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

半胱氨酸合成酶（Cysteine synthase，CSase）是高等植物中的一种酶，以O-乙酰丝氨酸和硫化氢为底物，催化生成L-半胱氨酸。4.2.99.8这种酶也负责二级非蛋白杂环β-取代的丙氨酸的生物合成，例如β-吡唑丙氨酸和含羞草素。我们长期研究的最终目标是揭示这些氨基酸生物合成的分子基础，并对产物进行工程改造。我们已经获得了两个cDNA克隆编码的细胞质CSaseA和叶绿体CSaseB从菠菜通过微测序的肽片段，然后合成寡核苷酸。CSaseB的cDNA含有52个氨基酸的转运肽序列。将12个保守的赖氨酸残基插入丙氨酸的定点突变实验，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。获得了整合有35 S-CSaseA cDNA的转基因烟草植株，对转基因植株的分析表明，有义构建体的植株中CSase活性增强，而反义构建体的植株中CSase活性降低。用pET载体构建了原核高效表达系统。通过向表达植物CSase的大肠杆菌培养物中饲喂吡唑，在碧波中产生西瓜的特异代谢产物β-Purazole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.