Gene Engineering and Protein Engineering of Secondaty Metabolism in Medicinal Plants.

高等植物半胱氨酸合成酶(O-乙酰丝氨酸(硫醇)裂解酶，EC 4.2.99.8)催化乙酰基-L-丝氨酸和硫化氢合成L-半胱氨酸。该酶还可用于次生非蛋白质杂环β-取代丙氨酸的生物合成，如β-吡唑丙氨酸和含草胺。UOR长期研究的最终目的是揭示这些氨基酸生物合成的分子基础，并设计其可生产性。以菠菜为材料，利用合成的寡核苷酸对多肽片段进行微测序，获得了两个编码细胞质CSaseA和叶绿体CSaseB的基因克隆。CSaseB的cDNA包含52个氨基酸的转运肽序列。通过定点突变实验，将12个保守的赖氨酸残基插入到ALA中，确定了一个与磷酸吡哆醛辅因子结合的功能性赖氨酸残基。获得了整合了35S-CSaseA基因构建体的转基因烟草植株，对转基因植株的分析表明，含有正义构建体的植株CSase活性增强，而反义构建体植株的CSase活性降低。利用pET载体构建了原核高效表达系统。用表达植物CSase的大肠杆菌培养物添加吡唑，在Bibo中生产西瓜的特异代谢产物--β-精氨唑丙氨酸。

Cysteine synthase (CSase) (O-acetylserine(thiol)-lyase, EC 4.2.99.8) in higher plants catalyxes the formation of L-cysteine from Oacetyl-L-serine and hydrogen sulfide as the substrates. This enzyme is also reponsible for the biosynthesis of secondary non-protein heterocyclic beta-substituted alamines, e.g. beta-pyrazole alanine and mimosine. The final aims of uor long-term study are to reveal molecular basis for the biosynthesis of these amino acids and to engineer the producibility. We have obtained two cDNA clones encoding cytoplasmic CSaseA and chloroplast CSaseB from spinach by means of the microsequencing of peptide fragments followed by synthetic oligonucleotide. The cDNA for CSaseB contained the transit peptide sequence of52 amino acids. The experiments of site-directed mutagenesis teplacing 12 conserved Lysresidues into Ala indentified a functional Lys residue respansible for the binding of pyridoxal phosphate cofactor. Transgenic tobacco plants, integrated with the constructs for 35S-CSaseA cDNA, were obtained.analysis of transgenic plants showed the enhanced CSase activities in the plants with the sense constructs and the decreased activity in the antisense-construct plants. Theprokaryotic high expression system was estabkished with pET vector. beta-Purazole alanine, a specific metabolite of watermelon, was produced in bibo by feeding pyrazole to E.coli cuture expressing plant CSase.