Constitutive expression of bergaptol O-methyltransferase in Glehnia littoralis cell cultures
Constitutive expression of bergaptol O-methyltransferase in Glehnia littoralis cell cultures
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DOI：
10.1007/s00299-008-0631-9
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发表时间：
2009-02
影响因子：
6.2
通讯作者：
A. Ishikawa;Tatsuya Kuma;H. Sasaki;Nobuhiro Sasaki;Y. Ozeki;N. Kobayashi;Y. Kitamura
中科院分区：
文献类型：
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作者：
A. Ishikawa;Tatsuya Kuma;H. Sasaki;Nobuhiro Sasaki;Y. Ozeki;N. Kobayashi;Y. Kitamura
我们研究了是否可以利用外源提供的佛手柑内酯的前体，即伞形酮，pepsilen和bergaptol，在北沙参细胞悬浮培养中无诱导产生佛手柑内酯。培养基中添加的补骨脂素和佛手柑酚的水平很快下降，随后在培养液和细胞中检测到佛手柑内酯。还掺入了伞形酮，但在这种情况下，没有产生佛手柑内酯;相反，检测到了脱脂蛋白，伞形酮单葡糖苷。为了确定是否通过前体进料的酶诱导将补骨脂素转化为佛手柑内酯，检测了催化佛手柑内酯合成的最后一步的佛手柑内酯O-甲基转移酶（BMT，EC 2.1.1.69）和催化类苯丙素生物合成途径的初始步骤并被称为引发标记酶的苯丙氨酸氨裂解酶（PAL，EC4.3.1.5）的转录物积累和酶活性。结果表明，BMT的表达和活性在所有的细胞中，包括对照细胞。由于PAL在有/无前体的细胞中被轻微诱导，因此在添加peptide之前将苯乙醇（PEA，PAL的竞争性抑制剂）应用于悬浮细胞。在1-5 mM浓度下，PEA有效地抑制PAL活性。在这些条件下，PEA不影响bergapten生产的细胞培养物与peptien在所有。这些结果表明BMT在G.细胞培养
We investigated whether exogenously supplied precursors of bergapten, namely umbelliferone, psoralen and bergaptol, could be utilized to produce bergapten without elicitation inGlehnia littoraliscell suspension cultures. The levels of added psoralen and bergaptol in the medium soon decreased, and this was followed by the detection of bergapten in both culture fluid and cells. Umbelliferone was also incorporated but in this case no bergapten was produced; instead, skimmin, umbelliferone monoglucoside, was detected. To determine whether conversion of psoralen to bergapten was due to enzyme induction by precursor feeding, the transcript accumulations and enzyme activities of bergaptolO-methyltransferase (BMT, EC 2.1.1.69), which catalyzes the last step of bergapten synthesis, and of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), which catalyzes the initial step of the phenylpropanoid biosynthetic pathway and is known as a marker enzyme of elicitation, were examined. The results showed that both the expression and the activity of BMT were always detected in all cells, including control cells. Since PAL was slightly induced in the cells supplied with/without precursors, phenylethyl alcohol (PEA, a competitive inhibitor of PAL) was applied to suspension cells prior to the addition of psoralen. PAL activity was effectively inhibited by PEA at 1–5 mM concentrations. Under these conditions, PEA did not affect bergapten production by cell cultures fed with psoralen at all. These results demonstrate that BMT is constitutively expressed inG. littoraliscell cultures.