综上所述,胡椒碱的生物合成途径可归纳如下:一方面,以PEP和E4P为最初反应底物,通过莽草酸途径合成胡椒酰胺辅酶A;另一方面,以赖氨酸为底物,经脱羧、环化等一系列反应生成六氢吡啶。最后,胡椒酰胺辅酶A和六氢吡啶在胡椒碱合成酶的催化下生成胡椒碱。根据文中所述,将胡椒碱生物合成途径中已经鉴定出的主要调控基因进行了汇总(表1)。

3 胡椒碱生物合成调控讨论及展望

胡椒属于典型的热带作物,其种植地区大多为地处热带、亚热带地区的不发达国家,分子育种等相关研究较为滞后。胡椒的消费国主要以发达国家为主,作为一味古老而著名的香料,发达国家在胡椒功能物质分离、保健医疗功效等方面开展了大量研究。胡椒碱作为胡椒最主要的功能物质,在促消化、抗炎症、抗抑郁、提高药效、降血脂、抑制肿瘤等方面具有广泛的作用,一直是研究的热点。因此,明晰胡椒中胡椒碱生物合成机理、克隆关键基因,通过生物工程技术提高胡椒碱含量,既是胡椒生产国品质育种的迫切需求,也是提高胡椒价值的有效途径。

本研究以胡椒碱生物合成的两个直接前体为切入点,参考模式植物莽草酸、赖氨酸代谢的研究进展,对胡椒碱生物合成研究进行了系统解析。在模式植物中,莽草酸代谢途径的研究已很清楚,从最初的呼吸作用产物PEP与E4P的富集到莽草酸、分支酸、香豆酸的合成,其催化反应的关键中间产物以及相关基因都已经被鉴定出来[18,20,43]。因此,通过对代谢途径中关键基因表达的调控,可以促进分支酸、香豆酸等中间产物的合成,以保证胡椒酰胺辅酶A合成有充分的前体物质。然而,从松柏醇到胡椒酰胺辅酶A合成需经过哪些酶促反应,仍然不清楚。笔者根据反应前体基团的变化,预测了反应类型,如claisen延伸反应,芳香族物质环化反应等,但这些反应在胡椒中是否真实存在、参与反应的基因等研究仍属空白。由于莽草酸途径的苯丙素类物质是次生代谢中间产物,不会在细胞内大量积累,不同物种下游的芳香物质不同,后续的合成调控也各不相同,代谢途径支路错综复杂,因此,如何定向调节胡椒酰胺辅酶A的生物合成还需进一步研究。鉴定松柏醇到胡椒酰胺辅酶A合成途径的中间产物、克隆参与该合成途径的关键基因是当前亟需解决的重要课题。

胡椒碱生物合成另一前体来源于赖氨酸代谢产生的六氢吡啶。根据赖氨酸代谢途径的特征,可通过3种方法提高六氢吡啶的合成效率,为胡椒碱合成提供充足前体。第一,从赖氨酸代谢的源头调控,抑制赖氨酸氧化酶,提高赖氨酸脱羧酶的活性,使赖氨酸经脱羧反应生成戊撑二胺,为下游胡椒碱的合成提供前体[23]。第二,控制赖氨酸脱羧产物戊撑二胺的下游反应。豆科植物中戊撑二胺会选择和丙酮酸发生反应,进入喹嗪烷类生物碱的合成途径[22]。而胡椒科植物中戊撑二胺则经过氧化反应进入哌啶环合成途径,为胡椒碱的合成提供底物,因此戊撑二胺下游的反应也是调节胡椒碱合成的重要途径[78]。第三,提高胡椒碱合成酶基因的表达水平及其酶活性,使得更多的哌啶环参与胡椒碱的合成。嘧啶环是植物体内多种生物碱的合成前体,胡椒碱并不是它唯一的路径,因此,克隆胡椒碱合成酶基因,通过基因工程技术超量表达该关键酶基因是提高胡椒碱合成水平的有效手段[79]。

目前,胡椒碱生物合成及其调控机制,主要基于模式植物以及离体实验的研究成果,胡椒中胡椒碱生物合成相关的基因及中间产物尚未得到鉴定。因此,克隆胡椒自身胡椒碱生物合成的关键基因及鉴定其特异的中间产物是后续研究的重点。

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