Metabolic engineering of glycine betaine synthesis: plant betaine aldehyde dehydrogenases lacking typical transit peptides are targeted to tobacco chloroplasts where they confer betaine aldehyde resistance

Certain higher plants synthesize and accumulate glycine betaine, a compound with osmoprotectant properties. Biosynthesis of glycine betaine proceeds via the pathway choline betaine aldehyde glycine betaine. Plants such as tobacco (Nicotiana tabacum L.) which do not accumulate glycine betaine lack the enzymes catalyzing both reactions. As a step towards engineering glycine betaine accumulation into a non-accumulator, spinach and sugar beet complementary-DNA sequences encoding the second enzyme of glycine-betaine synthesis (betaine aldehyde dehydrogenase, BADH, EC 1.2.1.8) were expressed in tobacco. Despite the absence of a typical transit peptide, BADH was targeted to the chloroplast in leaves of transgenic plants. Levels of extractable BADH were comparable to those in spinach and sugar beet, and the molecular weight, isoenzyme profile and K _m for betaine aldehyde of the BADH enzymes from transgenic plants were the same as for native spinach or sugar beet BADH. Transgenic plants converted supplied betaine aldehyde to glycine betaine at high rates, demonstrating that they were able to transport betaine aldehyde across both the plasma membrane and the chloroplast envelope. The glycine betaine produced in this way was not further metabolized and reached concentrations similar to those in plants which accumulate glycine betaine naturally. Betaine aldehyde was toxic to non-transformed tobacco tissues whereas transgenic tissues were resistant due to detoxification of betaine aldehyde to glycine betaine. Betaine aldehyded ehydrogenase is therefore of interest as a potential selectable marker, as well as in the metabolic engineering of osmoprotectant biosynthesis.Abbreviations BADH betaine aldehyde dehydrogenase - bp base pairs - FAB-MS fast atom bombardment-mass spectrometry - GAPDH NADP-linked glyceraldehyde-3-phosphate dehydrogenase We thank Dr. G. An for the gift of the vector pGA643 and Mr. Sylvain Lebeurier for help in maintaining plants. This work was supported, in part, by grants from the Natural Sciences and Engineering Research Council of Canada, the Rockefeller Foundation, and the U.S. Department of Agriculture, and by gifts from CIBAGEIGY Biotechnology.     

山菠菜胆碱单氧化物酶基因(CMO)的克隆与分析

甜菜碱是一类广泛存在于生物体内的渗透保护剂。高等植物中,甜菜碱的生物合成经由胆碱→甜菜碱醛→甜菜碱两步反应完成,其中第一步反应,也是甜菜碱生物合成的限速反应,由胆碱单氧化物酶(CMO)催化。本研究以耐盐植物山菠菜(Atriplex hortensis)为材料构建了盐胁迫下的cDNA文库,用菠菜CMO cDNA为探针从中筛选获得一个长1.77kb的cDNA克隆,测序结果表明该克隆包含一个完整的开放读码框,编码一个由438个氨基酸构成的多肽,与菠菜和甜菜CMO的氨基酸序列同源性分别为81%和72%。同菠菜和甜菜中的CMO序列相比,山菠菜CMO基因(AhCMO)也具有保守的RieskeType［2Fe2S］簇结合区和保守的多铁原子核结合域。对盐处理条件下山菠菜CMO基因转录水平的研究表明CMO基因在盐胁迫情况下表达量增加约3倍。将CMO与35S启动子连接后转化烟草(Nictiana tabacumvar.Xanthi),获得了具有一定耐盐性状的转基因植株,在1.2%NaCl的盐浓度下生长良好。     

Isolation of a choline monooxygenase cDNA clone from Amaranthus tricolor and its expressions under stress conditions

INTRODUCTIONAmaranth is a C4 dicotyledonous mesophytecrop plant. A. tricofor is a major variety for veg-etable and ornamental crops, and is widely culti-vated in the wor1d. Osmoprotectant glycine betaine(GB) was detected in Amaranthaceae, A. HyPochon-driacus L[2] and A. Caudatus L[3, 4]. GB iswidespread and an effective osmoprotectant in manyplants[3]. We studied the photosynthetic adaptationmechanism of A. trico1or under salt stress due to ac-cumulation of GB[5].GB is synthesized …     

中亚滨藜甜菜碱醛脱氢酶基因的表达特性

根据已发表的几种植物的甜菜碱醛脱氢酶（BADH）基因的同源保守区设计了一对兼并引物,通过RT-PCR方法从中亚滨藜中扩增出BADH基因的近5′端序列,共395bp,与菠菜、山菠菜、甜菜、千穗谷、大麦的BADHcDNA相应片段的同源性较高。以此片段为探针,对中亚滨藜的基因组进行Southern杂交分析,证明该基因可能是单拷贝的。Northern印迹杂交结果表明NaCl250mmol/L处理的植株的BADHmRNA水平比对照植株约高2倍,说明中亚滨藜中BADH基因的表达受盐诱导。     

菠菜甜菜碱醛脱氢酶基因的克隆和序列分析

以耐盐的菠菜mRNA为模板,经反转录合成甜菜碱醛脱氢酶(BADH)基因第一链cDNA。在人工合成的两端引物引导下,通过多聚酶链式反应(PCR),扩增获得双链cDNA。把重组有BADH基因的pUC19转化至E.coli DH5α菌株,亚克隆后测定了基因的全序列。所得到的BADH基因全长序列为1491bp,编码497个氨基酸。与文献报道的相比较,核苷酸序列同源性99.8％,氨基酸序列同源性达99.6％。在此基础上,构建了BADH基因的高等植物表达载体。     

Overproduction of spinach betaine aldehyde dehydrogenase in Escherichia coli. Structural and functional properties of wild-type, mutants and E. coli enzymes.

Betaine aldehyde dehydrogenase (BADH) catalyzes the last step in the synthesis of the osmoprotectant glycine betaine from choline. Although betaine aldehyde has been thought to be a specific substrate for BADH, recent studies have shown that human and sugar beet BADHs also catalyze the oxidation of omega-aminoaldehydes. To characterize the kinetic and stability properties of spinach BADH, five kinds of expression vectors encoding full length, mature, E103Q, E103K, and chimera BADHs were constructed. These enzymes together with Escherichia coli BADH were expressed in E. coli and purified. The affinities for betaine aldehyde were similar in the spinach and E. coli BADHs, whereas those for omega-aminoaldehydes were higher in spinach BADH than in E. coli BADH. A chimera BADH in which part of the Rossmann type fold in the spinach BADH was replaced with that of E. coli BADH, showed properties which resembled spinach BADH more than E. coli BADH. The spinach E103K mutant was almost inactive, whereas the E103Q mutant showed a similar activity for the oxidation of betaine aldehyde to that of wild type BADH, but a lower affinity for omega-aminoaldehydes. All spinach BADHs were dimers whereas E. coli BADH was a tetramer. E. coli BADH was more stable at high temperature than spinach BADHs. The E103Q mutant was most labile to high temperature. These properties are discussed in relation to the structure of spinach BADH.     

Isolation of cDNA and enzymatic properties of betaine aldehyde dehydrogenase from Zoysia tenuifolia

We isolated cDNAs encoding betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8) from the salt-tolerant Poaceae, Zoysia tenuifolia by polymerase chain reactions. Zoysia betaine aldehyde dehydrogenase 1 (ZBD1) is 1892bp long and codes for 507 amino acids. The deduced amino acid sequence of ZBD1 is 88% similar to the sequence of rice BADH. Ten cDNA clones were isolated from a cDNA Library of salt-treated Z. tenuifolia by using the ZBD1 fragment as a probe. The proteins coded in some clones were more homologous to BBD2, the cytosolic BADH of barley, than to ZBD1. To investigate their enzymatic properties, ZBD1 and spinach BADH were expressed in Escherichia coli and purified. The optimal pH of ZBD1 was 9.5, which was more alkaline than that of spinach BADH. ZBD1 was less tolerant to NaCl than spinach BADH. ZBD1 showed not only BADH activity but also aminoaldehyde dehydrogenase activity. The Km values of ZBD1 for betaine aldehyde, 4-aminobutyraldehyde (AB-ald), and 3-aminopropionaldehyde (AP-ald) were 291, 49, and 4.0 microM, respectively. ZBD1 showed higher specific activities for AB-ald and AP-ald than did spinach BADH.     

Transgenically Expressed Betaine Aldehyde Dehydrogenase Efficiently Catalyzes Oxidation of Dimethylsulfoniopropionaldehyde and [omega]-Aminoaldehydes

Tobacco (Nicotianum tabacum L.) plants engineered to express a sugar beet (Beta vulgaris L.) betaine aldehyde dehydrogenase (BADH) cDNA acquired not only BADH activity, but also three other aldehyde dehydrogenase activities (those measured with 3-dimethylsulfoniopropionaldehyde, 3-aminopropionaldehyde, and 4-aminobutyraldehyde, all of which are natural products). This shows that BADH is not, as believed up to now, a substrate-specific enzyme and that its role may not be limited to glycine betaine synthesis.