An efficient downstream box fusion allows high-level accumulation of active bacterial beta-glucosidase in tobacco chloroplasts
An efficient downstream box fusion allows high-level accumulation of active bacterial beta-glucosidase in tobacco chloroplasts
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DOI：
10.1007/s11103-011-9743-7
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发表时间：
2011-07-01
影响因子：
5.1
通讯作者：
Hanson, Maureen R.
中科院分区：
文献类型：
--
作者：
Gray, Benjamin N.;Yang, Huijun;Hanson, Maureen R.
已经提出在植物中生产用于木质纤维素水解的酶作为微生物生产的低成本替代方案，由于高外源蛋白产量，质体转化作为优选方法。叶绿体蛋白质产生的一个重要调节因子是下游盒（DB）区，位于起始密码子的下游。通过改变DB区域，蛋白质积累可以在几个数量级上变化。在细菌中的实验表明，这些蛋白质积累的差异可能是由翻译效率的变化引起的，尽管DB功能的精确机制尚不清楚。在这项研究中，三个DB区域与编码来自嗜热细菌嗜热裂菌（Thermobifida fusca）的β-葡萄糖苷酶的bglC ORF融合，并插入烟草（Nicotiana tabacum）质体基因组中。观察到BglC蛋白积累的差异超过两个数量级，这取决于DB融合体的特性。差异转录物积累解释了一些观察到的蛋白质积累的差异，但此外，在积累最多BglC酶的转基因植物中观察到较少的3'降解bglC转录物。叶绿体产生的BglC对纯纤维二糖和烟草木质纤维素都有活性。这些实验证明了转质体植物作为用于纤维素乙醇工业的异源β-葡糖苷酶生产的载体的潜在效用。
Production of enzymes for lignocellulose hydrolysis in planta has been proposed as a lower-cost alternative to microbial production, with plastid transformation as a preferred method due to high foreign protein yields. An important regulator of chloroplast protein production is the downstream box (DB) region, located immediately downstream of the start codon. Protein accumulation can vary over several orders of magnitude by altering the DB region. Experiments in bacteria have suggested that these differences in protein accumulation may result from changes in translation efficiency, though the precise mechanism of DB function is not known. In this study, three DB regions were fused to the bglC ORF encoding a beta-glucosidase from the thermophilic bacterium Thermobifida fusca and inserted into the tobacco (Nicotiana tabacum) plastid genome. More than a two order of magnitude of difference in BglC protein accumulation was observed, dependent on the identity of the DB fusion. Differential transcript accumulation explained some the observed differences in protein accumulation, but in addition, less 3' degradation of bglC transcripts was observed in transgenic plants that accumulated the most BglC enzyme. Chloroplast-produced BglC was active against both pure cellobiose and against tobacco lignocellulose. These experiments demonstrate the potential utility of transplastomic plants as a vehicle for heterologous beta-glucosidase production for the cellulosic ethanol industry.