PRODUCTION OF 2-KETO-L-GULONATE, AN INTERMEDIATE IN L-ASCORBATE SYNTHESIS, BY A GENETICALLY MODIFIED ERWINIA-HERBICOLA
PRODUCTION OF 2-KETO-L-GULONATE, AN INTERMEDIATE IN L-ASCORBATE SYNTHESIS, BY A GENETICALLY MODIFIED ERWINIA-HERBICOLA
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DOI：
10.1126/science.230.4722.144
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发表时间：
1985-01-01
期刊：
影响因子：
56.9
通讯作者：
ESTELL, D
中科院分区：
文献类型：
--
作者：
ANDERSON, S;MARKS, CB;ESTELL, D
在微生物Erwiniacidola 1中创造了一种新的代谢途径，使其能够产生2-酮基-L-古洛糖酸，这是合成L-抗坏血酸的重要中间体。首先，鉴定并纯化了一种能够立体选择性地将2，5-二酮基-D-葡萄糖酸还原为2-酮基-L-古洛糖酸的棒状杆菌酶。根据2，5-二酮-D-葡萄糖酸还原酶的氨基酸序列信息，用DNA探针从棒状杆菌基因组文库中分离该酶的基因。将2，5-二酮基-D-葡萄糖酸还原酶编码区与大肠杆菌的启动子和一个人工合成的核糖体结合位点融合，然后导入E.欧文氏菌通过葡萄糖氧化天然产生2，5-二酮基-D-葡萄糖酸，并且当表达质粒编码的还原酶的重组细胞在葡萄糖存在下生长时，产生2-酮基-L-古洛糖酸并释放到培养基中。这些数据证明了通过将来自不同来源的有用代谢特征结合在单个生物体中来创建用于合成重要特种化学品的新型体内途径的可行性。
A new metabolic pathway has been created in the microorganismErwinia herbicolathat gives it the ability to produce 2-keto-L-gulonic acid, an important intermediate in the synthesis of L-ascorbic acid. Initially, aCorynebacteriumenzyme that could stereoselectively reduce 2,5-diketo-D-gluconic acid to 2-keto-L-gulonic acid was identified and purified. DNA probes based on amino acid sequence information from 2,5-diketo-D-gluconic acid reductase were then used to isolate the gene for this enzyme from aCorynebacteriumgenomic library. The 2,5-diketo-D-gluconic acid reductase coding region was fused to theEscherichia colitrp promoter and a synthetic ribosome binding site and was then introduced intoE. herbicolaon a multicopy plasmid.Erwinia herbicolanaturally produces 2,5-diketo-D-gluconic acid via glucose oxidation, and when recombinant cells expressing the plasmid-encoded reductase were grown in the presence of glucose, 2-keto-L-gulonic acid was made and released into the culture medium. The data demonstrate the feasibility of creating novel in vivo routes for the synthesis of important specialty chemicals by combining useful metabolic traits from diverse sources in a single organism.